Download Chapter I Introduction Plant growth regulators or phytohormones

CHAPTER I
INTRODUCTION
Plant growth regulators or phytohormones, either naturally occurring or synthetic,
can increase yields of a target plant. These include auxins, cytokinins, gibberellins, ethylene
and abscisic acid (ABA) (Farooq et al 2009). The phytohormone abscisic acid (ABA)
controls many important aspects of plant growth and development including seed
development, desiccation tolerance of seeds, seed dormancy etc. Besides these, it plays
crucial role in the plant’s response to abiotic and biotic stresses (Chinnusamy et al 2004).
Plants accumulate increased amounts of ABA under drought, cold or salt stress conditions
where drought stress is having the most prominent effect (Finkelstein et al 2002).
Two ABA-dependent regulatory pathways have been proposed. One is bZip/ABRE
system and other is MYC/MYB system (Agarwal et al 2006), however there are several
ABA-independent pathways are also proposed to be working in plants where one pathway
includes DREB/CBF system (Fowler and Thomashow 2002). Ultimate effectors of these all
regulatory pathways are stress responsive genes which have been categorized into different
groups like genes encoding transcription factors, protein kinases (Kaur and Gupta 2005),
genes encoding proteins involved in the protection of membranes and proteins of the cell like
LEA proteins, HSPs, chaperons etc (Rodriguez et al 2005), genes encoding osmoregulatory
proteins (Bohnert et al 1995), genes encoding antioxidant and detoxifying proteins (Bartosz
1997), genes encoding ion transporters/channels (Bohnert et al 1995). ABA-responsive genes
are either containing ABRE (ABS Response Element) in their promoters region recognized
by bZip/ABF transcription factors or MYBRS/MYCRS (MYC Recognition site/ MYB
Recognition Site) recognized by MYC/MYB transcription factors. Many ABA-responsive
genes have been identified like genes encoding seed storage proteins, LEA and various other
proteins. Examples of ABA-responsive abiotic-stress related genes are Em in wheat, Em and
Rab16 in rice, Hva1, Hva2 and dehydrins in barley, Em and Rd29 in Arabidopsis thaliana
(Bray 1993, Zhang et al 2005).
LEA-proteins are Late Embryogenesis Abundant-proteins which are accumulated in
seeds during seed maturation and play roles in protection of cell membranes and enzymes
during dessication period after seed maturation. LEA-proteins have been classified into nine
groups based on their sequence similarities and conserved domains (Hundertmark and
Hincha 2008). Among nine groups, roles of only group 1, 2 and 3 have been investigated in
imparting abiotic stress tolerance. ABA is found to induce expressions of these genes during
seed development and abiotic stresses (Finkelstein et al 2002, Dalal et al 2009). Many LEA
genes are reported to be responsive to ABA but some are non-responsive to ABA also. As
LEA-proteins play important roles in protecting cells’ structures under stresses, hence these
represent a group of stress-responsive genes. These genes are the downstream genes of both
ABA-dependent and –independent signal pathways (Hong-Bo et al 2005, Kobayashi et al
2008a, 2008b). In wheat, reports related to such type of studies like elucidating the ABAdependency or –independency of LEA genes are very few except few reports like LEA genes
(belonging to group 2 and group 3) were studied in wheat seedlings under low temperature,
drought and ABA stresses, four of them were found to be responsive to ABA (Kobayashi et
al 2004, 2006) and later found to contain ABRE in their promoter regions (Kobayashi et al
2008a, 2008b).
One mode of ABA action is related to oxidative stress in plant cells. Role of ABA in
the induction of antioxidant defence has been the subject of extensive research. It has been
documented that ABA resulted in increased generation of ROS in maize (Guan et al 2000,
Jiang and Zhang 2001, 2002a, 2002b, 2002c, Hu et al 2005), Vicia faba (Zhang et al 2001);
induced the expression of antioxidant genes encoding SOD, CAT in maize (Guan et al 2000),
rice (Kaminaka et al 1999); enhanced the activities of antioxidant enzymes (SOD, CAT,
APX, GR) and increased contents of antioxidant metabolites (ascorbic acid, glutathione, tocopherol, carotenoids) in maize (Jiang and Zhang 2001), tobacco (Bueno et al 1998),
cotton (Bellaire et al 2000 ). However, all antioxidant genes are not responsive to ABA as
both ABA-dependent and ABA-independent signalling pathways are found to be involved in
expression of antioxidant genes under abiotic stresses (Bellaire et al 2000, Guan et al 2000).
LEA proteins and antioxidant mechanism play important roles in stress resistance
mechanism of plant. As ABA is involved in regulation of such enzymes/genes during abiotic
streses, elucidation of ABA-dependency/independency of these proteins/genes under stresses
will contribute to understanding of stress tolerance mechanism of wheat plant. Comparing
such responses in two wheat cultivars contrasting in drought tolerance for these
enzymes/genes will generate better understanding of such mechanisms. The present study
was aimed, to study such responses under water stress and to compare same in two wheat
cultivars PBW343 (drought susceptible) and C306 (drought resistant) with following
objectives:
1. To understand antioxidation mechanism of wheat seedlings at molecular level under
drought in the presence and absence of exogenous ABA.
2. To study role of LEA genes of wheat seedlings under drought and studying for
ABA-dependent or independent induction of these genes.
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CHAPTER II
REVIEW OF LITERATURE
2.1 Late embryogenesis abundant (LEA) proteins
2.1.1 Late embryogenesis abundant proteins
2.1.2 Desiccation induced folding of LEA
2.1.3 Classification
2.1.3.1 Group 1
2.1.3.2 Group 2
2.1.3.3 Group 3
2.1.3.4 Group 4
2.1.4 Localization of dehydrin in plants treated by different stresses
2.1.5 Association in expression of stress responsive genes
2.1.6 Expression of LEA genes in different plants at different developmental stages
2.1.7 Induction of expression of Lea genes
2.1.8 Promoter analysis of stress inducible genes
2.2 Antioxidant metabolism
2.2.1 Reactive oxygen species and their production sites
2.2.2 ROS scavenging enzymatic antioxidants
2.2.2.1 Superoxide dismutase
2.2.2.2 Catalase
2.2.2.3 Ascorabte peroxidase
2.2.2.4 Guaicol peroxidase
2.2.2.5 Glutathione reductase
2.2.3 Non-enzymatic antioxidants
2.2.3.1 Ascorbic acid
2.2.3.2 Reduced glutathione
2.2.3.3 Proline
2.2.4 Hydrogenperoxide (H2O2)
2.2.4.1 Cellular and extracellular sources of H2O2
2.2.4.1.1 Chloroplast
2.2.4.1.2 Peroxisomes
2.2.4.1.3 Mitochondria
2.2.4.1.4 Other sources
2.2.4.2 H2O2 in stress conditions and as signaling molecule
2.2.4.3 H2O2 signaling during growth and development
2.2.5
Malondialdehyde
2.3 Abscisic acid
2.3.1 ABA as a long distance signal mediating whole plant response to
drought and salt stress
2.3.2 Cell signaling from stress perception to ABA accumulation
2.3.3 ABA and antioxidant defense in plant cell
2.3.3.1
ABA –induced ROS generation
2.3.3.2
Expression of genes encoding antioxidant enzymes
induced by ABA
2.3.3.3
ABA induced modulation of metabolic and redox control
pathways
Plants have evolved two major mechanisms to cope up with water deficit: stress
avoidance and stress tolerance. Stress avoidance is achieved by specialized morphological
adaptations in plant architecture e.g. the development of specialized leaf structures to
decrease the rate of transpiration, the reduction of leaf area, sunken stomata or increase in
root length and density to use water more efficiently. Water stress tolerance appears to be the
result of co-ordination of physiological and biochemical alteration at cellular and molecular
level: i.e. the accumulation of various osmolytes and late embrogenesis abundant proteins
coupled with efficient antioxidant system (Ramanjulu and Bartels 2002).
2.1.1
Late Embryogenesis Abundant Proteins (LEA)
LEA proteins are the family of hydrophilic proteins that forms an integral part of
dessication tolerance of seeds. LEA proteins were first identified by Leon Dure III about 29
yrs ago when he observed the proteins accumulated in high level during maturation phase of
cotton (Gossypium hirsutum) embryogenesis. Subsequently LEA like proteins or their genes
were found in seeds of many other plants as well as in vegetative organs when plant exposed
to stress conditions such as cold, drought, high salinity or ABA. They are mainly localized in
cytoplasm and nuclear region. Their common structural features include a high
hydrophilicity, a lack or low proportion of Cys and Trp residues and a preponderance of
certain amino acid residues such as Gly, Ala, Glu, Lys/Arg and Thr which later led them to
be considered as a subset of hydrophilins (Garay-Arroyo et al 2000).
LEA proteins are class of hydrophilic proteins expressed in late seed development
and have been consistently associated with dessication tolerance because of their expression
profile and their hydrophilicity. LEA proteins are prominent in plants (there are > 50 lea
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genes in Arabidopsis Thaliana) but have also been found and related to dessication tolerance
in anhydrobiotes belonging to other kingdoms, such as bacteria and nematodes (Battista et al
2001, Browne et al 2002). A number of mechanisms have been proposed to justify their role
in dessiaction tolerance: water replacement, ion sequestering, macromolecules and
membrane stabilization. Experimentally, several lea proteins were shown to behave invitro as
cryoprotectants (Bravo et al 2003), stabilizes glassy states (Wolkers et al 2001), prevent
protein aggregation (Goyal et al 2005) and bind to lipid vesicles (Koag et al 2003) or actin
filaments (Abu-Abied et al 2006). The molecular mechanism of their action is rather
enigmatic. One likely reason for the slow progress in this area of structure-function
characterization of lea protein is that they are highly hydrophilic and lack well defined
structures (Goyal et al 2003, Mouillon et al 2006).
2.1.2
Dessication induced folding of LEA proteins
Lea proteins seem to be natively unfolded, at least in the hydrated state. Structural
disorder confers advantages onto the protein such as increased speed of interactions, the
combination of specificity with weak and reversible binding and able to carry out more than
one function (Tompa et al 2004). Recent experimental data showed conformational shift on
dehydration of group3 lea protein from nematode AavLEA1. Fourier transform infrared
spectronic analysis shows that protein become more folded, developing a significant alphahelical component when dried. This was extremely unusual observation because protein
dehydration is more often associated with a loss of structure, aggregation rather than increase
in structure, folding or subunit assembly.
2.1.3
Classification
Initially, LEA proteins were referred to according to their molecular weights-D-7, D-
11, D-19, D-29, D-34, D95, D113. Later LEA proteins were classified according to the
appearance of different sequence motifs/patterns or biased amino acid composition, into
seven distinctive groups or families. Groups 1, 2, 3, 4, 6 and 7 correspond to the hydrophilic
or ‘‘typical’’ LEA proteins, whereas those LEA proteins that show hydrophobic
characteristics (‘‘atypical’’) have been kept in group 5, where they could be subclassified
according to their homology A computational method called protein or oligonucleotide
probability profile (POPP) analysis has recently been developed in peptide composition
rather than in sequence (Wise 2003).
2.1.3.1 Group 1
Sequence motifs and peptide profiles
This set of LEA proteins, originally represented by the D-19 and D-132 proteins
from developing cotton seeds, were recognized by an internal 20-mer sequence
(TRKEQ[L/M]G[T/ E]EGY[Q/K]EMGRKGG[L/E]). They contain a very large proportion
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of charged residues, which contributes to their high hydrophilicity and a high content of Gly
residues (approximately 18%).
Secondary structure
They exist largely as random coils or unstructured in aqueous solution. Structural
analyses using circular dichroism (CD) strongly indicate that group1 members exhibit a high
percentage (70%–82.5% of their residues) of random coil configuration in aqueous solution,
with a small percentage of the protein exhibiting a left-handed extended helical or poly-(LPro)-type (PII) conformation (Soulages et al 2002).

Functions
Their possible role in the adaptation of different organisms to water scarcity is
supported by the fact that the transcripts of bacterial group 1 LEA-like proteins also
accumulate under stressful conditions such as stationary growth phase, Glc or phosphate
starvation, high osmolarity, high temperature, and hyperoxidant conditions. Further evidence
comes from the presence of these proteins in organisms with extreme habitats, such as
archaeons (uncultured like methanogenic RC1), as well as in some primordial saltwater
crustaceans such as Artemia (Wang et al 2007). Group 1 LEA-like proteins are particularly
abundant in the thick-shelled eggs of Artemia, whose encysted form can survive in a dried,
metabolically inactive state for 10 or more years while retaining the ability to endure severe
environmental conditions (Macrae 2005).
Direct evidence showing a function for group 1 LEA proteins is scarce. In vitro
experiments using recombinant versions of wheat (Triticum aestivum) Em protein suggested
their ability to protect citrate synthase or LDH from aggregation and/or inactivation due to
desiccation or freezing (Goyal et al 2005, Gilles et al 2007). A mutation in the predicted ahelical domains in the N terminus of the rEm protein suggested a role for this region in
providing protection from drying (Gilles et al 2007). Tolerance to stress conditions induced
by the constitutive expression of genes from this group has not been reported in plants;
however, the expression of wheat TaEm in Saccharomyces cerevisiae seems to attenuate the
growth inhibition of yeast cultures normally observed in high-osmolarity media. Also, the
absence of one of two group 1 members in Arabidopsis plants led to a subtle phenotype of
premature seed dehydration and maturation, suggesting a role during seed development
(Manfre et al 2006). The expression in vegetative tissues from plants grown under optimal
growth conditions of some of the group 1 LEA proteins implies that they may also have a
role during normal seed development.
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2.1.3.2 Group 2
Sequence motif and peptide profile
A distinctive feature of group 2 LEA proteins is a conserved, Lys-rich 15-residue motif,
EKKGIMDKIKEKLPG, named the K -segment which can be found in one to 11 copies
within a single polypeptide. An additional motif also found in this group is the Y-segment,
whose conserved consensus sequence is [V/T]D[E/Q]YGNP, usually found in one to 35
tandem copies in the N terminus of the protein . Many proteins of this group also contain a
tract of Ser residues, called the S-segment (Jiang and Wang 2004). The presence and
arrangement of these different motifs in a single polypeptide allow the classification of group
2 LEA proteins into five subgroups. Proteins that only contain the K-segment are in the Ksubgroup, and those that include the S-segment followed by K-segment are in the SKsubgroup. In addition, there are the YSK-, YK-, and KS-subgroups. Only K -segment is
present at least once in all dehydrins, making it the distinguishing feature of this group.
Functional significance of S-segment is known. It is phosphorylated leading to calcium
binding activity in some dehydrins.
Secondary structure
CD spectra of full length Arabidopsis dehydrins (COR47, LTI29, LTI30, RAB18) and
isolated peptides (K-, Y- and K-rich segments) showed mostly unordered structures in
solution, with a variable content of poly-Pro helices. However, neither temperature, metal
ions, nor stabilizing salts could promote ordered structures in either the peptides or the fulllength proteins. The motif studied most in depth was the K-segment of dehydrin. It can form
amphipathic hydrophilic alpha-helix structure which is probably involved in protein-protein
interactions and protein-lipid interactions (Mouillon et al 2006). Experimental evidence
showed that K-segment was important to stabilize other cellular components under stress
conditions. S-segment and RRKK segment were considered to be involved in nucleus
localization signal. Y-showed similarity to nucleic acid binding site of chaperon.
Role
Like group 1 LEA proteins, several studies of specific group 2 LEA proteins have
confirmed that they accumulate during seed desiccation and in response to water deficit
induced by drought, low temperature,or salinity (Nylander et al 2001).These proteins are also
present in nearly all vegetative tissues during optimal growth conditions (Rorat et al 2004).
A role in bud dormancy has also been attributed to group 2 LEA proteins (Karlson et al
2003a, 2003b). The ability to withstand freezing is highly developed in some trees, in which
the buds, which are critical for reassuming growth after winter, can build up tolerance to
temperatures as low as -196 ºC. Therefore, of particular interest is the fact that LEA proteins
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from this group are expressed in birch (Betula spp.) apices during wintertime dehydration, a
period in which buds become highly desiccated during endodormancy (Puhakainen et al
2004b). Similarly, the accumulation of chilling-responsive LEA proteins from this group was
detected in floral buds of blueberry (Vaccinium myrtillus), a woody perennial.
2.1.3.3 Group 3
Group3 lea proteins are characterized by multiple copies of an 11-amino acid motif
TAQAAKEKAGE (Battaglia et al 2008). The protein sequences have regularly spaced lysine
repeats. Peptide composition also showed that alanine and glutamine are overexpressed,
where as glycine is underrepresented (Wise 2003, Tunnacliffe and Wise 2007).

Secondary structure
Secondary structure prediction of group3 proteins by bioinformatics tool suggests
that 11-mer repeating unit principally form amphipathic alpha-helices, in the presence of
sucrose and glycerol after fast drying, which may dimerizes into an unusual right handed
coiled-coil arrangement (Battaglia et al 2008). Slow drying reversibly leads to both alphahelical and intermolecular extended beta-sheet structure (Wolkers et al 2001). E.g. of group3
in different species D-7 protein from Typhalatifolia, AavLEA1 protein from nematode
(Goyal et al 2003), GmPM16 protein from soyabean (Shih et al 2004) and PsLEAM protein
from pea (Tolleter et al 2007 ).

Functions
The propensity of group3 protein for alpha-helix formation enable them to contribute
to the formation of cytoplasmic glass or interact with membrane during dessication condition
thus stabilizing the cellular structure or maintains the activity of enzymes (Tolleter et al
2007). Group3 proteins hiC6 and hiC12 from Chlorella vulgaris have cryoprotectant activity,
through which hiC6 and hiC12 protect Lactate Dehydrogenase and Malate Dehydrogenase
against freeze-inactivation. The relationship between cryoprotection and the unit number of
11-mer amino acid motif in hiC6 was investigated (Honjoh et al 2000). Cryoprotectant
activity decreased with decrease in unit number of 11-mer motifs.
2.1.3.4 Group 4
Sequence motif and peptide profile
The LEA4 proteins are characterized by a high content of A, T, and G amino acid
residues, the latter highly represented in unstructured proteins. They have a conserved Nterminal domain of 70 to 80 residues, predicted to form amphipathic a-helices, and a less
conserved C-terminal region with variable size and random coil structure. Like other LEA
proteins, the LEA4 group is highly accumulated in all embryo tissues of dry seeds. LEA4
proteins are involved in the adaptive response of vascular plants to withstand water deficit.
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Role
These may act as protectors of macromolecules and some cellular structure during
water deficit, by preferentially interacting with the available water molecules and providing a
hydration shell to protect the target” integrity and function (Garay-Arroyo et al 2000,
Hoekstra et al 2001). An invitro dehydration assay showed that plant hydrophilins (LEA
proteins from group 2, 3, 4) were able to protect the activity of malate dehydrogenase and
lactate dehydrogenase under low water availability conditions (Reyes et al. 2005). Similarly
same invitro assay were used to assess the protective activity of group 4 LEA proteins under
dehydrations and cold.
2.1.4
Localization of dehydrins in plants subjected to different stresses
Cold-induced dehydrins were present in the epidermis and in the vascular cylinder
around xylem and phloem in Deschampsia Antarctica.This report is in agreement with
previous reports on dehydrins (group2) localization in other plant species. LTI30 was present
in the vascular tissue in Arabidopsis thaliana treated by low temperature (Nylander et al
2001); P-80, a dehydrin from barley, accumulates in vascular cylinders and epidermis in
response to low temperature; PCA60, a dehydrin from peach, was accumulated in epidermal,
cortical, phloem and xylem tissue. Early ice nucleation in vascular cylinder and epidermis
could induce a severe dehydration in adjacent cells. Therefore, the presence of cold-induced
dehydrins in these tissues could be related to preventing protoplasmic dehydration stress
associated with freezing and thus increasing freezing tolerance. In the cell surrounding the
xylem vessels dehydrins might function as water attractants during the transport of water
from xylem vessel to sink tissues. Accumulation of dehyrin constitutes an adaptive response
in plants to cope with further stress imposed by freezing. Dehydrin localization is extensive.
Dehyrins have also been identified in cytosol of dessicated leaf cells of Craterostigma
plantagiineum Hochst and the euchromatin, nucleolus and cytosol of various cells in tomato
seedlings and mature plant tissues (TAS 14). In Arabidopsis thaliana, tissue and cell type
specific accumulation of dehydrins have been reported as ERD14 in vascular tissue, LT129
in roots and stems, RAB18 provascular tissue of embryo (Nylander et al 2001). Pea (Pisum
sativum) seed mitochondria were indeed found to accumulate a LEA protein (LEAM) with
repeated motifs predicted to form amphipathic helices (Grelet et al 2005). There is also e.g.
of two closely related group3 LEA proteins located in endoplasmic reticulum. N -terminal
signal peptide suggesting translocation of ER membrane. In case of maize gp2 proteins,
DHN1/Rab17, distribution between nucleus and cytoplasm is controlled by phosphorylation
of serine stutter: removal of this sequence results in lack of phosphorylation and retention in
cytoplasm.
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2.1.5
Association between expressions of stress responsive genes
Based on Venn diagram analysis, differences and cross talk of gene expression
among cold-, drought-, and high-salinity stress responses and ABA response in rice has been
reported. As shown in Figure-(2.1) 36, 62, 57, and 43 genes are cold-, drought-, highsalinity-, and ABA-inducible genes identified by RNA gel- blot analysis, respectively. More
than 98% of the high-salinity- and 100% of ABA-inducible genes were also induced by
drought stress, which indicates a strong relationship not only between drought and highsalinity responses but also between drought and ABA responses. These results indicate the
existence of a substantial common regulatory system or a greater cross talk between drought
and high-salinity stress and between drought and the ABA signaling process than that
between cold and high-salinity stress or between cold and the ABA signaling process. These
results in rice are consistent with previous observations on the overlap of drought- and highsalinity- responsive gene expression in Arabidopsis (Shinozaki and Yamaguchi-Shinozaki
2000, Seki et al 2002a, 2002b) as well as in Brassica Napus (Chen et al 2010). However,
these results are contradictory to the observations made by Kreps et al (2002) in cold- and
high-salinity-stressed Arabidopsis and to those made by Ozturk et al (2002) in drought and
high-salinity-stressed barley (Hordeum vulgare). In their studies, the majority of stressregulated changes appeared to be stress specific and not part of a general stress response
common to cold, drought, and high-salinity stress. These discrepancies may be attributable to
the difference in plant species used, stress treatment, plant growth condition, array strategy,
or detection methodologies. During the identification of dehydrin gene fragment in
Deschampsia Antarctica northern blot analysis of LEA gene showed the transcript level in
osmotic stress was higher than in salt stress, suggesting that ionic effect of salt may reduces
its expression. Salt stress induces osmotic adjustment of the cytoplasm by balancing the
increased osmotic potential (lowered water potential) caused by sodium sequestration inside
the vacuole, where it can accumulate to concentration that have marked effect on the osmotic
balance of plant cell suggesting that salt and osmotic stress have a similar signal transduction
pathway.
Fig. 2.1
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2.1.6
Expression of LEA genes in various plant tissues and at different developmental
stages
Expression patterns could provide insight into the function of LEA genes.
Accumulation of LEA proteins is found to occur during the late stages of development. Some
LEA genes were expressed in non-seed tissues under normal or stress conditions. Most
peanut LEA genes showed high expression during seed development which is consistent with
expression of LEA genes from other plant species. In this work, semi-quantitative RT-PCR
was used for expression analysis of peanut LEA genes. The results showed that most AhLEA
genes were highly expressed in seeds from 30 to 90 DAP (days after pegging) and were not
detected in non-seed tissue, which was similar to the results observed from Arabidopsis
(Bies-Ethève et al 2008). The expression of AhLEA1, AhLEA4, AhLEA5 and AhLEA8
genes was only detected in seeds. The expression of seven LEA protein genes (AhDHN1,
AhLEA3-1, AhLEA3-2, AhLEA3-4, AhLEA3-6, AhLEA6-1 and AhLEA7-1) could be
detected both in seed and non-seed tissues including root, stem, leaf and flower. The
expression of AhLEA3-2 and AhLEA6-1 was observed in all the five non-seed tissues tested.
AhDHN1 was expressed weakly in leaf, flower and stem. AhLEA3-1 and AhLEA7-1 were
expressed in root, stem, leaf and flower. AhLEA3-6 was expressed in root, leaf and flower.
Interestingly, AhLEA3-4 was highly expressed in flower but not in other non-seed tissues. In
10 DAP seeds the expression of most LEA genes was undetectable during seed development.
However, the expression of AhLEA6-1, AhLEA6-2 and AhLEA7-1 was clearly detected in
10 DAP seeds. The expression of 17 LEA genes could be detected in 30 DAP seeds. The
results showed that different groups of LEA genes exhibited variable expression patterns
during seed development, for example, the genes of LEA1, LEA5, LEA6, LEA7 and LEA8
groups. Even different members from one group of LEA showed distinct expression patterns,
for instance, LEA6-1 and LEA6-2 (Su et al 2011).
2.1.7
Induction and regulation of expression of LEA genes
One characteristic feature of these proteins is that the expression of the genes that
encode them is transcriptionally induced by the plant growth regulator abscisic acid and this
induction is occur in highly seed specific manner. The LEA polypeptide is proteolytically
degraded rapidly following seed germination, and although its synthesis can be re-induced in
germinating embryos subjected to osmotic stress or ABA treatment, such stress- and
hormone induction does not occur in mature vegetative tissues. This highly seed specific
pattern of expression sets of group1 LEA proteins apart from members of LEA group2 and
group3, which additionally accumulate in ABA- and stress- induced vegetative tissues, and
likewise have an association with desiccation tolerance (Ramunjulu and Bartels 2002, Wise
and Tunnacliffe 2004).
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In higher plants, the Group 1 LEA genes are expressed in a strictly seed-specific
manner and transcription is induced in response to the plant growth regulator ABA and also
to the imposition of osmotic stress in the maturation and early germination phases of seed
development. As a bryophyte, Physcomitrella does not produce seeds. However, it is known
that the promoter of the wheat Em gene does exhibit an ABA- and stress-responsive pattern
of expression, when introduced as a transgene to Physcomitrella. It was therefore clearly of
interest to determine whether the bryophyte counterpart of this gene exhibited a similar
pattern of expression. Northern blot hybridization of RNA isolated from Physcomitrella
protonemal tissue showed unambiguously that PpLEA-1 transcript is induced to accumulate
rapidly to a high concentration upon incubation of tissue with ABA or following the
imposition of an osmotic stress (10% mannitol) sufficient to induce plasmolysis of the cells.
Additionally, the degree of accumulation of the PpLEA-1 transcript appears correlated with
the concentration of ABA applied to the tissue.
During seed maturation, the LEA gene expression is regulated by the coordinated
action of transcription factors (TFs), such as ABI3/VP1, ABI4, ABI5, LEC1, LEC2, and
FUS3. LEC1, LEC2, and FUS3 primarily regulate the transition from embryogenesis to
germinative growth. ABI3, ABI4, and ABI5 may regulate the ABA response, possibly
forming a regulatory complex that mediates seed -specific and/or ABA-inducible expression
(e.g., LEA synthesis). LEC1 and FUS3 also partly participate in these processes (Finkelstein
et al 2002). With abiotic stresses, such as salt, drought stress, the biosynthesis and
accumulation of ABA are increased in plant. The increased ABA interacts with the ABA
receptors PYR/PYL/RCAR of START proteins, which in turn bind to and inhibit the protein
phosphatase types 2C (PP2C) proteins (e.g., ABI1, ABI2, and HAB1) and activate the SNF1related kinase 2 (SnRK2 kinase). The activated SnRK2s phosphorylate downstream
effectors, such as the basic leucine-zipper (bZIP) TFs called ABFs/AREBs and ABI5, which
bind to ABA-responsive element (ABRE) in the promoters of many ABA-induced genes
(e.g., LEA genes), thereby switching on the stress-response programs (Fujii and Zhu 2009,
Ma et al 2009, Melcher et al 2009, Nakashima et al 2009, Park et al 2009).
2.1.8 Promoter analysis of stress-inducible genes
Promoter region of abiotic stress-responsive Cor/LEA genes contain several
conserved motifs as cis-acting elements functioning in stress-responsive transcription, such
regulatory system include both ABA –dependent and –independent signaling pathways
(Yamaguchi- Shinozaki and Shinozaki 2005). A conserved motif, PyACGTGGC, was first
identified as a Cis-acting element named ABRE (ABA- responsive element) in promoter
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region of ABA responsive genes such as wheat Em gene and rice Rab16 gene. A number of
ABRE-binding proteins are members of bZIP (basic-domain leucine zipper) -type DNAbinding proteins, such as Arabidopsis bZIP-type ABF (ABRE-binding factor)/ AREB (ABAresponsive element binding) proteins, which binds to ABRE and activate ABA-dependent
stress responsive gene expression (Choi et al 2000, Uno et al 2000). Another functional cisacting element of the Cor genes, i.e. the CCGAC motif known as CRT (C-repeat)/DRE
(dehydration responsive element) sequences play a critical role in promoter function of
Arabidopsis COR15A and RD29A. Arabidopsis CBF (CRT binding factor)/ DREB1 (DRE
binding protein1) and DREB2 recognize CRT/DRE. The transcription factor’s CBF/DREB1
and DREB2 gene expression can be induced either by drought stress or by low temperature
and both activate the expression of gene possessing a CRT/DRE cis-element. Overexpression
of Arabidopsis DREB2A results in significant drought stress tolerance but only slight
freezing tolerance in transgenic plants (Sakuma et al 2006a). DREB2A positively regulates
expression of many abiotic stress-responsive genes possessing DRE sequences in their 5’
upstream regions. Many cereals DREB2 homologs have been identified in rice, maize, pearl
millet, barley and wheat (Dubouzet et al 2003, Shen et al 2003, Aggarwal et al 2004, Xue
and Loveridge 2004, Egawa et al 2006, Qin et al 2007,). Although among these proteins
OsDREB2A, PgDREB2A and TaDREB1 bind to DRE sequences. The barley HvDRF1 gene
also belongs to the CBF/DREB family (Xue and Loveridge 2004). The HvDRF1
transcription factor binds preferentially to a CT-rich element called a DRF1E motif
(T(T/A)ACCGCCTT) rather than to CRT/DRE. A wheat DREB2 homolog Wdreb2, shows
quite a high similarity to HvDRF1 and its transcription is enhanced by low temperature,
drought, salt and ABA (Egawa et al 2006). Wheat ABA-responsive Cor/LEA genes have
been characterized. Expression pattern of four Cor/LEA Wdhn13, Wrab17, Wrab 18 and
Wrab 19 correspond well to the expression profile of Wdreb2 under low temperature,
drought, salt and ABA (Egawa et al 2006). Recently Kobayashi et al (2008) analyzed the
direct relationship between WDREB2 and the four Cor/LEA genes in development of abiotic
stress tolerance.
Recent approaches, especially the use of ABA-deficient and ABA-insensitive
mutants, have shown that signaling of water stress may be understood in two major
pathways: the ABA -dependent and -independent pathways. The ABA -dependent pathway
may also have two different routes: i.e. requiring new protein synthesis or not. In the route
where new protein synthesis is not required, the promoter domain in all ABA-responsive
genes has an ABA-responsive element ABRE (PyACGTGGC where Py indicates a
pyrimidine base, C or T). When ABRE is bound with its corresponding bzip family of
13
transcription factors such as EmBP-1, it can lead to ABA-induced gene expression. An
example is the ABA mediated induction in Arabidopsis of the dehydration-responsive rd29B
gene, which possesses two ABREs essential for its expression. Two bzip transcription
factors, AREB1 and AREB2, are involved in the ABA-mediated expression of rd29B
probably via phosphorylation mediated by an ABA activated protein kinase (Uno et al 2000).
In the route where new protein synthesis is required for the ABA induced gene expression,
de novo production of new proteins is the prerequisite. Such genes have no ABREs and its
ABA-responsive elements combine with the MYC family transcription factors. The
activation of the genes for MYC family transcription factors and the synthesis of the
transcription factors should precede the activation of any ABA-initiated genes. Some genes
may be induced by both the ABA-dependent and the ABA-independent pathways. Gene
rd29A is an example; its promoter has two types of regulatory cis- elements, one ABAresponsive and the other independent of ABA. The independent cis -element has a conserved
9-basepair motif, TACCGACAT, also known as DRE/C-repeat element.
2.2
Antioxidant metabolism
2.2.1
Reactive oxygen species and their production sites
Exposure of plant to unfavorable environmental conditions such as vicissitude of
temperature, high light intensity, water availability, salt stress can increase the production of
reactive oxygen species such as singlet oxygen, superoxide radical, hydrogen peroxide and
hydroxyl radical. Plant posses enzymatic and non –enzymatic mechanisms for scavenging
ROS. The enzymatic mechanism design to minimize the production of O2-. and production of
H2O2.The enzymes overproduced so far include superoxide dismutase (SOD), Ascorbate
peroxidase (APX), Glutathione reductase (GR). Superoxide radical is regularly synthesized
in chloroplast and mitochondria though small quantity is also reported to produced in
microbodies. Scavenging of superoxide by SOD results in the production of H2O2 which is
removed by APX and CAT. However both O2-.and H2O2 are not as toxic as OH- which is
formed by combination of O2-.and H2O2 in the presence of trace amount of Fe3+ by HaberWeiss reaction. Hydroxyl radical can damage chlorophyll, protein, DNA, lipids and other
macromolecules that fatally affecting plant metabolism and ultimately growth and yield.
A schematic presentation of production and scavenging of O2-. ,H2O2 and OH
mediated lipid peroxidation and glutathione mediated stabilization of lipids are present in
figure 2.2.
14
Fig. 2.2: Production and scavenging of ROS.
2.2.2
ROS scavenging enzymatic antioxidants
2.2.2.1 Superoxide dismutase (SOD)
SOD has been first line of defense against the toxic effects of elevated levels of
ROS. The SOD removes superoxide anion O2-. by catalyzing its dismutation, one O2-. being
reduced to H2O2 and another oxidized to O2 . It removes O2.- and hence decrease the risk of
OH. formation via metal catalyzed reaction. This reaction is 10,000 fold faster than
spontaneous dismutation (Mittler 2002). SODs are classified by their metal cofactors into
three known types, which are localized in different cellular compartments: The Mn-SOD is
found in mitochondria, Cu/Zn-SOD found in cytosolic fraction and also in chloroplast of
higher plants (Rio et al 2003). The Fe-SOD isozymes, often not detected in plants, are
usually associated with chloroplast compartments when present. There has been reported the
significant variation in the level of SOD under abiotic stress. Significant increase in SOD
activity under salt stress has been observed in various plants viz. mulberry (Harinasut et al
2003) Cicer arietinum (Kukreja et al 2005) and Lycopersicon esculentum (Gapinska et al
2008). Eyidogan and Oz (2005) noted three SOD activity bands (MnSOD, FeSOD and
Cu/ZnSOD) in Cicer arietinum under salt stress. Wang and Li (2008) studied the effect of
water stress on the activities of total leaf SOD and chloroplast SOD in Trifolium repens L.
and reported significant higher increase in SOD activity under water stress.
O2-. + O2-. + 2H+
O2 + H2O2
2.2.2.2 Catalase (CAT)
Catalase is a tetrameric heme containing enzyme with the potential to directly
dismutate H2O2 into H2O and O2 and is indispensable for ROS detoxification during stressed
15
conditions (Garg et al 2009). CAT has one of the highest turnover rates for all enzymes: one
molecule of CAT can convert 26 million molecules of H2O2 into H2O and O2 per minute.
CAT is important in the removal of H2O2 generated in peroxisomes by oxidases involved in
ß-oxidation of fatty acids, photorespiration and purine catabolism. The CAT isozymes have
been studied extensively in higher plants. Maize has 3 isoforms (CAT1, CAT2 and CAT3),
found on separate chromosomes. CAT1 and CAT2 are localised in peroxisomes and the
cytosol, whereas, CAT3 is mitochondrial. It has also been reported that apart from reaction
with H2O2, CAT also react with some hydroperoxides such as methyl hydrogen peroxide
(MeOOH). Pretreatment of rice seedlings with H2O2 under non-heat shock conditions
resulted in an increase in CAT activity. Similarly, increase in CAT activity in Cicer
arietinum roots following salinity stress was noted. Stoilova et al (2010) reported increased
CAT activity in wheat under drought stress but it was higher especially in sensitive varieties.
In another study, Sharma and Dubey (2005) reported a decrease in CAT activity in rice
seedlings under drought stress.
2H2O2
2H2O + O2
2.2.2.3 Ascorbate peroxidase (APX)
APX is thought to play the most essential role in scavenging ROS and protecting
cells in higher plants, algae, euglena and other organisms. APX is involved in scavenging of
H2O2 in water- water and ASH-GSH cycles and utilizes ascorbate (ASH) as the electron
donor. The APX family consists of at least five different isoforms including thylakoid
(tAPX) and glyoxisome membrane forms (gmAPX), as well as chloroplast stromal soluble
form (sAPX), cytosolic form (cAPX). APX has a higher affinity for H2O2 (mM). Significant
increase in APX activity was noted under water stress in three cultivars of Proteus vulgaris
(Zlatev et al 2006) and Proteus asperata (Yang et al 2008). Sharma and Dubey (2005) found
that mild drought stressed plants had higher chloroplastic- APX activity than control grown
plants but the activity declined at the higher level of drought stress.
H2O2 + Asc
D-Asc + 2 H2O
2.2.2.4 Guaiacol peroxidase (POD(GPX))
APX can be distinguished from plant-isolated guaiacol peroxidase (POD(GPX)) in
terms of differences in sequences and physiological functions. (POD(GPX))decomposes
indole-3-acetic acid (IAA) and has a role in the biosynthesis of lignin and defense against
biotic stresses by consuming H2O2. GPOX prefers aromatic electron donors such as guaiacol
and pyragallol usually oxidizing ascorbate at the rate of around 1% that of guaiacol. The
activity of GPOX varies considerably depending upon plant species and stresses conditions.
It increased in Cd exposed plants of Triticum aestivum (Milone 2003), Arabidopsis thaliana
16
(Cho et al 2005) and Ceratophyllun demersum (Arvind et al 2003). Radotic et al (2000)
noted an initial increase in GPOX activity in spruce needles subjected to Cd stress and
subsequent Cd-treatments caused a decline in the activity. A concomitant increase in GPOX
activity in both the leaf and root tissues of Vigna radiate.
H2O2 + 2GSH
GSSG + 2 H2O
ROOH + 2 GSH
GSSG + ROH + H2O
2.2.2.5 Glutathione reductase (GR)
GR is a flavo-protein oxidoreductase, found in both prokaryotes and eukaryotes
(Romero-Puertas et al 2006). It is a potential enzyme of the ASH-GSH cycle and plays an
essential role in defense system against ROS by sustaining the reduced status of GSH. It is
localized predominantly in chloroplasts, but small amount of this enzyme has also been
found in mitochondria and cytosol. GR catalyzes the reduction of GSH, a molecule involved
in many metabolic regulatory and antioxidative processes in plants where GR catalyses the
NADPH dependent reduction of disulphide bond of GSSG and is thus important for
maintaining the GSH pool (Reddy et al 2006, Chalapathi et al 2008). Actually, GSSG
consists of two GSH linked by a disulphide bridge which can be converted back to GSH by
GR. GR is involved in defense against oxidative stress, whereas, GSH plays an important
role within the cell system, which includes participation in the ASH-GSH cycle, maintenance
of the sulfhydryl (eSH) group and a substrate for GSTs (Reddy et al 2006). GR and GSH
play a crucial role in determining the tolerance of a plant under various stresses (Chalapathi
et al 2008).
GSSG + NADPH
NADP+ + 2 GSH
2.2.3 Non-enzymatic antioxidants
Different abiotic stress factors may provoke osmotic stress, oxidative stress and
protein denaturation in plants, which lead to cellular adaptive responses such as
accumulation of compatible solutes, induction of stress proteins, and acceleration of reactive
oxygen species scavenging systems (Zhu 2002). One of the most common stress responses in
plants is overproduction of different types of compatible organic solutes (Serraj and Sinclair
2002). Compatible solutes are low molecular weight, highly soluble compounds that are
usually nontoxic at high cellular concentrations. Generally, they protect plants from stress
through different courses, including contribution to cellular osmotic adjustment,
detoxification of reactive oxygen species, protection of membrane integrity and stabilization
of enzymes/proteins. Furthermore, because some of these solutes also protect cellular
components from dehydration injury, they are commonly referred to as osmoprotectants.
These solutes include proline, sucrose, polyols, trehalose and quaternary ammonium
17
compounds (QACs) such as glycine betaine, alaninebetaine, prolinebetaine, choline Osulfate, hydroxyprolinebetaine, and pipecolatebetaine.
2.2.3.1 Ascorbic acid
The small antioxidant molecule vitamin C (L-ascorbic acid, AsA) fulfils essential
metabolic functions in plants such as protection against oxidative damage (Smirnoff 2000),
maintain the photosynthetic apparatus (Chen and Murata 2002) delay premature senescence
of leaves and protect the chlorophylls. Several researchers provided that ascorbic acid plays
an important role as plant growth regulator, stimulates the synthesis of mRNA, involved in
flowering, and encourages the emergence of lateral buds. Recently it was shown that
ascorbic acid is very important for the regulation of photosynthesis, flowering and
senescence (Davey et al 2000, Barth et al 2006). It also fights against the negative effects of
salt stress on tomato (Shalata and Neumann 2001) and wheat (Al- Hakimi and Hamada 2001,
Athara et al 2008). In several studies conducted in the same way, it was found that only
tolerant plants best respond to the application of exogenous AsA (Athara et al 2008)
suggesting that the action of AsA to alleviate the harmful effects of salt stress is a variety
dependent. However, the positive effect of AsA on the growth of wheat plants stress is
caused by its action on the division and cell expansion (Smirnoff 2000). It seems that
ascorbic acid increased mitotic activity of cells including the transition from G1 to S phases.
Indeed several research conducted on the ascorbic acid, reported that they promote the cell
elongation and cell proliferation (Blokhina et al 2002). Due to the fact that AsA serves as an
important cofactor in the biosynthesis of many plant hormones, including ethylene,
gibberellic acid (GA), and abscisic acid (ABA), one has to assume that the endogenous level
of AsA will affect not only the biosynthesis, but also the levels and therefore the signaling of
these molecules (Barth et al 2006). So it is likely to say that modulation of growth by AsA is
may be induced by restoring the hormone equilibrium which is disturbed in salt stress
conditions. The effect of AsA on the content of proline and soluble sugars can suggest that
AsA probably improves growth of stressed plants, further to its antioxidant action, by
intensification of their potential for osmotic adjustment and activities of growth (cell division
and expansion). Indeed, several studies have shown that AsA plays an important role in
improving plant tolerance to abiotic stress (Al-Hakimi and Hamada 2001, Shalata and
Neumann 2001, Athara et al 2008). Moreover, the antioxidant effect exerted by the AsA on
treated plants showed through the determination of the content of H2O2 and carotenoids,
confirms the importance of antioxidant defence systems in plant tolerance to salinity
(Agarwal and Pandey 2004, Sairam et al 2005). Finally, the study of the effect of NaCl on
the leaf H2O2 content and analysis of correlations between the leaf H2O2 content and the
growth of leaves, in the presence and absence of vitamin C, can suggest that the AsA effect
18
on promoting the growth under stress is probably due to its role in improving the antioxidant
capacity (decrease of the H2O2 content). From the results mentioned above, it can be
concluded that ascorbic acid counteracts with salinity by enhancing antioxidant capacity,
improving growth and might also be by restoring the hormone balance which have to be
verified in the future study.
2.2.3.2 Reduced glutathione
Glutathione is a tripeptide (γ-glutamyl cysteinylglycine), which has been detected
virtually in all cell compartments such as cytosol, chloroplasts, endoplasmic reticulum,
vacuoles and mitochondria (Millar et al 2003) Glutathione is the major source of non-protein
thiols in most plant cells. The chemical reactivity of the thiol group of glutathione makes it
particularly suitable to serve a broad range of biochemical functions in all organisms. The
nucleophilic nature of the thiol group also is important in the formation of mercaptide bonds
with metals and for reacting with selected electrophiles. This reactivity along with the
relative stability and high water solubility of GSH makes it an ideal biochemical to protect
plants against stress including oxidative stress, heavy metals and certain exogenous and
endogenous organic chemicals (Millar et al 2003, Foyer et al 2005). Glutathione takes part in
the control of H2O2 levels (Shao et al 2006). The change in the ratio of its reduced (GSH) to
oxidized (GSSG) form during the degradation of H2O2 is important in certain redox signaling
pathways. It has been suggested that the GSH/GSSG ratio, indicative of the cellular redox
balance, may be involved in ROS perception. Reduced glutathione (GSH) acts as an
antioxidant and is involved directly in the reduction of most active oxygen radicals generated
due to stress. There was a report reporting that glutathione, an antioxidant helped to
withstand oxidative stress in transgenic lines of tobacco (Rio et al 2006).
2.2.3.3 Proline
Amino acid proline is known to occur widely in higher plants and normally
accumulates in large quantities in response to environmental stresses (Ozturk and Demir
2002, Hsu et al 2003, Kavi Kishore et al 2005). In addition to its role as an osmolyte for
osmotic adjustment, proline contributes to stabilizing sub-cellular structures (e.g. membranes
and proteins), scavenging free radicals, and buffering cellular redox potential under stress
conditions. It may also function as a protein compatible hydrotrope alleviating cytoplasmic
acidosis, and maintaining appropriate NADP+/NADPH ratios compatible with metabolism.
Also, rapid breakdown of proline upon relief of stress may provide sufficient reducing agents
that support mitochondrial oxidative phosphorylation and generation of ATP for recovery
from stress and repairing of stress-induced damages.
19
Furthermore, proline is known to induce expression of salt stress responsive genes,
which possess proline responsive elements (e.g. PRE, ACTCAT) in their promoters (Satoh et
al 2002, Ohno et al 2003, Chinnusamy et al 2005). In response to drought or salinity stress in
plants, proline accumulation normally occurs in the cytosol where it contributes substantially
to the cytoplasmic osmotic adjustment. Furthermore, It was determined that, in response to
water deficit, increased concentration of proline in maize root apical meristem was paralleled
with increased concentration of abscisic acid.
In plants, the precursor for proline biosynthesis is l-glutamic acid. Two enzymes,
pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR)
play major roles in proline biosynthetic pathway. Transgenic tobacco plants over-expressing
P5CS have shown increased concentration of proline and resistance to both drought and
salinity stresses.
Fig. 2.3: Biosynthetic pathway of proline in higher plants
Accumulation of proline under stress in many plant species has been correlated with
stress tolerance and its concentration has been shown to be generally higher in stress-tolerant
than in stress-sensitive plants In wheat, an assessment of the effects of drought stress on
proline accumulation in a drought-tolerant and a drought-sensitive cultivar revealed that the
rate of proline accumulation and utilization was significantly higher in the drought-tolerant
cultivar (Nayyar and Walia 2003).
In light of the available information, however, it seems that proline accumulation in
plants is mediated by both ABA-dependent and ABA -independent signaling pathways (Zhu
2001b, 2002). ABA is known to mediate signals in plant cells subjected to environmental
stresses. These signals can bring about expression of stress-related genes followed by
synthesis of compatible osmolytes such as proline (Kavi Kishore et al 2005). Furthermore,
ABA accumulation in plants in response to osmotic stress has been determined to regulate
expression of P5CS gene, which is involved in proline biosynthesis (Xiong et al 2001).
Despite the presence of a strong correlation between stress tolerance and accumulation of
proline in higher plants, this relationship may not be universal. For example, in rice plants
grown under salt stress, accumulation of proline in the leaf was deemed to be a symptom of
20
salt injury rather than an indication of salt tolerance. Similarly, assessment of proline
accumulation and distribution during shoot and leaf development in two sorghum genotypes
contrasting in salt tolerance suggested that proline accumulation was a reaction to salt stress
and not a plant response associated with tolerance (de-Lacerda et al 2003) and yet in another
study, under salt stress, sensitive rice cultivars accumulated greater amounts of proline than
did the tolerant genotypes. However, further studies are needed to determine whether the
relationship between stress tolerance and accumulation of proline is species-specific or if it
can be altered by experimental conditions.
2.2.3.3.1 Exogenous application of proline
Exogenous application of proline can play an important role in enhancing plant stress
tolerance. This role can be in the form of either osmoprotection or cryoprotection. Proline
can also protect cell membranes from salt-induced oxidative stress by enhancing activities of
various antioxidants (Yan et al 2000). For example, growth of tobacco suspension cells
under salt stress was promoted by exogenous application of 10mM proline, which was
proposed to be due to proline action as a protectant of enzymes and membranes (Okuma et al
2000). In soybean cell cultures maintained under salt stress, exogenous application of proline
increased activities of superoxide dismutase and peroxidase, which normally contribute to
increased salt tolerance (Yan et al 2000, Hua and Guo 2002). In barley embryo cultures
under saline conditions, exogenous application of proline resulted in a decrease in Na+ and
Cl− accumulations and an increase in growth. In contrast to the above findings on beneficial
effects of exogenous application of proline, there are a few reports cautioning its use. For
example, foliar application of proline to rice plants growing under saline conditions did not
change concentrations of either Na+ or Cl− in the leaves. Moreover, in Arabidopsis plants
exogenous application of proline was suggested to cause damages to ultra-structures of
chloroplast and mitochondria.
2.2.4
Hydrogen peroxide (H2O2)
H2O2 as one of the major and most stable ROS that regulate basic acclimatory,
defense and developmental processes in plants. The addition of single electron to the
molecular oxygen reduces it to the superoxide anion radical. The superoxide is a free radical.
It is relatively unstable, either converted back to O2 or in reaction with proton, to H2O2, either
spontaneously or in a reaction catalyzed by Superoxide dismutase (SOD). Because of this
and longer half life of H2O2 than that of superoxide anion radical, H2O2 is more likely to be a
long –distance signaling molecule.
21
2.2.4.1 Cellular and extracellular sources of H2O2
Hydrogen peroxide is produced not only through the disproportionation of
superoxide, but also due to the reduction of O2-. by reductant (X) such as ascorbate, thiols,
ferredioxins and others. Thus hydrogen peroxide at cellular levels is strictly linked with the
generation of superoxide. Nevertheless, other oxidases such as glycolate oxidase, glucose
oxidase, amino-acid oxidases release H2O2 following oxidation of their respective substrate.
2.2.4.1.1 Chloroplast
The photosynthetic electron transport (PET) chain in the chloroplast is responsible
for H2O2 production. The electron transport components of (PET) are auto-oxidizable which
cause limiting availability of NADP, superoxide anion radicals can be formed (Dat et al
2000, Foyer & Noctor 2003). ‘Mehler’ reaction’ is considered as primary and the most
powerful source of H2O2/ROS in chloroplast. Secondly, the H2O2 production in chloroplasts
is catalysed by SOD isoforms (Alsher et al 2002). The reduction of O2-. by ascorbate (Asc)
and reduced glutathione (GSH) does not contribute much to the production of H2O2 in
chloroplast.
2.2.4.1.2 Peroxisomes
The main function of peroxisomes in plant cell is photorespiration, which is
associated with generation of H2O2 (Dat et al 2000, Wingler et al 2000). Besides glycolate
oxidation H2O2 can be generated via ß-oxidation of fatty acids and oxidation of other
substrates (Dat et al 2000), but significance of this process in H2O2 production in
comparision to glycolate oxidation in C3 plants appear minor (Foyer and Noctor 2003).
2.2.4.1.3 Mitochondria
In plant mitochondria superoxide anion radical production occurs mainly at two sites
of the electron transport chain: NAD(P)H dehydrogenases (complex I) and cytochrome bc1
complex (complex III)
(Moller 2001). This process results in the formation of H2O2,
primarily through the action of a mitochondrion-specific manganese SOD (Rhoads et al
2006). The amount of H2O2 produced in plant mitochondria is less than that of chloroplasts
or peroxisomes when exposed to light (Foyer and Noctor 2003), but in dark or in non-green
tissues, mitochondria can be major source of ROS (Rhoads et al 2006).
22
Fig. 2.4: Production of ROS in mitichondria
2.2.4.1.4 Other sources of H2O2
In the cytoplasm, the electron transport chain associated with endoplasmic reticulum
is the main source of H2O2/ROS. The cytoplasm cannot be regarded as a major source of
H2O2 in plant cell but may act as sink for hydrogen peroxide leaking from other cellular
component. NADPH oxidase at the plasma membrane in the plant cell is the most intensively
studied oxidase system. The system catalyzes the production of O2- by one electron
reduction of O2 using NADPH as electron donor (Desikan et al 2003, Mahalingam &
Federoff 2003, Apel & Hirt 2004). The superoxide anion radical is most likely located in the
appoplastic space and is converted to H2O2 (Karpinska et al 2001, Bolwell et al 2002). Many
enzymes of extracellular membrane, such as pH-dependent cell wall peroxidases, germins,
germin like oxalate oxidases and amine oxidases have been proposed as a source of H2O2 in
apoplast (Bolwell et al 2002, Kacperska 2004).
2.2.4.2 H2O2 in stress conditions and as a signaling molecule
H2O2 play a central role in responses to both biotic and abiotic stresses in plants.
Most studies have reported increase in concentration after exposure to stress. The rate of
H2O2 depends upon the strength and exposure of imposed stress (Neill et al 2002a,
Kacperska 2004). Experiments with plant material have demonstrated that plant tissues can
tolerate high concentrations of H2O2 in the range 10–2 × 105 μM. Moreover, plants pre-treated
with H2O2 were more resistant to excess light and chilling stresses (Karpinska et al 2000, Yu
23
et al 2003). In the literature, the endogenous concentration of H2O2 is reported to lie in a
wide range, ranging from nanomoles to several hundred micromoles of H2O2 per gram fresh
mass (Veljovic-Jovanovic et al 2002). The relative stability and higher concentrations of
H2O2 in plant cells could point to the fact that H2O2 plays a key role as a signal transduction
factor. However, signal molecules are usually present in cells in very low concentrations; the
relatively high level of H2O2 in plant tissues supports the assumption that H2O2 is not only a
signaling molecule, but also plays a key role in primary plant metabolism. Moreover, H2O2
regulates the expression of various genes, including those encoding antioxidant enzymes and
modulators of H2O2 production (Neill et al 2002a, 2002b). Hydrogen peroxide has also been
shown to be an intercellular signal mediating systematic acquired resistance and systematic
acquired acclimation.
2.2.4.3 H2O2 signaling during growth and development
Recently, information on the role of H2O2/ROS as signal molecules regulating
growth and is not only a stress signal molecule, but may also be an intrinsic signal in plant
growth and development. H2O2 has shown to be involved in differentiation of cellulose rich
cell wall. Recently it has been demonstrated that diminished extracellular Cu/Zn SOD
expression in poplar trees regulate extracellular H2O2 level and plant development.
Fig. 2.5
Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are among the
important second messengers in abscisic acid (ABA) signaling in guard cells. To investigate
specific roles of H2O2 in ABA signaling in guard cells, the effects of mutations in the guard
cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-
24
aminotriazole (AT) on stomatal movement has been examined. The cat3 and cat1 cat3
mutations significantly reduced CAT activities, leading to higher basal level of H2O2 in
guard cells, when assessed by 2_,7_-dichlorodihydrofluorescein, whereas they did not affect
stomatal aperture size under non-stressed condition. In addition, AT-treatment at
concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size,
while basal H2O2 level increased extensively. In contrast, cat mutations and AT-treatment
potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was
observed in these mutants and wild type as well as in AT-treated guard cells. These results
suggest that ABA-inducible cytosolic H2O2 elevation functions in ABA-induced stomatal
closure, while constitutive increase of H2O2 does not cause stomatal closure (Jannat et al
2011).
Fig. 2.6
2.2.5
Malondialdehyde (MDA)
Estimation of malondialdehyde (MDA) amount, which is a secondary end product of
polyunsaturated fatty acid oxidation, is widely used to measure the extent of lipid
peroxidation as indicator of oxidative stress. Many studies have shown greater accumulation
of MDA and H2O2 caused by salinity and drought (Sairam and Srivastva 2001).
25
2.3
Abscisic acid
The plant hormone abscisic acid (ABA) was first named as such (Addicott et al
1968) a few years after its isolation from cotton fruits (Ohkuma et al 1963) as an abscission
accelerating factor, and from sycamore leaves (Cornforth et al 1965) during a search for
endogenous substances that induce dormancy. Although the role of ABA in fruit abscission
and dormancy of woody plants remains unclear (Schwartz et al 2003), a vast body of
evidence supports the implication of this ubiquitous hormone in essential plant processes
such as seed maturation, desiccation, dormancy and germination (Hoth et al 2002, Seki et al
2002, Leonhardt et al 2004). The plant hormone abscisic acid has long been known to be
involved in the responsiveness of plants to various environmental stresses including drought,
salinity, and low temperature. ABA production is increased in tissues during these stresses
and this cause a variety of physiological effects, including stomata closure in leaves.
Previous evidence showed that an elevation of cytisolic Ca, an increase in pH and a reduction
in K and Cl and organic solute content in both guard cells surrounding the stoamtal pore, are
downstream elements of ABA-induced stomatal closure. In addition, cADP-Rib,
phospholipase C and phospholipase D have been identified as siganlling molecules in the
ABA response and exerting their effects by regulating cytosolic Ca concentration and inward
K channels. Furthermore, Ca channels and anion channels at the plasma membrane of
stomatal guard cells are activated by hyperpolarization and ABA (Pei et al 2000) and
increase in Ca concentration resulting from activation of Ca channels leading to Ca influx is
known to inactivate inward-rectifying K channels, biasing the plasma membrane for solute
efflux, which drives stomatal closure.
2.3.1
ABA as a long distance signal mediating whole plant response to drought and
salt stresses
Stress ABA produced in dehydrated roots in drying soil is transported to the xylem
and regulates stomatal opening and leaf growth in roots and shoots. This mechanism is
modified by ionic conditions and pH in xylem. Hartung et al (2002) have shown that pH
changes play a central role in the ABA redistribution in leaf tissues and control the stomata at
times when no significant changes in ABA concentration are detected in the xylem. It
appears that plants have evolved two responses to soil drying. Initial soil drying may be
sensed by part of the root system and a root-sourced ABA in the xylem may regulate the
stomatal conductance such that water loss in the shoots may be reduced and leaf water deficit
can be avoided. When the soil drying is prolonged and becomes more severe, the shoot water
deficit becomes unavoidable and some older leaves may wilt, perhaps because of a weak
26
hydraulic link with the main stem or a weaker control of stomatal conductance. Such leaf
wilting should account for the much accelerated ABA concentration in the xylem and much
more severe stomatal inhibition that can be observed in the young leaves.
The effect of ABA on the regulation of plant growth is rather complex. Early results
showed that stress ABA is a regulator of shoot growth and development under water stress
and shoot growth can be inhibited when xylem ABA is increased as a result of soil drying.
More recent results have shown, however, differences in response exist between root and
shoot. Sharp and his group have shown that maintenance of root growth under low water
potential is a function of enhanced ABA accumulation in roots. They have shown that
ABA’s role may involve the inhibition of ethylene production, which is a growth inhibitor
under stress (Spollen et al 2000, Sharp and LeNoble 2002) even suggested that stress ABA
may also be helpful to maintain some limited shoot growth as well as root growth under
water deficit condition would enhance drought tolerance. More recent experiments have
supported the notion that ABA has dual roles in its physiological regulation (Cheng et al
2002, Finkelstein et al 2002). Its inhibitive role functions when it is accumulated in large
amount under stress to help plant survival through inhibition of processes such as stomatal
opening and plant size expansion. Its promoting role, when it is at low concentration and at
more ‘normal’ condition, has been shown essential for vegetative growth in several organs
e.g., primary root growth (Spollen et al 2000) and post-germination seedling development.
In addition to osmotic stress, salinity imposes on plants other stresses such as ion
toxicity, as a result of ion entry in excess of appropriate compartmentation, and nutrient
imbalances, as commonly seen in the displacement of potassium by sodium. The main
damage to plants, however, could result from osmotic stress imposed externally due to high
ion concentrations in the soil or internally when excess salt uptake resulted in high salt
accumulation in the intercellular spaces. In this regard, the damage caused by salinity is
mainly due to altered water relations; thus plant responses to salinity and water deficit are
closely related with overlapping mechanisms. Exposure of plants to salinity is known to
induce a proportional increase in ABA concentration that is in most cases correlated with leaf
or soil water potential, suggesting that salt-induced endogenous ABA is due to water deficit
rather than to a specific salt effects.
2.3.2
Cell signaling from stress perception to ABA accumulation
It has been proposed that for the cell signaling of ABA accumulation the perception
mechanism may be different not only between water and salt stress but also between leaf and
root tissues. The evidence for such a claim is that prevention of cell dehydration could
completely diminish the osmotic stress-induced ABA accumulation in leaf cells, while such
27
treatment had no visible effect on root tissues. As roots usually contain a lower level of
xanthophylls than leaves, ABA synthesis in roots is more likely to be limited by the size of
the precursor pools than in leaves. In addition, the osmosensor may be more abundant in
roots than in leaf cells (Jia et al 2002b), while the dehydration-induced production of ABA is
usually observed in shoots. Seo et al (2000) found that one of the key control points for ABA
accumulation, ABA-aldehyde oxidase, exists in different isoforms in the roots and leaves of
Arabidopsis and is encoded by different genes in the two tissues.
2.3.3
Abscisic acid and antioxidant defense in plant cells
2.3.3.1 ABA- induced ROS generation
In ABA-induced stomatal closure of guard cell of Arabidopsis, ABA treatments
induced a rapid increase in the production of H2O2 (Pei et al 2000). Treatment with 1
micromole/L ABA increased the production of H2O2 by 36.8% and treatment with 50
micromole/L ABA increased by 49%. Similar results were also observed in ABA-induced
stomatal closure of guard cell of Arabidopsis, Vicia faba (Zhang et al 2001) rice roots (Lin
and Kao 2001) and in maize embryo cells (Guan et al 2000).
Accumulation of apposplastic H2O2 is involved in the induction of the chloroplastic
and cytosolic antioxidant enzymes. These results suggest that H2O2 produced at a specific
cellular site could coordinate the activities of antioxidant enzymes in different subcellular
compartments (Hu et al 2005). Although recent studies have shown that ABA-induced ROS
production increase the total leaf activity of SOD, CAT, APX and GR in maize (Jiang and
Zhang 2002a, b), it is not clear whether ABA induced ROS production co-ordinates the
different subcellular activities of antioxidant enzymes.
2.3.3.2 Expression of Genes Encoding Antioxidant Enzymes Induced by ABA
It has been documented that ABA can induce the expression of antioxidant genes
encoding Cu/Zn-superoxide dismutase (SOD), Mn SOD and Fe SOD and catalase (CAT)
(Guan et al 2000) in plants. The expressions of Sod and Cat genes in response to ABA
depend on plant species or varieties. For example, in rice seedlings, ABA induced an
increase at the transcript levels of SodA1, which encodes Mn-SOD, SodB, which encodes FeSOD, and SodCc1 and SodCc2, which encodes cytosolic Cu/Zn-SODs, in a dose-dependent
manner. But in tobacco BY-2 cell suspensions, ABA treatments only increased the
accumulation at the transcript level of Mn-SOD gene, and did not change the transcript level
of Fe-SOD gene and reduced that of cytosolic Cu/Zn- SOD gene. In an inbred maize line, the
transcript of Cat3 was induced 3.6-fold by ABA in coleoptiles and 3.3-fold in mesocotyls. In
another inbred maize line, however, the Cat3 transcript decreased after 2h of ABA treatment
28
in leaves. Moreover, the expressions of Sod and Cat genes are different in response to ABA
at different developmental stages in plants. For example, in maize, the transcripts of Sod4
and Cat1 accumulated in response to ABA in developing and germinating embryos, and in
young leaves; the Sod4A transcript showed no increase in response to ABA in developing
and germinating embryos, but increased in young leaves; Cat2 and Cat3 transcripts were upregulated only at very high ABA concentrations (10-3 mol/L) during late embryogenesis and
in response to various concentrations of ABA in germinating embryos, and the Cat2
transcript increased in response to ABA and the Cat3 transcript decreased in young leaves.
These results suggest that the Sod and Cat genes in maize are regulated by ABA in a
multilayered fashion.
ABA not only induces the increase in activities of these antioxidant enzymes but also
induces the increase in contents of non-enzymatic antioxidants such as ascorbate, reduced
glutathione, alpha-tocopherol and carotenoids (Jiang and Zhang 2001, 2002a). These results
suggest that ABA can induce the capacity of whole antioxidant defense systems including
enzymatic and non-enzymatic constituents in plants. Pretreatment with ABA enhances the
capacity of antioxidant defense systems in plants exposed to environmental stress such as
chilling, high temperature, NaCl stress and water stress (Jiang and Zhang 2002a). The
increase in antioxidant defense system is closely related to stress tolerance. A recent study
provides a genetic evidence for involvement of ABA in the protection against oxidative
damage in Arabidopsis exposed to heat stress. ABA reduces the level of lipid peroxidation
and enhanced survival in Arabidopsis.
2.3.3.3 ABA induced modulation of metabolic and redox control pathways
Besides the up-regulation of pathways related to the biosynthesis of compatible
solutes as a response to ABA treatment, metabolic profiling indicated that specific
antioxidants, particularly alpha-tocopherol and L-ascorbic acid, were accumulated at high
level in ABA-treated seedlings compared to appropriate controls. The transcriptions of genes
involved in alpha-tocopherol biosynthesis were co-ordinately upregulated and appeared to be
integrated into a network of reactions controlling the level of reactive oxygen species. The
present study was aimed at providing evidence regarding metabolite levels that are affected
by the treatment with ABA and to provide comparison with metabolite pool that are
accumulated under cold and drought stress (which are possibly mediated by ABA). The
observed increase in the level of antioxidants alpha-tocopherol (1.7 fold increase) and Lascorbic acid (3.1 fold increase) is consistent with an early study reporting a dose-dependent
alpha-tocopherol formation in ABA treated maize seedlings (Jiang and Zhang 2001).
29
In guard cells of water-stressed seedlings, ABA causes the closing of stomata,
mediated by the second messenger Ca2+, thus reducing transcriptional water loss. ABAinduced increases in Ca2+ levels are facilitated by influx through plasma membrane Ca2+
channel and release from internal stores (MacRobbie 2000) Ca2+ channels are activated by
reactive oxygen species (Pie et al 2000 and Kohler et al 2003) the level of which increase as
the part of ABA signaling cascade (Guan et al 2000, Jiang and Zhang 2001, Zhang et al 2001
and Kwak et al 2003). ABA treatment is known to induce the transcription of the gene
encoding cytosolic ascorbate peroxidase (apx2), possibly to adjust the cytosolic redox poise
(Chang et al 2004, Davletova et al 2005), this finding was confirmed by quantitative realtime PCR analysis (approx. 4.5- fold up-regulated in ABA-treated seedlings at 24h) there
was up-regulation of genes encoding the cytosolic enzyme involved in ROS-scavenging
ascorbate-glutathione cycle (APX, up 4.5 fold), monodehydro ascorbate (MDAR, up 1.8
fold) reductase, glutathione reductase (GR, up 2-fold) and dehydro ascorbate reductase
(DHAR, up 1.6-fold). Based on gene expression observed in the experiments, the ABA
treatment led to the activation of cytosolic transcriptional redox control network in
Arabidosis seedling.
30
CHAPTER III
MATERIAL AND METHODS
3.1
Plant material
The experiments were conducted on roots and shoots of seedlings of two wheat
cultivars PBW343 (drought sensitive) and C306 (drought tolerant), exposed to different
treatments. Seeds were obtained from Department of Plant Breeding and Genetics, PAU,
Ludhiana.
Chemicals used for this study, were purchased from different companies like Bovine
serum albumin (BSA), glutathione (oxidized), NADPH, ascorbic acid, ninhydrin, Nitro blue
tetrazolium chloride (NBT), Diethylpyrocarbonate (DEPC), guaiacol, pyrogallol were
purchased from SRL Pvt. Ltd. Mumbai (India); bipyridyl, Thiobarbituric acid (TBA) were
purchased from Himedia Laboratories Pvt. Ltd. Mumbai (India); Hydrogen peroxide,
mannitol, sodium chloride from Fisher-Scientific Mumbai (India); Oligo(dT)18 and agarose
were purchased from G-Biosciences Pvt. Ltd. Noida (India). TRIsoln, M-MuLV Reverse
Transcriptase, Human Placental RNase Inhibitor, Taq DNA Polymerase, DNase I were
purchased from GeNei Pvt. Ltd. Banglore (India). Primers used in present study were
obtained synthesized by Sigma Aldrich Chemicals Pvt. Ltd. Banglore (India).
3.2
Germination of seeds under different treatments
For germination, seeds were washed by adding 2-3 drops of laboratory detergent and
then seeds were disinfected by immersing in 0.1% mercuric chloride solution for 4-5
minutes. Seeds were then washed 4-5 times with sterilized distilled water. After sterilization,
seeds were kept in incubator at 25 ºC for overnight for stratification (Tan et al 2008).
On next day, seeds were placed on moistened filter paper spreaded in sterilized petri
dishes (10 cm diameter). Autoclaved distilled water was used to moisten filter paper. Plates
were kept in dark at 25 ºC for 4 days. During this period, seeds were rewatered regularly
using autoclaved distilled water. Stress was applied on 4th day, where for ABA treatments,
filter paper was moistened with 20 µM ABA, for WS treatments filter paper was moistened
with 6% mannitol, for combined stress treatments filter paper was moistened with 20 µM
ABA plus 6% mannitol. Control seedlings were kept growing on autoclaved distilled water.
Plates were similarly incubated in dark at 25 ºC for another 1-3 days. Data was collected
from 3 stages; 24h, 48h, 72h after stress treatment and compared with similarly old control
seedlings grown on water as well as with 0h stage taken on 4th day before applying stress
treatment.
3.3
Growth measurement
Growth was measured as shoot length, fresh weight, dry weight and water contents
in roots and shoots of germinating seedlings.
3.3.1
Measurement of shoot length
Shoot length (cm) was measured with scale (of 50 seedlings) at 0h, 24h, 48h and 72h
stages.
3.3.2
Measurement of fresh weight and dry weight
Fresh roots and shoots of 25 seedlings were weighed separately in triplicates. Fresh
tissues were subjected to drying for 24hours at 80 ºC in oven and their dry weights were
recorded.
3.3.3
Measurement of water content (g H2O/g DW)
Water contents of roots and shoots were determined in triplicates by using equation:
WS=FW- DW/DW; where FW refers to fresh weight and DW refers to dry weight.
3.3.4 Determination of R/S ratio
Root to shoot ratio was determined in triplicates by using root and shoot dry weights
of 25 seedlings each.
Statistical analysis
Data was calculated as Mean ± S.D and analyzed by Duncan’s Multiple Test at P ≤
0.05 to test for statistical differences among samples using DSAASTAT software version
1.101.
3.4
Enzymatic Assays
3.4.1
Common extraction procedure for all antioxidant enzymes
All antioxidant enzymes studied, were extracted by using common extraction
procedure (as in Yang et al 2008) and were assayed by their specific procedures.
Fresh tissue (root and shoot) of 250 mg was homogenized in 1.5 ml of ice cold
buffer containing 75 µmole of potassium phosphate buffer (pH 7.0), 1.5 µmole of EDTA, 2%
PVP, 0.05% triton-X- 100 using pre-chilled pestle-mortar. Homogenate was passed through
layers of cheese cloth and then centrifuged at 10000 x g at 4 ºC for 15 minutes. Supernatant
after centrifugation was used as enzyme extract in following assays.
3.4.2
Assay for Glutathione reductase (GR)
Glutathione reductase catalyses the reduction of oxidized glutathione at the expense
of 2 molecule of NADPH+ H+.
GSSG + 2NADPH + H+
2GSH + 2NADP+
32
GR was assayed (as in Keles and Oncel 2002) in spectrophotometric cuvette at 25ºC
in a reaction volume of 3 ml containing 150 µmole of potassium phosphate buffer (pH 7.0),
1.75 µmole of glutathione oxidized (GSSG), 1.75 µmole of NADPH+H+. All contents were
mixed and reaction was started by adding 50-100 µl of enzyme extract. Change in
absorbance was recorded for 3 minutes at the interval of one minute at 340 nm. Decrease in
nmole of NADPH+H
+
min-1 was calculated using molar extinction coefficient of NADPH
(6.22 mM-1 cm-1). The glutathione reductase was expressed as nmole of NADPH+H+
disappeared min-1 g-1 DW and specific activity was expressed as nmole of NADPH+H+
disappeared min-1 mg-1 protein.
3.4.3
Assay for Superoxide dismutase (SOD)
SOD causes the conversion of superoxide anion to H2O2 in the presence of molecular
O2 hence scavenges toxic superoxide anion.
O2-. + O2-. + 2H+
O2 + H2O2
SOD was assayed (as in Gapinska et al 2008) at 25 ºC in spectrophotometric cuvette
containing 2.5 ml reaction volume of 1.42% Triton X-100, 125 µmole of Tris HCl buffer pH
8.5, 62.5 nmole of NBT to which, 0.01 µmole of pyrogallol was added. Reduction of NBT
was observed as change in absorbance at 550 nm for 2.5minutes at the interval of 30 seconds.
This was taken as blank. For enzymatic assay, 100-200 µl enzyme extract was added in
another similar above mentioned reaction after the addition of pyrogallol and change in
absorbance was recorded similarly at 550 nm. Enzyme activity was expressed in SOD units
where one SOD unit was defined as the amount of enzyme required to cause 50% inhibition
of NBT reduction as opposed to 0% observed in blank. SOD activity was expressed as SOD
units g-1 DW. SOD specific activity was expressed as SOD units mg-1 protein.
3.4.4 Assay for Ascorbate peroxidase (APX)
APX detoxifies H2O2 to H2O by using reducing power of ascorbate.
Ascorbate + H2O2
Monodehydroascorbate + H2O
APX was assayed (as in Yang et al 2008) in reaction volume of 2.5 ml containing
125 µmole of potassium phosphate buffer of pH 7.0, 0.25 µmole of EDTA, 0.75 µmole of
ascorbate, 2.91 µmole of H2O2 to which 50-100 µl of enzyme extract was added. Change in
absorbance was recorded at 290 nm in spectrophotometric cuvette at 25 ºC for 2.5 minutes at
the interval of 15 seconds. Decrease in µmole of ascorbate per minute was calculated using
molar extinction coefficient of ascorbate of 2.8 mM-1 cm-1. APX activity was expressed as
µmole of ascorbate disappeared min-1 g-1 DW and specific activity was expressed as µmole
of ascorbate disappeared min-1 mg-1 protein.
33
3.4.5 Assay for Guaiacol peroxidase (GOPX)
Guaiacol peroxidase detoxifies H2O2 by using reduction power of plant phenol
guaiacol.
Guaiacol + H2O2
tetraguaiacol + H2O
GOPX was assayed (as in Simova-Stilova et al 2008) in reaction volume of 2.5 ml
containing 250 µmole of potassium phosphate buffer of pH 6.5, 125 µmole of guaiacol, 80
µmole of H2O2, to which enzyme extract was added. Change in absorbace at 470 nm was
recorded in spectrophotometric cuvette at 25 ºC for 2.5 minutes at the interval of 30 seconds.
Change in µmole of tetraguaiacol per minute was calculated using molar extinction
coefficient of tetraguaiacol of 26.6 mM-1 cm-1. Activity was expressed in µmole of
tetraguaiacol appeared min-1 g-1 DW and specific activity was expressed as µmole of
tetraguaiacol appeared min-1 mg-1 protein.
3.4.6
Assay for Catalase
Catalase detoxifies H2O2 to H2O and O2 by following equation
2 H2O
2 H2O + O2
Catalase assay was performed (as in Yang et al 2008) in spectrophotometric cuvette
at 25 ºC in reaction volume of 2.5 ml containing 125 µmole of potassium phosphate buffer of
pH 7.0 and 62 µmole of H2O2, to which 50 µl of enzyme extract was added. Change in
absorbance was recorded at 240 nm for 2.5 minutes at the interval of 15 seconds. Change in
µmole of H2O2 per minute was calculated using molar extinction coefficient of H2O2 of
0.0394 mM-1 cm-1. Activity was expressed in µmole of H2O2 disappeared min-1 g-1 DW and
specific activity was expressed as µmole of H2O2 disappeared min-1 mg-1 protein.
Statistical analysis on enzymatic data:
All enzymes were extracted in triplicates. Mean ± SD was calculated. Data was
analyzed by Duncan’s Multiple Test at P ≤ 0.05 to test for statistical differences among
samples using DSAASTAT software version 1.101.
3.5
Measurement of metabolites involved in antioxidant response of plants
Ascorbate, H2O2, dehydroascorbate, proline, MDA contents were measured where
ascorbate and proline were important non-enzymatic antioxidants in plant under stress.
Ascorbate/ dehydroascorbate ratio, H2O2 level and MDA content were indicators of oxidative
levels of plant tissues under stresses.
3.5.1
Extraction and estimation of H2O2
For extraction, 250 mg of fresh tissue was homogenized in 1.5 ml of ice cold 0.1%
TCA. Homogenate was passed through layers of cheese cloth and then centrifuged at 10000
x g at 4 ºC for 15 minutes. Supernatant after centrifugation was used for estimation.
34
H2O2 was estimated (as in Alexieva et al 2001) by adding 0.5-1 ml of supernatant to
2 ml of reaction mixture containing 4 mmole of potassium iodide and 0.1 mmole of
potassium phosphate buffer of pH 7.0. Test tubes were incubated at room temperature in dark
for 1 hour. Absorbance was read at 390 nm against blank which carried distilled water in
place of supernatant. The amount of H2O2 was calculated by preparing standard curve of 50200 nmole of H2O2. H2O2 was expressed as nmole of H2O2 g-1 DW.
3.5.2
Extraction and estimation of ascorbic acid
250 mg of fresh tissue was homogenized in 1.5 ml of ice cold 5% TCA. Homogenate
was passed through layers of cheese cloth and then centrifuged at 10000 x g at 4 ºC for 15
minutes. Supernatant after centrifugation was used for estimation (as in Nobuhiko et al
1981).
500 µl of appropriately diluted supernatant was added to reaction volume of 2 ml
containing 1% H3PO4, 0.05% FeCl3, 0.25% bipyridyl in ethanol. Blank was prepared with
500 µl of 5% TCA in place of supernatant. The reaction mixture was incubated at 37 ºC for
40 minutes. Absorbance was measured at 525 nm against blank. The quantity of ascorbic was
determined by making standard curve of 5-25 µg of ascorbic acid. Ascorbic acid content was
expressed as µmole of ascorbate g-1 DW.
3.5.3
Extraction and estimation of MDA
1g of tissue was homogenized in 2.5ml of ice cold 0.1% TCA. Homogenate was
passed through layers of cheese cloth and then centrifuged at 10000 x g at 4 ºC for 15
minutes. Supernatant after centrifugation was used for estimation (as in Heath and Packer
1968).
For estimation, 1 ml of appropriately diluted supernatant was mixed with 4 ml of
solution containing 0.5% thiobarbituric acid and 20% TCA. Blank was prepared by taking 1
ml of 0.1% TCA in place of supernatant. Test tubes were kept at 100 ºC for 30 minutes then
cooled down to room temperature. Contents were centrifuged at high speed and then read at
532 nm and 600 nm against blank. Absorbance at 600 nm was subtracted from absorbance at
532 nm. The MDA content was calculated by using molar extinction coefficient of
malondialdehyde of 155 mM-1 cm1 and expressed as nmole of MDA g-1 DW.
3.5.4
Extraction and estimation of Dehydroascorbate
250 mg tissue was homogenized in 1.5 ml of 5% metaphosphoric acid and 1%
thiourea. Homogenate was passed through layers of cheese cloth and then centrifuged at
10000 x g at 4 ºC for 15 minutes. Supernatant after centrifugation was used for estimation (as
in Roe and Oesterling 1943).
35
For estimation 1 ml of appropriately diluted supernatant was mixed with 1 ml of
reagent (containing 2% 2,4-dinitrophenyl hydrazine (DNPH), 0.4% thiourea. 0.05%
CuSO4.5H2O in 9N H2SO4) and incubated at 37 ºC for 3 hours. After incubation the test
tubes were transferred immediately on ice bath and 5 ml of 85% H2SO4 (cold) was added and
test tubes were incubated at room temperature for another 30 minutes. The content was read
at 530 nm against blank containing 5% meatphosphoric acid in place of supernatant. The
content of dehydroascorbate was calculated by using standard curve of 20-80 µg of
dehydroascorbate and expressed as µg of dehydroascorbate g-1 DW.
3.5.5
Extraction and estimation of Proline
250 mg tissue was homogenized in 1.5 ml of ice cold 3% sulphosalicylic acid.
Homogenate was centrifuged at 10000 x g for 15 min at 4 ºC. Supernatant was collected and
used for estimation (Bates et al 1973).
Estimation was done by taking 1 ml of appropriately diluted supernatant with 3%
sulphosalicylic acid and then adding 1 ml of acid ninhydrin reagent (0.31 g ninhydrin, 7.5 ml
acetic acid and 5 ml 6M phosphoric acid) and 1 ml of glacial acetic acid. Test tubes were
incubated at 100 ºC for 1 hour. After 1 hour the reaction was terminated on ice bath. Reaction
mixtures were reacted with 4 ml toluene and mixed vigorously over vortex for 15-20 seconds.
Test tubes were placed at room temperature for phase separation. The pink-red upper layer of
chromophore containing toluene was read at 520 nm using toluene for blank. The quantity of
proline was determined by standard curve prepared by taking different concentrations of
proline ranging from 0.05 µM to 0.5 µM. Proline contents were expressed as µmole of proline
g-1 DW.
3.5.6
Estimation of Protein
Proteins were estimated in all samples extracted for enzymatic assays. Estimation was
done by using Lowry’s method (Lowry et al 1951).To 0.5 ml of appropriately diluted sample in
test tubes, 2.5 ml of reagent C (reagent A and reagent B in 50:1, mixed just before use, where
reagent A was 2% Na2CO3 in 0.1 N NaOH and reagent B was 0.5% CuSO4 in 1% sodium
potassium tartarate) was added. Contents of test tubes were mixed and allowed to stand for 10
minutes at room temperature. 0.25 ml of reagent D (Folin ciocalteu phenol reagent: Water in
1:1, mixed just before use) was added and mixed rapidly. The intensity of blue colour
developed was read after 30 minutes at 530 nm. The amount of protein was calculated from
standard curve of different concentrations of bovine serum albumin (20-100 µg).
3.6
Gene Expression Analysis
Gene expression analysis was done by semi-quantitative RT-PCR. In the present study,
LEA genes and genes involved in antioxidant response of the plants were analyzed for such
36
assays. Assays were done in shoots of both cultivars at 24h and 48h stages after applying stress
on 4th day after germination. Stress treatments were 20 µM ABA for ABA treatment, 6%
mannitol for WS treatment, 20 µM ABA plus 6% mannitol for combined stress treatment. The
results of stress treated samples were compared with similarly old grown control seedlings
grown on autoclaved distilled water. The protocol for analysis is mentioned below:
3.6.1
Database search
It was done for the availability of the gene sequences for above mentioned genes in
wheat plant. Nucleotide, mRNA, EST and protein databases were searched at NCBI
(www.ncbi.nlm.nih.gov/) for these genes in wheat. The results of this search were summarized
in Table 3.1. Primers were designed manually as well as with Primer-BLAST software
available at NCBI. Primer sequences corresponding to each gene are given in Table 3.1.
3.6.2
Total RNA isolation
Total RNA was isolated from shoots of all the four treatments in both cultivars at 24h
and 48h stages. Total RNA was isolated by using TRIsoln (GeNei Pvt. Ltd.) where tissue was
crushed in TRIsoln and then chloroform was added to separate the aqueous phase from organic
phase. RNA was precipitated from aqueous phase using isopropanol. Pellet obtained was
washed with 70% ethanol and then dissolved in DEPC-treated autoclaved distilled water.
Isolated RNA was treated with DNase I to remove contaminating DNA. The RNA was
specifically precipitated from DNase I treated RNA sample in about 3.5 M LiCl at -20 ºC. The
pellet obtained was washed in 70% ethanol and dissolved in DEPC-treated autoclaved distilled
water. RNA content and quality were estimated by UV spectrophotometer at 260/280 nm.
3.6.3
RT-PCR (Reverse Transcription-Polymerase chain reaction)
Approximately 1 µg of total RNA was used as a template to make cDNA using reverse
transcriptase. Approximately 1 µg of total RNA was mixed with 5-100 pmole of oligo-(dT)18
and denatured at 72 ºC for 5 minutes. The denatured mixture was mixed with 500 µM of each
dNTP, 20U of Recombinant Human Placental Ribonuclease inhibitor (GeNei Pvt. Ltd.), 70U of
M-MuLV Reverse Transcriptase (GeNei Pvt. Ltd.) in 1X transcription buffer in a total reaction
volume of 20 µl. The reaction mixture was incubated at 42 ºC for 1 hour and stored at -20 ºC.
PCR was performed in a total reaction volume of 25 µl with 1X buffer containing 1.5 mM
MgCl2, 200 µM of each dNTP, 0.4% PVP, 25 pmole of each primer and 5 Units of Taq
Polymerase (GeNei Pvt. Ltd.). PCR was performed under standard conditions (35 cycles of 40
sec at 94 ºC/ 40sec at 55 ºC/ 1-2 min at 72 ºC/ final extension of 10 min). RT-PCR of cytosolic
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also run on the same samples to
normalize the amount of template in each sample.
37
3.6.4
Agarose Gel Electrophoresis
PCR products were run on 1.5% agarose gel and then visualized in Gel DocTMXR
(BioRad Laboratories, Inc). Intensity of bands were measured by using Quantity One 1-D
Analysis software (BioRad Laboratories, Inc). Ratio was calculated by dividing intensity of
band of gene by the intensity of the band of GAPDH (constitutive gene used as internal
control).
38
Table 3.1: List of genes along with their primer sequences used in the present study
Antioxidant Genes
Gene ID
Primer sequence (5’ to 3’)
Gene name
EF555121
Peroxisomal APX
F- TAGGTCGTCCGCGATGGCGG
R- CCCCTTACTTGCTCCTCTTGG
X94352
CAT-2
F- GCTTTCTTCCTTCTTCCTCGCC
R- CAACTACTCTCCAGTTCTCCT
D86327
CAT-1
F- GCTGACCGTTCCTCCGTTCGC
R- TTGACGTAGTGCGCCTTGCC
Late Embryogenesis Abundant (LEA) Genes
Group
Gp2
Group
Gp3
Gene ID
Primer sequence (5’ to 3’)
Gene name
CV762802
Similar to X78429 (Td 16) of durum wheat
F- GCCAAGTGAGCAAGACAACA
R- ATGACCTTGCTGTCCGTAGG
DR739608
Similar to X78430 (Td 25a) of durum wheat
F- CGAGAAGAAGAGCCTCATGG
R- GTTTTCCCAGTCACGACGTT
AL815683
Similar to X78431 (Td 27e) of durum wheat
F- AAAGCCACAACCAAGTCCAG
R- GTAGGCTCCACCAGTTCCAG
U73211
WCOR410 (similar to AJ890140, Td 11 of durum wheat)
F- CGAGGAGGAGAAGAAAGGCT
R- CTCCCACCTTGACACCAACT
AB076807
Wdhn 13
F- TGAGGGCAAGATGGAGCACC
R- ATACCATGCACCGGTTGAACC
Gene ID
Primer sequence (5’ to 3’)
Gene name
AF255052
Wrab 19
F- GAGCAATACTAGCAGTGAGATTTAC
R- GTACTGTAGAAGGCTCGTGAAC
AB115913
Wrab 15
F- CGCGTCTCACGTCAGTCGGT
39
R- TAGGCGAGGGTATGCGTGG
Gp4
AF255053
Wrab 17
F- TCAACTTCAAAAATGTCTGGTTGGT
R- ATAGCGAAACAGAAGGAGGG
AB115914
Wrab 18
F- CTTCGTGTTTGTTGGTGAGAGAG
R- GCGAACGACCAAACGAGTAAAGG
AY148490
Ta LEA-3 like
F- AGGTCGTGTTCCAAGAAACC
R- TCGAAGGCAAACTTTTACACCA
GH729039
Similar to AJ890139 (Td 29) of durum wheat
F- ATTATTACGCCGTGCACACA
R- CTCGACATACCGGTGAAGGT
Constitutive Gene
Gene ID
EF592180
Primer sequence (5’ to 3’)
Gene name
Cytosolic Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)
40
F- GGATGCTCCCATGTTTGTC
R- CCTGTTGTCACCCTGAAAGT
CHAPTER IV
RESULTS AND DISCUSSION
4.1
Effect on growth
The seedlings of both cultivars were grown for four days in distilled water, stress
was applied on 4th day by 20 µM ABA for ABA treatment, 6% mannitol for WS treatment,
20 µM ABA plus 6% mannitol for combined stress treatment. Measurement of growth was
done at 24h, 48h, 72h after applying stress and was compared with similarly old control
seedlings growing on distilled water as well with 0h seedlings taken on 4 th day before
applying stress. Growth data was collected from root and shoot separately and different
growth parameters- shoot length (Table 4.1, Figure 4.1), water content (Table 4.2, Figure
4.2), fresh weight (Table 4.3, Figure 4.3), dry weight (Table 4.4, Figure 4.4) and root (dw)/
shoot (dw) ratio (Table 4.5, Figure 4.5) were studied.
Shoot length was determined with the help of ruler for 50 seedlings. Mean ± S.D was
calculated and data was analyzed by DSAASTAT by applying Duncan’s Multiple Test (p>
0.5). Decrease in shoot length was found under all stress treatments in both cultivars (Table
4.1; Figure 4.1). However, no significant variation for this parameter was found between
cultivars under all stress treatments.
Water contents were determined using FW-DW/DW formula. Water contents were
found to be affected in roots and shoots of both cultivars under water stress and combined
treatments. However water contents were not affected under ABA treatment in both
cultivars, except in PBW343 shoots at 24h stage where these were decreased (Table 4.2;
Figure 4.2). Comparing cultivars, water contents were decreased throughout the stress period
in C306 however water contents were regained after 24h of stress in PBW343.
It can be concluded from this study that shoot length but not water contents were
decreased by ABA which is also documented in literature that ABA inhibits shoot growth in
drying soil under conditions when shoot water deficit does not occur (Sharp and Le Noble
2002). Moreover low water content in C306 can also be correlated to its dehydration
tolerance as dehydration tolerance includes the ability of plant to maintain cell functions
under low water contents and hence a mechanism for dehydration tolerance (Passiosure et al
1993). Lower contents of RWC (Relative water content) were observed in leaves of drought
tolerant wheat cultivars than drought susceptible cultivars at different days after sowing
under field conditions (Lascano et al 2001).
Table 4.1: Shoot length (cm) of germinating wheat seedlings of two wheat cultivars PBW343 and C306 where stress was applied on 4 th day after
germination as ABA (20µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected
at 24h, 48h, 72h after stress treatment and compared with similarly aged control seedlings (Ct) grown on distilled water as well as
with 0h stage taken just before applying stress treatment.
PBW343
C306
Shoot
length
42
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
0h
2.79 ± 0.48a
2.79 ± 0.48a
2.79 ± 0.48a
2.79 ± 0.48a
2.40 ± 0.47a
2.40 ± 0.47a
2.40 ± 0.47a
2.40 ± 0.47a
24h
5.60 ± 1.26g
4.35 ± 1.03cdef
3.47 ± 0.89b
3.92 ± 0.97c
4.34 ± 1.21ef
4.13 ± 1.26def
2.93 ± 0.94ab
3.23 ± 0.92bc
48h
7.30 ± 1.24h
5.33 ± 1.27g
4.18 ± 0.95c
4.27 ± 1.15cd
7.18 ± 2.58h
4.77 ± 1.42f
3.58 ± 1.36cd
3.87 ± 0.98cde
72h
9.68 ± 1.45i
5.71 ± 1.24g
4.75 ± 1.11df
4.27 ± 1.35cde
9.58 ± 2.89i
5.92 ± 1.94g
4.30 ± 1.14ef
3.62 ± 1.00cd
*Shoot length was measured for 50 seedlings. Mean ± SD was calculated.
*Data in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of similar alphabets
indicate no significant difference among the values.
14
72h
48h
24h
0h
i
shoot length (cm)
12
i
h
10
h
8
gg
g
6
43
4
g
df
cdef
b
c
a
a
a
a
ef
cd cde
c
f
cde
cd
a
a
a
ef
def
ab
bc
cd
a
2
0
CT
Fig. 4.1:
ABA
PBW343
WS
ABA+WS
CT
ABA
WS
C306
ABA+WS
Shoot length of germinating wheat seedlings of two wheat cultivars PBW343 and C306 where stress was applied on 4 th day after
germination as ABA (20µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected
at 24h, 48h, 72h after stress treatment and compared with similarly aged control seedlings (Ct) grown on distilled water as well as
with 0h stage taken just before applying stress treatment. Shoot length was measured for 50 seedlings. Mean ± SD was calculated.
Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where
superscripts of similar alphabets indicate no significant difference among the values.
Table 4.2: Water contents of shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two cultivars PBW343 and C306
where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly aged control seedlings
(Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
PBW343
C306
WC
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
44
0h
10.21 ± 0.01de
10.21 ± 0.01de
10.21 ± 0.01de
10.21 ± 0.01de
12.31 ± 0.25h
12.31 ± 0.25h
12.31 ± 0.25h
12.31 ± 0.25h
24h
12.62 ± 4.73e
8.02 ± 1.21bcd
7.03 ± 0.40abc
7.06 ± 0.42abc
9.67 ± 0.01fg
10.13 ± 0.50g
7.17 ± 0.37cd
6.99 ± 0.33c
48h
8.74 ± 0.34bcd
9.44 ± 1.19cd
6.66 ± 0.50abc
6.39 ± 0.69ab
8.05 ± 0.10e
9.21 ± 0.29f
5.58 ± 0.06ab
6.01 ± 0.78b
72h
7.98 ± 0.19bcd
7.62 ± 0.043bcd
5.75 ± 0.47ab
4.63 ± 2.19a
7.77 ± 0.16de
7.64 ± 0.67cde
4.94 ± 0.14a
5.10 ± 0.47a
Root
0h
15.22 ± 0.91f
15.22 ± 0.91f
15.22± 0.91f
15.22 ± 0.91f
14.98 ± 3.03e
14.98 ± 3.03e
14.98 ± 3.03e
14.98 ± 3.03e
24h
13.62 ± 1.94ef
10.47 ± 1.33cde
8.67 ± 3.4abcd
6.44 ± 0.80c
13.91 ± 0.93de
11.68 ± 1.44cd
8.67 ± 1.28ab
7.54 ± 1.63a
48h
14.14 ± 0.14f
11.82 ± 1.84def
8.08 ± 2.56abc
5.72 ± 0.30b
11.32 ± 0.20c
10.24 ± 1.28bc
6.40 ± 1.51a
6.29 ± 0.88a
72h
10.23 ± 3.63cd
9.61 ± 1.12bcd
9.46 ± 0.87bcd
6.50 ± 1.59a
12.12 ± 0.42cd
10.65 ± 1.44bc
8.40 ± 0.52ab
8.26 ± 0.10ab
* Water contents were recorded in triplicates. Mean ± SD was calculated.
*Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
0h
A 20
48h
72h
e
18
Water content in shoot (g H2O/g DW)
24h
16
14
12
h
h
de
bcd
bcd
10
de cd
bcd
abc
abc
ab
bcd
8
fg
abcab
e
f
cde
de
c
cd
a
b
ab
6
a
a
4
2
0
CT
ABA
WS
ABA+WS
CT
ABA
PBW343
B 20
18
16
Water content in root (g H2O/g DW)
h
g
de
de
h
14
0h
24h
48h
f
f cd
f
72h
e
e
e
f
de
def
cde
12
ABA+WS
C306
e
f ef
WS
cd
abcd
bcd
10
c
abc
bcd
cd
bcbc
ab
ab
ab
8
a
ab
a ab
a
a
6
4
2
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.2: Water contents of shoot (A) and root (B) of germinating wheat seedlings of two
cultivars PBW343 and C306 where stress was applied on 4th day after
germination as ABA (20µM ABA), WS (6% Mannitol) and ABA+WS (20 µM
ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after
stress treatment and compared with similarly aged control seedlings (Ct)
grown on distilled water as well as with 0h stage taken just before applying
stress treatment. Water contents were recorded in triplicates. Mean ± SD was
calculated. Data of root and shoot in each cultivar was analyzed separately by
Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts
of similar alphabets indicate no significant difference among the values.
45
Table 4.3: Fresh weights (g) of shoot (upper panel) and root (lower panel) of 25 germinating wheat seedlings of two cultivars PBW343 and C306
where stress was applied on 4th day after germination as ABA (20µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly aged control seedlings
(Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
PBW343
C306
FW
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
46
0h
1.07 ± 0.07abcd
1.07 ± 0.07 abcd
1.07 ± 0.07 abcd
1.07 ± 0.07 abcd
1.13 ± 0.06ab
1.13 ± 0.06ab
1.13 ± 0.06ab
1.13 ± 0.06ab
24h
1.31 ± 0.04ef
1.24 ± 0.01de
1.08 ± 0.05abcd
1.10 ± 0.11bcde
1.44 ± 0.03de
1.40 ± 0.10de
0.93 ± 0.24a
1.19 ± 0.08bc
48h
1.48 ± 0.09f
1.19 ± 0.14cde
0.91 ± 0.18ab
1.01 ± 0.09abc
1.66 ± 0.02f
1.34 ± 0.14d
1.06 ± 0.12ab
1.00 ± 0.15ab
72h
1.72 ± 0.01g
1.17 ± 0.09cde
1.00 ± 0.09abc
0.89 ± 0.23a
1.74 ± 0.002f
1.55 ± 0.016ef
1.10 ± 0.16ab
1.17 ± 0.00bc
Root
0h
0.60 ± 0.10abc
0.60 ± 0.10 abc
0.60 ± 0.10 abc
0.60 ± 0.10 abc
0.73 ± 0.04abc
0.73 ± 0.04 abc
0.73 ± 0.04 abc
0.73 ± 0.04 abc
24h
0.72 ± 0.15cde
0.66 ± 0.20 abcd
0.64 ± 0.05 abcd
0.62 ± 0.02 abcd
0.83 ± 0.07cd
0.69 ± 0.18 abc
0.54 ± 0.07a
0.56 ± 0.08 ab
48h
0.94 ± 0.06e
0.62 ± 0.03 abcd
0.52 ± 0.14 abc
0.46 ± 0.05ab
0.99 ± 0.02d
0.71 ± 0.19 abc
0.73 ± 0.15 abc
0.51 ± 0.01a
72h
0.84 ± 0.15de
0.54 ± 0.20 abc
0.68 ± 0.07bcd
0.45 ± 0.10a
0.84 ± 0.15cd
0.76 ± 0.18 abcd
0.60 ± 0.09 abc
0.80 ± 0.20bcd
* Fresh weights of 25 seedlings were recorded in triplicates. Mean ± SD was calculated.
*Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
shoot fresh weight (g) per 25 seedlings
A 2
0h
48h
72h
g
1.8
f
f
1.6
1.4
24h
ef
decd
de
cde
de cde
abcd
bcde
abcd
abcd ababc abcd abca
1.2 abcd
f
ab
ef
ab
ab
ab aab
ab bc bc
ab
WS
ABA+WS
1
0.8
0.6
0.4
0.2
0
CT
ABA
WS
ABA+WS
CT
ABA
PBW343
B 1.2
0h
24h
C306
48h
72h
root fresh weight (g) per 25 seedlings
e de
d cd
1
cde
0.8
abc
cd
abcd
abc
abcd bcd
abc
abc
abcd
abc
abcd
abc
abc
0.6
ab
bcd
abcd
abc
abc
abc
abc
abc
abc
abc
ab
a
a
a
0.4
0.2
0
CT
ABA
WS
ABA+WS
PBW343
Fig. 4.3:
CT
ABA
WS
ABA+WS
C306
Fresh weights of shoot (A) and of root (B) of 25 germinating wheat seedlings of two
cultivars PBW343 and C306 where stress was applied on 4 th day after germination as
ABA (20µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and
compared with similarly aged control seedlings (Ct) grown on distilled water as well
as with 0h stage taken just before applying stress treatment. Fresh weights of 25
seedlings were recorded in triplicates. Mean ± SD was calculated. Data of root and
shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05
for test of significance, where superscripts of similar alphabets indicate no significant
difference among the values.
47
Table 4.4: Dry weights (g) of shoot (upper panel) and root (lower panel) of 25 germinating wheat seedlings of two cultivars PBW343 and C306
where stress was applied on 4th day after germination as ABA (20µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly aged control seedlings
(Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
DW
PBW343
CT
ABA
C306
WS
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
a
0.10 ± 0.006
a
0.10 ± 0.006
a
0.10 ± 0.006a
0.09 ± 0.006a
0.09 ± 0.006a
0.09 ± 0.006a
0.09 ± 0.006a
48
0h
0.10 ± 0.006
24h
0.10 ± 0.03a
0.14 ± 0.02bcd
0.13 ± 0.01bcd
0.14 ± 0.02bcd
0.14 ± 0.002bc
0.13 ± 0.015b
0.11 ± 0.04ab
0.15 ± 0.003bcd
48h
0.15 ± 0.01cd
0.12 ± 0.03ab
0.12 ± 0.016abc
0.14 ± 0.001bcd
0.18 ± 0.004ef
0.13 ± 0.01bc
0.16 ± 0.02cde
0.15 ± 0.04bc
72h
0.19 ± 0.005e
0.14 ± 0.009bcd
0.15 ± 0.02bcd
0.16 ± 0.02cd
0.20 ± 0.003f
0.18 ± 0.016def
0.19 ± 0.02ef
0.19 ± 0.014ef
Root
0h
0.04 ± 0.004a
0.04 ± 0.004a
0.04 ± 0.004a
0.04 ± 0.004a
0.05 ± 0.006a
0.05 ± 0.006a
0.05 ±0.006a
0.05 ± 0.006a
24h
0.05 ± 0.004ab
0.06 ± 0.010abc
0.07 ± 0.018bcd
0.08 ± 0.012d
0.06 ± 0.009ab
0.05 ± 0.008ab
0.06 ± 0.00ab
0.07 ± 0.004abc
48h
0.06 ± 0.004bcd
0.05 ± 0.004ab
0.06 ± 0.000abc
0.07 ± 0.004bcd
0.08 ± 0.003bcd
0.06 ± 0.010abc
0.10 ± 0.04d
0.07 ± 0.010abc
72h
0.08 ± 0.011cd
0.05 ± 0.013ab
0.06 ± 0.012bcd
0.06 ± 0.026bcd
0.06 ± 0.009abc
0.06 ± 0.007abc
0.06±0.006abc
0.09 ± 0.022cd
* Dry weights of 25 seedlings were recorded in triplicates. Mean ± SD was calculated.
*Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
A
0.25
0h
48h
72h
a
a
ef
def
ef
cd
bcd
cd
0.15
ef
f
e
0.2
shoot dry weight (g) per 25 seedlings
24h
bc
cde
bcd
bcd bcd
ab
bcd
abc
a
a
ab
b bc
bc
bcd
bcd
a
a
0.1
a
a
a
f
0.05
0
CT
ABA
WS
ABA+WS
CT
ABA
PBW343
B 0.16
0h
24h
WS
ABA+WS
C306
48h
72h
d
root dry weight (g) per 25 seedlings
0.14
0.12
cd
0.1
d
bcd
cd
bcd
0.08
bcd
abc
0.06
a
a
bcd
abc
ab
bcd
abc
ab
ab
ab
bcd
a
a
a
abcabc
ab
abc
aab
abc
abc
a
a
0.04
0.02
0
CT
ABA
WS
ABA+WS
PBW343
Fig. 4.4:
CT
ABA
WS
ABA+WS
C306
Dry weight of shoot (A) and of root (B) of 25 germinating wheat seedlings of two
cultivars PBW343 and C306 where stress was applied on 4 th day after germination
as ABA (20µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment
and compared with similarly aged control seedlings (Ct) grown on distilled water as
well as with 0h stage taken just before applying stress treatment. Dry weights of 25
seedlings were recorded in triplicates. Mean ± SD was calculated. Data of root and
shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P <
0.05 for test of significance, where superscripts of similar alphabets indicate no
significant difference among the values.
49
Table 4.5:
Root (dw)/shoot (dw) ratio of germinating wheat seedlings of two cultivars PBW343 and C306 where stress was applied on 4 th day
after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was
collected at 24h, 48h, 72h after stress treatment and compared with similarly aged control seedlings (Ct) grown on distilled water as
well as with 0h stage taken just before applying stress treatment.
PBW343
C306
50
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
0h
0.39 ± 0.07a
0.39 ± 0.07a
0.39 ± 0.07a
0.39 ± 0.07a
0.55 ± 0.12bc
0.55 ± 0.12bc
0.55 ± 0.12bc
0.55 ± 0.12bc
24h
0.50 ± 0.12ab
0.42 ± 0.13a
0.51 ± 0.084ab
0.61 ± 0.009b
0.41 ± 0.06ab
0.42 ± 0.02ab
0.51 ± 0.15abc
0.45 ± 0.03abc
48h
0.41 ± 0.063a
0.43 ± 0.14a
0.49 ± 0.067ab
0.50 ± 0.029ab
0.44 ± 0.003abc
0.48 ± 0.04abc
0.65 ± 0.32c
0.49 ± 0.06abc
72h
0.40 ± 0.07a
0.37 ± 0.07a
0.44 ± 0.009a
0.38 ± 0.11a
0.32 ± 0.05a
0.36 ± 0.07ab
0.34 ± 0.01a
0.45 ± 0.08abc
*Ratios were recorded in triplicates. Mean ± SD was calculated.
*Data in each cultivar was analyzed by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of similar alphabets indicate no
significant difference among the values.
Root dry weight/shoot dry weight ratio
1.2
0h
24h
48h
72h
1
c
0.8
bc
ab
0.6
a
a
a
a
ab
a
a
bc abc
bc
abc
ab
ab
a
bc
b
a
a
a
a
abc
ab
abc
0.4
ab
abc
abc
ab
a
a
51
0.2
0
CT
ABA
PBW343
WS
ABA+WS
CT
ABA
WS
ABA+WS
C306
Fig. 4.5: Root (dw)/shoot (dw) ratios of germinating wheat seedlings of two cultivars PBW343 and C306 where stress was applied on 4 th
day after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments.
Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly aged control seedlings (Ct) grown on
distilled water as well as with 0h stage taken just before applying stress treatment. Ratios were recorded in triplicates. Mean ±
SD was calculated. Data was analyzed separately in each cultivar by Duncan’s Multiple Test at P < 0.05 for test of significance,
where superscripts of similar alphabets indicate no significant difference among the values.
Fresh weights (Table 4.3, Figure 4.3) and Dry weights (Table 4.4, Figure 4.4) of
roots and shoots of 25 seedlings were measured in triplicates. Mean ± S.D. was calculated
and data was similarly analyzed by DMT. Both fresh weight and dry weight were affected
under all stress treatments in both roots and shoots of PBW343 only but not in C306.
In C306 shoots, dry weights were maintained at all three stages under all stress
treatments, except there was decrease in dry weight at 48h stage under ABA and combined
treatment. In C306 roots, dry weights were not affected under any treatment. In PBW343,
dry weights of shoots were affected more than roots. There was increase in the dry weights
of shoots of PBW343 at 24h under all stress treatments but this increase was followed by
decrease during later stages. In PBW343 roots, dry weights were decreased under ABA
treatment only otherwise these were maintained under other stress treatments.
Comparing ABA response and WS response in both cultivars, dry weights were
affected more by ABA than by WS. Moreover these effects of ABA were more pronounced
in shoots than in roots, which could be related to known function of ABA i.e. to maintain
more root growth under stresses hence to maintain root/shoot growth ratio (Vysotskaya et al
2008).
From above data, conclusion can be made that ABA affects dry weight accumulation
in plants but does not affect water content. ABA treatment has also been reported to increase
water content under drought stress in triploid Bermuda grass (Shaoyun et al 2009). Secondly,
under WS, dry weights were maintained better in roots than in shoots in both cultivars.
Extensive root growth under drought conditions is considered to be a major drought
avoidance mechanism (Shao et al 2008). This process is partly mediated through ABA
(Cutler et al 2010). Increased biomass and decreased water content under drought are
considered to be the drought tolerant features (Guoxiong et al 2002, Passiosure et al 1993,
Lascano et al 2001) and these features were found to be better shown in C306, drought
tolerant cultivar than in PBW343, drought sensitive cultivar in this study.
4.2
Effects on antioxidant response
In present study antioxidant response was determined in terms of H2O2, ascorbate,
ascorbate/dehydroascorbate ratio, malondialdehyde, proline contents and levels of
antioxidant enzymes, Ascorbate peroxidase (APX), Guaicol peroxidase (GPOX), Catalase
(CAT), Glutathione reductase (GR), Superoxide dismutase (SOD).
4.2.1
Hydrogen peroxides (H2O2) contents
H2O2 contents were found to be higher in roots and shoots of control seedlings of
PBW343 than of C306 (Table 4.6; Figure 4.6). H2O2 contents increased at 24h stage under all
stress treatments in roots of C306 but decreased in the roots of PBW343. These contents
were almost unaffected at 24h stage under all stress treatments in shoots of C306 but in
52
shoots of PBW343, it decreased under WS, unaffected under combined stress, increased
under ABA treatment at the same stage. H2O2 contents were brought down during later stages
in roots of C306 but in shoots of this cultivar, these contents were not affected by any stress
treatment and showed increasing trend during germination like as in control seedlings.
Comparing both cultivars for this feature, it was clear that there was no increase in
H2O2 contents in both root and shoot under WS in drought susceptible cultivar (PBW343).
However there was increase in H2O2 level by exogenous ABA in shoots of this cultivar when
ABA was applied either individually or under combined stress. Contrary to it, drought
tolerant cultivar C306 increased H2O2 level in its roots and maintained higher such contents
in its shoots during germination under all stress treatments. As exogenous supply of ABA
gave the feature of elevating H2O2 level in PBW343 which indicates that H2O2 production
might be the downstream effect of ABA in ABA -dependent pathway which was working in
C306 but might be lacking in PBW343 under WS.
Rise in H2O2 content in roots of C306 under stresses is understandable as roots of
plants are the first tissue to sense water deficit conditions (Selote and Khanna-Chopra 2010).
H2O2 content is reported to be increased under ABA and is a downstream effect of ABA. But
H2O2 contents have also been found to be decreased under stresses (Loggini et al 1999,
Simova-Stoilova et al 2008) similar to as observed in PBW343 in our study. So far H2O2
contents in literature are considered to be related to drought resistance mechanism where
lower levels of these contents under stresses were positively correlated to drought tolerance
levels as many reports reported lower levels of H2O2 in drought resistant cultivars than
drought susceptible cultivars when exposed to similar stresses (Guo et al 2006, KhannaChopra and Selote 2007, Wang et al 2009). These studies considered H2O2 as toxic molecule
in plants which causes oxidative damage. Hence its lower level under stresses favors growth
and avoids oxidative damage. But recently, another view is given, which considers H2O2 not
as toxic molecule rather a signaling molecule to initiate ROS signaling for producing stress
response. This is supported by observations that H2O2 content has not been found to be
related to lipid peroxidation (Jubany-Mari et al 2010); plants with lower cytosolic APX
activity or cAPX knockout were found to be more drought tolerant rather drought susceptible
(Ishikawa and Shigeoka 2008, Munns and Tester 2008, Jubany-Mari et al 2010). So higher
level of H2O2 contents in drought tolerant cultivar and lower level in drought susceptible
cultivar under WS might be related to such kind of observations.
53
Table 4.6: Amount of H2O2 (µmole of H2O2 g-1 DW) in shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two cultivars
PBW343 and C306 where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6% mannitol) and ABA+WS (20
µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly aged
control seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
H2O2
PBW343
CT
ABA
C306
WS
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
54
0h
1.53 ± 0.18abc
1.53 ± 0.180abc
1.53 ± 0.18abc
1.53 ± 0.18abc
1.12 ± 0.41a
1.12 ± 0.41a
1.12 ± 0.41a
1.12 ± 0.41a
24h
3.08 ± 0.58d
3.88 ± 0.49e
1.80 ± 0.09bc
2.99 ± 0.76d
1.29 ± 0.20ab
1.75 ± 0.02abcd
1.93 ± 0.23abcde
1.58 ± 0.12abc
48h
5.75 ± 0.18f
3.48 ± 0.04de
2.00 ± 0.15c
2.85 ± 0.32d
2.40 ± 1.18cde
2.09 ± 0.38bcde
2.57 ± 0.25def
2.50 ± 0.30def
72h
3.18 ± 0.32d
1.03 ± 0.08a
1.20 ± 0.40ab
1.79 ± 0.38bc
3.67 ± 0.79g
2.74 ± 0.39ef
3.30 ± 0.10f
1.79 ± 0.23abcd
cd
cd
cd
1.11 ± 0.24cd
1.20 ± 0.22ab
1.20 ± 0.22ab
1.20 ± 0.22ab
1.20 ± 0.22ab
Root
0h
1.11 ± 0.24
1.11 ± 0.24
1.11 ± 0.24
24h
2.37 ± 0.54e
1.28 ± 0.04cd
0.40 ± 0.05ab
0.81 ± 0.23bc
0.75 ± 0.18a
2.84 ± 0.88def
2.55 ± 0.03de
3.29 ± 0.06ef
48h
6.28 ± 0.70g
3.06 ± 0.35f
2.69 ± 0.07ef
1.41 ± 0.07d
3.57 ± 1.01f
1.65 ± 0.18bc
1.44 ± 0.37ab
1.45 ± 0.34ab
72h
0.19 ± 0.02a
0.23 ± 0.06a
0.16 ± 0.03a
0.11 ± 0.01a
4.89 ± 0.31g
*Estimations were done on samples extracted in triplicates. Mean ± SD was calculated.
2.36 ± 0.39cd
2.49 ± 0.13d
1.59 ± 0.11b
*Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
umole of H2O2 per gm DW
A
7
0h
24h
48h
72h
f
6
5
g
e
d
4
d
de
d
cde
d
ef
3
2
abc
bc
c
bc
abc
ab abc
abc
bcde
abcd
a
a ab
def
f
g
def
abcde
abcd
aabc
a
a
1
0
CT
ABA
WS
ABA+WS
CT
ABA
0h
24h
ABA+WS
C306
PBW343
B 8
WS
48h
72h
umole of H2O2 per gm DW
g
7
6
g
f
5
4
e
3
2
def
f
ef
cd
cdcd
1
a
cd
cd
a
cdbc
ab
ef
a
de
bc
d
ab
ab
d
ab
ab
ab b
ab
a
a
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.6: H2O2 contents (µmole of H2O2 g-1 DW) in shoot (A) and root (B) of germinating wheat
seedlings of two cultivars PBW343 and C306 where stress was applied on 4th day
after germination as ABA (20 µM ABA), WS (6% mannitol) and ABA+WS (20 µM
ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress
treatment and compared with similarly aged control seedlings (Ct) grown on
distilled water as well as with 0h stage taken just before applying stress treatment.
Estimations were done on samples extracted in triplicates. Mean ± SD was
calculated. Data of root and shoot in each cultivar was analyzed separately by
Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
55
4.2.2
Ascorbic acid contents and ascorbate/dehydroascorbate ratio
Ascorbate contents (Table 4.7; Figure 4.7) were found to be more in shoots than in
roots of both wheat cultivars. Ascorbate contents increased in shoots and roots of PBW343 at
48h stage following increased contents of H2O2 at 24h stage of shoot under ABA treatment,
accompanied by similar increase in ascorbate/dehydroascorbate ratio at the same stage
(Table 4.9; Figure 4.8). However under WS as well as under combined stress, such increases
in contents and ratios were not observed rather both decreased in roots as well as in shoots of
PBW343. But combined stress could be placed intermediate between ABA treatment and
WS treatment for contents as well as for ratios.
Shoots of C306 control plants had already high ascorbate/dehydroascorbate ratio at
the stage corresponding to 24h stage. Ascorbate contents of shoots of C306 increased under
ABA treatment from 24-48h accompanied by higher ascorbate/dehydroascorbate ratio during
same time period. Ascorbate contents were not altered under WS as well as under combined
stress in shoots of C306. But ascorbate/dehydroascorbate ratios first decreased at 24h stage
but were maintained higher during later stages compared to control seedlings of same stage
under WS and combined stresses. In C306 roots, ascorbate contents were not much affected
under all stress treatments but ratios were maintained higher compared to those of control
seedlings at 24h to 48h stages under all stresses.
Above results indicate that ABA increased H2O2 contents followed by increases in
ascorbic acid contents as well as in ascorbate/dehydroascorbate ratios in both cultivars. Such
type of ABA-dependent response might be lacked in PBW343 but working in C306 under
WS. In literature, there are many reports which correlate drought tolerance of plant with
higher ascorbate and ascorbate/dehydroascorbate ratio under stresses (Sairam and Shrivastva
2001, Jiang and Zhang 2002, Guo et al 2006, Khanna-Chopra and Selote 2007) but others
reports are also there where ascorbate contents were found to be decreased under stresses
(Selote and Khanna-Chopra 2006, Simova-Stilova et al 2008).
4.2.3
Malondialdehyde (MDA) contents
MDA contents (Table 4.10; Figure 4.9) were not much altered in roots and shoots of
C306 except its shoots showed higher contents at 24h stage under WS. In PBW343 shoots,
MDA contents were found to be higher at 24h stage under both WS and combined stress.
Roots of PBW343 showed higher level of MDA at 72h stage under all stress treatments and
at 48h stage under WS and combined treatments.
56
Table 4.7: Amount of ascorbic acid (µg of ascorbate g-1 DW) in shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two
cultivars PBW343 and C306 where stress was applied on 4th day after germination as ABA (20µM ABA), WS (6% Mannitol) and
ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with
similarly aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
Ascorbic
acid
PBW343
C306
ABA
WS
889.44 ±
153.14ab
490.21 ±
42.87a
2614.76 ±
753.00d
2620.90 ±
144.93d
889.44 ±
153.14ab
540.04 ±
21.98a
3405.77 ±
565.67e
1484.83 ±
89.31c
889.44 ±
153.14ab
639.45 ±
156.61ab
1180.99 ±
154.12bc
759.53 ±
231.41ab
ABA+WS
Shoot
CT
ABA
WS
ABA+WS
889.44 ±
825.33 ±
825.33 ±
825.33 ±
825.33 ±
153.14ab
103.87ab
103.87ab
103.87ab
103.87ab
868.44 ±
1257.20 ±
1470.11 ±
1370.74
1274.48 ±
24h
83.01ab
636.39bc
204.34cd
±476.82bcd
78.94bc
1648.97 ±
2414.57 ±
3979.59 ±
1893.88 ±
2388.96 ±
48h
430.96c
307.24e
608.88f
165.13de
106.74e
471.77 ±
866.84 ±
549.57 ±
946.92 ±
948.17 ±
72h
105.68a
145.18ab
47.26a
274.62abc
67.50abc
Root
1302.79 ±
1302.79 ±
1302.79 ±
1302.79 ±
1094.65 ±
1094.65 ±
1094.65 ±
1094.65 ±
0h
134.59d
134.59d
134.59d
134.59d
344.56c
344.56c
344.56c
344.56c
432.85 ±
412.98 ±
187.912 ±
227.64 ±
370.68 ±
410.46 ±
322.63 ±
218.99 ±
24h
ab
ab
a
a
ab
ab
ab
6.84
20.38
23.53
90.09
10.00
12.44
27.41
41.93a
1226.21 ±
1924.05 ±
512.17 ±
716.11 ±
865.43 ±
901.11 ±
327.61 ±
508.19 ±
48h
d
e
ab
bc
c
c
ab
148.94
665.56
40.95
62.08
114.25
54.88
51.96
26.37b
1230.43 ±
1052.53 ±
1127.81 ±
459.56 ±
858.83 ±
553.56 ±
507.76 ±
332.31 ±
72h
113.72d
166.84cd
132.39d
100.07ab
279.61c
80.80b
134.01b
109.35ab
*Estimations were done on samples extracted in triplicates. Mean ± SD was calculated.
*Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
0h
57
CT
A
5000
0h
24h
48h
ug of ascorbate per gm DW
4500
72h
f
e
4000
d
3500
d
3000
e
2500
e
bc
c
bcdde
cd
2000
1500
c
ab
1000
bc
ab
a
ab
ab
a
ab
ab
ab
ab
ab
bc
abc
ab
ab
ab
abc
a
a
500
0
Ct
ABA
WS
ABA+WS
Ct
ABA
3000
0h
24h
48h
ABA+WS
C306
PBW343
B
WS
72h
e
ug of ascorbate per gm DW
2500
2000
1500
d
d d
d
d
c
d
c
d
cd
c
c
c
c
1000
bc
ab
500
ab
ab
c
b
ab
ab
a
b
b ab
ab
abab
a
a
0
Ct
ABA
WS
ABA+WS
PBW343
Ct
ABA
WS
ABA+WS
C306
Fig. 4.7: Ascorbic acid (µg of ascorbate g-1 DW) in shoot (A) and root (B) of germinating wheat
seedlings of two cultivars PBW343 and C306 where stress was applied on 4 th day
after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM
ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress
treatment and compared with similarly aged control seedlings (Ct) grown on
distilled water as well as with 0h stage taken just before applying stress treatment.
Estimations were done on samples extracted in triplicates. Mean ± SD was
calculated. Data of root and shoot in each cultivar was analyzed separately by
Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
58
Table 4.8: Amount of dehydroascorbate (µg of dehydroascorbate g-1 DW) in shoot (upper panel) and root (lower panel) of germinating wheat
seedlings of two cultivars PBW343 and C306 where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6%
Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and
compared with similarly aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress
treatment.
PBW343
C306
Dha
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
59
0h
1587.64±214.83 1587.64±214.83 1587.64±214.83
1587.64±214.83
1884.08±254.93
24h
1788.36±649.39
1638.07±556.3
1217.64±240.42
1538.33±575.36
240.13±75.85
48h
1261.65±340.29
760.72±188.65
1796.00±318.54
1329.46±256.32
72h
2376.11±228.43 1201.52±354.58 2145.27±655.23
1085.10±350.36
1884.08±254.93 1884.08±254.93 1884.08±254.93
279.82±74.24
447.46±73.31
484.37±166.64
4127.69±1132.39 2203.67±182.76 1699.21±177.24 1562.92±260.69
563.07±64.82
429.32±27.18
735.21±243.73
466.28±26.34
Root
0h
1325±65.57
1325±65.57
1325±65.57
1325±65.57
1486.30±63.29
1486.30±63.29
1486.30±63.29
1486.30±63.29
24h
991.6±238.78
701.55±99.41
606.80±179.02
511.94±141.83
1249.26±106.69
577.59±39.47
1182.84±69.22
982.02±323.53
48h
790.85±134.21
605.63±206.13
1538.73±547.05
319.53±20.78
1287.41±214.44
1321.89±226.44
386.38±83.18
583.04±104.49
72h
1289.39±300.64
718.11±188.76
1799.58±5.00
965.84±5.00
873.18±220.64
551.72±5.00
879.86±170.74
339.95±94.26
*Estimations were done on samples extracted in triplicates. Mean ± SD was calculated.
*Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
A
6
ascorbate/dehydroascorbate
0h
24h
48h
72h
5
4
3
2
1
0
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
C306
PBW343
B
3.5
0h
24h
48h
72h
ascorbate/dehydroascorbate
3
2.5
2
1.5
1
0.5
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.8: Ascorbate/dehydroascorbate ratios in shoot (A) and root (B) of germinating
wheat seedlings of two cultivars PBW343 and C306 where stress was
applied on 4th day after germination as ABA (20 µM ABA), WS (6%
Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments.
60
Table 4.9: Ascorbate/Dehydroascorbate ratios in shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two cultivars
PBW343 and C306 where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS
(20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly
aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
Ascorbate/
Dehydroascorbate
PBW343
CT
ABA
C306
WS
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
61
0h
0.560
0.560
0.560
0.560
0.438
0.438
0.438
0.438
24h
0.274
0.330
0.525
0.565
5.236
5.254
3.063
2.631
48h
2.072
4.477
0.658
1.240
0.585
1.806
1.115
1.529
72h
1.103
1.236
0.354
0.435
1.539
1.280
1.288
2.033
Root
0h
0.983
0.983
0.983
0.983
0.736
0.736
0.736
0.736
24h
0.437
0.589
0.310
0.445
0.297
0.711
0.273
0.223
48h
1.550
3.177
0.333
2.241
0.672
0.682
0.848
0.872
72h
0.954
1.466
0.627
0.476
0.984
1.003
0.577
0.978
Table 4.10: Amount of malondialdehyde (nmole of MDA g-1 DW) in shoot (upper panel) and root (lower panel) of germinating wheat seedlings of
two cultivars PBW343 and C306 where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6% mannitol)
and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared
with similarly aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress
treatment.
MDA
PBW343
CT
ABA
C306
WS
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
62
0h
25.51±5.44ab
25.51±5.44ab
25.51±5.44ab
25.51±5.44ab
18.97±1.16bcde
18.97±1.16bcde 18.97±1.16bcde
18.97±1.16bcde
24h
25.10±6.92ab
31.24±0.51bc
38.12±10.77cd
46.15±11.3d
3.45±0.2a
12.46±5.41abc
22.30±9.51bcde
10.72±2.21ab
48h
23.98±1.46ab
15.43±1.71a
17.84±2.33a
16.71±2.53a
11.31±2.42ab
16.63±5.03bcd
13.25±4.01abc
11.06±0.87ab
72h
24.88±7.03ab
22.07±6.97ab
27.72±9.56abc
23.18±3.02ab
28.53±12.5e
29.17±10.3e
25.91±1.58de
23.78±8.47cde
Root
0h
23.06±1.97de
23.06±1.97de
23.06±1.97de
23.06±1.97de
16.69±6.03bc
16.69±6.03bc
16.69±6.03bc
16.69±6.03bc
24h
41.53±2.81f
27.61±1.65e
14.53±3.83bc
19.56±0.92cd
36.76±11.89def
41.90±0.81efg
46.69±8.26fg
6.67±1.01ab
48h
0.00±0.00a
0.00±0.00a
14.34±2.35bc
8.39±1.33b
25.60±8.72cd
17.60±0.41bc
6.54±0.50ab
3.41±0.50a
72h
0.00±0.00a
8.83±5.95b
17.84±9.90cd
8.11±5.73b
41.90±5.06efg
40.93±3.16efg
32.29±0.32de
50.70±13.96g
*Estimations were done on samples extracted in triplicates. Mean ± SD was calculated.
*Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
nmole of MDA per gm DW
A
70
0h
24h
48h
72h
d
60
cd
50
e
e
abc
40
ab
abab
30
bc
ab
ab
ab
ab
ab
a
a
20
cde
bcde
ab
de
bcde bcd
abc
bcde
a
bcde
abc
bcde
ab
10
abab
a
0
CT
ABA
WS
ABA+WS
CT
ABA
WS
C306
PBW343
nmole of MDA per gm DW
B 70
0h
24h
48h
ABA+WS
72h
g
60
fg
def
50
f
efg
efg efg
cd
40
de
30
e
de
cd
de
de
20
b
de
cd
bc
bcbc
bc
bc
bc
bc
b
b
ab
10
aa
a
ab
a
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.9: Malondialdehyde (nmole of MDA g-1 DW) in shoot (A) and root (B) of germinating
wheat seedlings of two cultivars PBW343 and C306 where stress was applied on 4 th
day after germination as ABA (20 µM ABA), WS (6% mannitol) and ABA+WS (20
µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after
stress treatment and compared with similarly aged control seedlings (Ct) grown on
distilled water as well as with 0h stage taken just before applying stress treatment.
Estimations were done on samples extracted in triplicates. Mean ± SD was calculated.
Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple
Test at P < 0.05 for test of significance, where superscripts of similar alphabets
indicate no significant difference among the values.
63
PBW343 showed elevated levels of MDA contents as compared to C306. Secondly,
ABA treatment produced less MDA contents as compared to WS and combined stress. ABA
was reported to alleviate drought- induced oxidative damage (Jiang and Zhang 2001,
Shaoyun et al 2009). There was no correlation between MDA content and H2O2 level (Table
4.6, 4.10; Figure 4.6, 4.9). In literature, MDA contents were reported to be increased under
stresses and lower levels of MDA was considered to be an important indice for evaluating
the redox status and drought resistance (Guo et al 2006). In most of these studies, MDA
contents were found to be positively correlated to H2O2 levels. But many recent studies
reported opposite to it where MDA contents decreased with increase in H2O2 contents
(Jubany-Mari et al 2010). Moreover it has been indicated that molecule responsible for lipid
peroxidation is singlet oxygen not H2O2 (Triantaphylides et al 2008).
4.2.4
Proline contents
Proline contents in roots and shoots of C306 were higher than of PBW343 for
control seedlings. In control seedlings of C306, proline contents were higher in roots than in
shoots (Table 4.11; Figure 4.10). Proline contents were found to be unaltered under ABA in
both cultivars, except it was increased in C306 roots at 72h stage and in PBW343 shoots at
48h stage. In literature, proline accumulation under WS was reported to be independent to
ABA, hence was a part of ABA independent stress response (de-Carvallo 2008, Sharma and
Verslues 2010, Verslues and Zhu 2004). Proline contents were found to be increased under
WS and combined stress in both cultivars and this increase was more prominent in shoots
than in roots of both cultivars.
In conclusions, one can say that Drought tolerant cultivar, C306 contained higher
amount proline mainly in roots but under WS, proline contents were more increased in
shoots than in roots and this increase in proline might be mediated through ABA independent
pathway. MDA contents and proline contents were negatively correlated in shoots of
PBW343 under WS and under combined stress at 24h and 48h stages (Table 4.10, 4.11;
Figure 4.9, 4.10). This could be correlated to other studies where proline was considered as
free radical scavenger (Kavi Kishore 2005) and proline accumulation under severe drought
had been found to be linked to the MDA contents and membrane protection among different
wheat varieties (Simova-Stilova et al 2008).
4.3
Antioxidant enzymes
Plants keep ROS under control by efficient scavenging systems which include
enzymatic antioxidants and non-enzymatic antioxidants. Enzymatic antioxidants are
superoxide dismutase (SOD), catalase (CAT) and enzymes involved in ascorbate glutathione cycle. These enzymes are located in different compartments of cell except
catalase which is located exclusively in peroxisomes. SOD is the front line enzyme in the
64
Table 4.11: Amount of proline (µmole of proline g-1 DW) in shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two
cultivars PBW343 and C306 where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6% Mannitol) and
ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with
similarly aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
PBW343
C306
Proline
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
65
0h
13.52 ± 0.79f
13.52 ± 0.79f
13.52 ± 0.79f
13.52 ± 0.79f
9.93 ± 3.05ab
9.93 ± 3.05ab
9.93 ± 3.05ab
9.93 ± 3.05ab
24h
7.46 ± 0.74abc
6.93 ± 0.81ab
7.73 ± 0.93abc
7.90 ± 1.56abc
7.65 ± 3.09ab
10.86 ± 0.73bc
10.56 ± 0.49abc
15.58 ± 0.67def
48h
8.48 ± 1.40bc
11.68 ± 1.24ef
18.52 ± 1.11g
18.48 ± 1.09g
15.29 ± 1.93de
13.7 ± 1.29cd
17.72 ± 1.16ef
19.38 ± 3.83f
72h
7.47 ± 2.11abc
5.75 ± 0.89a
11.24 ± 1.10de
9.47 ± 1.60cd
8.03 ± 1.04ab
9.93 ± 1.63ab
7.08 ± 0.71a
9.80 ± 1.95ab
Root
0h
13.0 ± 1.31de
13.0 ± 1.31de
13.0 ± 1.31de
13.0 ± 1.31de
9.09 ± 0.30a
9.09 ± 0.30a
9.09 ± 0.30a
9.09 ± 0.30a
24h
6.96 ± 0.98ab
5.34 ± 1.99a
8.59 ± 1.97b
5.55 ± 0.62a
24.18 ± 0.95e
16.81 ± 1.56bc
16.20 ± 2.26bc
15.00 ± 0.63b
48h
10.85 ± 0.53cd
5.97 ± 0.56a
13.48 ± 0.24e
10.91 ± 0.99cd
21.23 ± 3.13de
20.50 ± 1.64de
16.70 ± 2.41bc
15.43 ± 0.10b
72h
6.85 ± 1.80ab
8.31 ± 1.62b
12.82 ± 0.07de
8.95 ± 1.37bc
8.75 ± 3.37a
15.09 ± 2.86b
19.34 ± 3.06cd
13.26 ± 0.53b
*Estimations were done on samples extracted in triplicates. Mean ± SD was calculated.
*Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of
similar alphabets indicate no significant difference among the values.
umole of proline per gm DW
A 25
0h
24h
48h
f
72h
g
20
g
ef
de
15
10
f
f
f
ef
f
ab
de
cd
bc
abc
abc
ab
bc
ab
abc
abc
ab
def
cd
ab
ab
ab
ab
abc
ab
a
a
5
0
CT
ABA
WS
ABA+WS
CT
ABA
umole of proline per gm DW
0h
24h
48h
72h
e de
25
cd
de
20
15
bc
de
de
ab
b
ab
a
bcbc
b
bb
de
cd
10
ABA+WS
C306
PBW343
B 30
WS
e de
de
b
cd
bc
b
a
a
a
a
a
a
a
5
0
CT
ABA
WS
ABA+WS
PBW343
Fig. 4.9:
CT
ABA
WS
ABA+WS
C306
Proline (µmole of proline g -1 DW) in shoot (A) and root (B) of germinating wheat
seedlings of two cultivars PBW343 and C306 where stress was applied on 4 th day after
germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA
and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress
treatment and compared with similarly aged control seedlings (Ct) grown on distilled
water as well as with 0h stage taken just before applying stress treatment. Estimations
were done on samples extracted in triplicates. Mean ± SD was calculated. Data of root
and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P <
0.05 for test of significance, where superscripts of similar alphabets indicate no
significant difference among the values.
66
ROS attack. Since it rapidly scavenges superoxide which is the first ROS to be produced but
it produced another ROS (H2O2). H2O2 scavengers are CAT and APX but these enzymes
seem to have different roles because they are located in different compartments of the cell
i.e. APX is present in almost every compartment of the cell and CAT exclusively present in
peroxisomes and secondly, their affinity for H2O2 which is very high (Km in µM range) in
APX but lower (Km in mM range) in catalase. It indicates APX works fine regulator of ROS
under steady state level but catalase might be a bulk removal of H2O2 under stress conditions.
In the present study, activities of ascorbate peroxidase (APX), catalase (CAT),
guaiacol peroxidase (GPOX), superoxide dismutase (SOD), glutathione reductase (GR) were
determined in roots and shoots of similarly treated seedlings (as discussed previously) at
same four stages (0h, 24h, 48h and 72h). Besides determining activities for these enzymes,
database was also searched for availability of gene sequences corresponding to these
enzymes at NCBI (www.ncbi.nlm.nih.gov/). Nucleotide, mRNA, ESTs, protein databases
were searched for their genes sequences. Primers were designed from these sequences so as
to check the status of expression level of these genes by RT-PCR. Expression analysis by
RT-PCR was done in shoots of both cultivars at 24h and 48h stages. Gene IDs (in the form of
accession no.), gene names and primer sequences are presented in (Table 3.1)
4.3.1
Ascorbate peroxidase (APX)
The APX family consists of at least five different isoforms including thylakoid
(tAPX) and glyoxisome membrane forms (gmAPX), as well as chloroplast stromal soluble
form (sAPX), cytosolic form (cAPX).
APX activities were decreased or unaltered in roots of both cultivars under all stress
treatments (Table 4.12; Figure 4.11, 4.12). No changes in APX activities were found in
shoots of PBW343 under all stress treatments but increases in specific activities were found
at 24h stage under WS and at 48h stage under combined stress. In C306 shoots, increases in
APX activities as well as specific activities were mainly observed under ABA treatment,
however, unaltered under WS as well as under combined stress except 72h stage under
combined stress where it decreased. APX activities in both cultivars were concluded to be
increased mainly under ABA treatment and in C306 shoots only.
For expression studies, only one gene corresponding to peroxisomal ascorbate
peroxidase (EF556121, PER-APX) was used. This gene expression was higher in control
shoots of PBW343 but expressed very low in control shoots of C306 (Figure 4.13). Gene was
induced under all stress treatments in PBW343 mainly at 48h. However in C306, gene was
induced at 24h under ABA treatment as well as under WS treatment but continued to be
67
Table 4.12: Ascorbate peroxidase activity (µmole of ascorbate disappeared min-1 g-1 DW) and specific activity (µmole of ascorbate disappeared
min-1 mg-1 protein) in shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two cultivars PBW343 and C306
where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and
6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly aged control
seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
PBW343
C306
ABA+WS
CT
ABA
WS
ABA+WS
Shoot
0h
223.22±80.54def 223.22±80.54def 223.22±80.54def 223.22±80.54def
100.53±12.69ab 100.53±12.69ab
100.53±12.69ab
100.53±12.69ab
(0.81±0.25)abc
(0.81±0.25)abc
(0.81±0.25)abc
(0.81±0.25)abc
(0.46±0.04)a
(0.46±0.04)a
(0.46±0.04)a
(0.46±0.04)a
ef
cde
ef
f
cde
f
def
24h
230.58±20.28
179.27±15.99
233.73±16.26
248.43±12.13
164.46±27.50
231.37±39.32
184.34±28.52
129.12±16.48abc
abc
abc
d
cd
ab
ab
ab
(0.73±0.110)
(0.77±0.04)
(1.44±0.46)
(1.12±0.17)
(0.72±0.06)
(0.82±0.17)
(0.81±0.16)
(0.70±0.13)ab
bcd
cde
ab
abc
abc
abc
a
48h
171.94±20.29
180.41±30.52
121.15±14.05
151.51±29.51
119.46±12.04
128.83±45.61
88.03±3.72
125.97±12.47abc
ab
ab
bc
csd
ab
ab
ab
(0.63±0.15)
(0.65±0.09)
(0.99±0.23)
(1.12±0.19)
(0.89±0.12)
(0.72±0.38)
(0.82±0.15)
(0.83±0.15)ab
abc
a
a
a
f
ef
bcd
72h
132.56±28.88
96.64±14.66
115.37±20.40
98.75±18.02
209.33±28.32
197.63±16.75
142.14±22.80
103.61±4.93ab
abc
a
abc
abc
c
d
bc
(0.74±0.21)
(0.50±0.05)
(0.77±0.27)
(0.76±0.06)
(1.36±0.48)
(2.28±0.57)
(1.16±0.24)
(0.67±0.06)ab
Root
0h
263.85±14.56g
263.85±14.56g
263.85±14.56g
263.85±14.56g
217.34±40.31gh 217.34±40.31gh
217.34±40.31gh
217.34±40.31gh
bc
bc
bc
bc
abcd
abcd
abcd
(1.29±0.22)
(1.29±0.22)
(1.29±0.22)
(1.29±0.22)
(0.94±0.14)
(0.94±0.14)
(0.94±0.14)
(0.94±0.14)abcd
g
f
def
cd
fg
ef
def
24h
287.60±48.25
186.50±87.47
144.37±6.28
116.85±7.10
175.74±22.47
161.14±25.57
152.83±54.94
126.30±6.29cdef
d
cd
cd
cd
efg
def
fg
(2.09±0.50)
(1.70±0.68)
(1.58±0.07)
(1.67±0.23)
(1.82±0.03)
(1.61±0.36)
(2.02±0.90)
(1.77±0.06)efg
ef
cde
bcd
ab
cde
abc
a
48h
176.97±38.64
121.92±23.92
108.61±4.64
54.97±11.03
118.99±13.93
80.39±13.71
51.71±14.85
58.62±3.84ab
bc
b
bcd
bc
abcd
a
ab
(1.19±0.30)
(0.93±0.18)
(1.50±0.04)
(1.09±0.33)
(1.07±0.06)
(0.54±0.08)
(0.71±0.41)
(0.79±0.14)abc
bcd
a
bcd
abc
h
cdef
bcd
72h
92.77±23.15
26.737±8.96
92.66±1.69
63.39±6.38
249.22±53.62
127.10±19.01
105.71±14.66
100.77±24.70abcd
bc
a
bc
bc
g
def
cdef
(1.16±0.39)
(0.33±0.08)
(1.19±0.13)
(1.15±0.40)
(2.45±0.59)
(1.55±0.17)
(1.42±0.19)
(1.30±0.39)bcde
(1.16±0.39)bc
(0.33±0.08)a
(1.19±0.13)bc
(1.15±0.40)bc
(2.45±0.59)g
(1.55±0.17)def
(1.42±0.19)cdef
(1.30±0.39)bcde
*Values within parenthesis indicate specific activity and without parenthesis indicate activity. *Estimations were done on samples extracted in triplicates. Mean ± SD was
calculated. Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of similar
alphabets indicate no significant difference among the various samples.
CT
ABA
WS
68
A
350
umole of ascorbate disappeared /min/g DW
300
0h
24h
def
48h
def
72h
def
ef
250
f
f
ef
f
ef
cde
cde
bcd
200
def
cde
abc
abc
abc
ab a
150
a
a
bcd
abc
ab
ab
ab
a
100
abcabc
ab
ab
50
0
Ct
ABA
WS
ABA+WS
Ct
ABA
B400
0h
24h
48h
WS
ABA+WS
C306
PBW343
72h
g
350
umole of ascorbate disappeared /min/g DW
def
h
300
250
g
g
g
f
g
gh
ef
gh
fg
200
150
cde
bcd
def
bcd
bcd
100
cd
cde
gh
gh
def
ef
cdef
bcd
cdef abcd
abc
ababc
a
ab
a
50
0
Ct
ABA
WS
ABA+WS
PBW343
Ct
ABA
WS
ABA+WS
C306
Fig. 4.11: Ascorbate peroxidase activity (µmole of ascorbate disappeared min-1 g-1 DW) in shoot
(A) and root (B) of germinating wheat seedlings of two cultivars PBW343 and C306
where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6%
Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was
collected at 24h, 48h, 72h after stress treatment and compared with similarly aged
control seedlings (Ct) grown on distilled water as well as with 0h stage taken just
before applying stress treatment. Estimations were done on samples extracted in
triplicates. Mean ± SD was calculated. Data of root and shoot in each cultivar was
analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance,
where superscripts of similar alphabets indicate no significant difference among the
various samples.
69
A
0h
umole of ascorbate changed per min per mg
protein
3
24h
48h
72h
d
2.5
d
2
1.5
abc
abc
abcab
1
c
cdcd
bc
abc abc abc
abc
abc
abc
ab
a
bc
ab
ab
ab
ab
ab
a
0.5
ab
abab
a
ab
a
a
0
Ct
ABA
WS
ABA+WS
Ct
ABA
umole of ascorbate disappeared per min per mg
protein
3.5
0h
24h
48h
ABA+WS
C306
PBW343
B
WS
72h
g
fg
3
d
cd
2.5
cd
2
1.5
bc
bc bc
cd
bc bcd
bc bcbc
bc
bc
b
def
efg
abcd
abcd
efg
def
abcd ab
abcd
bcde
cdef
1
abcd
abc
a
a
0.5
0
Ct
ABA
WS
ABA+WS
PBW343
Ct
ABA
WS
ABA+WS
C306
Fig. 4.12: Ascorbate peroxidase specific activity (µmole of ascorbate disappeared min-1 mg-1
protein) in shoot (A) and root (B) of germinating wheat seedlings of two cultivars
PBW343 and C306 where stress was applied on 4 th day after germination as ABA (20
µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol)
treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared
with similarly aged control seedlings (Ct) grown on distilled water as well as with 0h
stage taken just before applying stress treatment. Estimations were done on samples
extracted in triplicates. Mean ± SD was calculated. Data of root and shoot in each
cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of
significance, where superscripts of similar alphabets indicate no significant
difference among the various samples.
70
PBW343
24hr
ABA+WS ABA
WS
C306
48hr
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
EF556121
(PERAPX)
GAPDH
2
24h
48h
PBW343
C306
Ratio
1.5
1
71
0.5
0
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
24h
1.05
1.22
0.105
0.023
0.08
0.35
1
0.11
48h
0.56
1.47
1.66
1.65
0.2
1.12
0
0.33
Figure 4.13: (A) Gel pictures of RT- PCR products of EF556121 (PERAPX) and GAPDH (internal control) where semiquantitative RT- PCR was done on total
RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4 th day of germination with ABA (20 µM),
WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and compared with
shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of EF556121 (PERAPX) in the form of ratio of intensity of band obtained with gene specific primer to the
intensity of band obtained with GAPDH specific primer.
induced under ABA treatment but not under WS treatment. While comparing the expression
data of this gene with activities and specific activities (Table 4.12) of APX obtained in same
samples, only higher induction of gene under ABA treatment in C306 might be correlated to
higher activities and specific activities of enzymes obtained in same samples. Otherwise this
gene expression does not follow the pattern obtained with activity data. Reason might be the
contribution of other APX isoforms in activities besides this gene or there might be the
regulation at other steps rather than at transcriptional level.
4.3.2
Catalase (CAT)
Catalases are tetrameric heme containing enzymes which directly dismutate H2O2 into
H2O and oxygen. Catalases play important role in removal of H2O2 generated in peroxisomes by
oxidases involved in ß-oxidation of fatty acids, photorespiration and purine catalases.
Higher CAT specific activities were found in C306 than PBW343 in both roots and
shoots of control seedlings. In roots of both cultivars, catalase activities as well as specific
activities were mostly decreased under all stress treatments (Table 4.13; Figure 4.14, 4.15).
Catalase was found to be maintained at higher levels (at activities and specific activities)
mainly under ABA treatment in shoots of C306 only but not in shoots of PBW343. Under other
stress treatments (WS and combined stress), catalase was not found to be altered in shoots of
both cultivars. Catalase activities and H2O2 contents were in positive correlation in C306 shoots
of control as well as stress treated seedlings which are in agreement with other reports which
suggests that catalase gene expression and activity are controlled by H2O2 through H2O2 signal
transduction pathway initiated under dehydration signal (Bailly et al 2008).
Two catalase genes CAT-1 and CAT-2 (D86327, CAT-1; X94352, CAT-2) were used
for gene expression analysis. Expression of CAT-1 was slightly induced at 24h stage under
ABA treatment in shoots of PBW343 only but not expressed in any other samples (Figure
4.16(a), 4.16(b)). CAT-2was also induced under ABA treatment in both cultivars but
expression level was higher in PBW343 than in C306. Comparing (CAT-1 and CAT-2) gene
expression with activities and specific activities of catalases obtained in the same samples
(Table 4.13); a good correlation was not found between two, though catalase activities were
higher under ABA treatment. There can be many reasons for it like catalase isoforms
contributing to activities in these samples might be different than the forms encoded by these
two genes (CAT-1 and CAT-2). Secondly, catalase enzyme is suggested to be controlled at
other levels (translational and protein turnover number) rather than at transcriptional level
(Luna et al 2004). There are reports in literature where catalase (CAT-1 and CAT-2) gene
expression was reduced but activities increased in the same samples as studied in leaves of
wheat plant under drought ( Luna et al 2004) and these genes were suggested to be regulated
through circadian control and by light. Our observations for catalase gene expression and
activities in this study might be due these factors.
72
Table 4.13:
Catalase activity (µmole of H2O2 disappeared min-1 g-1 DW) and specific activity (µmole of H2O2 disappeared min-1 mg-1 protein)
in shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two cultivars PBW343 and C306 where stress
was applied on 4th day after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly aged control
seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
PBW343
0h
24h
73
48h
72h
0h
24h
48h
72h
C306
CT
ABA
WS
752.70±113.05a
(2.82±0.78)a
6147.62±779.19fg
(20.10±4.79)bcde
6978.87±1748.79g
(15.22±5.42)bc
4335.98±1255.26de
(23.19±1.88)cde
752.70±113.05a
(2.82±0.78)a
531.64±52.86a
(2.73±0.06)a
1t241.78±244.58c
(8.98±2.38)bc
1759.76±534.39d
(12.16±3.81)bcd
1836.65±541.24d
(23.02±7.26)e
CT
ABA
WS
ABA+WS
752.70±113.05a
(2.82±0.78)a
ABA+WS
Shoot
752.70±113.05a
(2.82±0.78)a
695.56±102.26a
(2.93±0.27)a
695.56±102.26a
(2.93±0.27)a
4350.13±32.71de
(18.89±2.32)bcde
5363.81±1137.34ef
(19.41±4.88)bcde
5165.66±131.40ef
(27.19±2.55)e
4449.53±599.99de
(27.29±8.58)e
2696.73±354.51bc
(21.70±0.33)bcde
3966.98±522.94cde
(26.20±7.38)de
3177.38±118.22bcd
(14.17±2.68)b
2457.94±266.41b
(18.41±1.56)bcd
2493.75±503.81b
(20.09±2.55)bcde
3592.25±1311.03b
(16.07±6.87)b
4720.88±406.16bcd
(35.14±4.48)ef
5376.84±190.55cd
(30.48±7.11)de
6208.23±739.72d
(21.58±1.66)bcd
5353.06±951.68cd
(35.12±7.20)ef
4226.50±318.38bc
(45.72±8.34)f
695.56±102.26a
(2.93±0.27)a
4530.66±1549.42bc
(19.64±5.12)bc
3768.07±655.35b
(34.65±0.75)e
3346.98±562.31b
(27.15±4.53)cde
695.56±102.26a
(2.93±0.27)a
3443.31±527.43b
(18.56±4.09)bc
3629.56±462.47b
(23.50±1.50)bcd
3771.95±1231.40b
(19.93±8.48)bc
531.64±52.86a
(2.73±0.06)a
1097.49±305.06abc
(9.93±2.79)bcd
531.64±52.86a
(2.73±0.06)a
Root
531.64±52.86a
(2.73±0.06)a
474.75±80.12a
(2.08±0.42)a
474.75±80.12a
(2.08±0.42)a
474.75±80.12a
(2.08±0.42)a
474.75±80.12a
(2.08±0.42)a
2972.55±510.02c
(27.03±3.56)g
2491.50±154.22c
(21.74±3.46)fg
1725.97±402.22b
(16.04±3.75)de
817.72±363.58a
(8.25±1.99)bc
535.14±136.47a
(7.33±1.96)bc
554.12±86.28a
(7.93±1.45)bc
2471.64±539.37c
(16.31±2.87)de
995.56±181.29a
(12.11±1.62)bcd
1604.85±76.86b
(20.40±4.86)ef
1520.28±127.23b
(19.73±2.39)ef
978.15±294.19a
(12.58±4.34)cd
2045.43±468.46d
(12.72±5.21)bcd
1038.46±395.77abc
(11.29±4.07)bcd
511.34±177.03a
(6.71±1.78)ab
806.12±11.48abc
(11.38±0.68)bcd
629.38±122.07ab
(11.81±0.28)bcd
747.53±76.29abc
(10.59±1.62)bcd
1169.41±225.95bc
(14.93±4.07)cd
751.50±146.22abc
(16.65±3.72)de
534.58±66.55a
(7.09±0.68)b
*Values within parenthesis indicate specific activity and without parenthesis indicate activity. *Estimations were done on samples extracted in triplicates. Mean ± SD was
calculated. Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of similar
alphabets indicate no significant difference among the various samples.
umole of H2O2 disappeared /min/g DW
A 10000
0h
24h
48h
72h
g
9000
8000
7000
fg
d
ef
6000
de
cd
ef
5000
cd
bcd
b
de
de
cde
bc
b
bc
b
b b
b
4000
bc
bcd
b
3000
b
2000
1000
a
a
a
a
a
a
a
a
0
CT
ABA
WS
ABA+WS
CT
ABA
4000
0h
24h
48h
ABA+WS
C306
PBW343
B
WS
72h
c
3500
umole of H2O2 disappeared /min/g DW
c
3000
c
d
dd
2500
b
2000
b
c
1500
a
abc
1000
a
a
b
abc bc
abc
a
a
abcab
a
a
a
abc
a
a a
a
a
a
a
500
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.14: Catalase activity (µmole of H2O2 disappeared min-1 g-1 DW) in shoot (A) and root (B) of
germinating wheat seedlings of two cultivars PBW343 and C306 where stress was
applied on 4th day after germination as ABA (20 µM ABA), WS (6% Mannitol) and
ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h,
72h after stress treatment and compared with similarly aged control seedlings (Ct)
grown on distilled water as well as with 0h stage taken just before applying stress
treatment. Estimations were done on samples extracted in triplicates. Mean ± SD was
calculated. Data of root and shoot in each cultivar was analyzed separately by Duncan’s
Multiple Test at P < 0.05 for test of significance, where superscripts of similar alphabets
indicate no significant difference among the various samples.
74
umole of H2O2 disappeared per min per mg protein
A 60
0h
24h
48h
72h
f
50
ef
ef
40
e
de
de
e
cde
e
30
bcde
cde
bcde
bcde
bcde
bc
b
bcd bcde
20
bcd
10
a
a
a
a
a
a
a
a
0
ABA
WS
ABA+WS
CT
ABA
35
0h
24h
48h
WS
ABA+WS
C306
PBW343
umole of H2O2 disappeared per min per mg protein
bc
b
CT
B
bcd
bc
72h
g
e
30
fg
ef
25
ef
20
cd
bcd
bcd
15
bc
de
de
cd
bcd
bcd
bcd bcd
bcd bcd
bc
ab
10
5
de
a
a
a
a
a
bc
a
a
bc
b
a
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.15: Catalase specific activity (µmole of H2O2 disappeared min-1 mg-1 protein) in shoot
(A) and root (B) of germinating wheat seedlings of two cultivars PBW343 and C306
where stress was applied on 4th day after germination as ABA (20 µM ABA), WS
(6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was
collected at 24h, 48h, 72h after stress treatment and compared with similarly aged
control seedlings (Ct) grown on distilled water as well as with 0h stage taken just
before applying stress treatment. Estimations were done on samples extracted in
triplicates. Mean ± SD was calculated. Data of root and shoot in each cultivar was
analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance,
where superscripts of similar alphabets indicate no significant difference among the
various samples.
75
(A)
PBW343
C306
24hr
ABA+WS ABA
48hr
WS
CT
ABA+WS ABA
24hr
WS
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
X94352 (CAT-2)
GAPDH
(B)
24h
PBW343
48h
C306
0.8
0.6
0.4
76
0.2
0
Figure 4.16(a):
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
24h
0.045
0.69
0
0
0
0.08
0
0
48h
0
0
0
0
0
0
0
0
(A) Gel pictures of RT- PCR products of X94352 (CAT-2) and GAPDH (internal control) where semiquantitative RT- PCR was done on total
RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4th day of germination with ABA (20
µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and
compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of X94352 (CAT-2) in the form of ratio of intensity of band obtained with gene specific primer to the
intensity of band obtained with GAPDH specific primer.
PBW343
(A)
C306
24hr
ABA+WS ABA
WS
48hr
CT
ABA+WS ABA
24hr
WS
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
D86327(CAT-1)
GAPDH
24h
PBW343
48h
C306
0.08
(B)
77
Ratio
0.06
0.04
0.02
0
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
24h
0
0.076
0
0
0
0
0
0
48h
0
0
0
0
0
0
0
0
Figure 4.16(b): (A) Gel pictures of RT- PCR products of D86327 (CAT-1) and GAPDH (internal control) where semiquantitative RT- PCR was done on total
RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4 th day of germination with ABA (20
µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and
compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of D86327 (CAT-1) in the form of ratio of intensity of band obtained with gene specific primer to the intensity
of band obtained with GAPDH specific primer.
4.3.3
Peroxidases
Peroxidases have a role in biosynthesis of lignin and in defense against biotic and
abiotic stresses by consuming H2O2. Increased peroxidase activities are reported under abiotic
stresses (Gill and Tuteja 2010). Peroxidases and ascorbate peroxidases (APX) can be
distinguished from each other in terms of their gene sequences and their functions. Peroxidases
prefer aromatic electron donor such as guaiacol and pyrogallol but oxidise ascorbate at the rate
of around 1% to that with guaiacol. While ascorbate peroxidases have high degree of specificity
for ascorbate as electron donor. Secondly, peroxidases participate in a number of processes like
biosynthesis of lignin and are involved in processes of various plant responses like senescence,
wounding of pathogens. APX has elemental role in scavenging H2O2 in chloroplast mainly.
In this study, POX activities were determined using guaiacol as substrate hence enzyme
was named as guaiacol peroxidases (GPOX). GPOX specific activities were not altered (Table
4.14, Figure 4.17, 4.18) but activities were decreased in roots of both cultivars under all stress
treatments. Under ABA treatment, GPOX specific activities were increased in shoots of both
cultivars but these increases were more prominent in shoots of C306. Under WS and combined
stress, GPOX was almost unaltered in shoots of both cultivars. Higher APX, POD, CAT
activities were reported to be related to drought tolerance in different plants (Guo et al 2006,
Khanna-Chopra and Stelote 2007, Hameed et al 2011). These enzymes were also reported in
downstream effect of ABA under WS in maize leaves where sequential effects downstream to
ABA lead to these increases following increased levels of superoxide radicals and H2O2 (Jiang
and Zhang 2002). In this study, higher CAT, APX, and POD level were found under ABA
treatment in shoots of both cultivars but the effect was more pronounced in shoots of C306.
Similar to results obtained with contents (H2O2, ascorbic acid, ascorbate/dehydroascorbate ratio)
(Table 4.9) measurements, these three enzymes followed almost same pattern. It could be
concluded that ABA led to increased contents of H2O2 at 24h mainly followed by increased
contents of ascorbate as well as increased ascorbate/dehydroascorbate ratio and higher levels of
these three antioxidant enzymes (CAT, APX, POD), can be ABA-dependent pathway under
WS.
4.3.4
Glutathione reductase (GR)
GR is a flavoprotein oxidoreductase which is potential enzyme in ascorbate-glutathione
cycle. Secondly, it produces reduced glutathione (an antioxidant), hence maintains ROS. It is
localized predominantly in chloroplast and small amount in cytosol and mitochondria.
In roots of PBW343, GR activities were mostly decreased under all stress treatments
(Table 4.15, Figure 4.19, 4.20). However in roots of C306, GR activities were not altered under
almost all stress treatments, which might be the reason for maintaining higher
ascorbate/dehydroascorbate ratio in these tissues (Table 4.9). In shoots of both cultivars, GR
78
79
Table 4.14: Guaiacol peroxidase activity (µmole of tetraguaicol appeared min -1 g-1 DW) and specific activity (µmole of tetraguaicol appeared min -1 mg-1 protein) in
shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two cultivars PBW343 and C306 where stress was applied on 4th day
after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h,
48h, 72h after stress treatment and compared with similarly aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken just
before applying stress treatment.
GPX
Shoot
PBW343
C306
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
0h
140.22±47.42a
140.22±47.42a
140.22±47.42a
140.22±47.42a
84.44±15.66a
84.44±15.66a
84.44±15.66a
84.44±15.66a
(0.51±0.13)a
(0.51±0.13)a
(0.51±0.13)a
(0.51±0.13)a
(0.36±0.09)a
(0.36±0.09)a
(0.36±0.09)a
(0.36±0.09)a
24h
576.92±27.92e
397.86±103.55bcd 374.79±6.07bcd
371.85±83.02bcd
290.32±60.01b
442.71±54.75d
342.30±46.22bc
317.52±20.52b
bcde
bcd
cde
bc
ab
bc
bc
(1.90±0.49)
(1.69±0.29)
(2.30±0.63)
(1.63±0.33)
(1.28±0.28)
(1.55±0.22)
(1.52±0.29)
(1.70±0.20)bc
48h
313.27±138.99bc
469.28±96.62de
419.52±91.58cd
267.06±62.62b
459.00±33.58d
575.52±66.33e
413.51±59.33cd
431.32±23.63cd
(1.13±0.47)ab
(1.66±0.09)bc
(3.38±0.67)f
(1.97±0.49)cde
(2.00±0.05)bcd
(1.94±0.36)bcd
(1.87±0.38)bcd
(1.76±0.21)bcd
cd
de
bcd
bc
cd
bcd
b
72h
412.57±32.30
474.83±24.11
392.21±26.06
295.21±5.24
412.40±77.72
385.73±62.69
309.66±50.51
292.40±43.46b
(2.29±0.42)cde
(2.50±0.20)de
(2.57±0.56)ef
(2.26±0.31)cde
(2.80±1.28)de
(3.25±1.28)e
(2.49±0.24)cde
(1.89±0.23)bcd
Root
0h
800.00±26.20defg
800.00±26.20defg
800.00±26.20defg
800.00±26.20defg 634.33±132.57def 634.33±132.57def 634.33±132.57def
634.33±132.57def
(3.94±0.48)a
(3.94±0.48)a
(3.94±0.48)a
(3.94±0.48)a
(2.78±0.61)a
(2.78±0.61)a
(2.78±0.61)a
(2.78±0.61)a
24h
1461.94±336.20h
976.35±250.82g
954.26±13.92fg
825.09±155.38defg
710.02±10.39f
626.91±82.30def
458.84±126.77ab
423.39±41.20a
ef
def
ef
f
bc
bc
b
(10.35±1.38)
(8.90±2.59)
(10.42±0.39)
(11.59±1.66)
(7.16±1.11)
(5.72±0.37)b
(6.70±0.49)
(5.73±1.07)
48h
868.64±74.48efg
665.23±138.62bcde 425.42±150.92ab
333.67±46.58a
1310.49±129.07h 1114.84±40.48g 495.85±146.85abcd
520.36±135.79abcde
(5.99±0.48)abc
(5.19±1.12)ab
(5.72±2.06)abc
(6.49±1.87)bcd
(10.73±1.33)d
(9.58±1.51)cd
(8.69±2.60)bcd
(9.65±4.12)cd
cdef
bcd
bcde
abc
f
ef
abc
72h
704.26±8.50
588.60±34.79
682.08±4.02
464.91±16.51
749.26±37.05
676.59±36.96
459.88±15.19
447.25±78.42a
(8.76±0.23)de
(7.93±0.27)cde
(8.64±0.87)de
(8.24±1.55)cde
(7.15±0.58)bc
(5.70±0.96)b
(8.29±0.87)bcd
(6.12±0.32)b
*Values within parenthesis indicate specific activity and without parenthesis indicate activity.*Estimations were done on samples extracted in triplicates. Mean ± SD was
calculated. Data of root and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of similar
alphabets indicate no significant difference among the values.
A 700
0h
24h
48h
72h
e
umole of tetraguaicol produced /min/g DW
e
de
600
500
cd
bcd de
bccd
d cd
d
bcd
bcd
bcd
400
b
b
b
bc
300
a
a
a
200
a
a
a
100
a
a
0
ABA
WS
ABA+WS
CT
ABA
2000
1800
0h
24h
48h
ABA+WS
72h
h
1600
h
1400
g
g
1200
1000 defg
800
WS
C306
PBW343
umole of tetraguaicol produced /min/g DW
cd
bc
b
b
CT
B
cd
bcd
efg
fg
defg
bcde defg
cdef
bcd
defg
defg
bcde
ab
deff
f
defdef ef def
def
ababcd
abcde
abc
600
abc
a
a
a
400
200
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.17: Guaiacol peroxidase activity (µmole of tetraguaicol appeared min -1 g-1 DW) in shoot
(A) and of root (B) of germinating wheat seedlings of two cultivars PBW343 and
C306 where stress was applied on 4th day after germination as ABA (20 µM ABA),
WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data
was collected at 24h, 48h, 72h after stress treatment and compared with similarly
aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken
just before applying stress treatment. Estimations were done on samples extracted in
triplicates. Mean ± SD was calculated. Data of root and shoot in each cultivar was
analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance,
where superscripts of similar alphabets indicate no significant difference among the
values.
80
umole of tetraguaicol produced per min per mg protein
A
5
0h
24h
48h
72h
e
de
4.5
f
4
ef
3.5
de
cde
3
cde
2.5
ab
cde
cdecde
bcde
bcdbc
bc
a
a
a
a
a
1
bcbcd
bc
bc
ab
2
1.5
bcd
bcd
bcd
a
a
bcd
a
0.5
0
CT
ABA
WS
ABA+WS
CT
ABA
umole of tetraguaicol produced per min per mg
protein
0h
24h
48h
ABA+WS
C306
PBW343
B 16
WS
72h
cd
f
14
ef
12
d
def
cd
ef
10
de
de
cde
8
abc
bcd
cde
bcd
bcd
abc
bc
ab
bc
bc
b
b
b
b
6
a
a
a
a
a
4
a
a
a
2
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.18: Guaiacol peroxidase specific activity (µmole of tetraguaicol appeared min -1 mg-1
protein) in shoot (A) and of root (B) of germinating wheat seedlings of two
cultivars PBW343 and C306 where stress was applied on 4 th day after germination
as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment
and compared with similarly aged control seedlings (Ct) grown on distilled water as
well as with 0h stage taken just before applying stress treatment. Estimations were
done on samples extracted in triplicates. Mean ± SD was calculated. Data of root
and shoot in each cultivar was analyzed separately by Duncan’s Multiple Test at P
< 0.05 for test of significance, where superscripts of similar alphabets indicate no
significant difference among the values.
81
activities were almost unaffected under all stress treatments. GR was reported to be induced
under water stress through ABA-dependent pathway (Jiang and Zhang 2002). In this study,
unlike other antioxidants-enzymes (APX, POD, CAT), GR activities were not found to be
increased under ABA. Secondly higher GR and glutathione contents are assumed to be related
to drought resistance of plant under various stresses (Gill and Tuteja 2010). However in this
study, GR in stressed samples was not found to be significantly different from unstressed
samples. But one conclusion can be drawn that GR activities were decreased in roots of drought
susceptible cultivar, PBW343 under stresses but these decreases were not observed in roots of
C306, drought tolerant cultivar where higher ascorbate/ dehydroascorbate ratios were also
maintained under stresses as compared to the ratios observed in roots of control seedlings
(Table 4.9).
4.3.5
Superoxide dismutase (SOD)
Three types of SOD have been reported in plants; Mn-SOD, Cu/Zn-SOD, Fe-SOD.
Mn-SOD are located in mitochondria of eukaryotic cells and peroxisomes. Cu/Zn-SOD
isozymes are located in chloroplast as well as in cytosol of higher plants. Fe-SOD is often not
detected in plants if present then located in chloroplast. All SOD enzymes are encoded by
nuclear genes and targeted to different compartments. SOD enzymes act on superoxide radical
and dismutate it to H2O2 . Its activity has been found to be increased under stresses in various
tissues of various plants (Gill and Tuteja 2010).
In our studies, SOD activities and specific activities were determined (Table 4.16;
Figure 4.21, 4.22). SOD specific activities increased in roots as well as in shoots of C306 under
all stress treatments. However in PBW343, its specific activity increased at 48h and 72h stages
under WS, at 48h stage under combined stress, unaltered under ABA treatment in shoots and
increased under combined stress only in roots. It was clear that SOD level was higher in C306
than in PBW343 under all stress treatments. SOD is the only enzyme among all other studied in
this study, which act on ROS particles other than H2O2, however all other were peroxidases
which removes H2O2. SOD is usually considered to be the first line of defense against oxidative
stress. SOD has also been reported to be increased under ABA treatment as well as under WS
through ABA dependent pathway (Jiang and Zhang 2002). So higher level of SOD found in
drought tolerant cultivar C306 might be related to its drought tolerance mechanism.
In control seedlings of both cultivars, there were increases or decreases of antioxidant
enzymes during growth of seedlings. Reason for such changes might be related to increasing or
decreasing contents of H2O2 at the corresponding stages. As out of five antioxidant enzymes,
four enzymes i.e. APX, CAT, GPOX and GR are involved in the regulation of H2O2 among
other ROS species and H2O2 contents are related to metabolic activities of the cells, so
changing trends of antioxidant activity might be controlling changing levels of H2O2 in these
tissues.
82
Table 4.15: The glutathione reductase activity (nmole of NADPH+H+ disappeared min-1 g-1 DW) and specific activity (as nmole of NADPH+ H+
disappeared min-1 mg-1 protein) in shoot (upper panel) and root (lower panel) of germinating wheat seedlings of two cultivars
PBW343 and C306 where stress was applied on 4th day after germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS
(20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly
aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
PBW343
CT
ABA
C306
WS
ABA+WS
CT
ABA
WS
ABA+WS
5785.24±2117.55abc
(23.52±4.39)ab
5785.24±2117.55abc
(23.52±4.39)ab
5785.24±2117.55abc
(23.52±4.39)ab
83
0h
5119.10±1446.33a
(18.90±5.42)a
5119.10±1446.33a
(18.90±5.42)a
5119.10±1446.33a
(18.90±5.42)a
Shoot
5119.10±1446.33a 5785.24±2117.55abc
(18.90±5.42)a
(23.52±4.39)ab
24h
9458.01±1716.62b
(30.66±6.46)abc
6035.39±1299.27a
(25.81±3.38)ab
4829.16±1298.69a
(29.25±8.90)abc
6096.53±1632.93a
(26.60±4.97)ab
6314.62±1110.95abcd
(27.81±3.71)ab
9063.40±459.11d
(31.66±3.05)b
5411.47±2104.59abc
(24.57±11.88)ab
5188.37±283.44abc
(27.76±1.76)ab
48h
17370.55±4999.64c
(64.11±26.28)d
7784.88±766.69ab
(28.26±6.05)abc
5799.16±2004.15a
(47.18±18.59)cd
6485.86±75.31ab
(47.76±2.29)cd
5222.56±557.78abc
(22.77±1.89)ab
5576.97±1626.84abc
(18.44±4.39)ab
4483.11±1515.95abc
(19.72±4.80)ab
4025.58±945.71a
(16.18±2.73)a
72h
5652.02±572.79a
(28.05±8.09)abc
4643.79±730.23a
(24.50±5.06)a
5046.04±723.81a
(28.69±2.34)abc
5864.60±600.41a
(44.55±1.92)bcd
7317.43±2801.31cd
(45.35±7.93)c
6867.32±1693.13bcd
(56.49±20.57)c
3533.45±1062.17a
(28.03±5.59)ab
4550.53±1214.44abc
(29.35±6.82)ab
5739.84±983.47c
5739.84±983.47c
5739.84±983.47c
(25.03±3.77)abc
2105.57±764.39a
(25.03±3.77)abc
3391.18±215.59ab
(34.49±5.66)abc
(25.03±3.77)abc
2464.09±713.21a
0h
bcd
3734.43±794.47
(18.29±4.90)a
24h
4486.06±956.59d
48h
(31.79±2.70)abc
6300.71±1405.41e
(43.51±10.19)cd
72h
5660.00±508.21
f
e
3734.43±794.47
bcd
3734.43±794.47
bcd
(18.29±4.90)a
3082.92±898.77bc
(18.29±4.90)a
2648.69±247.27ab
(28.28±10.38)abc
3317.78±546.98bcd
(28.90±2.05)abc
2844.70±233.06abc
(26.18±6.18)ab
3096.53±20.99bc
(38.21±1.21)bcd
3958.59±373.22cd
(51.68 ± 11.84)de
cd
Root
3734.43±794.47bcd
5739.84±983.47c
(18.29±4.90)a
(25.03±3.77)abc
1734.66±56.76a
4858.33±165.83bc
ab
(24.49±1.92)
(44.30±0.27)cd
3354.50±470.58bcd
(63.62±22.8)ef
3179.71±210.69bc
(62.08 ± 6.77)ef
2645.80±1089.82a
(21.63±9.07)ab
6641.13±2007.03
(63.82 ± 22.03)d
c
(24.47±7.57)abc
2048.67±168.45a
(17.47±0.69)a
5047.16±1628.35c
1754.30±442.76a
(32.88±6.16)abc
1771.12±725.09a
(31.27±10.81)abc
3167.60±280.58a
(33.27±18.79)abc
3040.74±687.10a
(70.79±7.88)
(43.73±1.95)
(61.93±20.36)d
(42.05±2.10)bcd
(38.60±7.68)abc
*Values within parenthesis indicate specific activity and without parenthesis indicate activity.
*Estimations were done on samples extracted in triplicates. Mean ± SD was calculated. Data of root and shoot in each cultivar was analyzed separately by Duncan’s
Multiple Test at P < 0.05 for test of significance, where superscripts of similar alphabets indicate no significant difference among the values.
nmole of NADPH2 disappeared /min/g DW
A
25000
0h
c
24h
48h
72h
20000
15000
b
cd
ab
10000
a
a
aa
a
a
a
a
a
a
a
abc
abcd
ab a
abc
d
abc
bcd
abc abc
abc
abc
a
abc
abca abc
5000
0
CT
ABA
WS
ABA+WS
CT
ABA
0h
24h
48h
72h
c
9000
nmole of NADPH2 disappeared /min/g DW
ABA+WS
C306
PBW343
B 10000
WS
e
8000
c
7000
c
c
c
c
e
6000
d
5000
bcd
bc
bcd
bcbcd
4000
bc
3000
bcd
cd
ab
abc
bcd
bcd
bc
a
ab
a
a
a
a
2000
a
a
a
a
1000
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.19: The glutathione reductase activity (nmole of NADPH+H + disappeared min-1 g-1 DW)
shoot (A) and root (B) of germinating wheat seedlings of two cultivars PBW343 and
C306 where stress was applied on 4th day after germination as ABA (20 µM ABA),
WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data
was collected at 24h, 48h, 72h after stress treatment and compared with similarly
aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken just
before applying stress treatment. Estimations were done on samples extracted in
triplicates. Mean ± SD was calculated. Data of root and shoot in each cultivar was
analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance,
where superscripts of similar alphabets indicate no significant difference among the
values.
84
nmole of NADPH2 disappeared per min per mg
protein
A 100
0h
d
24h
48h
72h
c
90
80
cd
70
c
60
cd
bcd
50
40
abc abc
30
a
abc
abc
ab a
a
abc
a
ab
a
ab ab
b
ab
ab ab
ab
ab
ab
a
10
0
ABA
WS
ABA+WS
CT
ABA
0h
100
90
24h
48h
ABA+WS
72h
ef
f
80
d
d
ef
de
70
cd
60
cd
50
abc
ab
a
a
abc
a
abc
abc
abcbcd
abc
abc
cd
bcd
abc
40
30
WS
C306
PBW343
nmole of NADPH2 disappeared per min per mg protein
abab
20
CT
B
ab
ab
ab
abc
ab
a
abc
abc
abc
abc
a
20
10
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.20: The glutathione reductase specific activity (as nmole of NADPH+ H + disappeared
min-1 mg-1 protein) in shoot (A) and root (B) of germinating wheat seedlings of two
cultivars PBW343 and C306 where stress was applied on 4 th day after germination as
ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and
compared with similarly aged control seedlings (Ct) grown on distilled water as well
as with 0h stage taken just before applying stress treatment. Estimations were done
on samples extracted in triplicates. Mean ± SD was calculated. Data of root and shoot
in each cultivar was analyzed separately by Duncan’s Multiple Test at P < 0.05 for
test of significance, where superscripts of similar alphabets indicate no significant
difference among the values.
85
Table 4.16: SOD activity (SOD units g-1 DW) and specific activity (SOD units mg-1 protein) in shoot (upper panel) and root (lower panel) of germinating wheat
seedlings of two cultivars PBW343 and C306 where stress was applied on 4 th day after germination as ABA (20 µM ABA), WS (6% Mannitol) and
ABA+WS (20 µM ABA and 6% Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with similarly aged
control seedlings (Ct) grown on distilled water as well as with 0h stage taken just before applying stress treatment.
PBW343
0h
24h
48h
72h
C306
ABA
WS
00.00±0.00
(0.00±0.00)
145.47±41.99a
(0.50±0.27)ab
80.94±23.27a
(0.31±0.13)a
98.26±39.30a
(0.76±0.26)b
00.00±0.00
(0.00±0.00)
134.03±64.37a
(0.56±0.23)ab
149.83±40.39ab
(0.53±0.08)ab
123.93±32.38a
(0.65±0.15)ab
00.00±0.00
(0.00±0.00)
135.60±17.07a
(0.83±0.20)b
95.64±7.21a
(0.78±0.14)b
227.61±71.98b
(1.46±0.36)c
00.00±0.00
(00.00±0.00)
201.69±52.86c
(1.48±0.57)bc
97.61±12.58ab
(0.67±0.10)ab
99.75±24.25ab
(1.24±0.33)abc
00.00±0.00
(00.00±0.00)
197.72±110.23c
(1.74±0.86)cd
84.18±3.04ab
(0.66±0.07)ab
53.31±31.80a
(0.69±0.34)ab
00.00±00.00
(00.00±0.00)
144.00±17.55bc
(1.57±0.16)c
43.25±2.32a
(0.58±0.07)a
96.11±12.54ab
(1.21±0.04)abc
86
CT
0h
24h
48h
72h
ABA+WS
Shoot
00.00±0.00
(0.00±0.00)
114.55±51.54a
(0.51±0.25)ab
96.46±9.74a
(0.71±0.09)b
96.36±45.14a
(0.71±0.25)b
Root
00.00±0.00
(00.00±0.00)
168.69±19.45c
(2.39±0.38)de
48.75±22.84a
(0.97±0.58)abc
75.91±39.23ab
(2.97±0.97)e
CT
ABA
WS
ABA+WS
00.00±0.00
(0.00±0.00)
144.02±59.24c
(0.61±0.17)cd
66.32±28.68ab
(0.30±0.15)ab
45.02±6.23a
(0.26±0.10)a
00.00±0.00
(0.00±0.00)
116.80±70.79bc
(0.42±0.27)abc
109.01±46.31abc
(0.36±0.15)abc
278.89±47.50d
(1.14±0.09)e
00.00±0.00
(0.00±0.00)
82.87±41.85abc
(0.39±0.23)abc
57.19±2.33ab
(0.26±0.03)a
106.39±16.46abc
(0.88±0.25)de
00.00±0.00
(0.00±0.00)
44.76±3.90a
(0.25±0.001)a
83.76±34.10abc
(0.35±0.18)abc
89.79±16.77abc
(0.60±0.15)bcd
00.00±0.00
(00.00±0.00)
154.316±14.88cd
(1.45±0.10)bcd
127.44±12.71bc
(1.04±0.13)abc
122.16±20.70bc
(1.17±0.23)abc
00.00±0.00
((00.00±0.00))
197.91±53.43d
(2.04±0.12)e
116.27±7.54bc
(1.00±0.19)ab
55.17±9.75a
(0.65±0.09)a
00.00±0.00
(00.00±0.00)
95.78±9.59ab
(1.04±0.10)abc
100.79±51.32ab
(1.70±0.66)de
112.99±42.73bc
(1.51±0.60)bcde
00.00±0.00
(00.00±0.00)
77.82±7.37ab
(1.11±0.07)abc
88.35±18.69ab
(1.57±0.06)cde
89.94±23.03ab
(1.16±0.35)abc
*Values within parenthesis indicate specific activity and without parenthesis indicate activity.
*Estimations were done on samples extracted in triplicates. Mean ± SD was calculated. Data of root and shoot in each cultivar was analyzed separately by
Duncan’s Multiple Test at P < 0.05 for test of significance, where superscripts of similar alphabets indicate no significant difference among the values.
A
350
0h
24h
48h
72h
d
b
300
SOD units /g DW
250
200
a
a
c
ab
a
a
150
bc
a
a
a
abc
a
abc abc
a
a
abc
abc
ab
100
ab
a
a
50
0
CT
ABA
WS
ABA+WS
CT
ABA
0h
24h
48h
ABA+WS
C306
PBW343
B 350
WS
72h
c
300
SOD units / g DW
c
d
250
c
200
cd
bc
bcbc
150
ab
ab
ab
ab a
100
a
ab
ab bc
bc
ab
ab
ab
ab
a
a
50
0
CT
ABA
WS
ABA+WS
CT
ABA
WS
ABA+WS
C306
PBW343
Fig. 4.21: SOD activity (SOD units g-1 DW) in shoot (A) and root (B) of germinating wheat seedlings of
two cultivars PBW343 and C306 where stress was applied on 4 th day after germination as
ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6% Mannitol)
treatments. Data was collected at 24h, 48h, 72h after stress treatment and compared with
similarly aged control seedlings (Ct) grown on distilled water as well as with 0h stage taken
just before applying stress treatment. SOD activity could not be detected at 0h stage in any of
above mentioned samples. Estimations were done on samples extracted in triplicates.
Mean ± SD was calculated. Data of root and shoot in each cultivar was analyzed
separately by Duncan’s Multiple Test at P < 0.05 for test of significance, where
superscripts of similar alphabets indicate no significant difference among the values.
87
A
2
0h
24h
48h
72h
c
SOD units per mg protein
1.8
1.6
1.4
e
1.2
b
b
1
0.8
b
b
ab ab
ab
de
ab
b
cd
bcd
abc
ab
0.6
abc
ab
a
abc
a
0.4
abc
a
a
0.2
0
CT
ABA
WS
ABA+WS
CT
ABA
4.5
0h
24h
48h
ABA+WS
C306
PBW343
B
WS
72h
e
SOD units per mg protein
4
3.5
de
3
2.5
2
cd
bc
c
abc
abc
abc
1.5
1
de
bcde
e
ab
ab
abc
ab
cde
bcd abc
ab
abc
abc
abc
a
a
0.5
0
CT
ABA
WS
ABA+WS
PBW343
CT
ABA
WS
ABA+WS
C306
Fig. 4.22: SOD specific activity (SOD units mg-1 protein) in shoot (A) and root (B) of germinating wheat
seedlings of two cultivars PBW343 and C306 where stress was applied on 4 th day after
germination as ABA (20 µM ABA), WS (6% Mannitol) and ABA+WS (20 µM ABA and 6%
Mannitol) treatments. Data was collected at 24h, 48h, 72h after stress treatment and
compared with similarly aged control seedlings (Ct) grown on distilled water as well as with
0h stage taken just before applying stress treatment. SOD activity could not be detected at 0h
stage in any of above mentioned samples. Estimations were done on samples extracted in
triplicates. Mean ± SD was calculated. Data of root and shoot in each cultivar was
analyzed separately by Duncan’s Multiple Test at P < 0.05 for test of significance,
where superscripts of similar alphabets indicate no significant difference among the
values.
88
4.4
Late Embryogenesis Abundant Protein (LEA)
LEA (Late Embryogenesis Abundant Protein) proteins are expressed at high levels
during later stages of embryo development (post abscission) in plant seeds as at this stage in
the developmental process, orthodox seeds acquire ability to withstand extreme dehydration.
LEA proteins have been associated with dessication tolerance (Tunnacliffe and Wise 2007).
LEA proteins have been classified into 9 groups based upon sequence similarities and
conserved domains (Hundertmark and Hincha 2008). Among nine groups, role of only group
1, 2 and 3 has been investigated in imparting abiotic stress tolerance. ABA induces the
expression of LEA genes during seed development and abiotic stresses (Finkelstein et al
2002, Dalal et al 2009). However all LEA genes were not responsive to ABA , they represent
a major group of downstream genes involved in ABA-dependent and –independent signal
pathways (Hong-Bo et al 2005, Kobayashi et al 2008a, 2008b).
In the present study, LEA genes belonging to three groups; group 2, group 3, group 4
were studied for their expression level by semi-quantitative RT- PCR, where internal control
of the constitutive gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used.
This work was done in shoots of both cultivars at 24h and 48h after applying stress. All four
similar treatments were used i.e. 20µM for ABA stress, 6% mannitol for WS and 20µM for
ABA plus 6% mannitol for combined stress and shoots of control seedlings growing on
distilled water was used as control to compare the expression level under stresses versus
unstressed. This work was done by designing primers from known sequences of LEA genes
available in database. Database was searched for nucleotide, mRNA, ESTs and protein
sequences for LEA genes/proteins in wheat plant at NCBI (www.ncbi.nlm.nih.gov/). Primers
were designed from sequences either manually or using Primer BLAST available at NCBI.
Gene ID (NCBI Accession no.), gene name and primer sequences are summarized in Table
3.1. Some of these sequences belong to cDNA sequences isolated from Triticum aestivum
and found to be 100% similar to dehydrin sequences isolated from Triticum durum. SemiQuantitative RT-PCR was done on total RNA isolated from shoots of above mentioned
samples. RT-PCR products were run on 1.5% agarose gel and were pictured using Gel
DocTM XR (Biorad). The intensities of bands were measured using Quantity One software
(Biorad Laboratories Inc.). The ratio for each band was calculated by dividing the intensity
of that band with intensity of band obtained with GAPDH primers in same sample. Results
are presented in Figure 4.23- 4.33.
89
(A)
PBW343
C306
24hr
ABA+WS ABA
48hr
WS
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
Td16
GAPDH
(B)
3.5
24h
48h
PBW343
C306
3
Ratio
2.5
2
90
1.5
1
0.5
0
24h
48h
CT
1.52
0.39
ABA
1.37
1.68
WS
3.26
1.59
ABA+WS
2.4
1.92
CT
2.22
0
ABA
1.81
1.6
WS
1.52
2.71
ABA+WS
2.22
0.88
Figure 4.23: (A) Gel pictures of RT- PCR products of CV762802 (similar to X78429 (Td16) of durum wheat) and GAPDH (internal control) where
semiquantitative RT- PCR was done on total RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306
treated on 4th day of germination with ABA (20 µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at
24h and 48h after stress applications and compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of CV762802 (similar to X78429 (Td16) of durum wheat) in the form of ratio of intensity of band obtained with
gene specific primer to the intensity of band obtained with GAPDH specific primer.
PBW343
(A)
C306
24hr
ABA+WS ABA
WS
48hr
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
Td25a
GAPDH
(B)
1.2
24h
48h
PBW343
C30
1
91
Ratio
0.8
0.6
0.4
0.2
0
24h
48h
Figure 4.24:
CT
1.07
0
ABA
0.563
0.36
WS
0.369
0
ABA+WS
0.161
0
CT
0.31
0
ABA
0.12
0.18
WS
1.09
0.79
ABA+WS
0.39
0.14
(A) Gel pictures of RT- PCR products of DR739608 (similar to X78431 (Td25a) of durum wheat) and GAPDH (internal control) where
semiquantitative RT- PCR was done on total RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306
treated on 4th day of germination with ABA (20 µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at
24h and 48h after stress applications and compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of in the form of ratio of DR739608 (similar to X78431 (Td25a) of durum wheat) intensity of band obtained
with gene specific primer to the intensity of band obtained with GAPDH specific primer.
(A)
PBW343
C306
24hr
ABA+WS ABA
WS
48hr
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
Td27e
GAPDH
(B)
3
24h
48h
C306
PBW343
2.5
92
Ratio
2
1.5
1
0.5
0
24h
48h
CT
1.12
0.69
ABA
0.81
1.41
WS
2.15
1.33
ABA+WS
1.81
1.31
CT
1.45
0.77
ABA
1.27
1.38
WS
1.04
2.57
ABA+WS
1.59
0.52
Figure 4.25: (A) Gel pictures of RT- PCR products of AL815683 (similar to X78431 (Td27e) of durum wheat) and GAPDH (internal control) where
semiquantitative RT- PCR was done on total RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306
treated on 4th day of germination with ABA (20 µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at
24h and 48h after stress applications and compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of in the form of ratio of AL815683 (similar to X78431 (Td27e) of durum wheat) intensity of band obtained with
gene specific primer to the intensity of band obtained with GAPDH specific primer.
(A)
PBW343
C306
24hr
ABA+WS ABA
48hr
WS
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
Td11
GAPDH
3
(B)
24h
48h
PBW343
C306
2.5
93
Ratio
2
1.5
1
0.5
0
24h
48h
Figure 4.26:
CT
1.49
1.66
ABA
1.08
1.6
WS
2.67
1.56
ABA+WS
2.22
1.49
CT
1.85
0.96
ABA
1.54
1.39
WS
1.42
2.27
ABA+WS
1.89
1.1
(A) Gel pictures of RT- PCR products of U73211 (WCOR410 (similar to AJ890140, Td11 of durum wheat) and GAPDH (internal control) where
semiquantitative RT- PCR was done on total RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306
treated on 4th day of germination with ABA (20 µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at
24h and 48h after stress applications and compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of U73211 (WCOR410 (similar to AJ890140, Td11 of durum wheat) in the form of ratio of intensity of band
obtained with gene specific primer to the intensity of band obtained with GAPDH specific primer.
PBW343
(A)
C306
24hr
ABA+WS ABA
48hr
WS
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
Wdhn 13
GAPDH
24h
2.5
48h
(B)
PBW343
C306
94
Ratio
2
1.5
1
0.5
0
24h
48h
Figure 4.27:
CT
1.72
1.31
ABA
1.43
1.35
WS
1.19
1.61
ABA+WS
2.33
1.4
CT
0.77
0.44
ABA
1.51
1.04
WS
1.11
0.77
ABA+WS
0.92
0.55
(A) Gel pictures of RT- PCR products of AB076807 (Wdhn 13) and GAPDH (internal control) where semiquantitative RT- PCR was done on
total RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4 th day of germination with ABA
(20 µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and
compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of AB076807 (Wdhn 13) in the form of ratio of intensity of band obtained with gene specific primer to the
intensity of band obtained with GAPDH specific primer.
Among five LEA genes belonging to group 2 (Figure 4.23- 4.27), Wdhn 13 LEA
gene was induced more under ABA than under WS in C306 otherwise other LEA genes were
induced more under WS than under ABA in both cultivars. This gene was neither responsive
to ABA nor induced under WS in PBW343 but expressed in control shoot. Among other four
LEA genes; two LEA genes CV762802 (similar to Td16 of durum plant) and AL815683
(similar to Td27e of durum plant) were responsive to ABA and other two, DR739608
(similar to Td25a of durum plant) and U73211 (WCOR410, similar to Td11 of durum plant)
were weakly responsive to ABA in both cultivars. Under WS treatment, all four were
induced under WS in both cultivars but DR739608 (similar to Td25a of durum plant) was
expressed only in C306 and Al815683 (similar to Td27e of durum plant) was induced by
more amounts in C306 than PBW343. Td16, Td27e, Td25a were reported to be more
expressed under WS, while Td11 responded to cold, salt stress, ABA application but not to
WS in wheat (Ali-Benali et al 2005). In another study, WCOR410 (Td11) was found to
expressed only in freezing tolerant cultivar (Danyluk et al 1994, 1998). Among these five
group 2 Lea genes; CV762802 (similar to Td16 of durum plant) and AL815683 (similar to
Td27e of durum plant) and U73211 (WCOR410 similar to Td11 of durum plant) were
induced earlier (24h) in PBW343 but later (48h) in C306. Accumulation of Td27e, Td25a
was reported to be delayed (67h) in drought tolerant cultivar but earlier (30h) in drought
susceptible cultivar in both root and shoot, moreover these two genes were found to be
responsive to ABA and induced by more amount in drought tolerant cultivar than drought
susceptible cultivar (Labhili et al 1995). In present study, these two genes (Td25a and
Td27e) were similarly found to be more induced or induced only in drought tolerant cultivar
but for their sensitivity towards ABA, Td27e was more responsive to ABA than Td25a in
drought tolerant cultivar and Td27e was induced earlier (24h) in drought susceptible cultivar,
PBW343 and later (48h) in drought tolerant cultivar, C306 where Td25a was induced only in
tolerant cultivar not in drought susceptible cultivar.
Comparing cultivars for ABA response in terms of expression status of group 2 LEA
genes in shoots, Wdhn13 gene was responsive to ABA as well as induced under WS in
drought tolerant cultivar C306 but was neither responsive to ABA nor induced under WS in
drought susceptible cultivar, PBW343 (though expressed in its control shoots), hence it
might be involved in ABA- dependent pathway of drought tolerance. In literature, it is found
to be ABA-responsive in Triticum aestivum (Kobayashi et al 2006) as well as in Ae. Tauschii
(Kurahashi et al 2009) and its level of expression was also correlated to freezing tolerance,
drought tolerance as well as ABA-sensitivity. It is also reported to be induced under salt
stress besides drought and low temperature (Ohno et al 2003).
95
(A)
PBW343
C306
24hr
ABA+WS ABA
WS
48hr
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
AF255052
(Wrab 19)
GAPDH
(B)
3
24h
48h
PBW343
C306
96
Ratio
2.5
2
1.5
1
0.5
0
24h
48h
CT
1.35
0
ABA
0.63
0.68
WS
1.5
1.19
ABA+WS
1.18
0.74
CT
1.18
0
ABA
1.33
0.69
WS
0.93
2.7
ABA+WS
1.77
0
Figure 4.28: (A) Gel pictures of RT- PCR products of AF25505 (Wrab 19) and GAPDH (internal control) where semiquantitative RT- PCR was done on total
RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4 th day of germination with ABA (20 µM),
WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and compared with
shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of AF255052 (Wrab 19) in the form of ratio of intensity of band obtained with gene specific primer to the
intensity of band obtained with GAPDH specific primer.
PBW343
(A)
C306
24hr
ABA+WS ABA
48hr
WS
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
AB115913
(Wrab 15)
GAPDH
2.5
(B)
24h
48h
PBW343
C306
97
Ratio
2
1.5
1
0.5
0
24h
48h
Figure 4.29:
CT
1.43
0.56
ABA
0.8
0.88
WS
1.85
1.76
ABA+WS
1.92
1.68
CT
0.54
0
ABA
1.56
0.68
WS
1.27
2.01
ABA+WS
1.94
1.08
(A) Gel pictures of RT- PCR products of AB115913 (Wrab 15) and GAPDH (internal control) where semiquantitative RT- PCR was done on total
RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4 th day of germination with ABA (20
µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and
compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of AB115913 (Wrab 15) in the form of ratio of intensity of band obtained with gene specific primer to the
intensity of band obtained with GAPDH specific primer.
(A)
PBW343
C306
24hr
ABA+WS ABA
WS
48hr
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
AF255053
(Wrab 17)
GAPDH
(B)
2
24h
48h
PBW343
C306
1.8
1.6
98
Ratio
1.4
1.2
1
0.8
0.6
0.4
0.2
0
24h
48h
CT
1.21
0.75
ABA
0.89
0.82
WS
1.87
1.36
ABA+WS
1.28
0.98
CT
1.05
1.46
ABA
1.07
1
WS
1.03
0.31
ABA+WS
0.8
0
Figure 4.30: (A) Gel pictures of RT- PCR products of AF255053 (Wrab 17) and GAPDH (internal control) where semiquantitative RT- PCR was done on total
RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4 th day of germination with ABA (20 µM),
WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and compared with
shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of AF255053 (Wrab 17) in the form of ratio of intensity of band obtained with gene specific primer to the
intensity of band obtained with GAPDH specific primer.
PBW343
(A)
C306
24hr
ABA+WS ABA
WS
48hr
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
AB115914
(Wrab18)
GAPDH
(B)
3
24h
48h
PBW343
C306
99
Ratio
2.5
2
1.5
1
0.5
0
24h
48h
CT
1.39
0
ABA
0.64
0.76
WS
1.66
2
ABA+WS
1.22
1.63
CT
1.18
0
ABA
1.39
0.17
WS
1.01
2.49
ABA+WS
1.54
0
Figure 4.31: (A) Gel pictures of RT- PCR products of AB115914 (Wrab18) and GAPDH (internal control) where semiquantitative RT- PCR was done on total
RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4 th day of germination with ABA (20 µM),
WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and compared with
shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of AB115914 (Wrab18) in the form of ratio of intensity of band obtained with gene specific primer to the intensity
of band obtained with GAPDH specific primer.
(A)
PBW343
C306
24hr
ABA+WS ABA
48hr
WS
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
AY148490
GAPDH
(B)
1.6
24h
48h
PBW343
C306
1.4
10
0
Ratio
1.2
1
0.8
0.6
0.4
0.2
0
24h
48h
CT
1.5
0
ABA
0.044
0
WS
0.881
0.69
ABA+WS
0.312
0.79
CT
0.61
0
ABA
1.42
0.31
WS
0.78
1.34
ABA+WS
1.43
0
Figure 4.32: (A) Gel pictures of RT- PCR products of AY148490 (TaLEA-3 like) and GAPDH (internal control) where semiquantitative RT- PCR was done on
total RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306 treated on 4 th day of germination with ABA (20
µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at 24h and 48h after stress applications and compared
with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of AY148490 (TaLEA-3 like) in the form of ratio of intensity of band obtained with gene specific primer to the
intensity of band obtained with GAPDH specific primer.
(A)
PBW343
C306
24hr
ABA+WS ABA
48hr
WS
CT
ABA+WS ABA
WS
24hr
CT
ABA+WS ABA WS
48hr
CT
ABA+WS ABA
WS
CT
AY148490
GAPDH
(B)
3.5
24h
48h
PBW343
C306
3
10
1
Ratio
2.5
2
1.5
1
0.5
0
24h
48h
Figure 4.33:
CT
1.75
0.9
ABA
1.89
1.76
WS
3.28
1.71
ABA+WS
2.72
1.67
CT
1.7
0
ABA
1.62
1.22
WS
1.31
2.94
ABA+WS
1.92
0.95
(A) Gel pictures of RT- PCR products of GH729039 (similar to AJ890139 (Td29) of durum wheat) and GAPDH (internal control) where
semiquantitative RT- PCR was done on total RNA isolated from shoots of germinating wheat seedlings of two cultivars PBW343 and C306
treated on 4th day of germination with ABA (20 µM), WS (6% mannitol), ABA+ WS (20 µM and 6% mannitol). Experiment was performed at
24h and 48h after stress applications and compared with shoots of control seedling (CT) growing on distilled water for similar time period.
(B) Quantitative expression level of GH729039 (similar to AJ890139 (Td29) of durum wheat) in the form of ratio of intensity of band obtained
with gene specific primer to the intensity of band obtained with GAPDH specific primer.
LEA Group3 Wrab genes (Wrab15, Wrab17, Wrab18, Wrab19) were observed as weakly- or
non-responsive to ABA e.g. Wrab17 and Wrab18 were non-responsive (rather
downregulated under ABA treatment), Wrab19 was weakly responsive to ABA in both
cultivars but Wrab 15 and another gp3 gene (LEA gp 3-like gene) were responsive to ABA
mainly in C306 cultivar. As opposed to ABA treatment, these genes were induced under WS,
where Wrab19, Wrab15, Wrab18 and Ta LEA-3 like were more induced in C306 than in
PBW343, while Wrab17 was induced only in PBW343 not induced in C306. All five LEA
gp3 genes were induced earlier (24h) in PBW343 while later (48h) in C306.
In literature, Wrab genes were reported to be responsive to ABA, low temperature,
drought (Kobayashi et al 2004, 2006, 2008, Kurahashi 2009, Egawa et al 2006) but almost
all of this work was done in two wheat cultivars M808 (winter wheat, freezing tolerant) and
CS (spring wheat freezing susceptible) where expression levels of these genes were more in
tolerant cultivar, M808 than in susceptible cultivar, CS under ABA treatment but were not
different between cultivars under low temperature (Egawa et al 2006, Kobayashi et al 2004,
2006, 2008). But these genes (except Wrab17) were actually not so good responsive to ABA,
if these expressed, stayed for 2-5 hr after the treatment and not stayed like as under low
temperature treatment (Kobayashi et al 2004, 2006). Reasons for such temporary response
under ABA but stable response under stress treatment were not clear though suggested to be
probable metabolization of exogenously applied ABA (Kobayashi et al 2004). In this study,
adding exogenous ABA with WS (combined stress) gave response more similar to ABA
treatment than to WS in drought tolerant C306 cultivar as 5of 11 LEA genes (belonging to all
three gps 2,3,4) as these were induced under WS and remain un-induced or less induced
under combined stress (Figures 4.23 to 4.33). It indicated that ABA might be inhibiting or
down-regulating the expression of these genes in C306 cultivar. Our observations agreed to
the results (Kobayashi et al 2006) of these genes expression in another wheat cultivar
Kitakei1354 (ABA hypersensitive and dormant winter wheat) where unlike M808 cultivar,
these genes were downregulated under ABA treatment while in another cultivar EH47-1
(derived from Kitakei1354 by single mutation, which became ABA-sensitive and nondormant but more freezing tolerant than Kitakei1354), these genes were responsive to ABA
like as observed in freezing tolerant M808 cultivar. Under low temperature, these genes were
almost equally induced in both EH47-1 and Kitakei-1354. Drought tolerant cultivar, C306
used in this study was also found to be higher sensitive to ABA than drought susceptible
cultivar, PBW343 (16% germination in C306 as opposed to 81% in PBW343 in the presence
of 20 µM ABA on 6th day of germination, unpublished results). It can be concluded that LEA
genes’ expressions are controlled by both ABA-dependent and ABA-independent pathways
under water stress but dominating effects of ABA over the effects of WS when applied both
102
together in combined stress observed in C306 not in PBW343, might be due to higher ABAsensitivity of C306 and ABA-insensitivity of PBW343.
Only one gene GH729039 (similar to AJ890139 (Td29) of durum wheat) of LEA gp
4 was studied. Response of this gene was similar to gp2 LEA genes (studied in the present
study) than to gp3 LEA genes. This gene was poorly responsive to ABA but induced under
WS in both cultivars, however induction level was higher in C306 than in PBW343. It was
induced earlier in PBW343 (24h) and later in C306 (48h).
103
Chapter V
SUMMARY
Two wheat cultivars contrasting in drought tolerance were compared for their
response to exogenous ABA, water stress (WS) and combined stress (ABA plus WS) during
germination. Their response was studied in the form of growth and antioxidant potential in
roots and shoots at different hours (0h, 24h, 48h, 72h) and in the form of LEA gene
expression in shoots at 24h and 48h after stress treatment where stress was given on 4th day of
germination. Responses were compared with similarly old control seedlings growing on
distilled water and with 0h stage taken on 4th day of germination before applying stress
treatment. Such responses were also compared among stresses and between cultivars. ABAdependent maintenance of dry weights under WS as well as combined stress was found in
shoots and roots of drought tolerant cultivar, C306 but lacked in drought susceptible cultivar,
PBW343. Drought tolerant cultivar, C306 showed lower water contents throughout its stress
period while water contents were higher and regained after 24h of stress in drought
susceptible cultivar, PBW343. Exogenous application of ABA led to increased H2O2 contents
in roots of C306 at 24h, followed by increase in ascorbate content, ascorbated/
dehydroascorbate ratio, activities of catalase, ascobate peroxidase and guaicol peroxidase in
shoots; decreased membrane toxicity in both shoots and roots. Such types of responses were
poorly given by PBW343 under exogenous ABA supply. Under WS, both cultivars behaved
differently where above mentioned ABA-dependent responses were given by C306 but were
lacked in PBW343. Under combined stress, above mentioned responses were similarly
produced as under WS and as under ABA supply in C306, however in PBW343, such
responses were more similar to as under ABA than to as under WS. It indicated the existence
of different pathways for ABA and for WS where ABA-dependent pathway was working in
C306 but lacked in PBW343 under WS. Drought tolerant cultivar showed lesser level of
membrane oxidation and higher levels of SOD. MDA contents were not related to H2O2
contents. Proline contents were higher in roots of drought tolerant cultivar, C306 under
unstressed conditions and on exposure to WS, this cultivar accumulated more proline contents
in its roots. However drought susceptible cultivar, PBW343 contained lesser contents of
proline under unstressed conditions and on exposure to WS, it accumulated proline contents
mainly in its shoots. Catalases and H2O2 content were positively correlated in shoots of C306
in control as well as in stressed samples.
Eleven LEA genes, five of gp2, five of gp3 and one of gp4, were studied for their
expression status in shoots of both cultivars at 24h and 48h after stress treatment under same
stresses (ABA, WS, ABA plus WS). Results were compared with control shoots of same age,
among stresses and between cultivars. All 11 LEA genes were more induced under WS than
under ABA in both cultivars except Wdhn13 which was more induced under ABA than under
WS in C306. All LEA genes were responsive to ABA in both cultivars except Wdhn13
(responsive to ABA in C306 only but not in PBW343) and Wrab17 (ABA-responsive in
PBW343 not in C306). Among all 11 LEA genes, 6 (three of gp2, Td25a, Td27e, Wdhn13;
two of gp3 Wrab1, Ta LEA gp3-like; one gp4, Td29) were more induced or induced only in
C306, one LEA gene of gp3 (Wrab17) was induced only in PBW343 and others four (two of
gp2, Td11, Td16; two of gp3, Wrab18, Wrab15) were equally induced in both cultivars under
WS. Gp2 and Gp4 LEA genes were induced earlier (24h) in PBW343 and later (48h) in C306
while gp3 genes were induced mainly at 48 h in both cultivars under WS.
105
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