Download OsPRP3, a flower specific proline-rich protein of rice, determines

Plant Mol Biol (2010) 72:125–135
DOI 10.1007/s11103-009-9557-z
OsPRP3, a flower specific proline-rich protein of rice, determines
extracellular matrix structure of floral organs
and its overexpression confers cold-tolerance
Kodiveri Muthukalianan Gothandam •
Easwaran Nalini • Sivashanmugam Karthikeyan
Jeong Sheop Shin
•
Received: 8 August 2008 / Accepted: 2 October 2009 / Published online: 15 October 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Proline-rich protein (PRP), a cell wall protein
of plant, has been studied in many plant species. Yet, none
of the PRPs has been functionally elucidated. Here we
report a novel flower-specific PRP designated OsPRP3
from rice. Expression analysis showed that the OsPRP3
transcript was mainly present in rice flower and accumulated abundantly during the late stage of the flower
development. To study the function of OsPRP3, we constructed and transformed a binary vector containing a full
clone of OsPRP3 in sense orientation and also an RNAi
vector to achieve overexpression and knockout of the gene,
respectively. Our overexpression plants showed a significant increase in cold tolerance than the WT plants which is
conferred by the accumulation of OsPRP3 protein during
cold treatment. Further the microscopic analysis revealed
that OsPRP3 enhances the cell wall integrity in the cold
tolerant plant and confers cold-tolerance in rice. Microscopic analysis of the RNAi mutant flower revealed that
blocking OsPRP3 function caused significant defects in
floral organogenesis. Taken together, the results suggested
that OsPRP3 is a cell wall protein, playing a crucial role in
determining extracellular matrix structure of floral organs.
Electronic supplementary material The online version of this
article (doi:10.1007/s11103-009-9557-z) contains supplementary
material, which is available to authorized users.
K. M. Gothandam (&) E. Nalini S. Karthikeyan
School of Bio Sciences & Technology, VIT University, Vellore
632 014, Tamil Nadu, India
e-mail: [email protected]
J. S. Shin
School of Life Sciences & Biotechnology, Korea University,
Seoul 136-701, Korea
Keywords Cold tolerance Proline rich protein Rice Flower development
Introduction
Plant cell walls are complex structures that consist of
carbohydrates, proteins, lignin, cellulose microfibrils, and
also incrusting substances such as cutin and suberin
(Showalter 1993). The composition and structure vary
depending on different cell types due to their functional
specializations and also can be modified as plants adapt to
environmental signaling such as biotic and abiotic stresses
(Showalter 1993). The cell walls are composed of about
10% proteins, including extensins, glycine-rich proteins,
proline-rich proteins, the solanaceous lectins, and the
arabinogalactan proteins (Showalter 1993; Lamport 1965).
These proteins have their own unique distribution patterns
among the various organs, tissues, and cell of plants,
playing an integral role in the extracellular matrix structure
of many plant cells (Vaner and Lin 1989).
Proline-rich proteins (PRPs) are one of the structural cell
wall proteins in plant (Chen and Varner 1985; Tierney
et al. 1988) and these proteins have been shown to be
expressed in many plant species. PRPs were first identified
as the proteins in response to physical damage (Chen and
Varner 1985; Tierney et al. 1988) and later the proteins
were found during plant development. During the plant
development, expression of PRP genes often appears to be
regulated temporally and spatially. For instance, in soybean
individual PRP genes were spatially expressed in different
types of plant organs (Hong et al. 1989; Kleis-San Francisco and Tierney 1990; Lindstrom and Vodkin 1991;
Wyatt et al. 1992). PRP gene expression was associated
with root nodule formation, seedling growth (Sheng et al.
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1991), and with fruit development (Santino et al. 1997). A
potato gene, StGCPRP, was expressed in a highly differentiated cell type such as guard cells (Menke et al. 2000)
and AtPRP3 in Arabidopsis was detected exclusively in
root (Fowler et al. 1999). Expression of PRPs is also
influenced by factors associated with biotic and abiotic
stresses, suggesting that the synthesis of the proteins is
sensitive to external stimuli (Tierney et al. 1988; Sheng
et al. 1991; Marcus et al. 1991). To date, the precise
functions of PRPs are unknown. However, there are
increasing evidences that the proteins may have important
roles in normal development, conferring the integrity of
plant cell wall and the structure maintenance of organs
(Sheng et al. 1991; Nicholas et al. 1993; Carpita and Gibeaut 1993). Localization studies suggest that PRPs may
function in determining cell-type-specific wall structure
during plant development (Menke et al. 2000).
To our knowledge, two PRP genes have been studied in
rice so far (Akiyama and Pillai 2003; Wu et al. 2003; Wang
et al. 2006). Yet, none of these genes has been functionally
elucidated. Here we report a novel rice PRP gene designated OsPRP3 (Oryza sativa Proline-Rich Protein 3) which
accumulates during cold stress and confers cold-tolerance
in the rice. We found that the OsPRP3 gene is regulated
during flower development in rice. Also, our knockout
mutant generated by RNAi demonstrated that the gene
plays an important role in floral organ formation.
Materials and methods
Isolation and sequence analysis
OsPRP3 clone was obtained from the rice anther cDNA
library (Choi et al. 2000). Nucleotide and deduced amino
acid sequence were analyzed with the Basic Local Alignment Search Tool (BLAST) at the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov)
and the Soft berry programme (http://www.softberry.com).
The signal peptide cleavage site was predicted by the
SignalP programme (Nielsen et al. 1997). Sequence comparisons were conducted using Clustal W (Altschul et al.
1990).
Southern blot analysis
Genomic DNA was isolated from rice seedlings as previously described (Gothandam et al. 2005). Fifteen micrograms of the genomic DNA were digested with EcoRI,
HindIII and XbaI. The digested DNA was subjected to
electrophoresis on a 0.8% agarose gel and transferred to
Hybond-N membrane (Amersham). The blot was hybridized with 32P labeled C terminal region of OsPRP3 probe
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at 65°C for 16 h. Following incubation, the blot was
washed two times with 2 9 SSC, 0.5% of (w/v) of SDS for
5 min and two times with 2 9 SSC, 0.1% of (w/v) of SDS
for 5 min at 65°C.
Expression analysis
Total RNA was isolated from flower, leaf, and different
stages of flower using TRIzol reagent (Manufacturer’s
method). Five microgram of total RNA was converted to
single stranded cDNA. PCR was performed in a 20 ll total
volume containing 1 ll (25 ng) of cDNA, 10 pm of each
primer, 1 unit of taq polymerase, 500 mM KCl, 100 mM
Tris–HCl (pH 9), 1% Triton X-100, 15 mM MgCl2 and
2.5 mM dNTPs. The PCR amplification was carried out in
a 25 cycle setup (94°C, 5 min; followed by cyles of 94°C,
30 s; 62°C, 45 s; 72°C, 1 min; followed by 72°C,
5 min).The primers for OsPRP3 were 50 -CTT GCT GGT
GAA CGT GCT CGC CGT TG-30 (forward) and 50 -CCA
TTG CTT AAT TCG CCG GAG G-30 (reverse). The
constitutively expressed OsActin gene was used as normalization control. Primers for cold inducible genes; Lip5
50 -GAA GAC GAG CAC AAG AAG GAG-30 (forward)
and 50 -TAT TAC AAG GCA CCG TGC AG-30 (reverse);
Lip9 50 -CTC CTG CTC CCG TGG TGA C-30 (forward)
and 50 -GTA CCC CAC ACG AAA CAC AAA C-30
(reverse); COR413 50 -CTG GTG GGC TGT TCT CTC
TG-30 (forward) and 50 -CAT CAG GAG GCA GGA GGT
C-30 (reverse); Mapk2 50 -GAT GCT CAC CTT CAA CCC
GCT G-30 (forward) and 50 -CAA TGT TCA GTC TAC
CCG GCT CTC-30 (reverse); CDPK7 50 -CTG GAG CGA
GAG GAA CAT CTT G-30 (forward) and 50 -CAT CCG
CGG ACA TCT GAC AAC-30 (reverse).
Construct and transformation analysis
A binary vector, pGA1611, was used for over expression
construct. The full cDNA clone of OsPRP3 was inserted in
between the SacI and KpnI site of pGA1611 in the sense
orientation. pANDA vector was used for RNAi mediated
gene silencing. A 300-bp OsPRP3 fragment was used to
make the RNAi construct following the procedure as previously described (Miki and Shimamoto 2004). A japonica
rice variety, Dongjin, was used for transformation by the
Agrobacterium co-cultivation method (Jeon et al. 1999).
Agrobacterium tumefaciens LBA4404, containing Ach5
chromosomal background and a disarmed helper-Ti plasmid pAL4404 was used for rice transformation (Hoekema
et al. 1983). Calli were induced from mature seeds on N6
medium. Agrobacterium-mediated transformation of rice
callus was performed according to the method of Lee et al.
(1999). Transformed calli were selected by hygromycin
resistance, and the transgenic plants were regenerated from
Plant Mol Biol (2010) 72:125–135
the transformed calli. Regenerated transgenic rice plants
were grown in a greenhouse.
Immuno-blot analysis
Leaves and flowers were collected from wild type and
transgenic plants. Total proteins were isolated and concentration of the protein samples were determined by the
Bio-Rad DC protein assay. About 30 lg of each protein
sample were separated on 10% SDS–polyacrylamide gel,
and transferred to polyvinylidene difluoride (PVDF)
membrane. The membrane was incubated with OsPRP3
rabbit polyclonal antiserum (diluted 1:10,000) for 2 h in
TBS buffer, containing 0.5% BSA [Primary antibody
against OsPRP3 was produced from rabbit by injecting a
synthetic oligopeptide derived from the OsPRP3 sequence
(TPAYSHPTPVYKQPLPT)]. After two rinses in TBST
(TBS ? 0.5% Tween 20), the membrane was incubated
with anti-rabbit IgG-conjugated with horseradish peroxidase (diluted 1:10,000) for 1 h in TBS buffer containing
0.5% BSA. After two rinses in the TBST, OsPRP3 was
visualized on the blot using the Alkaline Phosphate conjugate substrate kit (Bio-Rad).
Cold tolerance, proline content and real time
quantitative RT PCR analysis
To assay cold tolerance, young and mature transgenic and
wild type plants were grown in a cold room (set to 4°C)
under 10 h day/14 h night cycle. Leaf samples were collected from cold treated and control plants and used for
proline content and expression analysis. Proline content was
determined by the method of Bates et al. (1973) using acid
ninhydrin reagent and acetic acid. Samples were incubated
in a boiling water bath for 1 h, cooled at room temperature
and mixed with 1 ml toluene. After phase separation, the
absorbance of the organic phase was measured at 520 nm
and compared with standard curve generated with L-Pro.
Amounts of proline were expressed as lmol g-1 initial
fresh weight. Real-time quantitative RT-PCR analysis was
performed on a Bio-Rad iCycler using SYBR green method.
Thermal cycling conditions consisted of 10 min at 95°C and
then 40 cycles of 30 s at 95°C, 45 s at 58°C and 45 s at
72°C. The primers for OsPRP3 were 50 -GAG GAG AAG
AAG GTG GCG ATG-30 (forward) and 50 -CAG GCA GGC
AAC AGA CCA AG-30 (reverse). OsPRP3 expression was
normalized to Actin expression according to the following
formula: 2DCt ; DCt ¼ CtOsPRP3 CtActin :
Cytological analysis
Rice samples from wild type, overexpression and RNAi
plants were fixed in a solution containing 2.5%
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glutaraldehyde, 2% paraformaldehyde, and 0.1 M Phosphate buffer (PBS) (pH 7.4) overnight at 4°C. The samples
were then rinsed in 0.1 M PBS (pH 7.4) and further fixed in
1% (w/v) osmium tetroxide (OsO4) at 4°C overnight. After
rinsing again in PBS buffer, the samples were dehydrated
with an ethanol series and embedded in acrylic resin. The
resin-embedded flower samples were sliced into 1-lm
sections with an ultra-microtome (LKB, Bromma 2088)
and stained with 0.5% toluidine blue containing 0.1%
sodium carbonate. The tissue sections were then observed
under a light microscope (Zeiss). For electron microscopy,
thin sections (40–50 nm thickness) of rice leaves were
prepared with an ultra-microtome (LKB, Bromma 2088)
and were collected on nickel grids (1-GN, 150 mesh).
These sections were then stained with uranyl acetate and
lead citrate and examined under a transmission electron
microscope (JEM—100CX-1). For immunolocalization,
thin sections prepared as described above were etched with
10% hydrogen peroxide for 30 min, rinsed in a deionized
water, and then incubated in 0.56 mM sodium meta periodate. The sections were incubated in a PBS buffer containing 1% BSA for 1 h, followed directly by incubation
overnight in the PBS buffer containing the primary antibody diluted to 1:100 at 4°C. The sections were rinsed
several times with PBS-BSA and continued to incubate in
the PBS buffer containing an anti-rabbit IgG conjugated to
20 nm gold particle for 1 h. The sections were then stained
and examined as described above. Preimmune serum was
used as a negative control. Tetrazolium staining was done
by using a solution containing a 1% (w/v) aqueous solution
of 2,3,5-triphenyltetrazolium chloride in 50% sucrose at
28°C in darkness for 1 h.
Results
Molecular characterization of OsPRP3
OsPRP3 is a cDNA clone obtained from a rice anther
cDNA library (Choi et al. 2000). Sequence analysis
revealed that the clone contained an open reading frame of
271 amino acid residues with a predicted molecular mass
of 28.8 kDa and encoded a protein homologous to a series
of proline rich protein from plants. BLAST search indicated that OsPRP3 had 48% identity to both OsPRP1 and
OsPRP2, 36% identity to NgGPP1 of Nicotiana glauca and
32% identity to AtPRP2 of Arabidopsis thaliana (Supplementary Fig. 1A). Signal P analysis revealed that OsPRP3
contained a signal peptide of 25 amino acid residues and a
putative cleavage site at Ala26. We found that there were
ten proline residues conserved with both monocot and dicot
PRPs. Previously, among the two rice PRP genes, OsPRP1
and OsPRP2, OsPRP1 was analyzed in a detailed manner
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(Wang et al. 2006). The report described that the OsPRP1
formed a gene family of four members and these four genes
were tandemly organized within a 20 kb range in the
chromosome 10. Our investigation in a rice genome database revealed that the OsPRP3 also lies in the same
chromosome (Supplementary Fig. 2A). Phylogentic analysis, however, showed that the gene aligned apart from the
two rice PRPs (Supplementary Fig. 1B). The results suggested that the gene contained an evolutionary divergence.
DNA blot analysis was done to investigate organization of
OsPRP3 in the rice genome and the blot hybridization
using a gene-specific probe revealed that OsPRP3 exists as
a single copy gene in the rice genome (Supplementary
Fig. 2B). In our DNA blot hybridization probed with a full
length of OsPRP3 cDNA clone at a high stringent condition, however, two or three hybridized bands were detected
(data not shown), indicating that the gene belongs to a
small gene family. Expression analysis was done by RTPCR (Fig. 1a, b). The result revealed that OsPRP3 transcript was accumulated in flower and not in leaf (Fig. 1a).
RT-PCR analysis with the rice flowers at different developmental stages showed that OsPRP3 was highly expressed in mature flower (Fig. 1b). These results suggested that
OsPRP3 expression was not only spatially but also temporally regulated.
Generation of overexpression and knockout mutant
plants
To study function of OsPRP3 gene in rice, we constructed
and transformed a binary vector containing a full clone of
OsPRP3 in sense orientation and also an RNAi vector to
achieve overexpression and knockout of the gene, respectively (Supplementary Fig. 3A). After transformation, we
examined expression levels of OsPRP3 gene in a total of
nineteen independent transgenic lines to check over-
Fig. 1 Expression analysis of OsPRP3. a Expression of OsPRP3 in
rice flower and leaf. The result indicated that the gene is expressed
only in the flower, b expression of OsPRP3 at the different
developmental stages of flower 1 young flower; 2 immature flower;
3 mature flower. OsActin, rice actin used as a positive control
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expression and knockout of the gene by RT-PCR (Fig. 2a,
b). The result indicated that leaves from the transgenic plants
carrying overexpression of OsPRP3 appeared to accumulate
the transcript at higher level (Fig. 2a) whereas flowers from
RNAi plants showed either suppression or a complete
knockout of the gene transcription (Fig. 2b). We also analysed the OsPRP1 and OsPRP2 mRNA levels in the RNAi
flowers, the result showed that those mRNA levels were not
reduced in the RNAi plants (data not shown). In addition, we
also performed immuno-blotting with the total protein
extracts. To do this, a polyclonal antibody was raised to
recognize the OsPRP3 protein and we found that the antibody detected a protein band of approximately 28 kD,
demonstrating its reliability. Our immunoblot assay confirmed that the OsPRP3 protein was expressed in the leaf of
the overexpression plant whereas the protein was absent in
the flower of the knockout plants (Fig. 2c). Taken together,
the result indicated that the transformation strategy was
successfully achieved. Also, the immunoblot study suggested that the OsPRP3 was flower-specific (Fig. 2c).
Moreover, our immunolocalization showed that OsPRP3
proteins were localized in the cell wall of transgenic plant
leaf (Fig. 3a). The result was consistent with our sequence
analysis in which OsPRP3 protein showed common features
with cell wall proteins (Supplementary Fig. 1A). This suggests that OsPRP3 is a cell wall protein of rice flower.
Cold-tolerance conferred by overexpression of OsPRP3
In our observation with the overexpression plants grown
under normal conditions, we found that their growth was
perfectly normal as wild-type showed. However, when the
transgenic plants were transferred to a growth chamber in
which the growing temperature was set to 4°C, the transformants showed a significantly increased cold-tolerance
than the wild-type plant (Fig. 4). In the first week of cold
stress, the wild-type plant leaf became curling and then the
leaf showed wilting symptoms in the second week (Fig. 4a).
In 4 weeks of cold stress, the wild-type plant displayed a
severe freeze-induced dehydration which eventually leaded
to plant death, whereas the transgenic plants were consistently resistant to the cold-stress and survived at such low
temperature (Fig. 4a). RNAi plants were exposed to cold
treatment; the result was similar to that of wild type plants
(Supplementary Fig. 4). To gain insight into the effect of
OsPRP3 overexpression in conferring cold-tolerance, we
measured expression levels of the cold-regulated genes such
as CDPK7, MAPK2, COR413, LIP5 and LIP9 (Saijo et al.
2000; Xiong and Yang 2003; Lee et al. 2004; Aguan et al.
1991) in the overexpression plant (Fig. 4b). Our RT-PCR
analysis, however, showed that increased OsPRP3 did not
alter transcript levels of these cold inducible genes. The
result suggests that the cold tolerance conferred by
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Fig. 2 OsPRP3 expression analysis in transgenic rice a accumulation
of OsPRP3 mRNA in the leaves of overexpression transgenic plants.
1–9 Individual overexpressed transgenic plants; WT, wild type leaf.
OsActin, rice actin gene used as a control, b transcript accumulation
of OsPRP3 in the flowers of RNAi transgenic plants. The Gus linker
indicates RT-PCR products of the gus linker region, indicative of the
expression of the trigger dsRNA. 1–10, Individual RNAi transgenic
plants; WT, wild type flower. OsActin, rice actin gene used as a
control, c immunoblot analysis of OsPRP3. Top panel shows the
immunoblot probed with OsPRP3 specific antibody. Bottom panel
shows the corresponding SDS–PAGE gel (silver stained). L leaf; F
flower
overexpression of the OsPRP3 gene did not involve the
pathway related to those cold-regulated genes.
We further investigated the correlation between the
cold-tolerance phenotype and the proline content of the
transgenic plant (Fig. 5). Previously, proline was reported
to be an effective cryoprotectant (Mahajan and Tuteja
2005). Furthermore, a correlation between freezing tolerance and increase in proline content during cold acclimation was also studied (Li et al. 2004; Wanner and Junttila
1999; Yelenosky 1979). In this experiment, thus, we used
6 weeks old T1 plants (after selection) in the cold-stress
assay since young plants are more chilling sensitive than
mature plants. To do this, the plants were treated continuously at 4°C growth condition and we measured expression of OsPRP3 gene and accumulation of proline in the
leaf at a time course level. As we already observed in the
mature plants, the T1 plants showed cold tolerance at the
4°C growth condition (Fig. 5c, e) whereas wild-type plants
showed freeze induced symptoms such as wilting and
dehydration within 9 days of cold treatment (Fig. 5d) and
afterward in 15 days of cold treatment the plant was
completely dehydrated (Fig. 5f). Real-time RT-PCR analysis and measurement of free proline content were effectively done to indicate the correlations with the coldtolerance of the transgenic plant. The real time RT-PCR
result revealed that OsPRP3 mRNA level in the coldtreated transgenic plants was increased constantly
(Fig. 5g). The transgenic plant also showed a continuous
increase of proline content, reaching more than four fold
within a week of cold treatment compared to the free
proline accumulation in the control plant (data not shown).
The result suggested that level of free proline accumulation
of the transgenic plants was correlated to the OsPRP3
expression. To investigate structural injuries that could
occur to the leaf tissue resulted from the chilling, we performed a cytological analysis (Fig. 6). Our microscopic
observation showed that all mesophyll cells of wild type
and RNAi plant leaves lost their cell wall integrity which
allowed solute leakage and cell lysis (Fig. 6c, d, g, h),
indicating that the cold-stress caused severe damage to the
leaf tissue and thus the plant did not survive. Whereas the
mesophyll cells of the overexpression plant well maintained their cell structure, retaining the cell wall integrity
and thus the plant survived against the freeze induced
injury (Fig. 6b, f). Taken together, we propose that the cell
wall protein, OsPRP3, confers cold tolerance by stabilizing
the cell wall integrity.
Role of OsPRP3
Detailed function of the OsPRP3 gene during flower
development was studied in the knockout mutants generated by the RNAi strategy. Looking into the overall
structure of the RNAi plant during growth and development, we did not observed any abnormal phenotype in
vegetative organs as we expected from the result of
expression analysis; i.e., the OsPRP3 gene was flowerspecific (Fig. 1a). The knockout plants, however, showed
defects in the spikelet on which floral buds appeared to be
wilty and some were shrunken and white (Fig. 7a). The
floral buds also lost their ability of opening. Staining with
tetrazolium of the anther from the mutant flower revealed
that the anther produced non-viable pollen (Fig. 7l). We
also found that many flowers showed a complete loss of
pollen production, resulting in male-sterility (Fig. 7m).
Microscopic observation of anther locule of the knockout
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cell layers (Fig. 7n–p) or a complete loss of the cell layers
except for the outermost epidermal cell layer in many cases
(Fig. 7o, q). Taken together, our data suggested that OsPRP3 plays a crucial role in determining the extracellular
matrix structure of anther, palea and lemma. No significant
alterations were observed in the overexpression transgenic
plant (Fig. 7h–j).
Discussion
Fig. 3 Immunolocalization of OsPRP3. a, b Immunolocalization of
OsPRP3 in the leaves from the overexpression transgenic and the
wild-type plants, respectively. Arrowhead indicates the OsPRP3
localized on cell wall, indicating that the OsPRP3 is a cell wall
protein. CH Chloroplast; CW cell wall, scale bar = 1 lm
mutant showed that the anther did not contain tapetum,
middle layer, and endothecium (Fig. 7q) whereas the
anthers at the same developmental stage of wild-type and
overexpression plants showed normal development of
those tissue layers (Fig. 7f, k). Our microscopic observation revealed that blocking of the OsPRP3 function affected not only the anther development but also other floral
organs, such as palea and lemma (Fig. 7n–p). Palea and
lemma of the wild-type flower contained an outermost
epidermal cell layer, three to six layers of fibrous sclerenchyma and spongy parenchyma cells, and then an
innermost cell layer (Fig. 7c–e). The knockout florets,
however, showed a severe reduction in formation of those
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PRPs are cell wall proteins in plant (Chen and Varner
1985; Tierney et al. 1988). These proteins can be classified
into five groups on the basis of motifs, domains and biochemical characters (Wang et al. 2006). The first group is
characterized by PRPs which contain tandem copies of the
pentapeptide PPVXK/T (X is often H, Y or E) as appeared
in MtPRP2, SbPRP2, (Wilson and Cooper 1994; Hong
et al. 1987, 1989). The second group is characterized by
two domain proteins, which contain a Proline-rich N-terminal domain with tandem repeats of PPYV motifs, and a
C-terminal domain that lacks Proline-rich sequences, such
as the sequence of AtPRP1/3 (Fowler et al. 1999). The
third group also consists of two domains but its N-terminal
domain is non-repetitive and its C-terminal domain contains proline-rich repeats which are often to be either PPV
or PV/IY. This group of PRPs contains the Cys-rich motif,
KKPCPP, as found in AtPRP2 and AtPRP4 (Fowler et al.
1999). The fourth group is characterized by the tandem
repeats of PEPK motifs in whole protein sequence, as
appeared in OsPRP (Akiyama and Pillai 2003) and TaPRP
(Raines et al. 1991). The fifth group of PRP characterized
by the tandem repeats of PKPE, P(V/E)PPK in the C terminal of the protein sequence, as found in OsPRP1.1-4
(Wang et al. 2006). OsPRP3 contains three PPXY (X = S,
V, I) and 2 P(V/I)YK motifs in the C terminal region
indicating that OsPRP3 belongs to the third group of PRP
(Supplementary Table 1 and Supplementary Fig 1A). Our
phylogenetic analysis also confirmed that OsPRP3 aligned
under the third group of PRPs (Supplementary Fig. 1B).
Wang et al. (2006) described that OsPRP1 formed a gene
family with tandem duplication and the duplication showed
a expression divergence in spatial specificity. Alignment of
OsPRP3 with the rice PRPs and also with PRPs from other
plant species showed a low sequence identity (Supplementary Fig. 1A). Taken together, the results suggest that
plant PRP genes are evolved in diversity. Expression
analysis of OsPRP3 gene revealed that the gene was regulated during the plant development (Fig. 1). This regulation was not surprising because PRP genes from plants
were often appeared to be regulated spatially and also
temporally. For instance, transcripts of wheat WPRP and
maize ZmPRP were detected in the growing tissues like
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Fig. 4 Phenotype and coldregulated gene expression in
transgenic and wild type plants
under 4°C cold stress. a
Phenotype of the Ubi:OsPRP3
overexpression transgenic and
wild type plants grown at 4°C.
Overexpression transgenic plant
was more tolerant to the cold
stress than the wild type plant,
b expression analysis of cold
regulated genes in rice leaves
after 0, 1, 2, 3, 7 and 14 days
under 4°C cold-stress. WT wildtype; OsPRP3, OsPRP3
overexpression transgenic plant.
A constitutive expression of
OsPRP3 in the transgenic rice
leaf was shown whereas the
gene transcript was hardly
detected in wild-type leaf,
indicating that the transgene
was over-expressed in the
transgenic plant
Fig. 5 Cold-stress assay. a, c,
e, Overexpression plant (T1),
b, d, f, wild-type plant as
control. 9 and 15 days after cold
treatment were shown in the
pictures, g real-time quantitative
RT-PCR analysis of OsPRP3 in
4°C cold treated leaves
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Fig. 6 Cytological analysis of
cold-treated transgenic and
wild-type leaves. a–d Cross
section of control (untreated),
cold-treated transgenic
(overexpression), cold treated
wild type and cold treated
(RNAi) leaves, respectively,
e–h magnification of mesophyll
cells from the cross sections
(a–d) of the leaves,
respectively. Arrows indicates
the mesophyll cells that lost
their cell wall in the wild-type
plant with cold-induced injury.
UE upper epidermis; LE lower
epidermis; VB vascular bundle;
MC mesophyll cells; CP
chloroplast. Scale bar = 30 lm
root and meristem (Raines et al. 1991) and in the xylem
and epidermis (Vignols et al. 1999), respectively. This
suggests that PRPs are encoded by a number of different
genes whose mRNAs are accumulated preferentially in
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distinct plant tissues and thereby the proteins play a crucial
role in the tissues (Tierney et al. 1988). Arabidopsis contains four PRPs (designated AtPRP1-4) and also shows
different expression patterns (Fowler et al. 1999).
Plant Mol Biol (2010) 72:125–135
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Fig. 7 Characterization of OsPRP3 RNAi transgenic plants. a
Spikelet of RNAi plant. Inset box shows magnification of an
abnormal flower appeared in the spikelet. Microscopic observation
of wild type (b–f), OsPRP3 overexpression (h–k), and knockout (m–q)
flowers. le lemma; pa palea; ep epidermis; sl sclerenchyma layer; ie
inner epidermis; ms microspore; t tapetum; a anther; vb vascular
bundle. c, h, p Magnification of the interlocking of lemma and palea.
d, j ,o Palea histology showing different cell layers. The knockout
plant showed a severe reduction in sclerenchyma layer and inner
epidermis. f, k, q anther locule at micropsore stage of anther
development. The knockout plant showed a severe abnormality in
anther development. Tetrazolium staining of anther from wild type (g)
and knockout (l) plants. Scale bar = 20 lm
Expression divergence in spatial specificity of PRPs has
been studied in rice (Wang et al. 2006). Our study showed
that transcript of OsPRP3 is accumulated most abundantly
during the late stage of flower development, suggesting that
it may contribute to the cell wall integrity during the flower
maturation.
In order to elucidate function of the OsPRP3, we
transformed the binary constructs to bring either overexpression or knockout of the gene in rice (Supplementary
Fig. 3A). After transformation, we confirmed that the
protein was successively synthesized in the leaf of the
overexpression plant whereas the protein was absent in the
flower of the knockout plant (Fig. 2c). From our cold
treatment study, we found that the overexpression plant
tolerated to cold stress whereas the wild-type plant showed
chilling sensitivity (Figs. 4, 5, 6). It has been reported that
chilling sensitive plants characteristically exhibit structural
injuries and may suffer from metabolic dysfunction when
chilled (Kacperska 1999). Chilling ultimately results in loss
of membrane integrity, which leads to solute leakage.
Integrity of intracellular organelles is also disrupted leading to the loss of compartmentalization, reduction and
impairing of photosynthesis, protein assembly and general
metabolic processes. The primary function of cold acclimation is to stabilize the integrity of cellular membranes
against freezing induced injury (Mahajan and Tuteja 2005).
Cold acclimation also results in enhancement of the antioxidative mechanisms and increased cellular sugar levels
as well as accumulation of cryoprotectants (Xin and
Browse 2000). All these modifications help the plant to
withstand and surpass the severe dehydration associated
with cold stress. In our primary observation using T0
mature plants shown in the Fig. 5, we found that the leaf of
the transgenic plants expresses the OsPRP3 in the leaves
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and the expression was increased during the cold treatment
(Fig. 4b) and also the transgenic plants accumulated proline at a high level (data not shown). Based on this, we
measured the amount of OsPRP3 and free proline content
in the T1 plants during cold treatment. Expression of OsPRP3 in the overexpression plant is by Ubiquitin promoter.
Whereas increased expression of OsPRP3 under cold stress
is also because of ubiquitin promoter. Recently Perales
et al. (2008) elucidated that ubiquitin promoter responds to
various environmental stresses by enhancing the expression
of any gene under its control. Proline rich protein/glycoproteins are thought to play an integral role in extracellular
matrix structure of many plant cells that adds mechanical
strength to the cell wall and assists in proper wall assembly
(Vaner and Lin 1989). Free proline is known to be one of
the compatible osmolytes preventing dehydration in
response to freezing and drought stress (Delauney and
Verma 1993). Increase in proline content occurs in many
plant species during cold acclimation (Koster and Lynch
1992). Proline is also known to protect membranes and
proteins against the adverse effects of temperature
extremes (Paleg et al. 1984; Rudolph et al. 1986; Santarius
1992). Rapid catabolism of proline upon relief of stress
may provide reducing equivalents that support mitochondrial oxidative phosphorylation and the generation of ATP
for recovery from stress and repair of stress-induced
damage (Hare and Cress 1997; Hare et al. 1998). All of
those reports support that proline plays an important role in
plant cold-tolerance. Furthermore, our cytological analysis
of the leaf tissue from the transgenic plant revealed that the
leaf cells maintained their cell wall integrity against the
chilling induced injury (Fig. 6e). It is well reported that
PRP proteins accumulated in response to wounding,
infection and other stresses (Sheng et al. 1991; Showalter
1993; Cassab 1998; Bernhardt and Tierney 2000). Their
accumulation during theses processes has been associated
with a possible role as structural proteins within the
extracellular matrix to add mechanical strength to the wall
and assist in proper wall assembly. It is also proposed that
these kinds of proteins are secreted into the wall, where
eventually they become insolublised in response to a
hydrogen peroxide burst elicited by environmental signals
(Marshall et al. 1999; Somerville et al. 2004). Further the
insolubilization of the PRPs may lead to the formation of
protein/protein or protein/carbohydrate linkages within the
cell wall and contributes to the stability of the extracellular
matrix (Fowler et al. 1999). Therefore, it is possible that
the overexpression of OsPRP3 is related to remodeling of
plant cell wall components during cold stress and indirectly
confers cold tolerance in the rice plant.
Knockout of OsPRP3 gene showed a severe disruption
of the cell walls in the floral organs (Fig. 7m–q). The
extracellular matrix has a central role both in plant
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Plant Mol Biol (2010) 72:125–135
development and in the interactions with pathogenic
microorganisms. Although the matrix is mainly composed
of polysaccharide, the less abundant proteins, plays a crucial role in differentiation of different tissues (Knox 1995;
Davis et al. 1997). Moreover proline rich protein/glycoproteins are thought to play an integral role in extracellular
matrix structure of many plant cells and the structural cell
wall proteins form an independent structure-determining
network within the extracellular matrix that adds to the
mechanical strength of the wall and assists in proper wall
assembly (Vaner and Lin 1989). Our microscopic observation revealed that the knockout mutant displayed loss of
cell layers in the palea, lemma, and anther due to the cell
wall collapse (Fig. 7n, q). Taken together, our study suggested that loss of OsPRP3 function resulted in the failure
of determining cellular structure during the floral organ
formation in rice.
In summary, we report a novel PRP gene from rice. Our
study strongly suggests that the gene is involved in cell
wall assembly during flower maturation in rice and accumulation of this protein in leaf during cold stress conferred
cold tolerance in rice. Characterization of the biochemical
properties of the PRP protein should be done to gain more
insight into the diverse physiological role in the cell wall
assembly.
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