Download Ashraf M, Akram NM. 2009- Improving salinity tolerance of plants

Screening for Salt Tolerance of Some Romanian Local Vegetable Landraces for
Conservation of Genetic Potential and Biodiversity
Part I - METHODS AND THEORETICAL APPROACHES
Radu-Liviu Sumalan1*, Brigitta Schmid, Giancarla Velicevici and Carmen Beinsan,
Dept. of Plant Physiology, Banat’s University of Agricultural Sciences and Veterinary
Medicine ”King Michael I st of Romania” from Timisoara, Calea Aradului 119, 300645,
Timisoara, Romania
Correspondence to: [email protected]
Earth is a saline planet. 71% of its surface is covered by salt water, and in addition,
circa 6% of its total area and 20% of the irrigated land is affected by salinity, which means
more than 800 millions hectares of land are affected by salinity worldwide (FAO. 2008. FAO
Land and Plant Nutrition Service Management. http://www.fao.org/ag/agl/agll/spush).
Either in Europe the situation is not better, where are important areas with saline
soils, as in the hollow of Caspian Sea, Ukraine, Carpathian Basin, Pannonian Plain and
Iberian Peninsula. In European Union soil, salinity affects around 1 million hectares and is
the main reason of desertification. Romania is one of the European countries with vast areas
of low productivity soils containing toxic salts, being frequently associated with poverty. The
problems determined by salinity are associated mostly with other abiotic stress factors, as
drought and phosphorus deficit. Quality improvement of these soils by using amendments
associated with irrigation and drainage needs much too high investments for semisubsistence agriculture, specific to Romanian rural areas. Although, there are viable
solutions, accessible to farmers, as identification of valuable local germplasm and its
breeding, which can be realized due to technological evolution and improvement of modern
biochemical, physiological and molecular analytical instruments, enabling to understand and
use the genetic basis of cultivated species tolerant to salinity. The role of our researches
would be the collection, identification and (biochemical, physiological and molecular)
characterization of salt-tolerant landraces from the saline areas from Northern and Western
Romania, from species of vegetables with still have local genetic resources in culture: tomato
(Solanum lycopersicum L.), bean (Phaseolus vulgaris L.), onion (Allium cepa L.) and garlic
(Allium sativum L.).
Scientific background
In the last 100 year, European agriculture suffered significant changes. At the
beginning of the XXth century, agriculture was based mainly on traditional cultural systems,
the main part of inputs and products (including seeds) deriving from the farm itself. In the
present, most of the traditional cultivars were changed with commercial breeds and hybrids,
with high productivity and genetic uniformity.
It is difficult to measure exactly the level of losses produced by the reduction of
genetic biodiversity of plant cultures, associated with the loss of some valuable landraces,
but it is well-known that both on global and European level, there were massive losses of old
breeds and landraces, which had a negative impact on genetic variability.
This erosion of agricultural biodiversity’s resources, which can represent a danger for
future’s food safety and security, was acknowledged in a series of international legal
documents, such as the Convention for biological diversity and the International Treaty for
the Plant Genetic Resources for food and agriculture. By joining these treaties, Romania is
obliged to take all the measures for the protection and conservation of own plant genetic
resources for use as food and agriculture (http://www.fao.org/ag//CGRFA/itpgr.htm).
Starting with the objectives of treaty, the scope of our researches is the identification,
characterization, conservation and sustainable use of salt tolerant vegetal genetical
resources for some horticultural species, for use as food and agriculture, based on complex
biochemical, physiological and molecular biology methods.
In the twentieth century, the green revolution resulted in plant varieties with improved
productivity under favorable conditions. However, the progress in producing crop varieties
with acceptable yield under saline conditions is still limited, perhaps due to the lack of genetic
variation in the gene pools of crop plants (Ashraf and Akram 2009) and the fact that salt
tolerance is a complex trait governed by large numbers of genes and its inheritance modes
are difficult to expect (Flowers and Flowers 2005). Over the past two decades, many studies
(hundreds) have reported the improvement of salt tolerance of glycophytic crop (and noncrop) plants by overexpressing single genes. The vast majority of those did not provide
quantitative measurements of growth of the transformants under salt stress (Flowers 2004)
but rather provided metabolic changes that are supposed to contribute to salt tolerance such
as accumulation of osmolytes, activities of antioxidant enzymes, etc. Furthermore, in most
cases the salt tolerance of transformants has been tested in the lab by cultivating the plants
in different concentrations of salt rather than in the field, where many environmental
conditions such as light intensity and spectral composition, temperature, wind speed, water
availability, etc interact to intensify salt stress (He et al. 2005). It is not unfair to conclude that
in most published reports, an increase in salt tolerance of the transformants has been
marginal. Thus, such overexpression studies have provided insights into the metabolic
functions of the overexpressed genes but the field performance of the transformants is yet to
be verified.
The main objective of our research is the identification of salt tolerant local landraces
of tomato, bean, onion and garlic, for utilization in breeding programs and as seeds for
farmers.This objective will be accomplished by following a few steps: biochemical,
physiological, genetic and molecular characterization of collected genotypes; Conservation
with conventional and non-conventional methods of valuable genotypes, for present and
future utilization; creation of an on-line database with researches of tested genotypes and
dissemination of results and, very important, initiation of breeding programs for obtaining new
genotypes, with tolerance to salinity and stable production (http://www.s-stress.ro/).
State of the art: The yield capacity of cultivated plant species can be totally exploited
only with a consortium of favorable environmental conditions, stable through the entire
growing period. Negative environmental conditions act as stress factors and disorders the
normal life processes, producing metabolic, functional and structural modifications, which
alters the bioproductive capacity of plants (Flowers and Yeo, 1995).
The transformation of soils into saline ones is a frequent phenomenon in our country
and also worldwide, due to the accumulation of highly soluble salts at the surface or in the
soil profile. The area covered by saline soils in Romania is 614 thousand ha, which
represents 2,7% of the total area, 4,2% of agricultural land and 6,6 % of arable area
(Serbanescu I. Cited by Cozma Catalina 2006).
Generally, due to the natural conditions, in Romania saline soils are positioned in
lowlands, in depressions with reduced natural drainage. High levels of precipitations over a
short period of time, as well as irrigation caused the increase of ground water level. Thus the
risk of soil salinization appears mostly where ground water is near to surface and in drought
conditions (http://www.icpa.ro/proiecte/ramsol/51-031_raport_4.pdf)
The sampling and analyzed area for soils and local landraces adapted for saline
conditions will cover 5 from the 10 highly affected counties, such as Timis, Arad, Bihor, Iași
and Botosani, those situated in the Northern and Western Romania, with a total area of
saline soils of 224.000 ha.
In most of the saline soils the main stress factor is represented by the sodium ions,
which through ion change processes can replace the potassium, calcium and magnesium
ions from soil’s colloidal complex, blocking their absorption by the plants’ root system
(Sumalan, 2009). Thus, the germination of seeds and growing of the root system are blocked,
with a negative impact on plantlets at even lower concentrations toxic to mature plants.
Generally, the term for salinity includes all the problems caused by the presence of
salt in soil, but strictly these soils are divided into two categories: sodic (or alkaline) alkaline
soils, but some consider the third type, the saline-alkaline soils (Singh et al., 2001).
The intensity that the salt affected soils were and is still unknown, but is estimated to
340 to 1200x106 ha. In spite of many arid areas or with very low productivity, there are some
examples where tolerant cultivars developed. The reason is the complex treat and its
complicated components, which are probably controlled by poli-genes (Gu et al., 2000).
Though the salt tolerance in cultivated species is low, there are some
genotypes which present an acceptable level of adaptability to moderate saline conditions. In
legumes, these genotypes are represented especially by local landraces breeded over the
time by local farmers form areas with saline soils. Identification of these cultivars represents
a priority in breeding programs of legumes all over the world. The better we understand the
response of plants to stress factors, the better we will manage the natural and anthropic
ecosystems (Mandre, 2002).
Though in case of Solanum lycopersicum L. there are genotypes with relatively high
tolerance to salinity, the studies demonstrated that is difficult to improve an elite line with
genes from wild species with would offer salt tolerance, due to the high number of genes
implied in this process, most of which have low effects and high costs for the recovery of
gene pool of the receptor cultivar. The conventional breeding programs for the improvement
of salt tolerance in elite genotypes using wild species as donors are inefficient in the
selection strategies (Cuartero et al., 2006).
SALINITY TOLERANCE ISSUES
The accumulation of high salt concentrations in soil can have multiple causes:
-irrigation with water rich in salts that determines the accumulation of carbonates,
especially those of calcium and magnesium,
-soils are rich in NaCl in areas with marine geological history or where sea salts have
been deposited for a very long time, and where the precipitations are low and the salts
cannot infiltrate
-besides NaCl, saline soils frequently have high concentrations of acids, like boric
acid, with an unknown absorption, transport and toxicity mechanism.
All of the salts affect the growth of plants, but they don’t inhibit this process. The salts
don’t react alone in soil, but in synergism with other nutritional elements and with the
multitude of biotic and abiotic components. Also the interaction effect manifests in the inside
of the plant, too, some of these being simple (like interaction between Na+ and Ca2+),
meanwhile some are complex (for example, the carbonates and their effect on growth).
Symptoms. Saline solutions exert both ionic and osmotic stress in plants. This stress
can be observed at different levels. In plants sensitive to salinity, the shoots and the growth
of roots is permanently inhibited during stress periods, and this effect doesn’t seem to
depend on Na+ concentrations in growing tissues, but mostly are a response to osmolarity of
external solution (Munns et al., 2000; Munns, 2002). The injury specific to Na+ is associated
with the accumulation of Na ions in leaf tissues. The symptoms are necrosis of senescent
leaves, shoots and buds from the top to the leaf till its bottom. The reduction of growing
intensity and productivity appears as a result to short life period of individual leaves, clearly
reducing the productivity of the cultures (Munns, 1993, 2002). The period until the
manifestation of the injuries specific to Na+ depends on the rate of its accumulation in leaves
and on the efficiency of Na+ compartmentalization inside of leaf tissues and cells. These
effects specific to Na+ are overlapped with the NaCl’s osmotic effects and presents a higher
variation the species level than the osmotic effects (Munns, 2002).
Main bases of na+ toxicity and salinity effects
In shoots, the high concentrations of Na+ can cause a series of osmotic and
metabolic problems for plants. The leaves are more vulnerable to Na+ than roots, simply
because Na+ and Cl- are accumulated in higher concentrations in shoots than in roots. The
roots tend to maintain constant the level of NaCl in time, and they can regulate the level of
salts by exporting then into the soil or shoots. Na+ is transported inside the roots by quick
transport in xylem, due to intensification of transpiration, but it can turn back to the roots via
phloem. The proof regarding the extent recirculation of Na+ from shoots to roots are few,
suggesting that Na+ transport is basically in one direction and results from the progressive
accumulation of Na+ in the older leaves.
Figure 1. Na+ fluxes in plant cells (Pardo and Quintero 2002)
Sodium ions penetrate the root cells using the HKT proteins, which are independent
of the cation channels, some of them (labeled CNGC) are inactivated by cyclic nucleotides
(cAMP and GMPc) (fig.1). Though there is no direct experimental evidence of this fact, the
ion transport or selectivity of HKT protein could be regulated by a process of Ca2+
independently to SOS3 for preventing the excessive absorption of Na+. SOS3 associated
with SOS2 kinase protein, positively regulates the activity of ceroplastic membrane’s activity
of antiporter SOS1 Na+ / H+, which mediates the elimination of Na+ and eventually, the longrange distance of Na+ from roots to shoots. HAK is a K+ / H+ simporter, which can transport
Na+ with low affinity. Na+ ions are compartmentalized in vacuole membranes (Pardo and
Quintero, 2002).
Metabolic toxicity of Na+ is mostly the result of its ability to compete with K+, with a
compulsory essential position for the cell functioning. More than 50 enzymes are activated by
K+, and Na+ cannot substitute its role (Bhandal and Malik, 1988). Thus, the high level of Na+,
or of the Na+:K+ ratio can break many of cytoplasmic enzymatic processes. The block of
protein synthesis due to the growing of Na+ concentration can be an important cause for the
injuries produces by the excess of Na+ ions.
The osmotic injuries can be a result of the increasing Na+ concentrations in the leaf
apoplast, the main transport being through the xylem. This mechanism of Na+ toxicity was
proposed for the first time by Öertli (1968), and the direct proof was supported by the
measurements and microanalysis with X-rays of Na+ concentrations in the apoplast of rice
leaves (Flowers et al., 1991). These authors calculated that the level of saline stress in the
leaf apoplast was moderated, of circa 600mM.
The cellular toxicity of Na+ causes another type of osmotic problems. The plants have
to maintain a low water potential compared to soil for keeping the turgescence and the water
absorption capacity for growing. This needs an osmotic increase, by absorbing water from
the soil solution or by synthesizing metabolically active solutions. This component of salinity
represents a dilemma for plants: the most insignificant solution in saline soils are those of
Na+ and Cl2-, but these are toxic in the cytosol.
Physiological bases of plant tolerance to Na+. The physiological studies of salt
tolerance had taken a benefit of knowledge in molecular and cellular techniques, but
experimental methods can be optimized by improving the growing and handling methods of
plants. For example, the determinations regarding the immediate reaction of plants after a
major and rapid change in salinity are not recommended to mark out the physiological
reactions regarding plant growing in saline conditions, where the rhythm of modifications is
relatively slow.
Furthermore, is the subject of the study is Na+ specific toxicity, then it must be
outlined that there is a difference between the specific effects of osmotic stress components.
For example, the plants grown in very low transpiration conditions, like in vitro conditions,
could be more sensitive to the osmotic component of salinity (circulation of Na+ in shoots will
be lower). Also, determination of radicular elongation intensity doesn’t represent an important
index in the evaluation of Na+ specific toxicity, because it is not correlated to Na+ transport in
shoots (Munns, 2002).
It is proven that there is no link between the accumulation of Na+ and sensitivity to
salt in Arabidopsis thaliana L. Mutations which determine the increased sensitivity to saline
excess, most of the time don’t determine the intensification of Na+ accumulation. More, even
if the addition of Ca2+ reduces the Na+ influx and accumulation in Arabidopsis, it does not
diminish the inhibition of growing (Essah, 2000; Davenport and Tester, 2000). This does not
stop the utilization of this model for studying the Na+ transport, but using it as a model for the
study of salt tolerance in crop plants can be limited.
Studies regarding the physiological effects of saline stress. Improving the salt
tolerance of crop and pasture species requires new genetic diversity (either natural or
transgenic), and efficient techniques for identifying salt-tolerance. International collections
must contain a considerable genetic diversity in salinity tolerance that is not discovered or
utilized (Munns and James, 2003).The negative effect of salt excess on plants manifests
mainly in two directions, osmotic and toxic. The osmotic effect has a direct consequence the
limitation of the provision with water and tissue dehydration. Physiological drought appears
just when the external solution has compounds that cannot penetrate the cells and create a
high osmotic pressure.
The inhibition of growing in above-ground organs, with the growing of radicular
system is considered an important morphological adaptation to saline or hydric stress
(Creelman et al., 1990). Plants grown in saline stress conditions during their vegetative
period remain short, with thin leaves which presents small cells, with a high content of lignin
in cell walls and a high number of stomata of leaf area unit.
Salinity starts specific reactions in cells, like osmotic regulation, modifications of
metabolic flux, lignifications of cell walls, connected with the defense mechanisms specific to
the whole plant, as reduction of growing rate, modifications of biomass phenology, leaf
senescence, and finally, plant death (Munns, 2005).
As a result, the tolerance to salinity can be improved by avoiding or delaying the
senescence, permitting to plants the accumulation of resources on a long term, to grow and
maintain the defense mechanisms, as ion regulation.
Many researches demonstrated the capacity of senescent leaves to accumulate
important quantities of salt, for the protection of meristematic and active growing tissues
(Flowers and Yeo 1995). This fact determines the huge reduction of chlorophyll in plants
grown in saline stress (Chen and Murata, 2002). The senescence of leaves is correlated to
the increased permeability of cytoplasmic membranes at high salt concentrations.
The salinity reduces the bioproductivity of plants, first affecting the growing intensity
during the osmotic stress phase, then producing leaf senescence in the toxicity phase, when
excessive salt is accumulated in leaves (Munns et al. 2002a). Despite of the positive effect of
Na+ on vacuoles for maintaining its turgescence (Flowers, 2004), the early senescence due
to salinity appears faster in glycophille plants, which accumulate more Na+ (Fortmeier and
Schubert, 1995; Munns et al. 2002a). Anyhow, as mentioned earlier, the inhibition of growing
and metabolic modifications in osmotic faze of salinity are similar to those produces by
drought, which also conduct to early senescence (Munns et al. 2002 a,b). The senescence
determined by the osmotic component of salinity has probably the same physiological events
as that induced by drought (Pic et al., 2002). The initial reduction of shoot growing followed
by the fast senescence is probably determined by hormonal signals, produced as a response
to stress.
The action of saline stress on plants can manifest in every development phase, from
germination to fruiting. The high salt solution in soil inhibits germination and morphogenetic
of radicular system. The highest sensitivity is in young plants.
The limit of salinity tolerance is enhanced by the stopping of growing, the forming of
necrosis on leaves or the loss of leaves (Toma and Robu, 2000, cited by Nicolae, 2008).
Studies regarding the biochemical effects of saline stress. At cell level, the high
concentration of salts produces a toxic action on protoplasm, manifested in disorder of the
microscopic structure of cytoplasm. The plant tissues become succulent, soluble sugars and
mineral ions are deposited in vacuoles, enhancing the osmotic pressure of vacuolar juice,
with benefic effects on endosmosis.
Researches on biochemical and physiological mechanisms involved in
enhancing plants’ tolerance to salinity
The „salt tolerance” term implies the survival and productive growth of a plant
adapted to support a certain level of salts in the conditions when a similar genotype, which is
„sensitive to saline excess” stops growing, development and dies.
The most optimistic approaches of the future regarding plant tolerance to salinity
sometimes appeal an impossible situation when plants grow and produce fruit abundantly in
highly saline soils. It is known that there are important differentiations both morphologically
and physiologically between halophytes and most of the crop plants. At genetic level, the salt
tolerance is considered a quantitative trait (Fooland and Jones, 1993). As a result, it is
necessary to realize an equilibrum between productivity and salinity, creating some
genotypes with relatively high productivity. Plant tolerance to saline excess depends mostly
on some characters, which can be grouped as follows:
- physical processes of absorption and elimination of salts, followed by transport and
distribution in plants;
- morphological adaptation and distribution of biomass between root system and
above-ground organs, which can include the regulation of transpiration intensity by closing
and opening the stomatal osceola;
-physiological and metabolic mechanisms, which antagonize the presence of salt
excess at cellular level.
The tolerance mechanisms are part of the category that minimize the effects
generated by hyperosmolarity, ion imbalance and other side effects. In conditions of shortterm osmotic stress, the plant cell has the ability to prevent water loss, but long term
exposure affects plant development, and continuous culture in saline stress condition implies
the preservation of the osmotic stress and exclusion and/or ion transport-compartmentalizing
(Jurcoane Ştefana et al., 2006).
Salt tolerance is thus positively correlated with: capacity of osmotic regulation by
accumulating organic solutions in cytoplasm; efficient absorption o potassium ions; capacity
of accumulating sodium and chlorine ions in vacuole; capacity of eliminating sodium and
chlorine ions from shoots; capacity of synthesizing non-toxic active solutions for enzyme and
membrane protection. As a result, it can be considered that there are mechanisms actioning
at cellular level, and other at whole plant – in salt stress conditions. Researches on soil salt
tolerance of cultivated vegetable species regarded many directions, like understanding the
way how plant cells stop the accumulation of ions and synthesis of organic solutions, as an
important adaptation factor (Munns, 2002).
Other researchers linked saline stress with some deficiencies in macroelement
absorption, for example, high concentrations of NaCl determine in tomato deficiencies of
phosphorus and potassium absorption and metabolizing. As an alternative to reducing the
saline stress could be a supply of growing medium or soil solution with P and K.
Cellular dehydration begins when water potential difference is so high that it can be
compensated and results in loss of turgescence and plasmolysis of cells (Yeo and Flowers,
1986). The cellular response to loss of turgescence is osmotic balancing. Anyway, the Na
and Cl ions are efficient osmoltes, useful in osmotic regulation, being accumulated and
isolated in vacuoles for reducing their cytotoxicity. In these conditions the cellular growth is
determined by the enhancement of vacuolar volume, the accumulation of Na and Cl
facilitating the osmotic water exchanges, essential to cell growth.
The plant cells respond to osmotic stress with: ion elimination, ion export, modification
of cell wall, osmotic adjusting, osmo-protection, catching of reactive oxygen species.
Synthesis of osmoprotectant compounds. Osmoregulating compounds are
synthesized in cells or taken from the external environment, and this case they are named
osmoprotectants (proline and betaine). In cells, the osmolyte has a double role, playing an
important role in stress response, but they can be used in other ways too, like carbon and
energy source. From structural point of view, the osmoregulating and osmoprotectant
compounds represent a high diversity. From chemical point of view, they are organic
compounds with low molecular weight (in most cases), soluble in water in high
concentrations (Jurcoane Ştefana et al., 2006).
The accumulation of proline was studied for the first time at perennial raigrass and
later it was demonstrated to be one of the most common physiological answers of higher
plants to stress factors. Accumulation of proline was present in plants exposed to high
salinity, drought, heavy metals, low temperature, UV radiation, pathogens (Abraham et al.,
2010).
Proline accumulates during abiotic and biotic stress, like high salinity (Ben Hassine et
al., 2008; Yoshiba et al., 1995), drought (Ben Hassine et al., 2008; Huang and Cavalieri,
1979; Rhodes et al., 1986), UV radiations (Saradhi et al., 1995), heavy metals (Mehta and
Gaur, 1999; Schat et al.,1997; Singh et al., 2010) and oxidative stress (Yang et al., 2009).
Furthermore, it was observed that proline accumulates in plants infected by non-virulent
bacterium (Fabro et al., 2004) or agrobacterium (Haudecoeur et al., 2009), cited by
Szabados et al., 2011.
The proline’s biosynthesis pathway was well described in Escherichia coli,
Arabidopsis thaliana L., as well as in some plant and animal species. There are two
alternative ways of proline biosynthesis in higher plants: L-ornithine and L-glutamate
pathways. As in plants, both ornitine and glutamate are precursors or proline biosynthesis in
microorganisms
and
mammals
(http://www.arabidopsis.org:1555/ARA/NEWIMAGE?type=PATHWAY&object=PWY-3341).
Proline biosynthesis starts with glutamic acid, which is reduced with the formation of
γ-semialdehyde of glutamic acid. This can derive from ornithine too, by a transamination
reaction (Neamtu et al., 1995).Glutamic acid is easily converted to proline. First of all, the γcarboxyl group is reduced to aldehydes, obtaining glutamate semialdehyde. The aldehyde
reacts with the α-amino group, releasing water and forming Schiff-alkali (Δ1-pyrroline 5carboxylate).
The
second
step,
this
is
reduced
to
proline
(http://www.biology.arizona.edu/biochemistry/problem_sets/aa/proline.html#proline).
The glutamate pathway in plants differs from that in bacteria and humans; a bifunctional enzyme catalyzes the conversion of glutamate -5-semialdehyde (GSA) in a single
reaction. Many research activities were focused on the understanding of the relative
contribution of the alternative ways in increasing the proline accumulation during stress.
Regarding the proline biosynthetic mechanisms in plants grown in salt stress, the most of the
researches demonstrated that besides the fact that it is a major building block of the proteins,
L-proline plays an important role as osmotic protector in bacteria, plants and animals.
In plants, proline is synthesized (fig.2.) from glutamate in cytosol and probably, also in
chloroplasts, due to the action of delta-1-pyrrolin-5-carboxylate sintetaze (P5CS) and of P5C
reductase (P5CR). P5CS produces glutamate semialdehyde, which is unstable and is
converted into pyrroline-5-carboxylate (P5C). P5CR reduces P5C to proline, reaction which
takes place in the cytosol and also in chloroplasts (see figure below (Delauney and Verma,
1993; Hu et al., 1992; Rayapati et al., 1989; Strizhov et al., 1997; Szabados and Savoure,
2010; Szoke et al., 1992; Verbruggen et al., 1993; Yoshiba et al., 1995, cited by Szabados et
al., 2011).
Szabados explain that “another source for P5C is the pathway of mitochondrial
decomposition of arginine, which first reaction was catalyzed by arginase, which forms
ornithine and urea. In the second reaction, the ornithine-amino-transferase (OAT) produces
P5C by desamination of ornithine in mitochondria” (Szabados et al. 2011).
Figure 2. Essential role of tissue specific proline synthesis and catabolism in growth and
redox balance at low water potential (Sharma et.al. 2011, www.plantphysiol.org, copyright
American Society of Plant Biologists).
The role of this pathway in proline biosynthesis was recently questioned because the
proline level was not modified in OAT mutant (Funck et al., 2008). Though the P5C
transporters were not identified in mitochondrial or chloroplast membrane of plants, the
biochemical evidences prove that the transport o P5C from mitochondria to cytosol of human
and plant cells, which permit the reduction of P5C produced in mitochondria to proline in
cytosol (Miller et al., 2009; Yoon et al., 2004, cited by Szabados et al., 2011).
The precise role of proline accumulation still represents a highly discussed area,
especially in higher plants. For many authors, the proline accumulation represents a
beneficial function and importance for adaptation of plant cell to environmental stress
conditions. Alternatively, it was thought that proline accumulation could represent only a
symptom of more profound metabolically disorders, produces by the low level of water
activity (Jones, 1986, cited by Sumalan et al., 2007).
The amount of proline accumulated during saline stress conditions represents a
depositing component for chlorophyll formation, after removing the stress factor. This permits
the tomato genotypes to accumulate high quantities of proline in leaves, and to become
metabolically active immediately after the removal of the stress.
Most of the authors think that proline level is a valuable index for the determination of
tolerance to salinity.
As a result of high salinity levels at Vicia faba plants putrescine is accumulated. It is
missing form Helianthus and Hordeum, where is possible that the putrescine is decomposed
and ammonia is released, phenomenon that produces proline. The putrescine which appears
due to reduction of diaminoxidase activity, the cadaverine, and at the end, tetramethylputrescine, toxic for plants, can produce the plant death, after they accumulate in a certain
level in cells. It was observed that there is a considerable enhance of peroxide activity, which
is associated with reduction of catalase activity, determining the accumulation of hydrogen
peroxide in cells and tissues, also explaining the plant death when is in high concentration.
In biological systems, betaine serves as an organic osmolyte, substance synthesized
or taken from the environment by the cells for the protection against osmotic stress, drought,
high salinity or temperatures. The intracellular accumulation of betaine doesn’t perturb the
enzyme functions, protein structure or membrane integrity, permitting the cells to retain water,
protecting the organism from the effect of dehydration (http://en.wikipedia.org/wiki/Betaine).
Pro and Gly betaine are the most diverse nitrogenized osmolites, accumulated in
osmotic stress conditions in plants. Gly betaine is produced is high amounts in many plant
families, especially in Chenopodiaceae, Amaranthaceae and Gramineae. Pro betaine in
Medicago sativa L. plants is in high quantities in stems, roots and nodules only after three
weeks of exposure to saline stress (Trinchant et al. 2004).
Modern methods for increasing salt tolerance. Besides the conventional breeding
methods, the modern techniques, which imply in vitro culture of cells and tissues, were
proven as useful methods of producing biological material for practice. Some of these
methods are: protoplast fusion, utilization of molecular markers and recombinant-DNA
techniques, as well as the in vitro culture for stress factor testing and producing somaclonal
variability (Fooland and Jones, 1993; Maas and Poos, 1989).
Biochemical methods are frequently used for determination of some biochemical
product, which synthesis is correlated to breeding of stress tolerance. Some of these are:
determination of proline content, of glycine betaine or of other amino acids, of some
polysaccharides, polyols or specific proteins, or increasing the activity of some enzymes, like
peroxidases (Bartles and Nelson, 1994; Bohnert and Nelson, 1995).
The need for tolerant cultivars – worldwide the researches for overcoming the salinity
problems are based on two proposals: (i) the modification of the environment, to be proper
for crop production; (ii) or selection of cultures and/or alteration of genetic structure of plants,
thus enabling its culture in the affected areas. The first proposal implies huge engineering
and soils amendments, which supposes many resources, which are inaccessible to small
farmers. The second proposal, such as; reproduction of varieties tolerant to salinity seems a
promising option, with a smaller amount of economical and social resources. The plant’s
ability to tolerate saline stress until a certain value has a major importance for the optimal
management of resources, which is also the reason for developing crops with high tolerance
to salinity, adapted to saline stress (Singh et al., 2002).
Genetic fingerprinting for the collected genotypes and correlation with
the phenotypic and physiological traits
The differences between plants are encoded in the genetic material, the
deoxyribonucleic acid (DNA). Even if the entire genome sequence is decoded for a few
number of species such as Arabidopsis thaliana (The Arabidopsis Genome Initiative, 2000)
and rice (The Rice Genome Mapping Project, 2005), most scientists use genetic markers to
identify the specific genes located on a specific chromosome. King and Stansfield (1990)
define a genetic marker as: (a) a chromosomal landmark or allele that allows for the tracing
of a specific region of DNA; (b) a specific piece of DNA with a known position; or (c) a gene
whose phenotypic expression is usually easily discerned, used to identify an individual or a
cell that carries it, or as a probe to mark a nucleus, chromosomes, or locus.
Scientists can create genetic linkage maps based on the position of markers on
chromosomes and the distance between the markers and the specific genes, as it was
discovered that the markers and the genes they mark are located on the same chromosome
and stay together as each generation of plants is produced. These genetic maps are used in
the study of economically important traits and genes or quantitative trait loci (QTLs). In
breeding programs, these techniques are used for the introgression of desirable genes or
QTLs through marker-assisted selection (King and Stansfield, 1990).
Molecular markers usually do not have any biological effect but can be considered
constant landmarks in the genome. We can define them as identifiable DNA sequences,
found at specific locations of the genome, and transmitted by the standard laws of
inheritance from one generation to the next. The molecular markers are now widely used for
marker-trait association studies, genetic mapping, genetic diversity studies and markerassisted selection programs.
The analysis of genetic diversity and relatedness between the different landraces
collected in this project is a central task of this phase.
Traditional methods of cultivar identification frequently are based on the evaluation of
sets of morphological characteristics. Although it is usually cost-effective, morphological
assessments may have their limitations, including (1) insufficient variation among cultivars
(especially if the cultivars to be compared share a closely related pedigree), (2) subjectivity in
the analysis, (3) influence of the environment and management practice, and (4) expression
of some characters only in certain developmental stages. These considerations triggered the
exploration of alternative means of cultivar identification, including allozyme analyzes,
cytogenetics, analysis of secondary metabolites, and DNA profiling (Camlin et al., 2003)
In the RAPD technique, 10 bp oligonucleotide primers are used for a DNA sample
then PCR is applied. Random polymorphic segments with sizes from 100 to 3000 bp are
resulting. Even if this method is sensitive, fast and easily performed, RAPD markers have
limited usefulness because many closely linked markers are needed to insure reliable
comparisons among plant populations (Williams et al., 1990).
Inter-simple sequence repeat (ISSR) PCR uses the variation in the regions between
microsatellites. This method has a wide range of uses, including the characterization of
genetic relatedness among populations and genetic fingerprinting (Pradeep et al., 2002).
ISSR method is based on the amplification of DNA segments positioned at an
amplifiable distance between two identical microsatellite regions oriented in opposite
direction. To amplify inter simple sequence repeats of different sizes, ISSR uses
microsatellites as primers in a single primer PCR reaction and is targeting multiple genomic
loci.
The microsatellite used as primers for ISSRs can be di-nucleotide, trinucleotide, tetranucleotide or penta-nucleotide. ISSRs use longer primers (15–30 mers), which make
possible the use of high annealing temperatures leading to higher stringency. The primers
can be synthesized by anyone but usually show high polymorphism. Reproducibility,
dominant inheritance and homology of comigrating amplification products are the main
limitations of ISSRs. Usually ISSRs segregate mostly as dominant markers, but also codominant segregation has been reported in some cases (Sankar and Moore, 2001). tive,
2000) and rice (The Rice Genome Mapping Project, 2005), most scientists use genetic
markers to identiy the specific genes located on a specific chromosome. King and Stansfield
(1990) define a genetic marker as: (a) a chromosomal landmark or allele that allows for the
tracing of a specific region of DNA; (b) a specific piece of DNA with a known position; or (c) a
gene whose phenotypic expression is usually easily discerned, used to identify an individual
or a cell that carries it, or as a probe to mark a nucleus, chromosomes, or locus.
Scientists can create genetic linkage maps based on the position of markers on
chromosomes and the distance between the markers and the specific genes, as it was
discovered that the markers and the genes they mark are located on the same chromosome
and stay together as each generation of plants is produced. These genetic maps are used in
the study of economically important traits and genes or quantitative trait loci (QTLs). In
breeding programs, these techniques are used for the introgression of desirable genes or
QTLs through marker-assisted selection (King and Stansfield, 1990).
Molecular markers usually do not have any biological effect but can be considered
constant landmarks in the genome. We can define them as identifiable DNA sequences,
found at specific locations of the genome, and transmitted by the standard laws of
inheritance from one generation to the next. The molecular markers are now widely used for
marker-trait association studies, genetic mapping, genetic diversity studies and markerassisted selection programs.
The analysis of genetic diversity and relatedness between the different landraces
collected in this project is a central task of this phase.
Traditional methods of cultivar identification frequently are based on the evaluation of
sets of morphological characteristics. Although it is usually cost-effective, morphological
assessments may have their limitations, including (1) insufficient variation among cultivars
(especially if the cultivars to be compared share a closely related pedigree), (2) subjectivity in
the analysis, (3) influence of the environment and management practice, and (4) expression
of some characters only in certain developmental stages. These considerations triggered the
exploration of alternative means of cultivar identification, including allozyme analyzes,
cytogenetics, analysis of secondary metabolites, and DNA profiling (Camlin, 2003)
In the RAPD technique, 10 bp oligonucleotide primers are used for a DNA sample
then PCR is applied. Random polymorphic segments with sizes from 100 to 3000 bp are
resulting. Even if this method is sensitive, fast and easily performed, RAPD markers have
limited usefulness because many closely linked markers are needed to insure reliable
comparisons among plant populations (Williams et al., 1990).
Inter-simple sequence repeat (ISSR) PCR uses the variation in the regions between
microsatellites. This method has a wide range of uses, including the characterization of
genetic relatedness among populations and genetic fingerprinting (Pradeep et al., 2002).
ISSR method is based on the amplification of DNA segments positioned at an
amplifiable distance between two identical microsatellite regions oriented in opposite
direction. To amplify inter simple sequence repeats of different sizes, ISSR uses
microsatellites as primers in a single primer PCR reaction and is targeting multiple genomic
loci.
The microsatellite used as primers for ISSRs can be di-nucleotide, trinucleotide, tetranucleotide or penta-nucleotide. ISSRs use longer primers (15–30 mers), which make
possible the use of high annealing temperatures leading to higher stringency. The primers
can be synthesized by anyone but usually show high polymorphism. Reproducibility,
dominant inheritance and homology of comigrating amplification products are the main
limitations of ISSRs. Usually ISSRs segregate mostly as dominant markers, but also
codominant segregation has been reported in some cases (Sankar and Moore, 2001).
Conclusions:
Soil salinity affects important areas both at global and regional level, having direct
negative effects on food security at many countries.
Improvement actions of saline soils are long term procedures and need major
investments which are not affordable for most of the counties and farms.
Identification of crop plant genotypes with a certain level of salt stress tolerance
represents a viable, fast and relatively cheap procedure.
Old cultivars and local landraces, adapted to saline stress represents a valuable
germplasm source which can be directly used in production or in plant breeding programs.
Collection, characterization and depositing of valuable germplasm should be a priority
in plant breeding programs, mostly because of the pressure of presently having many
modern cultivars and hybrids with very high productivity but low tolerance to abiotic stress
factors.
It is necessary to optimize a unitary protocol for each crop species in order to identify
the degree of salt tolerance of different genotypes.
ACKNOWLEDGEMENT: This work was supported by a grant of the Romanian
National Authority for Scientific Research, CNDI-UEFISCDI, project number PN-II-PT-PCCA2011-3.1-0965.
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