Genes that could increase salt tolerance fall into three main functional groups: (1) those that control salt uptake and transport; (2) those that have an osmotic or protective function; and (3) those that could make a plant grow more quickly in saline soil.
1. Genes that control salt uptake and transport
This section focuses on the transport of Na+ rather than Cl−. The control of Na+ uptake is probably more difficult and energy-demanding than Cl− because plant cells have a negative electrical potential (about −180 mV). The prevention of Na+ uptake from the apoplast is more demanding in terms of ion selectivity and energy costs than the prevention of Cl− uptake: Cl− would not enter a cell passively unless the cytosolic Cl− concentration was very low (Appendix S2, available online as supplementary material). Furthermore, genetic variation in salt tolerance of the whole plant correlates with the degree to which the plant can limit the rate of Na+ transport to leaves. However, in some dicotyledonous species salinity tolerance correlates with reductions in Cl− accumulation in the leaves. For these species, Cl− uptake and transport mechanisms may be particularly relevant. Reviews by Tyerman & Skerrett (1999) and White & Broadley (2001) cover Cl− transport mechanisms.
Membrane proteins controlling Na+ transport Proteins controlling the uptake of Na+ from the soil and the subsequent transport of Na+ within the plant are embedded in the lipid bilayer of the membrane, which otherwise is relatively impermeable to ions. Na+ probably enters cells through nonselective cation channels, and at high salinity, possibly through K+ channels or transporters that are incompletely selective for K+ (Amtmann & Sanders, 1999; Schachtman & Liu, 1999; Tyerman & Skerrett, 1999; Tester & Davenport, 2003). These are summarized in Table 3.
Table 3. Cloned genes with likely relevance for controlling K+ or Na+ uptake by roots and transport within the plant, and which are candidates for overexpression studies
|K+ channel||Shaker type (single pore, tetramer) inward channel|| AKT1, AKT2, KAT1|| AKT1 (Arabidopsis K+transporter) is an inward-rectifying K+ channel expressed in roots. It is highly selective for K+ over Na+ (PNa/PK = 0.05). However, at high salinities this channel could transport Na+. AKT2 and KAT1 are related. These are expressed in leaf phloem tissue and guard cells, respectively, in Arabidopsis, but may function in other cell types in other species.|
|K+ channel||Shaker type, outward channel|| SKOR || SKOR (stelar K+outward rectifier) is important in maintaining K+ homeostasis in both roots and shoots. SKOR mutants have lower K+ concentrations in shoots but not roots, indicating that SKOR influences xylem loading of K+. SKOR is probably located on the plasma membrane.|
|K+ channel|| KCO family (two pore channel)|| KCO1 || KCO1 (K+channel outward) rectifier is expressed in leaf cells, probably on the tonoplast.|
|Nonselective cation channel|| CNGC and GLR families|| CNGC1–20, GLR1–20||Some members of the CNGCs (cyclic nucleotide-gated channels) and GLRs (glutamate receptors) families are predicted to have a similar permeability to Na+ and K+, and to be regulated by Ca2+ (Demidchik et al., 2002).|
|K+ antiporter||K/H antiporter|| KEA or CPA (CHA) family||K+ antiporters may be important in K+ homeostasis by loading K+ into vacuoles. KEA (K+exchange antiporter) is present in the plant genome, but its function is unknown. It is possible that these could carry Na+, just as Na+/H+ antiporters can carry K+.|
|K+ transporter|| KUP/HAK/KT family|| HAK1–10, KUP1–4||A very large family of K+ transporters, similar to the K+ uptake family in bacteria and high-affinity K+ transporters in fungi. There are many variants in all higher plants, and they are likely to be very important in control of K+ homeostasis. Their selectivity for K+ over Na+ in higher plants is not known.|
|K+ transporter|| HKT family|| HKT1 ||K+ starvation induces HKT1 (high-affinity K+transporter) expression in wheat, indicating that it functions in high-affinity K+ uptake, but it also transports Na+. In Arabidopsis it is likely that AtHKT1 is important in regulating Na+ and K+ homeostasis, as mutations that disrupt its function alter the transport of Na+ from root to shoot, and the K+/Na+ ratio in the root (Rus et al., 2004).|
|Cation antiporter|| CHX family|| CHX10, 15|| Cation hydrogen exchangers such as CHX10 and CHX15 regulate K+ uptake by vacuoles. They may carry Na+, and their expression is down- regulated under salt stress (Maathuis et al., 2003).|
|Na+ antiporter|| NHX family|| NHX1 || AtNHX1 (Na+/H+exchanger) is an Na+/H+ antiporter on the tonoplast membrane. It is expressed in roots and leaves, and selectively transports Na+ into the vacuole, as well as K+ in nonsaline conditions (Apse et al., 2003).|
| NHX2–5 || AtNHX2–5 are expressed in specific cell types, transport Na+ or K+ into the vacuole, and have a likely role in K+ or pH regulation.|
| SOS1 || SOS1 (AtNHX7) is an Na+/H+ antiporter on the plasma membrane. SOS1 (salt overly sensitive) is expressed in root cells (Shi et al., 2002). SOS1 would efflux Na+ from cells and may be important in Na+ extrusion from roots into the external medium.|
|Proton pump||AHA P-type H ± ATPase|| AHA2 ||H+ transport across plasma membrane.|
|Proton pump||H ± PPase|| AVP1 ||H+ transport across tonoplast.|
Passive ion transport occurs through channels, which are membrane proteins with ion-selective pores that allow ion movement down an electrochemical gradient. They are usually highly selective for a specific ion, but there is a class of nonselective cation channels that transport Na+, K+, Ca2+ and NH4+ (Demidchik et al., 2002). Some nonchannel transporters, such as HKT1, can allow passive transport under certain conditions (Tester & Davenport, 2003).
Active ion transport occurs through symporters and antiporters that can transport ions against an electrochemical potential gradient. Transport is driven by the electrochemical potential difference of a coupled solute, usually H+. Transporters undergo a cycle of conformational changes while transporting ions, the rate of transport being much lower than that of channels. Most are highly selective for an ion other than Na+, but the NHX family of antiporters (Na+/H+ exchangers) are selective for Na+.
Proton pumps are essential to provide the electrical potential difference that drives Na+ symporters or antiporters. Active Na+ uptake into the cell across the plasma membrane is driven by P-type H+-ATPases, which hydrolyse ATP to pump H+ into the cell wall (Reinhold & Guy, 2002); active ion transport into the vacuole is driven by V-type H+-ATPases, which hydrolyse ATP to pump H+ into the vacuole (Binzel & Ratajczak, 2002). Also important is the H+-PPiase on the tonoplast, which hydrolyses pyrophosphate to pump H+ into the vacuole (Gaxiola et al., 2001; Binzel & Ratajczak, 2002).
There are no classical Na+ pumps in higher plants. In fungi and mosses there is an Na+ pump, ENA1, which hydrolyses ATP to pump Na+ out of the cell (Benito & Rodriguez-Navarro, 2003). Also in yeast are ‘HAL’ genes (Serrano et al., 1999), with uncertain function other than the control of K+ or Na+ transport. Many of these do not have orthologues in higher plants. The expression of these in plants may introduce new mechanisms for control of Na+ or K+ transport: for example Rus et al. (2001) have shown that HAL1, which controls K+ transport, improves salt tolerance of tomato.
Na+ that enters a cell will have one of three fates (presuming it cannot be retained in the cytoplasm, where it would be toxic). Na+ can move symplastically into an adjacent cell via plasmadesmata; it can be effluxed back into the cell wall; or it can be ‘compartmentalized’ (i.e. transported into the vacuole). Efflux occurs through Na+/H+ antiporters on the plasma membrane such as SOS1 (Zhu, 2003). Compartmentalization occurs through vacuolar Na+/H+ antiporters such as NHX1 (Blumwald et al., 2000).
The processes that maintain low Na+ in organelles such as chloroplasts and mitochondria are less well known, although the maintenance of pH in organelles, as well as their cation composition, is important. A cation/proton antiporter, CHX23, which is specific to the chloroplast envelope membrane, has been identified by Song et al. (2004). Such proteins are probably essential for pH control of chloroplasts.
Candidate genes for improving control of Na+ uptake and transport Many genes are important in maintaining K+ or Na+ homeostasis in higher plants, and could be considered candidates for genetic manipulation. These are listed in Table 3. The K+ channels and transporters may regulate Na+ transport – either directly, because they may be incompletely selective for K+ and transport Na+ when presented with a high Na+ concentration or a high Na+/K+ ratio; or indirectly, because they may buffer the cell against Na+ uptake by maintaining rigorous K+ homeostasis. Single amino acid changes in the pore region of a channel or transporter can change the selectivity for cations, so a single base-pair mutation can have a large effect.
Many of these transporters have a large number of variants, both within and between species. In cereals there is a particularly large redundancy in the HKT and the HAK/KUP/KT families (Damien Platten, pers. comm.). These transporters control K+ uptake and K+/Na+ selectivity. The function of the redundancy is unclear. Are some variants active and others silent? Are some expressed under different Na+ or K+ concentrations, or in different tissues, or at different cell developmental stages? The function of individual genes in such large gene families will be difficult to fully understand.
Not listed in Table 3 are regulatory genes such as kinases and phosphatases that provide post-transcriptional regulation of ion transporters. The SOS pathway appears to be regulated in this way (Zhu, 2003). It is activated by a sodium-induced rise in Ca2+ which binds SOS3, leading to a cascade which activates the kinase SOS2, which phosphorylates and thereby activates SOS1 (Zhu, 2003). Other regulatory pathways are summarized by Tester & Davenport (2003, p. 512).
There is little unequivocal information on the cell or tissue specificity of these cation transporters, most information being based on in situ localization. Microscopic images can be difficult to interpret, and as reporter genes are expressed only in the cytoplasm, the intensity of the expression in different cell types may reflect differences in cytoplasmic volumes in different cell types. For example, the visual expression of SOS1 promoter-GUS expression pattern in root epidermal cells near the root tip only, and in the cells lining the xylem throughout the plant (Shi et al., 2003), may be influenced by the higher cytoplasmic volumes in these cell types. Furthermore, expression patterns from gene constructs may vary from one species to another, and it is difficult to know whether this is caused by real differences in expression in heterologous systems, or by different tissue anatomy.
How to evaluate the function of cation transporters in salt tolerance It is not easy to measure the activity of individual ion transporters, particularly in specific cell types. Measurement of ion concentrations at a tissue or organ level is easy, and direct measurement or calculation of ion concentrations in xylem sap is simple although tedious (Table 1), but suprisingly few measurements are made of ion concentrations in tissues or xylem sap. More commonly, salt tolerance of the whole plant is measured, as either plant biomass or plant survival.
In some studies, rather than growing plants for a long time at moderate salinity, the severity of the stress is increased until the more sensitive plants die. This has the advantage of showing up individuals with high tolerance of the stress, but can wipe out the chance of finding the mechanism, as the experimental control (e.g. the wild-type genotype) may have died.
Results of transformation studies Transformation experiments with the greatest successes have come from manipulating tonoplast Na+ transport. An antiporter cloned from Arabidopsis, AtNHX1, when overexpressed in that plant, increased its salt tolerance so that plants could then grow and set seed at 200 mm NaCl, whereas before they were limited to 100 mm NaCl (Apse et al., 1999). Similar results with AtNHX1 were presented for tomato and brassica (Zhang & Blumwald, 2001; Zhang et al., 2001). In those experiments the death of control plants (plants transformed with vector only) at high salinity does not enable us to understand the function of the gene at the whole-plant level. The death of tomato plants at 200 mm NaCl is puzzling, as in other studies the same tomato cultivar, Moneymaker, could grow and set seed at 200 mm NaCl (Flowers, 2004). When AtNHX1 was overexpressed in wheat in moderately saline soil, with an ECe of 10 dS m−1, the grain yield of the best transgenic line was reduced by only 50%, compared with 65% in the untransformed control plants (Xue et al., 2004). Na+ concentrations in the root were unchanged by the treatment, but Na+ in the shoot decreased. It is therefore difficult to know how the antiporter was acting, as one might expect higher tissue concentrations to be tolerated. When the cotton orthologue, GhNHX1, was overexpressed in tobacco there were more dramatic increases in salt tolerance (Wu et al., 2004).
Overexpression of the plasma membrane Na+/H+ antiporter SOS1 in Arabidopsis increased the tolerance of Arabidopsis (Shi et al., 2003). Roots grew more quickly at 100 mm NaCl, and the percentage of plants that survived 5 d at 200 mm NaCl was increased from 17 to 43%. This was probably the result of the 50% reduction in Na+ uptake by plants (Shi et al., 2003).
The activity of Na+/H+ antiporters could be limited by their number, or by the H+ difference across the membranes. In that case, increasing the capacity of a proton pump would increase the salt tolerance of the plant. The vacuolar H+-PPiase, AVP1, which may be important to energize the vacuole membrane under salt stress, was overexpressed in Arabidopsis and increased its salt tolerance (Gaxiola et al., 2001). Transformed plants were able to grow in 250 mm NaCl, whereas the wild type died. The enhanced performance of transformed plants may have been caused by their higher accumulation of Na+ and K+.
HKT transporters are probably very important in regulation of K+ and Na+ transport from root to shoots, but the mechanism is not known. Transgenic studies have yielded somewhat inconsistent results with TaHKT1 (Laurie et al., 2002) and AtHKT1 (Rus et al., 2004). However, a knockout mutant of AtHKT1 clearly increased salt sensitivity (Rus et al., 2004), which shows that the function of HKT1 in the control of K+ or Na+ transport is important.
The HAL1 gene from yeast controls K+/Na+ selectivity and salt tolerance of yeast cells. Expression in tomato increased fruit yield and enhanced K/Na+ selectivity in leaves (Rus et al., 2001). The exact function of this gene in higher plants is not known.
The experiments described above all measured plant growth; studies with cells or calluses are not included, nor are studies that present only qualitative data. A complete list of transformation experiments is given by Flowers (2004). He suggests that claims of improved salt tolerance are not substantiated in many of these cases because of poor experimental designs, for example a single high-salinity treatment for 3 d, and inappropriate choices of methods to evaluate tolerance, such as photographs or survival numbers. A checklist of methods is suggested at the end of this review.
2. Genes with an osmotic or unknown protective function
Molecules having a protective function include small organic compounds that are variously called osmolytes, osmoprotectants or compatible solutes. These have two functional roles: at high concentrations, osmotic adjustment; and at low concentrations, an unknown protective role. Other gene products include enzymes that ‘mop up’ free radicals, and proteins that protect the formation or stability of other proteins. None of these is confined to salinity: all occur under drought as well as salinity stress, and sometimes under other stresses that also reduce growth, such as low temperature.
Protective molecules are referred to in this section as the gene product, rather than the gene, for brevity and familiarity.
Organic solutes and proteins with osmotic or other protective functions Compatible solutes or osmolytes would be essential for coordinated regulation of vacuolar and cytoplasmic volumes. The term ‘compatible solute’ was advanced by Wyn Jones et al. (1977) to describe nontoxic solutes that could increase in high concentrations in the cytosol and be compatible with metabolic activity. They would be important to adapt plants to drought, as they could enhance osmotic adjustment and allow turgor maintenance of cells that would otherwise dehydrate. Tissue dehydration is less likely in salinity than drought, as the uptake of NaCl promotes osmotic adjustment, but compatible solutes would be essential to balance the osmotic pressure of the cytoplasm where salts accumulated preferentially in the vacuole.
In addition, certain solutes could have a metabolic protective role – they could stabilize soluble or membrane proteins, and so maintain growth at high salinity. The term ‘osmoprotectant’ has arisen for this function (reviewed by Rhodes et al., 2002). Specific functions are uncertain in higher plants; their putative function is based on that in microorganisms. The metabolic pathways and genes controlling their synthesis or accumulation have been summarized in comprehensive reviews by Hare et al. (1998); Nuccio et al. (1999); Chen & Murata (2002) and, with particular reference to salinity, Rhodes et al. (2002). There are four main classes of solute that could have an osmotic or protective role: N-containing solutes such as proline and glycine betaine; sugars such as sucrose and raffinose; straight-chain polyhydric alcohols (polyols) such as mannitol and sorbitol; and cyclic polyhydric alcohols (cyclic polyols) (Table 4). Many of these may be important as compatible solutes during freezing.
Table 4. Major categories of solutes with possible function as osmolytes or protectants, and results of key overexpression transformation studies, summarized from reviews by Hare et al. (1998); Nuccio et al. (1999); Chen & Murata (2002); Rhodes et al. (2002); Smirnoff (1998)
|N-containing solutes such as proline, glycine betaine, trigonelline, proline betaine||Turgor maintenance.||1–50 mm||proline|| P5CS mod||tobacco||60 mm|| Hong et al. (2000)|
|Possibly redox regulation, to buffer cellular redox potential as metabolic activities slow down during stress, and so to promote recovery from stress.|| ||glycine betaine|| codA ||rice||5 mm|| Sakamoto et al. (1998)|
|Trehalose||Signalling, possibly through hexose sensing.||5–10 µm||trehalose|| otsA, otsB||rice||300 µm|| Garg et al. (2002)|
|Straight-chain polyhydric such as mannitol and sorbitol||Turgor maintenance, particularly in the cytosol.||1–50 mm||mannitol|| mt1D ||wheat||2 mm|| Abebe et al. (2003)|
|Mannitol may be involved in hexose sensing or in mopping up hydroxyl radicals.|| ||mannitol|| mt1D ||tobacco||6 mm|| Tarczynski et al., 1993)|
| || ||sorbitol||S6PDH||persimmon||60 mm|| Gao et al. (2001)|
|Cyclic polyhydric alcohols such as myo-inositol, ononitol and pinitol||Turgor maintenance in certain halophytes.||1–200 mm||ononitol|| imt1 ||tobacco||35 mm|| Sheveleva et al. (1997)|
|Might also mop up hydroxyl radicals.|| || || || || || |
|Sugars such as fructose, sucrose, glucose and raffinose||Turgor maintenance, carbohydrate storage.||1–200 mm||–||–||–||–||–|
If the concentrations of compatible solutes were high enough to contribute to turgor maintenance in the cell as a whole, production of these solutes would have an energy cost and hence a growth penalty. Their synthesis would divert substrates from metabolic pathways essential to growth, such as protein synthesis and cell-wall synthesis (Munns, 1988). However, they could contribute to leaf longevity when salt concentrations start to rise. Hence they may not make the plant grow more quickly when the salt is first applied, but would allow it to avoid wilting, to avoid toxicity in the presence of high internal concentrations of salt, and possibly to survive a period of stress.
Proteins with protective roles of unknown function include late-embryogenesis-abundant proteins (LEAs) and their close relatives, dehydrins. These are produced as cells lose water, and confer either tolerance to dehydration or recovery on subsequent hydration, but the biochemical mechanism is uncertain. They have properties similar to chaperonins, and may help to stabilize protein structure when cells have high salt concentrations or start to lose water. However, cell dehydration is not a common event in salt-treated plants, as leaf tissue is usually turgid (because of high salt content) and loses water only when the sodium rises to toxic levels. Many of these protective proteins are expressed only after osmotic shocks.
Enzymes that detoxify reactive oxygen species (ROS) may have an essential role in adapting plants to salinity stress. Free oxygen radicals (oxygen molecules or hydroxyl groups with an extra electron) are highly reactive and can oxidize essential compounds, particularly membrane lipids, unless detoxified by antioxidants. Important pathways of detoxifying ROS involve superoxide dismutase, catalase, and the ascorbate–glutathione cycle (Foyer et al., 1994). These enzymes are induced under drought and salinity, and during other stresses such as high light or extremes of temperature. The activities of the antioxidant defence systems are important in limiting photo-oxidative damage and in detoxifying ROS produced in excess of those normally required for signal transduction or metabolism (Foyer et al., 1994). Any environmental condition that suppresses photosynthesis (such as low temperature, salinity or drought) while plants are in high light could allow the production of excessive and potentially toxic ROS.
How to evaluate the function of protective compounds in salt tolerance As the function of most of these compounds is unknown, their benefit can be measured only by their effect on either leaf injury or plant growth rate. Many solutes accumulate under abiotic stress simply because the supply of assimilate by source leaves has exceeded its demand by growing tissues (Munns, 1988), so an increase in any one solute may not make a plant grow more quickly. It might even make it grow more slowly if there is a high nitrogen or energy cost for its synthesis. Should growth rate be measured over the short or long term? That is, while osmotic stress dominates the growth response (phase 1), or when internal salt toxicity can occur in some genotypes (phase 2)?
Compatible solutes would exert their effect in phase 1, but could also function in phase 2. The effect of higher proline accumulation, or of other compounds that enhance turgor maintenance, could be seen on the rate of elongation of new leaves and roots. High turgor does not necessarily make plants grow more quickly (Termaat et al., 1985; Munns, 2002), but they cannot grow at all if they are wilted. If compatible solutes function to maintain turgor as the leaves age, then longer-term measurements, taking in whole-plant growth, are appropriate.
The effect of osmoprotectants that accumulate to only low concentrations would be seen only in phase 2. These would protect metabolic function in cells in which salt concentrations are high. They are therefore most likely to function in photosynthetic tissues of older leaves, and to affect growth in the long term. Proteins with protective or chaperonin-like functions are also most likely to function in the long term.
Enzymes that detoxify ROS would feature in the long term to protect metabolic function in cells in which salt concentrations are high, and to prevent premature senescence. They could also be very important in the short term, particularly in tissues exposed to high light, as they would help photosynthesizing leaves to remain functional despite low stomatal conductance, which occurs soon after plants are exposed to salinity because of the osmotic effect of salt outside the roots (James et al., 2002).
Results of transformation studies Many papers have reported positive results for salt tolerance when plants are transformed with genes for osmoprotectants or protective proteins, summarized by Hare et al. (1998); Nuccio et al. (1999); Chen & Murata (2002); Gorham & Wyn Jones (2002); Rhodes et al. (2002); Flowers (2004). A few cases are highlighted here and in Table 4.
Some species do not accumulate glycine betaine under stress, including the model species Arabidopsis, rice and tobacco. Overexpression of genes for glycine betaine synthesis in these species enhanced salt tolerance in terms of biomass production or survival, despite the fact that the glycine betaine level in the transgenic plants was much lower than in species which naturally accumulate this compound, and too low to have a significant effect on osmotic pressure even if confined to the cytoplasm (reviewed by Chen & Murata, 2002). Thus improved salt tolerance in these transgenics must have been via a protective effect of the low levels of glycine betaine, rather than an osmotic effect bringing about more favourable water relations. The level of glycine betaine found in transformed plants was no more than 5 mm in the leaves (Chen & Murata, 2002), much lower than in other solutes such as proline or sugars.
All species accumulate proline under stress, and the gene for its synthesis, P5CS (Δ-1-pyrroline-5-carboxylate synthetase), is induced rapidly by stress in all tissues (Hong et al., 2000). However this enzyme is subject to feedback control, and overexpression with this gene increases the concentration of proline in leaves only twofold (Hong et al., 2000). Tobacco carrying a modified P5CS gene from Vinca aconitifolia to avoid feedback inhibition had a fourfold increase in proline (up to 60 mm compared with the control, with vector only, of 15 mm) in 200 mm NaCl (Hong et al., 2000). These plants had a higher germination rate, a greater seedling weight at 2 wk, and produced fewer free radicals. That is, they were more tolerant to the osmotic stress.
Overexpression with the bacterial gene mt1D for mannitol synthesis has resulted in increased salinity tolerance, for example in wheat (Abebe et al., 2003). Shoot fresh weight in 150 mm NaCl was reduced by 77% of plants lacking the mt1D gene, but by only 50% in transformed plants. The transformed plants had only 2 mm mannitol, too little to influence their water relations, requiring some other explanation for the improved growth.
Overexpression with the bacterial genes for trehalose synthesis increased salt tolerance in rice: there was fourfold greater dry weight after 4 wk in 100 mm NaCl in transformed than in untransformed plants (Garg et al., 2002). Trehalose levels increased in transformed plants, but were still only 0.3 mm in leaves after salt treatment, so the trehalose may have altered growth through signalling pathways (Hare et al., 1998).
In summary, overproduction of many of these solutes through gene transformation has convincingly increased growth of plants in saline soil – but we do not know how.
3. Genes that control cell and tissue growth rates
Genes controlling cell growth and leaf function Genes that could increase the growth rate of plants in saline soil would influence the rate of production of new leaves and roots (by controlling the rate of cell division; the development of new primordia for shoot or root branching; the rate of cell wall expansion; or the dimensions of differentiated cells), or they would influence the rate of photosynthesis (by controlling stomatal aperture; or the dimensions of mesophyll cells, which influences leaf morphology and transpiration efficiency).
Candidate genes controlling growth are probably involved in signalling pathways that start with a sensor and involve hormones, transcription factors, protein kinases, protein phosphatases and other signalling molecules such as calmodulin-binding proteins. It is highly likely that such genes are common to drought stress (reviewed by Chaves et al., 2003), and are common to other stresses such as cold, and soil conditions that reduce growth such as soil hardness caused by compaction or sodicity. Many are induced by treatment with ABA. The sensor may be a metabolite that changes in concentration, or a membrane protein that changes in configuration, as the cell shrinks in response to a salt, drought or cold stress, or a long-distance signal moving from roots to shoots in the transpiration stream.
Progress on the discovery of transcription factors and signalling pathways is fast, and is being covered by a series of comprehensive reviews and updates (Shinozaki & Yamaguchi-Shinozaki, 2000; Knight & Knight, 2001; Zhu, 2002; Wang et al., 2003; Zhang et al., 2004).
Few transcription factors have known physiological functions, and all are likely to be tissue- or cell-specific, but (apart from guard cells) no tissue or cell specificity has been investigated. This point is discussed further in the following section. Trehalose is considered to have signalling functions in growth, but again no tissue specificity has been investigated. Most of the transcription factors are induced by rapidly imposed and severe stress, so their function in adapting a plant to a long-term salinity stress is not yet known.
How to evaluate the function of genes controlling leaf growth in salt tolerance Whether experiments are short or long term would depend on the function of the genes. Genes affecting the rate of cell division and elongation would be seen soon after the salinity is applied (in phase 1). Genes involved in leaf morphology, leading to enhanced transpiration efficiency, would take longer to show their effect, through enhanced carbon supply to growing regions. With overexpression studies of transcription factors, and without knowing exactly what a given transcription factor ‘does’, long-term growth experiments are appropriate. Comparisons with other stresses that reduce growth, such as water stress, low temperature and soil hardness, would indicate whether or not there are salt-specific factors involved.
Results of transformation studies Studies on overexpression of 13 transcription factors are summarized by Zhang et al. (2004), although some of these studies are liable to the criticisms pointed out by Flowers (2004). So far no studies have proved that the enhanced responses to abiotic stresses are salt-specific.