Gene functions expected in roots Genes for salt tolerance expected to function in roots would be involved in the control of ion uptake and transport to the shoots, and the regulation of root ion homeostasis and water status.
Roots have a remarkable ability to control their own Na+ and Cl− concentrations. Their ion concentrations do not increase with time, and at high salinities they can have a much lower Na+ and Cl− concentration than the external solution. For example, in a number of bread wheat genotypes growing in 150 mm NaCl, Na+ in the roots was only about 30 mm, with a small variation about that value with different genotypes (Gorham et al., 1990). Similarly low values for both Na+ and Cl− with different durum and bread wheat genotypes were found in salinities from 50 to 150 mm NaCl (Husain et al., 2004; one genotype shown in Fig. 5). Barley also has a remarkably low Na+ concentration in roots: for example, plants grown at 250 mm NaCl had only 120 mm NaCl in their roots (1.8 mmol g−1 d. wt; Storey & Wyn Jones, 1978). Roots would therefore be accumulating organic solutes to maintain turgor, so enzymes that synthesize or transport organic solutes such as proline or sucrose should increase in activity.
Figure 5. Effect of a range of NaCl concentrations on Na+ concentrations in the roots of durum landrace Line 141 after 14 d treatment, taken from two experiments with overlapping ranges of NaCl (Husain et al., 2004). Values are means (n = 4) ± SE. Cl− concentrations were similar to Na+ (Husain et al., 2004). Dotted line, 1 : 1 relation between internal and external Na+ concentration.
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The activity of transporters that control the net uptake of Na+ or Cl− from the external solution, the compartmentation of these ions in vacuoles, and the loading of the xylem should be increased by NaCl treatments. As Na+ will enter root cells passively from a saline soil (Appendix S2), Na+ efflux might increase dramatically, requiring an antiporter such as SOS1 and a plasma membrane proton pump. K+ channels and symporters such as HKT1 may also be upregulated to maintain K+ homeostasis. Channels that are likely to carry Na+, such as nonselective cation channels, may be downregulated. Cation/H+ antiporters that control the ion homeostasis of organelles may be upregulated. Na+ will not enter the xylem passively unless there is a large membrane depolarization (Appendix S2), but may enter through K+ transporters, as K+ loading of the xylem could occur through active rather than passive transport (Appendix S2). The types of K+ transporter with the greatest selectivity for K+ over Na+ may be upregulated.
Genes involved in signal transduction pathways that regulate the rate of cell division and elongation in roots might be affected. The activity of processes that synthesize and deliver hormonal signals to the root tip could change, and ‘root signals’ that travel in the transpiration stream, and cause stomatal closure and reductions in leaf expansion rates, might be induced.
Genes found in roots: results of expression studies Only one gene expression study has revealed the induction of relevant ion transporters in roots by salt treatment. A microarray was designed specifically to represent the major transporter genes from Arabidopsis that could be associated with cation excess or deficiency, and roots were examined over 96 h following salt stress (80 mm NaCl), Ca2+ deficiency and K+ deficiency (Maathuis et al., 2003). Salt stress induced the expression or repression of a number of transporters that could be involved in control of cation uptake across the plasma membrane, or in the storage of cations in vacuoles. Some particularly interesting genes included proton pumps (V-type H+-ATPases, AHA2) Na+/H+ antiporters (NHX1, NHX3) and cation/H+ antiporters (CHX10, CHX15). This paper is a valuable reference for future studies on transporters. However, there was no change in transcript level of SOS1. A possible reason is that the plants were grown on medium already containing 2 mm NaCl, and with the K+ concentration in the medium being less than 2 mm many transporters controlling the uptake and efflux of Na+ may have already been induced before the treatment started.
In the other studies that analyse roots separately, few K+ or Na+ transporters were found. The exception was the expression of a putative Na+/H+ exchanger, CHX17, but this occurred under cold and mannitol treatment, as well as under salt treatment (Kreps et al., 2002).
The reason for the absence of changed expression levels of transporters in microarray studies could be simply that the sequences were absent from the chips. Alternatively, it could be that their regulation was post-translational, that the genes were constitutively expressed, or that they had already been induced before NaCl treatment. They could be fully induced by the basal nutrient solutions commonly used. Hoagland and Arnon's solution contains 6.5 mm K+ and ≈ 0.2 mm Na+. Murashige and Skoog's medium contains 20 mm K+ and ≈ 0.8 mm Na+. The K+ and Na+ concentration might be high enough to induce all transporters regulating K+ and Na+ uptake and intracellular transport.
Genes in roots that are often upregulated by salt stress are involved in antioxidant responses, such as ascorbate peroxidase and glutathione reductase (Kawasaki et al., 2001; Kreps et al., 2002; Ueda et al., 2004). These are usually also expressed under other stresses, to different degrees.
Many transcription factors are altered by stress. An extensive and thorough study was reported by Chen et al. (2002), who examined the mRNA levels of 402 transcription factors in various plant tissues after different abiotic and biotic stresses, in highly replicated experiments with plants at different stages of development. About 15% were highly expressed in roots, and some were root-specific (Chen et al., 2002). However, few of these were induced by abiotic stress alone, and none was salt-specific.
In most studies the genes that are upregulated or downregulated have little relevance to salinity, and are expressed under unrelated stresses such as low temperature. The most likely explanation is the nature of the treatments, which (whether salt, cold, or dehydration) are sudden and severe, and plants are harvested within 1–2 h of treatment. The earlier time points are taken on the basis that the earliest response is the most important, but it is likely that most of these are transitory and are involved in repairing membranes, reinstating the volume of cell organelles, and metabolic adjustments to the sudden increases in metabolites caused by the sudden withdrawal of water.
Genes that peak over the first 2 h after salt stress are undoubtedly involved in repair of damaged membranes, particularly in plants that are exposed to concentrations of 150 mm NaCl or higher. Roots in solution will plasmolyse. The turgor of young root cells is in the range 0.5–0.7 MPa (Pritchard et al., 1991; Frensch & Hsiao, 1994). The osmotic pressure of a solution of 150 mm NaCl is ≈ 0.7 MPa, so cells will plasmolyse if plants are transferred in one step to 150 mm NaCl. If cell turgor is only 0.4 MPa, which is likely for older cells in roots (Pritchard et al., 1991), then a one-step transfer to a solution of 100 mm NaCl (0.5 MPa) will plasmolyse cells. With 200 mm NaCl, the protoplast would shrink to half its original volume to achieve equilibrium (Munns, 2002). It will regain its original volume with time by accumulation of solutes, by the process known as osmotic adjustment.
This process of recovery from osmotic shock possibly explains the findings that many genes expressed within a few hours of osmotic shock are in common with those expressed under other shock treatments. For example, in a study with Arabidopsis that compared responses to the relatively moderate shock treatments of 100 mm NaCl, 200 mm mannitol and 4°C, at 3 h, the number of shared responses was reduced tenfold over time, between 3 and 27 h after treatment (Kreps et al., 2002). That is, there was a progression with time towards stimulus-specific responses.
Ca2+ deficiency is a common problem with salinity studies (Cramer, 2002). It is well known that Ca2+ activity (the concentration of Ca which is fully ionized and hydrated) is lowered by adding salts to the medium, and that this causes effects on Ca2+ uptake, membrane selectivity of other cations, and ultimately growth of the plant (reviewed by Cramer & Läuchli, 1986; Reid & Smith, 2000; Tester & Davenport, 2003). For example, when half-strength Hoagland's solution (which contains 2 mm Ca) is used as the basal nutrient medium to which is added 100 mm NaCl or above, the chemical activity of Ca2+ will be about one-third of the concentration, which is below the level adequate for biological requirements. Few of the gene expression studies added supplemental Ca2+. Many effects of salt treatment on membrane transporters, including those for organic solutes, could be caused by impaired root membrane function caused by low Ca2+ activity.