ENGINEERING SALT TOLERANCE IN CROP PLANTS
(From ISB News Report)
Throughout civilized history, environmental stress due to high concentrations of salt in soils has endured as one of the most serious factors limiting productivity of agricultural crops, which are particularly sensitive to soil salinity. Currently, elevated soil salinity affects agricultural production in a large proportion of the world's terrestrial areas. It is estimated that more than a third of all irrigated land is presently affected, exclusive of regions classified as arid and desert lands, which comprise 25% of total land surface of the planet. This continual loss of farmable land due to salinization is directly in conflict with the needs of the world population, projected to increase by 1.5 billion in the next 20 years, and poses a formidable challenge to the task of maintaining world food supplies.
Wild plants that tolerate salt and grow in saline environments have high intracellular salt levels. A major component of the osmotic adjustment in these cells is accomplished by ion uptake. The utilization of inorganic ions for osmotic adjustment would suggest that salt-tolerant plants must be able to tolerate high levels of salts within their cells. However, enzymes extracted from these plants show high sensitivity to salt, suggesting that these plants are able to keep Na+ ions away from the cytosol.
Plants can use three strategies for the maintenance of a low cytosolic sodium concentration: sodium exclusion, compartmentation, and secretion. One mechanism for sodium transport out of the cell is through operation of plasma membrane-bound Na+/H+ antiports, as confirmed by the characterization of SOS1, a putative plasma membrane Na+/H+ antiport from Arabidopsis thaliana.1 The efficient compartmentation of sodium is likewise accomplished through operation of vacuolar Na+/H+ antiports that move potentially harmful ions from cytosol into large, internally acidic, tonoplast-bound vacuoles.2 These ions, in turn, act as an osmoticum within the vacuole to maintain water flow into the cell.3 Antiports use the protonmotive force generated by vacuolar H+-translocating enzymes, H+-adenosine triphosphatase (ATPase) and H+-inorganic pyrophosphatase (PPiase), to couple downhill movement of H+ (down its electrochemical potential) with uphill movement of Na+ (against its electrochemical potential).
Eduardo Blumwald and coworkers have been engineering crop plants with improved salt tolerance. In 1999, they successfully engineered transgenic Arabidopsis plants that overexpress AtNHX1, a vacuolar Na+/H+ antiport, which allowed the plants to grow in 200 mM NaCl.3 In the August 2001 issue of Nature Biotechnology, they reported the genetic modification of tomato plants to overexpress the Arabidopsis thaliana AtNHX1 antiport, which likewise allowed those plants to grow in the presence of 200 mM NaCl .4
|
|
|
Figure 1. Salt tolerance of wild-type tomato plants and transgenic plants overexpressing AtNHX1 grown in the presence of 200 mM NaCl. (A) wild-type plants grown in the presence of 5 mM NaCl. (B) transgenic plants overexpressing AtNHX1, grown in the presence of 5 mM NaCl. (C) Western blots from leaf membrane proteins (5 mg) tested with antibodies raised against AtNHX1. Upper panel: Lanes 1 and 4, tonoplast-enriched fraction; lanes 2 and 5, Golgi/ER-enriched fractions; 3 and 6, plasma membrane fraction. Lanes 1,2,3 correspond to membranes from wild-type plants while lanes 4,5,6 correspond to membranes from transgenic plants. Relative molecular masses are indicated on the left; lower panel: Enrichment of the fractions with tonoplast membranes was assessed with antibodies raised against the vacuolar H+-PPiase. (D) wild-type plants grown in the presence of 200 mM NaCl. (E) transgenic plants overexpressing AtNHX1, grown in the presence of 200 mM NaCl. Plants shown after 11 weeks of growth. Bar = 25 cm. (Used with permission4–Copyright 2001 Nature Biotechnology). |
A salt concentration of 200 mM is equivalent to 40% of the salt concentration of seawater and will inhibit the growth of almost all crop plants. The growth of the wild-type plants in this study was severely inhibited by the presence of 200 mM NaCl in the growth solution, and most of the plants died or were severely stunted (Fig 1D). On the other hand, the transgenic plants grew, flowered, and produced fruit (Fig 1E). The high sodium and chloride content in the leaves of transgenic plants grown in salty water demonstrated that enhanced vacuolar accumulation of Na+ ions, mediated by the Na+/H+ antiport, allowed transgenic plants to ameliorate the toxic effects of Na+. Most notable was the production of fruit by these transgenic plants grown in the presence of 200 mM NaCl. While the transgenic leaves accumulated Na+ to almost 1% of their dry weight, the fruits displayed only a marginal increase in Na+ content and a 25% increase in K+ content.
A similar strategy was used by Blumwald et al. to genetically modify Brassica napus,5 commonly known as Canola or rapeseed and one of the most important oilseed crops cultivated worldwide. A construct containing the AtNHX gene, coding for a vacuolar Na+/H+ antiport from Arabidopsis thaliana, was introduced into the genome of Brassica napus cv Westar. Overexpression of the vacuolar Na+/H+ antiport did not affect the growth of transgenic plants since similar growth was observed when wild-type and transgenic plants were grown in the presence of 10 mM NaCl. While growth of wild-type plants was severely affected by the presence of 200 mM NaCl in the growth solution, transgenic plants grew, flowered, and produced seeds .5
|
|
|
Figure 2. Salt tolerance of wild-type plants and transgenic Brassica plants overexpressing AtNHX1grown in the presence of 200 mM NaCl. Wild-type (wt) and homozygous plants showing high (X1OE1), medium (X1OE2) and low (X1OE3) levels of expression were grown in the presence of 200 mM NaCl. Plants shown after 10 weeks of growth. Inset: Western blots of leaf tonoplast-enriched membrane fractions isolated from wild-type and transgenic plants with low, medium and high levels of expression of AtNHX1. Blots were probed with antibodies raised against the C-terminus of AtNHX1. Equal amounts of protein (20 mg) were loaded in each lane. Relative molecular masses are indicated on the left. (Used with permission--5-Copyright 2001 National Academy of Sciences). |
Notably, transgenic plants grown at 200 mM NaCl produced seed numbers similar to those of wild-type plants grown at low salinity. Moreover, qualitative and quantitative analyses of oil content showed no significant differences between seeds from wild-type plants grown at low salinity and transgenic plants grown at high salinity.
Twenty years ago, Epstein6 argued for development of crops in which the consumable portion is botanically a fruit, such as grain, berries, or pomes, and that have a truly halophytic response to salinity. In these plants, Na+ ions would accumulate mainly in leaves, and since water transport to fruits and seeds is primarily through the phloem pathway (i.e., the intercellular connections), much of the salt from these organs would be screened. The results obtained with transgenic salt-tolerant tomato and Canola clearly support Epstein's argument.
Degradation of agricultural land and water supplies is a result of intensive agricultural practices employed in developed and developing countries for a long time. Ideally these practices should be changed to a more sustainable use of land and water resources. For example, mixed cropping with perennials and trees would alleviate the accumulation of sodium and other salts in upper soil layers. Nonetheless, this type of change in farming systems and the development of new products is likely a long and difficult process, since it will require the use of new land and will not address the problem of growing crops in land that is already compromised. Development and use of crops that can tolerate high levels of soil salinity is a practical solution, at least for the time being. Although conventional breeding for salt tolerance has been attempted for a long time, the lack of success in generating tolerant varieties (given the low number of varieties released and their limited salt tolerance) would suggest that conventional breeding practices are not enough, and that, in order to succeed, a breeding program should include the engineering of transgenic salt-tolerant crops.
Remarkably, Blumwald's transgenic Canola plants grown in high salinity conditions accumulated sodium up to 6% of total dry weight. Taking into consideration that a mature Brassica plant in the field can weight 2 kg fresh weight or 300 grams dry weight, each plant could accumulate 18 grams of sodium when grown in the presence of 200 mM NaCl.6 This significant amount of sodium taken up by transgenic plants would suggest that, in addition to value as an agronomic crop, these plants could be used as one component needed to reclaim saline soils.
Sources
1. Shi H, Ishitani M, Kim C, and Zhu J-K. 2000. The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter. Proceedings of the National Academy Sciences USA 97: 6896–6901.
2. Apse MP, Aharon GS, Snedden WS, and Blumwald E. 1999. Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285: 1256–1258.
3. Glenn E, Brown JJ, and Blumwald E. 1999. Salt-tolerant mechanisms and crop potential of halophytes. Critical Reviews in Plant Sciences 18: 227–255.
4. Zhang H-X and Blumwald E. 2001. Transgenic salt tolerant tomato plants accumulate salt in the foliage but not in the fruits. Nature Biotechnology 19: 765–768.
5. Zhang H-X, Hodson J, Williams JP, and Blumwald E. 2001. Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. Proceedings of the National Academy of Sciences USA 98: 12832–12836.
6. Epstein E. 1983. Crops tolerant to salinity and other mineral stresses. In Better Crops for Food, Ciba Foundation Symposium, eds. J Nugent and M O'Connor, pp 97, 61-82. London: Pitman.
Eduardo Blumwald
Department of Pomology
University of California
Eblumwald@ucdavis.edu