Using Transgenesis to Create Salt-Tolerant Plants

Crop agriculture has succeeded because growers have identified and cultivated useful plant variants through selective breeding and environmental alterations. Transgenic technology improves the precision of agriculture, modifying crops in ways that are uniquely useful that probably would not have arisen naturally. Salt tolerance is one such coveted trait. Recent research on promoting salt tolerance through transgenesis focuses on boosting salt-sequestering physiological mechanisms within species, and transferring this ability from the model organism Arabidopsis thaliana to selected crop species. Underlying all of the approaches to making salt-tolerant crops is a nod to nature. "Organisms solved problems using their genomes for eons, through evolution. Rather than trying to outguess them, we let organisms tell us what mechanisms they can use to overcome stress," explains Ry Wagner, vice president for research at Exelixis Plant Sciences Inc. in Portland, Ore. If going the genetically modified (GM) route to making crops salt tolerant can withstand anti-technology scrutiny, the benefits would be considerable, researchers say. "Salt tolerance is one of a number of traits that can have a major impact in both the developed and developing world by allowing crops to be grown where they normally can't, with less water, or with poor quality water," says George Scangos, president and chief executive officer of Exelixis. About one quarter of irrigated land worldwide has sustained salt damage to plants, to the tune of 60 million hectares, according to Eduardo Blumwald, the Will W. Lester endowed chair at the University of California, Davis. The problem has plagued ancient as well as modern growers, he adds. Sequestering Salt Few plant species are true halophytes, which thrive in the saltiness of oceanfront or estuarial habitats. Inland species can nonetheless face high salinity from extended irrigation, when water evaporates and is pulled upwards into roots. This action leaves behind salt in the soil that can accumulate to toxic levels. Such salt build-up impairs plants in two ways: It disrupts the roots' ability to draw water up, causing osmotic stress and enzyme damage in the cytoplasm, an insult called ion toxicity. It can destroy the enzymes that are vital for protein synthesis and photosynthesis. Many crop plants are sensitive to salt. Corn, citrus, lettuce, onion, pecans and beans are among the most vulnerable, withering into uselessness when grown in high salinity soil. Barley and cotton can withstand moderate salt levels; date palms and sugar beets are particularly salt-tolerant. A few halophytes, such as salt cedar (Tamarix species) and the salt bush (Atriplex species), route the sodium and chloride ions of salt to specialized glands on their leaf surfaces, where they combine to form nontoxic crystals. It is more common for plants to ship sodium ions to vacuoles, the huge storage compartments that are a hallmark of plant cells. (Chloride ions are excluded at the roots of salt-sensitive plants because their concentration is greater inside the root cells.) Like a threatened human body that sends blood to the organs that carry out a "fight or flight" response, a plant flooded with salt manages to keep it away from tender meristem tissues and young leaves at the starts of their photosynthetic careers. "Arabidopsis, canola, and tomato plants store toxic salts mainly in their older leaves, trying to avoid at any cost the migration of salt into the reproductive parts," explains Roberto Gaxiola, assistant professor of plant molecular genetics in the department of plant sciences at the University of Connecticut in Storrs. At a cellular level, shipping sodium ions to vacuoles shelters vulnerable enzymes in the cytoplasm. But this protection requires energy. Since more sodium ions are continually coming in, the plant cell must actively transport it into the vacuole; a protein called an antiport is the escort. One intensely studied antiport protein is AtNHX1. The antiport's power comes from another protein that functions as a hydrogen pump, jettisoning hydrogen ions (protons) to provide cellular fuel. Both the antiport and two forms of the proton pump span the vacuole's membrane. Genetically modifying salt tolerance targets these two membrane members. Boosting Salt Tolerance Courtesy of Eduardo BlumwaldDon't Hold the Salt: Wild-type plants grown in the presence of 200 mM NaCl (left) contrast with transgenic plants overexpressing AtNHX1, again in the presence of 200 mM NaCl (right). The plants are 11 weeks old. Gaxiola's research focuses on one of two proton pumps, called vacuolar H+-translocating pyrophosphatase, or AVP1.1 Reasoning that equipping a plant cell with more copies of AVP1 would rev up the proton-out, sodium-in traffic at the vacuole's interface with the cytoplasm, Gaxiola modified Arabidopsis to overexpress the gene that encodes the proton pump. The result: enhanced salt tolerance. "AtNHX1 is like a revolving door that exchanges sodium for hydrogen ions," explains Gaxiola. "The gene we are overexpressing is a proton pump that will generate the gradient that revolving doors such as AtNHX1 require to function." Other transport proteins tap into the energy that the proton pump provides, altering the ion balance so that the plant can retain more water. "As a result, our plants are not just salt-tolerant, but they are drought-tolerant too," Gaxiola adds. As a model organism, Arabidopsis is commonly used to prove principles, and Gaxiola is optimistic that the ability to render the small plant relatively salt-tolerant will have important applications. "The up-regulation of vacuolar proton pumps such as AVP1 in economically important crops holds promise for the reclamation of farmlands lost to salinization and lack of rainfall," he says. Evidence that Arabidopsis' abilities can indeed be transferred to crop species comes from Blumwald's laboratory, where the target is the antiport protein AtNHX1. The team has created tomato and canola plants that harbor and overexpress the Arabidopsis version of the antiport gene.2,3 Both transgenic crops grew, flowered, and produced seeds in a high-salt environment. The altered tomato had high sodium concentrations in the leaves, but not in the fruits, suggesting that the genetically modified plant could be of agricultural value. The transgenic canola plants were up to 6% dry weight sodium, yet their seed oil quality and seed yields were normal. Gene Discovery The work on the Arabidopsis antiport and protein pump indicates that the potentially powerful effects of manipulating individual genes can control salt tolerance. But other genes are probably important too. "Although this gene is necessary (for salt tolerance) it is not sufficient, and more traits have to be acquired in order to obtain true salt tolerance, such as compounds that help the plant to deal with stress-induced oxidative damage," says Blumwald. Some of those putative genes that contribute to salt tolerance just might turn up at Exelixis, where its "ACTTAG trait discovery technology" is being used to find a numerous genes that enable plants to survive stress. Exelixis' proprietary technology introduces DNA sequences that insert next to, and enhance, the expression of particular genes in plant species of interest. The high-throughput screening protocols use the fast-growing microtomato, Arabidopsis, and rice as experimental systems. "We are building a very large collection—a botanical library—of independent transgenic events," says Wagner. "For Arabidopsis, we have more than 250,000 transgenic lines, each with a tag in a different part of the genome." The researchers, assisted by robots, collect seeds from each line, then grow the plants and look for interesting variants. "For Arabidopsis, we are screening for eight targeted traits. One is abiotic stress tolerance, such as drought or high salinity. The drought tolerance screens have yielded a number of interesting leads, and one line may help regulate salt stress in a novel way when the gene is upregulated," he says. Wagner identifies the mechanism only as "a pump that causes the plant to excrete high salt, when in a high salt environment." The goal, he says, "is to identify genes in these species that we can later transfer to crop species." Crop plants that are transgenic for salt tolerance may face the same consumer backlash that has greeted other GM plants. But many researchers feel that with time and improved communication, current opponents may eventually feel that the benefits outweigh the risks. "The resistance to GM crops is at some level an elite, middle-class privileged point of view," says Scangos. "People who don't have enough to eat don't have that resistance. [Biotechnologically]-derived crops hold unbelievable promise and some risks, even if those risks are exceedingly small. But there must be more open dialogue about the risks of the technology, as well as the risks of not using the technology." Meanwhile, research can also focus on altering the environment to temper salt damage to plants. Concludes Gaxiola, "Salt tolerance of plants will work if at the same time we take care of the problem of water quality. Low quality water will increase the level of salt in soil to levels that not even transgenic plants can help. Treatment of water sources is as important as generating transgenic plants." Ricki Lewis (rickilewis@nasw.org) is a contributing editor. References 1. R. Gaxiola et al., "Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump," Proceedings of the National Academy of Sciences (PNAS), 98:11444-9, Sept. 25, 2001. 2. H. Zhang and E. Blumwald, "Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit," Nature Genetics, 19:765-70, August 2000. 3. H. Zhang et al, "Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation," PNAS, 98:12832-6, Oct. 23, 2001.

Using Transgenesis to Create Salt-Tolerant Plants

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