Recent advances in plant and microbial genomics have provided new and exciting molecular tools that allow scientists to dissect the biochemical and molecular mechanisms of stress tolerance or resistance. This information can be utilized to develop new crop plants that are protected from the impact of abiotic stresses. Since many of these major abiotic stresses arise as a result of a common biochemical phenomenon, efforts to improve tolerance to one
abiotic stress have potential to confer tolerance to other abiotic stresses. Recently, new scientific efforts have focused on modifying higher plants with stress tolerance-conferring genes. These efforts have demonstrated enhanced abiotic stress tolerance to drought, salinity, temperature, oxidative, pH, heavy metal and flooding stress, and have also provided validation to the concept of enhancing abiotic stress tolerance in crop plants via biotechnology.
Drought and salinity are the major and most widespread environmental stresses that substantially constrain crop productivity in both non-irrigated and irrigated agriculture (Epstein et al., 1980). The detrimental impact of salinity in agriculture is exacerbated by irrigation management practices used to increase crop outputs. Cellular water deficits due to salinity, drought or temperature can result in changes in cell volume, including loss of turgor, solute concentration, ion displacement, membrane structure and integrity and protein denaturation, as well as alterations of various cellular processes (Bray, 1997). Due to the large economic impact of these abiotic stresses, much of the initial biotech-nological research has centred on drought and salinity tolerance. Recent metabolic engineering reports have demonstrated that the accumulation of low molecular weight osmoprotectants in the form of sugars (fructan), poly-hydroxylated sugar alcohols (polyols), amino acids such as proline, and quartenary amines such as glycinebetaine provide a mechanism for osmoprotection and osmotic adjustment in plants.
Pilon-Smits et al. (1995) demonstrated that the drought tolerance of tobacco plants could be improved by engineering the plants to produce increased fructan sugar levels. Tobacco (Nicotiana tabacum) plants engineered with the bacterial sacB gene, which encoded a fructosyl transferase enzyme targeted to the plant vacuole, exhibited a 55% increase in growth rate and a 59% increase in biomass production relative to wild-type control plants under water-deficit conditions. The researchers concluded that the introduction of fructans into non-fructan-producing species will potentially enhance resistance to drought stress.
Trehalose, a non-reducing disaccharide, has been implicated as a component in the protective role in plants that can withstand complete dehydration (Drennan et al., 1993). Overproduction of trehalose in transgenic tobacco was achieved by introducing the trehalose-6-phosphate synthase gene of yeast under the control of the Rubisco small subunit promoter (Holstrom et al., 1996). Trehalose-producing plants exhibited both increased water retention and drought tolerance at various stages of development, though the cellular levels of trehalose were small (0.5 mM in cytosol).
Many photosynthetic organisms accumulate proline naturally in response to osmotic stress. The accumulation of proline is believed to serve as a redox sink, radical scavenger, pH stabilizer and protective solute that provides drought and salinity tolerance to the cells (Kishor et al., 1995). The mothbean structural gene for D1-pyrroline-5-carboxylate synthase (a bifunctional enzyme that catalyses the conversion of glutamate to A1-pyrroline-5-carboxylate, the immediate precursor to proline) has been introduced into tobacco (Kishor et al., 1995). The transgenic plants produced 10- to 18-fold more proline and exhibited a relatively higher osmotic potential in the leaf sap than controls under drought stress. In addition, the proline-overproducing plants had enhanced biomass and flower development under salinity-stress conditions.
The ability to engineer production of the osmoprotectant mannitol in higher plants has been demonstrated in both Arabidopsis (Thomas et al., 1995) and tobacco (Tarczynski et al., 1993). Seeds from Arabidopsis plants overexpressing the bacterial mltd gene encoding mannitol 1-phosphate dehydrogenase exhibited enhanced germination rates in the presence of elevated salt concentrations. Similarly, tobacco plants expressing the mltd gene produced a maximum concentration of mannitol of 100 mM, which led to increased biomass production under high saline conditions relative to the control.
Overproduction of cyclic sugar alcohols (cyclitols, such as ononitol) by expression of the lmt1 gene that encodes a myo-inositol o-methyl transferase in tobacco was used by Vernon et al. (1993) as a model system to test the role of cyclitol overproduction in conferring tolerance to osmotic stress. However, no conclusive evidence was presented to correlate overproduction of cyclitols and stress tolerance in these transgenic plants.
Saneko et al. (1995) demonstrated that glycinebetaine-containing maize (Zea mays L.) lines were less sensitive to salinity stress than were mutants deficient in glycinebetaine production, thus providing evidence of the protective potential of this osmolyte. By introducing the Escherichia coli betA gene that encodes choline dehydrogenase into tobacco, Lullis et al. (1996) demonstrated increased salinity tolerance in the transgenic plants. Tobacco plants expressing the betA gene exhibited increased glycinebetaine production that resulted in an 80% increase in salt tolerance over wild-type plants, as measured by dry weight accumulation. Similar results have been achieved in Arabidopsis by overexpressing the bacterial codA gene for choline oxidase (Hayashi et al., 1997). Arabidopsis plants overexpressing codA have also shown tolerance to cold stress and exhibited stability of photosystem II and normal germination under high salt.
In addition to small molecules playing a role in plant stress tolerance, a series of proteins known as late embryogenesis abundant (LEA) proteins, first identified in plant embryos undergoing desiccation, have been implicated as having a role in stress tolerance. An LEA protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice (Oryza sativa L.) plants under the regulation of the rice actin promoter (Xu et al., 1996). Transgenic HVA1 rice plants showed significant increases in tolerance to water deficits and salinity stress. Increased tolerance was exhibited by increased growth rates under stress, delayed development of damage symptoms, and improved recovery upon removal of the stress.
Although the ability of the aforementioned effectors to increase water-deficit tolerance in plants has been demonstrated, it is possible that some of these single effectors may not be fully adequate to confer substantial stress tolerance to a wide range of plants. The ability to control several effectors in a coordinated manner may be required to achieve consistent stress tolerance. To this end, recent engineering efforts have focused on modulating signal transduction cascades and transcriptional regulators as a means of altering plant endogenous stress-coping mechanisms. Pardo et al. (1998) demonstrated that overexpression of a truncated form of calcineurin (CaN, a Ca2+-calmodulin-dependent protein phosphatase involved in salt-stress signal transduction in yeast) mediated salt adaptation in transgenic tobacco plants. The modified plants exhibited less perturbation of root growth and increased shoot survival. The increased tolerance of plants with the CaNtransgene in the segregating population provides evidence that a common signal pathway exists in yeast and plants, and that heterologous protein is capable of modulating stress tolerance.
Genetic engineering studies with melon cultivars (Cucumis melo) to express the yeast salt-tolerance gene HAL1 (a gene encoding a water-soluble protein that modulates monovalent ion channels) showed that placing trans-genic melon plants under high salt conditions reduced root and vegetative growth. Plants containing this trait consistently had a higher level of tolerance than the control plants without this trait (Bordas et al., 1997).
Plants vary tremendously in their ability to survive both high and low temperature extremes. What determines the ability of plants to survive or adapt to temperature extremes is only partially understood, but the need to alter crop plants to withstand temperature extremes is evident. As weather patterns change due to human impact, it will be increasingly important to develop crop species that function in broader geographical and temperature ranges.
Low temperature is one of the major environment stresses that limits plant growth. Significant advances in understanding cold tolerance and modifying cold susceptibility in plants have been made using biotechnology. The increase of plastid membrane fatty acid desaturation has been correlated with increased cold tolerance in plants (Steponkus et al., 1993). Several attempts have been made to modify cold tolerance via altering the fatty acid composition of plants. Transgenic tobacco overexpressing the Arabidopsis w-3 fatty acid desaturase (Fad7) gene were analysed for altered cold tolerance (Kodama et al., 1994). When exposed to 1°C temperatures for 7 days and then returned to optimal temperatures, transgenic Fad7 plants did not exhibit the growth suppression and chlorosis that was displayed by the wild-type controls. Thus, the transgenic alteration appeared to have protected the tobacco from cold injury. The cold tolerance was increased due to the overexpressing of the Fad7gene.
A broad specificity D9-desaturase gene (Des9) from Anacystis nidulans was fused to a plant chloroplast-targeting sequence, and was introduced into tobacco by Ishizaki-Nishizawa et al. (1996). They demonstrated a 17-fold increase of D9-monosaturated fatty acids in transgenic plants. When these plants were exposed to 1°C for 11 days, they showed no signs of chlorosis. This suggests that plants with the increased D9-monosaturated fatty acids were protected from low-temperature injury.
Since freezing temperatures modify the cellular water balance, plants that normally show drought or salinity tolerance also have tolerance to subfreezing temperatures. Transgenically modified plants that have increased drought and salinity tolerance, apparently because of increased amounts of low molecular weight osmolytes, also have increased low-temperature tolerance. Arabidopsis plants overexpressing the codA gene exhibited elevated levels of glycine-betaine and proved to have both improved salinity tolerance and enhanced cold tolerance (Hayashi et al., 1997). When the bacterial betA gene for choline dehydrogenase was introduced into potato (Solanum tuberosum L.), a cold-tolerant phenotype was observed (Holmberg and Bulow, 1998).
Many of the genes induced in cold-acclimated plants encode hydrophilic proteins (COR proteins) of undetermined function and these are believed to play a key role in cold tolerance (Thomashow, 1998). Transgenic Arabidopsis plants expressing one of the COR proteins (COR15am) constitutively showed that chloroplasts were 1—2 °C more tolerant than non-transgenic control plastids (Artus et al., 1996).
Formation of ice crystals in plant cells exposed to transient freezing temperatures leads to a mechanical disruption that kills the cells and can impact both crop productivity and harvested produce quality. Many freezing-tolerant plants contain antifreeze proteins that lower the temperature at which cellular ice forms (Thomashow, 1998). Antifreeze proteins in the blood of polar fishes has been shown to inhibit ice nucleation. Hightower et al. (1991) demonstrated that introduction of a gene encoding a fish antifreeze protein (AFP) into tobacco inhibited ice recrystallization in the transgenic tissue. Although no clear demonstration of freezing tolerance was demonstrated in these transgenic plants, these experiments provide some validation for using AFPs to confer tolerance to rapid freezing in plants.
Less effort has been put into engineering high-temperature tolerance in plants than low-temperature tolerance. Often, breeders and physiologists have associated heat stress with drought stress when assessing yield stability and crop performance in the field. It is believed that high temperature stress on plant growth and development occurs in photosynthetic functions and reproductive processes such as flowering, pollination and seed set. Plant cells respond to heat stress by rapidly accumulating heat shock proteins (HSPs). Although there is only correlative evidence for HSPs protecting cells from high-temperature stress, attempts have been made with some success to modify plant thermotolerance by overexpressing HSP—protein fusions. Lee et al. (1995) and Hinderhofer et al. (1998) have demonstrated that the basal thermotolerance of Arabidopsis can be increased when HSP-reporter fusions genes are introduced into plants.
Jaglo-Ottosen et al. (1998) demonstrated the ability to engineer freezing tolerance in Arabidopsis by constitutively expressing the Arabidopsis low-temperature transcription activator protein CBF-1. By modifying the expression of this protein, identified as one of the master switches of cold-tolerance inducing genes, the researchers demonstrated that it is possible to redirect the genetic resources of a plant to be cold tolerant in the absence of an acclimation period. This lends credence to the ability to modify thermo-tolerance and other stress responses via engineering of plant signal and regulatory pathways.
Over the past decade, modern agricultural practices such as the use of sewage sludge have resulted in contamination of arable soils with heavy metals. In addition, runoff from mining and smelting operations has produced large areas of land contaminated with copper (Cu) and zinc (Zn) (Petalino and Collins, 1984). Aluminium (Al) is the most common metal in the earth's crust (predominantly in the form of insoluble aluminosilicates). When present in the solubilized form (primarily as Al3+) in acid soils with pH of 5.5 or less, Al becomes toxic to many crop plants and presently is believed to represent a major obstacle to crop yield and productivity on 40% of the world's arable soils (Ryan et al., 1995). Soil acidification is accelerated by certain farming practices and by occurrence of acid rain. In order to maintain productivity in soils that are adversely affected by increased levels of these pollutants, consistent applications of lime are required. This remedial practice may itself create runoff pollution, thus generating additional water quality issues. The increasing levels and areas of contaminated soils require the development of crop plants that are capable of growth in the presence of heavy metals and the sequestration of heavy metals in non-edible plant parts. This must be done through either conventional breeding or genetic engineering.
The primary effect of Al toxicity on plants is the inhibition of root growth. This results in nutrient deficiency and reduction of growth, and thus results in a negative impact on crop yield (Ryan et al., 1995). One mechanism that some Al-tolerant plant species are postulated to utilize is the release of organic acids such as citric acid, which chelates Al outside the cell plasma membrane, thus preventing its uptake (Miyasaka et al., 1991). De la Fuente et al. (1997) demonstrated that transgenic tobacco and papaya (Carica papaya L.) plants, engineered to overproduce citrate, resulted in tolerance to significant levels of Al. Tobacco and papaya plants were engineered with the bacterial gene that encodes citrate synthase (CSb) under the regulation of a constitutive promoter. Plants expressing the CSb gene exhibited a tenfold higher level of citrate in their roots and exhibited root growth at concentrations of 300 ||M Al, whereas the controls showed severe inhibition at 50 ||M.
An alternative solution to heavy metal tolerance is to bind and sequester the heavy metals within the plant to prevent their toxic effects, thus imparting heavy-metal resistance. Several researchers have utilized this approach by engineering plants with low molecular weight metal-binding proteins known as metallothioneins. The metallothioneins (MTs) are found in mammals and fungi and function to detoxify heavy metals in these systems. Transgenic brassica and tobacco plants have been created that constitutively express a mammalian MT-II gene (Misra and Gedamu, 1989). Plants bearing these constructs were shown to grow on toxic concentrations of cadmium (Cd) up to 100 ||M, whereas controls exhibited severe root/shoot stunting and chlorosis. This approach was also used to engineer tobacco plants with the mouse (MT-I) gene under the control of a constitutive promoter (Pan et al., 1994). Transgenic MT-1 tobacco plants were unaffected by concentrations of cadmium up to 200 mM, whereas control plants exhibited chlorosis at 10 ||M. These early experiments provide promising evidence that crop plants can be engineered to grow in these heavy-metal-laden soils.
A frequent consequence of environmental stress is the phenomenon of oxidative stress. These oxidative events arise from the disruption of various cellular responses that generate reactive oxygen species which cause oxidative damage in the cell. These oxidative stresses can also arise directly from exposure to ozone and pesticides. Although the plant system for protection against secondary oxidative stress damage is complex, the key enzymatic defences are superoxide dismutases (SODs) (Bowler et al., 1992). Three different classes of SODs have been identified in plants and are separated on the basis of their cofactor metal: (i) copper/zinc SOD in cytosol and chloroplast; (ii) iron SOD in chloroplast; and (iii) manganese SOD in the mitochondria (Bowler et al., 1994). Several researchers have reported improved performance in tobacco and lucerne (Medicago sativa L.) plants under oxidative stress by overexpressing distinct SOD genes targeted to plant organelles (Van Camp et al., 1994; Aono et al., 1995; Slooten et al., 1995; Mckersie et al., 1996; Van Camp et al., 1996). In addition, Aono et al. (1995) demonstrated that a combination of SOD and the gene for glutathione reductase exhibited greater protection against oxidative stress than either gene alone.
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