Annex II Genetic Engineering for Abiotic Stress Tolerance in Plants

SDR/TAC:IAR/01/27

CONSULTATIVE GROUP ON INTERNATIONAL AGRICULTURAL RESEARCH
TECHNICAL ADVISORY COMMITTEE

TAC SECRETARIAT
FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS
September 2001

Genetic Engineering for Abiotic Stress Tolerance in Plants^[1]

1. Introduction

A number of abnormal environment parameters such as drought, salinity, cold, freezing, high temperature, anoxia, high light intensity and nutrient imbalances etc. are collectively termed as abiotic stresses.

Abiotic stresses lead to dehydration or osmotic stress through reduced availability of water for vital cellular functions and maintenance of turgor pressure. Stomata closure, reduced supply of CO2 and slower rate of biochemical reactions during prolonged periods of dehydration, high light intensity, high and low temperatures lead to high production of Reactive Oxygen Intermediates (ROI) in the chloroplasts causing irreversible cellular damage and photo inhibition.

In response to dehydration or osmotic stress a series of compatible osmolytes are accumulated for osmotic adjustment, water retention and free radical scavenging. Similarly, overexpression of certain enzymes such as superoxide dismutase, ascorbate peroxidase and glutathione reductase has been implicated in free radical detoxification and scavenging of free radicals under oxidative stress.

2. Complexed stresses by osmoticum, dehydration and salinity

Proline has been recognized as a potent and compatible osmoprotectant which is accumulated in high concentrations in glycophytes and halophytes in response to osmotic stress such as drought and high salinity. Two important enzymes for the biosynthesis of proline i.e. Ä1 - pyrroline-5-carboxylate synthetase (P5CS) and Ä1 - pyrroline-5-carboxylate reductase (P5CR) have been cloned from several plants and their expression studied under various abiotic stresses and ABA application.

Some transgenic plants expressing a high level of P5CS mRNA also accumulated high level of P5CS protein. The transgenic plants produced 10 to 18-fold more proline than the control plants. Under drought stress the proline content increased from about 80mg/g fresh leaf (before stress) to about 3000mg/g (after stress) in control and from 1000mg/g to an average of 6500mg/g in transgenic lines. Wilting in the transgenic plants was less severe and delayed by 2-3 days in transgenic plants as compared with the wild type (WT) control plants. Their results demonstrated that proline acts as an osmotic protectant and its increased production in the transgenic plants increased tolerance to drought and salt stress.

Glycinebetaine, a quaternary amine, is another important compatible solute, which is widely distributed among plants and protects plants on exposure to salt and cold stress. In plants like spinach and barley betaine is synthesized from choline by oxidation of choline to betaine aldehyde and then to betaine. The first step is catalyzed by choline mono-oxygenase while the second by nuclear coded gene for betaine aldehyde dehydrogenase. The transformed plants grew slowly at 200 mM NaCl whereas none of the WT plants grew. The accumulation of glycine betaine through genetic engineering in Arabidopsis enhanced its ability to tolerate salt and cold stress.

3. Anaerobiosis/anoxia

Most plants are highly sensitive to anoxia during submergence. An important aspect of the adaptation to oxygen limitation include metabolic changes such as avoidance of self poisoning and cytoplasmic acidosis, maintenance of adequate supplies of energy and sugar. During anoxia, ATP and NAD+ are generated not in the Krebs cycle and the respiratory chain but via glycolysis and fermentation. A number of enzymes of the anaerobic pathways such as alcohol dehydrogenase and pyruvate decarboxylase induced during anoxia have been cloned and characterized.

4. Heavy metal

Optimum growth and productivity and even cultivation of most of the plants is severely restricted in soils with elevated levels of one or more inorganic ions such as sodium in saline soils; Al, and Mn in acidic soils and heavy metals Cu, Zn Pb, Ni, Cd etc. due to mining, industrial affluents and other human activities.

In plants with genetic resistance to Al toxicity, the Al exclusion and uptake from root tips have been found to be correlated to their increased capacity to release organic acids such as citric acid which chelates Al3+ outside the plasma membrane. Transgenic tobacco and papaya that overexpressed a citrate synthase gene (CSb) from Pseudomonas aeruginosa in their cytoplasm. Tobacco lines expressing CSb had up to 10-fold higher level of citrate in their root tissues and one of the lines released 4-fold citrate extracellularly whereas in papaya there was only 2 to 3-fold increase of citric acid production. Increased production of citric acid was shown to result in Al tolerance in both the species.

5. Heat and Cold

Temperate and subtropical plants are highly susceptible to high temperature during early tillering, flower initiation, anthesis and grain filling stages leading to substantial reduction in their productivity. In response to high temperature all organisms, including plants, synthesize a set of proteins called as heat shock proteins (HSPs) which have been classified into several families according to their molecular masses. The induction of HSPs at permissive temperatures have been associated with the acquisition of thermotolerance to withstand short periods of an otherwise lethal temperature.

The chilling sensitivity of plants is closely correlated with the degree of unsaturation of fatty acids in the phosphatidylglycerol of chloroplast membranes. Plants with a high proportion of cis-unsaturated fatty acids, such as spinach and Arabidopsis, are resistant to chilling, whereas species like squash with only a small proportion are not. The chloroplast enzyme glycerol-3-phosphate acyltransferase seems to be important for determining the level of phosphatidylglycerol fatty acid unsaturation. Thus they demonstrated for the first time that the level of fatty acid unsaturation of phosphatidylglycerol and the degree of chilling sensitivity of tobacco can be manipulated by transformation with cDNAs for glycerol-3-phosphate acyltransferases from squash and Arabidopsis.

6. Shading

Optimum supply of nutrients and efficient photosynthesis are conducive to biomass production but the allocation of assimilates within the developing plant determines the harvest index and economic yield. In pure stand canopy as well as in mixed cropping, competition for light energy invokes shade avoidance syndrome manifested by rapid growth and extension of stem and petiole at the expense of leaves, storage and reproductive organs thus predisposing plants to lodging, susceptibility to diseases and insect pests and a lower harvest index. Although the development of semi-dwarf varieties of wheat and rice in the 60s has led to their higher harvest index and grain yield by overcoming some of the defects of the tall genotypes yet the competition among plants for light energy continues to operate in canopies under intensive cultivation practices. The photosynthetic pigments in plants absorb the visible radiation (400-700 nm) and reflect and transmit far red (FR) radiation beyond 700 nm. The FR wave band between 700-800 nm predominating in the dense plant stands have been implicated in proximity perception for initiating shade avoidance syndrome. The FR reflection signals are perceived by the photoreceptors called phytochromes which possess distinct photo sensory functions. Phytochrome (phyA) mediating the inhibition of stem growth on etiolated plants in response to FR wave length 710-720 nm is rapidly degraded and down regulated in light grown plants. Transgenic tobacco lines expressing a high level of heterologous oat phyA apoprotein have been produced.

The level of growth inhibition of transgenic plants correlated with the level of phyA production. Under field trials at various planting densities from 20 to 100 cm, the transgenic plants were indistinguishable from the WT plants at the lowest plant density but became progressively shorter as the plant density increased. This phenomenon termed as “proximity conditional dwarfing” led to a 15 to 20% increase in harvest index (expressed as leaf biomass as a proportion of total biomass) in transgenic plants under high plant density thus demonstrating the suppression of shade avoidance response under high level of phyA expression. Further understanding of the molecular basis of interaction of various phytochromes among themselves and with R: FR rations in natural light environment may help to change crop plant architecture to avoid shade stress and obtain maximum production under high plant density, mixed cropping and agroforestry.

7. UV - B

The high influxes and absorption of UV-B radiation affects terrestrial plants through damage to DNA directly or indirectly through formation of free radicals, membranes by peroxidation of unsaturated fatty acids, photosystemII, phytohormones and even symbiotic relationship of plants with micro-organisms.

A number of secondary metabolites such as flavonoids, tannins and lignins are increased at elevated levels of UV-B radiation which screen UV-B and protect the cellular components against the UV-B damage.

8. Oxidative stress

A number of abiotic stresses such as extreme temperatures, high light intensity, osmotic stresses, heavy metals and a number of herbicides and toxins lead to over production of reactive oxygen intermediates (ROI) including H2O2 causing extensive cellular damage and inhibition of photosynthesis.

9. Perspectives and strategies for improving tolerance

The work on genetic engineering of tolerance to abiotic stresses began piece meal within a decade of the molecular understanding of pathways induced in response to one or more of the abiotic stresses. In most of the cases the transgenes expressed faithfully but only a limited level of tolerance was provided under stress conditions as compared to the non-transformed wild type plants. In many cases the transgenic plants had morphological abnormalities and slower growth under nonstressed environment. The level of many compatible osmolytes responsible for osmotic adjustment was too low to be effective per se in providing the required water retention and osmotic adjustment.

The use of multiple tolerance mechanisms for one or more of the abiotic stresses through stepwise or co-transformation may help to achieve high levels of tolerance for commercial exploitation. The QTL mapping of stress tolerance in certain species, comparative mapping and map based cloning in plants may be used to screen genes which function under stress as well as those induced and expressed in response to stress.

Molecular understanding of the stress perception, signal transduction and transcriptional regulation of abiotic stress responsive genes may help to engineer tolerance for multiple stresses.

Understanding the molecular mechanism for providing protection against biotic and abiotic stresses may lead to a generalized master mechanism for stress tolerance. Optimum homeostasis is always a key to living organisms for adjusted environments. Thus, abiotic stress accompanying a number of biological phenomena must be precisely investigated by consideration of plant homeostasis.

Table 1. Genetic engineering of plants for tolerance to abiotic stresses

Table 2. Genetic engineering of plants for tolerance to heavy metal