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ENHANCED TOLERANCE TO ABIOTIC STRESS IN TRANSGENIC TOBACCO BY OVEREXPRESSION OF A ZINC-FINGER PROTEIN GENE FROM RICE
Plants are constantly challenged by changes in environmental conditions. The increasing need for food and consumer preferences necessitates that crop plants are grown in regions where they are not naturally adapted, leading to stress. Reclaiming lands from the sea, seepage of saline water, and excess irrigation lead to increased salinity that plants have to tolerate. Excessive rainfall, flash flooding, or prolonged flooding causes hypoxia and anoxia. A paucity of water due to infrequent rain and inadequate irrigation leads to drought stress. Most of these stresses produce oxidative stress in the plant cells. Man's activity also produces xenobiotic pollutants such as factory wastes, aerosols, refrigerants, aviation and motor fuel exhaust gases, as well as biotic pollutants such as sewage, which constantly challenge plants. Plant response to stress is complex, as stress may occur at different stages of plant development or plants may experience more than one stress at a time1. Stress in all its forms has negative effects on plant development and productivity. Plants respond to salinity by reducing leaf growth through inhibition of cell division and expansion. The decrease in the osmotic potential of root cells causes inhibition of water uptake and dehydration of the plants, leading to the death of tissues, organs, and eventually the whole plant. Chilling stress stunts plant growth, brings about cellular autolysis and senescence, and has detrimental effects on flower induction, pollen production, and germination. Chilling and desiccation damage cell membranes while oxidative stress targets membranes, proteins, and DNA. A serious challenge today is to sustain and improve crop yields to feed the growing world population. Rice is a staple food in much of the world and hence increasing its production is of special interest. Though rice production has increased over the years, it will need to increase another 60% by 2025 to feed the growing population2. Traditional breeding strategies have been used to exploit natural genetic variation in improving crop varieties, but until now, very few plants showing enhanced tolerance to stresses and better yields have actually made it to the fields. Thus, genetically engineering plants to increase stress tolerance is a desirable alternative to breeding. Several genes have been reported to be up- or down-regulated in response to different stresses. These genes might generate products either directly involved in protection against environmental stress or that play a role in stress regulation. The first category would constitute genes coding for osmoprotectants, scavengers of reactive oxygen species, or stress proteins such as COR or LEA with an undefined mechanism of action. In the latter category would be genes that code for regulatory proteins such as transcription factors or components of signal cascades. These proteins would regulate the expression of a set of genes involved in stress. Both categories of genes have been shown to impart tolerance when overexpressed in plants. However, a desirable candidate gene of possible use in crop improvement would likely be a regulatory gene since it would have potential to play a broader role in stress tolerance, imparting tolerance to several stresses. The OSISAP1 gene was cloned via the differential screening of an indica rice cDNA library in an attempt to identify genes that show organ-specific and/or stress inducible expression3. OSISAP1 was expressed at a higher level in the root and the prepollination stage spikelet as compared to shoot. Further, expression analysis of OSISAP1 revealed that the gene is expressed in response to several abiotic stresses like cold, salt, drought, submergence, mechanical wounding, and heavy metals. OSISAP1 codes for a zinc-finger protein, which shows homology in the AN1-type zinc-finger region to the human and mouse PRK-1-associated protein AWP1, Phaseolus vulgaris pathogenesis-related protein PVPR3, human and mouse zinc-finger protein ZNF216, Xenopus laevis ubiquitin-like fusion protein XLULFP, and the ascidian posterior end mark protein PEM6. OSISAP1 also shows homology in the A20-like zinc-finger region to AWP1, hZNF216, and mZNF216. A part of the encoded protein also shows homology to human transcription factor NF-kB p65 subunit. The gene was overexpressed in tobacco under the control of a constitutive CaMV35S promoter to understand its function, and especially to determine whether the gene has a role to play in stress response. Transgenic lines were analyzed for cold, dehydration, and salt stress tolerance in the T1 generation. Germination, fresh weight gain, and bleaching were used as main parameters for comparing the response of nontransgenic and transgenic lines to different abiotic stresses3. Fresh weight of nontransgenic and transgenic seedlings was measured after 15 days of a recovery period following 15 days of cold stress. Nontransgenic seedlings could gain only 35% fresh weight in comparison to 67—92% fresh weight gained by stressed transgenics when compared to unstressed seedlings. Morphologically, transgenics show better recovery since the third or fourth leaf had already emerged, whereas in the nontransgenic seedlings only the first two leaves could be observed. The transgenic lines were evaluated for dehydration stress tolerance by germinating the nontransgenics and transgenics on 0.3 M and 0.4 M mannitol. Only 60% of nontransgenics germinated while all the transgenics with the exception of one line showed » 90% germination. The OSISAP1 overexpressing lines were also analyzed for tolerance to salt stress. Salt stress was given for 4 days and the difference between the fresh weight of nontransgenics and transgenics was recorded on day 8 of recovery. The transgenics performed better both in terms of fresh weight and retention of green color3. The exact mechanism of how OSISAP1 overexpression confers tolerance to different abiotic stresses remains to be worked out. Several other stress responsive genes with unknown function have been reported to confer tolerance to plants upon overexpression. Overexpression of COR15a from Arabidopsis leads to increased freezing tolerance4. Similarly, HVA1 from barley was overexpressed in rice and found to confer stress tolerance5. Like COR15a and HVA1, OSISAP1 is also a hydrophilic protein that may contribute an increased tolerance to abiotic stress. It may also act as a signal cascade component using the zinc fingers for protein-protein or protein-DNA interactions. Furthermore, OSISAP1 shows homology to mammalian A20 protein. The mammalian counterpart has been shown to inhibit tumor necrosis factor-induced apoptosis through inhibition of NF-kappa B mediated gene expression6. Thus, it could be possible that OSISAP1 overexpression leads to less stress-associated injuries like chlorosis and cell death, and hence the transgenics performed better than the nontransgenics. OSISAP1 could be a promising target for producing stress tolerant crops because it is inducible by different kinds of abiotic stresses and, upon ectopic overexpression, the transgenics show improved tolerance to cold, dehydration, and salt stress. It is quite possible that the gene may have a role in imparting tolerance to other stresses, which causes an increase in its transcription in rice. References1. Chinnusamy V, Schumaker K, & Zhu J-K. (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signaling in plants. J. Exp. Bot. 55:225-236. 2. Khush GS. (1997) Origin, dispersal, cultivation and variation of rice. Plant Mol. Biol. 35, 25-34. 3. Mukhopadhyay A, Vij S, & Tyagi AK. (2004) Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco. Proc. Natl. Acad. Sci. USA 101:6309-6314. 4. Artus NN et al. (1996) Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Proc. Natl. Acad. Sci. USA 93:13404-13409. 5. Xu D et al. (1996) Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol. 110: 249-257. 6. De Valck D et al. (1997) A20 inhibits NF-kappaB activation independently of binding to 14-3-3 proteins. Biophys. Biochem. Res. Commun. 238: 590-594. Akhilesh K. Tyagi, Arnab Mukhopadhyay and Shubha Vij
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