Thomashow, M; Stockinger, E; Jaglo-Ottosen, K; Zarka, D

US Patent: 5929305, , 27 Jul 1999

A gene, designated as CBF1, encoding a protein, CBF1, which binds to a region regulating expression of genes which promote cold temperature and dehydration tolerance in plants is described. CBF1 is used to transform microorganisms and can be used to transform plants.

Kasuga, M; Liu, Q; Miura, S; Yamaguchi-Shinozaki, K*; Shinozak, K

Nature Biotechnology [Nat. Biotechnol.], vol. 17, no. 3, pp. 287-291, Mar 1999

Plant productivity is greatly affected by environmental stresses such as drought, salt loading, and freezing. We reported previously that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses. The transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic plants activated the expression of many of these stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter.

Sakamoto, A; Murata, N*

Plant Molecular Biology [Plant Mol. Biol.], vol. 38, no. 5, pp. 1011-1019, Dec 1998

Genetically engineered rice (Oryza sativa L.) with the ability to synthesize glycinebetaine was established by introducing the codA gene for choline oxidase from the soil bacterium Arthrobacter globiformis. Levels of glycinebetaine were as high as 1 and 5 mu mol per gram fresh weight of leaves in two types of transgenic plant in which choline oxidase was targeted to the chloroplasts (ChlCOD plants) and to the cytosol (CytCOD plants), respectively. Although treatment with 0.15 m NaCl inhibited the growth of both wild-type and transgenic plants, the transgenic plants began to grow again at the normal rate after a significantly less time than the wild-type plants after elimination of the salt stress. Inactivation of photosynthesis, used as a measure of cellular damage, indicated that ChlCOD plants were more tolerant than CytCOD plants to photoinhibition under salt stress and low-temperature stress. These results indicated that the subcellular compartmentalization of the biosynthesis of glycinebetaine was a critical element in the efficient enhancement of tolerance to stress in the engineered plants.

Apse, MP; Aharon, GS; Snedden, WA; Blumwald, E*

Science (Washington) [Science (Wash.)], vol. 285, no. 5431, pp. 1256-1258, 20 Aug 1999

Agricultural productivity is severely affected by soil salinity. One possible mechanisms by which plants could survive salt stress is to compartmentalize sodium ions away from the cytosol. Overexpression of a vacuolar Na super(+)/H super(+) antiport from Arabidopsis thaliana in Arabidopsis plants promotes sustained growth and development in soil watered with up to 200 millimolar sodium chloride. This salinity tolerance was correlated with higher-than-normal levels of AtNHX1 transcripts, protein, and vacuolar Na super(+)/H super(+) (sodium/proton) antiport activity. These results demonstrate the feasibility of engineering salt tolerance in plants.

Siddiqui, NU; Chung, H; Thomas, TL; Drew, MC

Plant Physiology [Plant Physiol.], vol. 118, no. 4, pp. 1181-1190, Dec 1998

We studied the expression of three promoter 5' deletion constructs (-218, -599, and -1312) of the LEA (late embryogenesis abundant)-class gene Dc3 fused to beta -glucuronidase (GUS), where each construct value refers to the number of base pairs upstream of the transcription start site at which the deletion occurred. The Dc3 gene is noted for its induction by abscisic acid (ABA), but its response to other plant hormones and various environmental stresses has not been reported previously for vegetative cells. Fourteen-day-old transgenic tobacco (Nicotiana tabacum L.) seedlings were exposed to dehydration, hypoxia, salinity, exogenous ethylene, or exogenous methyl jasmonate (MeJa). GUS activity was quantified fluorimetrically and expression was observed by histochemical staining of the seedlings. An increase in GUS activity was observed in plants with constructs -599 and - 1312 in response to dehydration and salinity within 6 h of stress, and at 12 h in response to hypoxia. No increase in endogenous ABA was found in any of the three lines, even after 72 h of hypoxia. An ABA-independent increase in GUS activity was observed when endogenous ABA biosynthesis was blocked by fluridone and plants were exposed to 5 mu L L super(-1) ethylene in air or 100 mu M MeJa. Virtually no expression was observed in construct -218 in response to dehydration, salinity, or MeJa, but there was a moderate response to ethylene and hypoxia. This suggests that the region between -218 and -599 is necessary for ABA (dehydration and salinity)- and MeJa-dependent expression, whereas ethylene- mediated expression does not require this region of the promoter.

Grover, A; Pareek, A; Singla, SL; Minhas, D; Katiyar, S; Ghawana, S; Dubey, H; Agarwal, M; Rao, GU; Rathee, J; Grover, A

Current Science [Curr. Sci.], vol. 75, no. 7, pp. 689-696, 10 Oct 1998

Plant genetic engineering took birth in the mid-eighties when, for the first time, plants were successfully engineered for improved virus, herbicide and insect resistance. This sphere has been ever-increasing since then. Abiotic stresses (such as high salt levels, low water availability leading to drought, excess water leading to flooding, high and low temperature regimes, etc.) adversely affect crop plants. The genetic responses of plants to these stresses are complex involving simultaneous expression of a number of genes. Till the early-nineties it was inconceivable that there would be any success in attaining the goal of improving resistance of crop plants to abiotic stresses. Continuing efforts of the stress biologists have resulted in engineering of plants resistant to low temperature, high temperature and excess salinity. A satisfactory progress has also been achieved in the area of generating plants resistant to water stress and flooding. While what has been achieved is impressive, it is still a challenging task to pyramid useful genes for high-level resistance to such stresses. The limiting factor in extension of biotechnology to abiotic stresses is the lack of information on what are the `useful genes'--genes which would lead to better stress tolerance. We have reviewed how these genes are being searched to enable further development of strategies for stress management in crop plants. This is important because the strategies for coping with the abiotic stresses (and also for several other applications in plant biotechnology) have also come through the research work of scientists working on as diverse organisms as bacteria and fish.

Hayashi, Hidenori; Alia; Sakamoto, Atsushi; Nonaka, Hideko; Chen, Tony HH; Murata, Norio

Journal of Plant Research [J. Plant Res.], vol. 111, no. 1102, pp. 357-362, Jun 1998

Arabidopsis thaliana was transformed previously with the codA gene from the soil bacterium Arthrobacter globiformis. This gene encodes choline oxidase, the enzyme that converts choline to glycinebetaine. Transformation with the codA gene significantly enhanced the tolerance of transgenic plants to low temperature and high-salt stress. We report here that seeds of transgenic plants that expressed the codA gene were also more tolerant to salt stress during germination than seeds of non-transformed wild-type plants. Seedlings of transgenic plants grew more rapidly than those of wild-type plants under salt-stress conditions. Furthermore, exogenously applied glycinebetaine was effective in alleviating the harmful effects of salt stress during germination of seeds and growth of young seedlings, a result that suggests that it was glycinebetaine that had enhanced the tolerance of the transgenic plants. These observations indicate that synthesis of glycinebetaine in transgenic plants in vivo, as a result of the expression of the codA gene, might be very useful in improving the ability of crop plants to tolerate salt stress.

Karakas, B; Ozias-Akins, P; Stushnoff, C; Suefferheld, M; Rieger, M

Plant, Cell & Environment [PLANT, CELL ENVIRON.], vol. 20, no. 5, pp. 609-616, May 1997

Tobacco plants (Nicotiana tabacum L.) were transformed with a mannitol-1-phosphate dehydrogenase gene resulting in mannitol accumulation. Experiments were conducted to determine whether mannitol provides salt and/or drought stress protection through osmotic adjustment. Non-stressed transgenic plants were 20-25% smaller than non-stressed, non-transformed (wild-type) plants in both salinity and drought experiments. However, salt stress reduced dry weight in wild-type plants by 44%, but did not reduce the dry weight of transgenic plants. Transgenic plants adjusted osmotically by 0.57 MPa, whereas wild-type plants did not adjust osmotically in response to salt stress. Calculations of solute contribution to osmotic adjustment showed that mannitol contributed only 0.003-0.004 MPa to the 0.2 MPa difference in full turgor osmotic potential ( pi sub(o)) between salt-stressed transgenic and wild-type plants. Assuming a cytoplasmic location for mannitol and that the cytoplasm constituted 5% of the total water volume, mannitol accounted for only 30-40% of the change in pi sub(o) of the cytoplasm. Inositol, a naturally occurring polyol in tobacco, accumulated in response to salt stress in both transgenic and wild-type plants, and was 3-fold more abundant than mannitol in transgenic plants. Drought stress reduced the leaf relative water content, leaf expansion, and dry weight of transgenic and wild-type plants. However, pi sub(o) was not significantly reduced by drought stress in transgenic or wild-type plants, despite an increase in non-structural carbohydrates and mannitol in droughted plants. We conclude that (1) mannitol was a relatively minor osmolyte in transgenic tobacco, but may have indirectly enhanced osmotic adjustment and salt tolerance; (2) inositol cannot substitute for mannitol in this role; (3) slower growth of the transgenic plants, and not the presence of mannitol per se, may have been the cause of greater salt tolerance, and (4) mannitol accumulation was enhanced by drought stress but did not affect pi sub(o) or drought tolerance.

Bordas, M; Montesinos, C; Dabauza, M; Salvador, A; Roig, LA; Serrano, R; Moreno, V*

Transgenic Research [TRANSGEN. RES.], vol. 6, no. 1, pp. 41-50, Jan 1997

An Agrobacterium-mediated gene transfer method for production of transgenic melon plants has been optimized. The HAL1 gene, an halotolerance gene isolated from yeast, was inserted in a chimaeric construct and joined to two marker genes: a selectable-neomycin phosphotransferase-II (nptII)-, and a reporter- beta -glucuronidase (gus)-. The entire construct was introduced into commercial cultivars of melon. Transformants were selected for their ability to grow on media containing kanamycin. Transformation was confirmed by GUS assays, PCR analysis and Southern hybridization. Transformation efficiency depended on the cultivar, selection scheme used and the induction of vir-genes by the addition of acetosyringone during the cocultivation period. The highest transformation frequency, 3% of the total number of explants cocultivated, was obtained with cotyledonary explants of cv. 'Pharo'. Although at a lower frequency (1.3%), we have also succeeded in the transformation of leaf explants. A loss of genetic material was detected in some plants, and results are in accordance with the directional model of T-DNA transfer. In vitro cultured shoots from transgenic populations carrying the HAL1 gene were evaluated for salt tolerance on shoot growth medium containing 10 gl super(-1) NaCl. Although root and vegetative growth were reduced, transgenic HAL1-positive plants consistently showed a higher level of tolerance than control HAL1-negative plants.