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.

Kenward, KD; Brandle, J; McPherson, J; Davies, PL*

Transgenic Research [Transgenic Res.], vol. 8, no. 2, pp. 105-117, Apr 1999

Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfide-bonded 14 kDa protein was targeted for accumulation in its pro- and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% of the total protein present in the apoplast. Plant-produced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wild-type AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for large-scale production: but no difference in frost resistance was observed between transgenic and wild-type plants during the onset of early fall frosts.

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.

McKersie, BD; Bowley, SR; Jones, KS

Plant Physiology [Plant Physiol.], vol. 119, no. 3, pp. 839-848, Mar 1999

To test the hypothesis that enhanced tolerance of oxidative stress would improve winter survival, two clones of alfalfa (Medicago sativa) were transformed with a Mn-superoxide dismutase (Mn-SOD) targeted to the mitochondria or to the chloroplast. Although Mn-SOD activity increased in most primary transgenic plants, both cytosolic and chloroplastic forms of Cu/Zn-SOD had lower activity in the chloroplast SOD transgenic plants than in the nontransgenic plants. In a field trial at Elora, Ontario, Canada, the survival and yield of 33 primary transgenic and control plants were compared. After one winter most transgenic plants had higher survival rates than control plants, with some at 100%. Similarly, some independent transgenic plants had twice the herbage yield of the control plants. Prescreening the transgenic plants for SOD activity, vigor, or freezing tolerance in the greenhouse was not effective in identifying individual transgenic plants with improved field performance. Freezing injury to leaf blades and fibrous roots, measured by electrolyte leakage from greenhouse-grown acclimated plants, indicated that the most tolerant were only 1 degree C more freezing-tolerant than alfalfa clone N4. There were no differences among transgenic and control plants for tetrazolium staining of field-grown plants at any freezing temperature. Therefore, although many of the transgenic plants had higher winter survival rates and herbage yield, there was no apparent difference in primary freezing injury, and therefore, the trait is not associated with a change in the primary site of freezing injury.

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.

Guy, C; Haskell, D; Hofig, A; Neven, LA

US Patent 5837545, , 17 Nov 1998

Described are proteins having molecular weights of 85 and 160 kDa, which proteins are responsive to cold acclimation or drought stress in plants. The cDNA for the 85 and 160 kDa proteins, designated CAP85 and CAP160, are disclosed. Transgenic cells, including microorganisms and plants, can be produced which express the CAP85 and CAP160 proteins and thereby advantageously enhance the cold or water stress tolerance in the transgenic organism. Freeze and desiccation damage can also be prevented by applying a cold acclimation protein to the organism needing such protection.

Wallis, JG; Wang, Hongyu; Guerra, DJ*

Plant Molecular Biology [PLANT MOL. BIOL.], vol. 35, no. 3, pp. 323-330, Oct 1997

A synthetic antifreeze protein gene was expressed in plants and reduced electrolyte leakage from the leaves at freezing temperatures. The synthetic AFP was expressed as a fusion to a signal peptide, directing it to the extracytoplasmic space where ice crystallization first occurs. The gene was introduced to Solanum tuberosum L. cv. Russet Burbank by Agrobacterium-mediated transformation. Transformants were identified by PCR screening and expression of the introduced protein was verified by immunoblot. Electrolyte-release analysis of transgenic plant leaves established a correlation between the level of transgenic protein expression and degree of tolerance to freezing. This is the first identification of a phenotype associated with antifreeze protein expression in plant tissue.

Duman, JG

US Patent 5633451, , 27 May 1997

The present invention is directed to transgenic plants having nucleic acid sequences encoding Dendroides canadensis thermal hysteresis proteins. The THPs of Dendroides have significantly greater thermal hysteresis activity than any other known anti-freeze protein. The thermal hysteresis activity of the purified THPs can be further enhanced by combining the THPs with various "activating" compounds.

Vazquez-Thello, A; Yang, Li Jun; Hidaka, M; Uozumi, T

Plant Cell Reports [PLANT CELL REP.], vol. 15, no. 7, pp. 506-511, 1996

Improvement of Hibiscus rosa-sinensis for increased frost tolerance has been attempted through somatic hybridization with the frost tolerant Lavatera thuringiaca. Cell suspensions from Hibiscus and Lavatera were transformed with A. tumefaciens harboring plasmids containing selectable genes coding for kanamycin and hygromycin resistance, respectively. We provided evidence that H. rosa-sinensis and L. thuringiaca were transformed by strong selection of transformed calluses in medium containing antibiotics, by GUS activity determination in protein extracts and by molecular confirmation of chromosomal integration and expression of the selectable genes. Protoplasts isolated from a kanamycin-resistant Hibiscus callus and from a hygromycin-resistant Lavatera callus were fused and selected in medium containing both antibiotics. We determined unambiguously that the regenerated double-antibiotic resistant clones obtained are indeed somatic hybrids through analysis of acid phosphatase zymograms and nuclear DNA content. Plant regeneration through somatic embryogenesis was accomplished from both isolated protoplasts and transgenic calluses of L. thuringiaca. However, regeneration from the double-antibiotic resistant fusant calluses was unsuccessful. Analysis of the somatic hybrids at the callus level showed that chilling and freezing tolerance are governed by independent genetic components. The somatic hybrids displayed significant improvement for chilling tolerance at conditions lethal to H. rosa-sinensis, although frost tolerance was not expressed.

Sun, Xiuying; Griffith, M; Pasternak, JJ; Glick, BR*

Canadian Journal of Microbiology/Revue Canadienne de Microbiologie [CAN. J. MICROBIOL./REV. CAN. MICROBIOL.], vol. 41, no. 9, pp. 776-784, 1995

The plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was originally isolated from the rhizosphere of plants growing in the Canadian High Arctic. Here we report that this bacterium was able to grow and promote root elongation of both spring and winter canola at 5 degree C, a temperature at which only a relatively small number of bacteria are able to proliferate and function. In addition, the bacterium survived exposure to freezing temperatures, i.e., -20 and -50 degree C. In an effort to determine the mechanistic basis for this behaviour, it was discovered that following growth at 5 degree C, P. putida GR12-2 synthesized and secreted to the growth medium a protein with antifreeze activity. Analysis of the spent growth medium, following concentration by ultrafiltration, by SDS-polyacrylamide gel electrophoresis revealed the presence of one major protein with a molecular mass of approximately 32-34 kDa and a number of minor proteins. However, at this point it is not known which of these proteins contains the antifreeze activity.