Low temperature-inducible wheat WCS120 gene promoter

Details Low temperature-inducible wheat WCS120 gene promoter Low temperature-inducible wheat WCS120 gene promoter

1998-12-28

2003-09-30

Mehta, Ashwin (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S069100, C435S320100, C435S468000, C536S024100, C800S289000, C800S298000, C800S306000, C800S320000

Reexamination Certificate

active

06627793

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the identification and characterization of a promoter of the wheat wcs120 gene. This promoter is inducible by low temperatures in both moncotyledoneous and dicotyledoneous species. It can therefore be used as a universal promoter for genes that are involved in the improvement of low temperature or freeze tolerance in plants.
BACKGROUND OF THE INVENTION
During the exposure of plants to low temperature (LT), and in the process of cold acclimation (CA) of plants, many physiological and biochemical changes occur, leading in some plants to the development of freezing tolerance (FT). The survival of these tolerant plants at freezing temperatures depends on the timely modulation of specific sets of genes, for which the accumulation of both mRNA and encoded proteins correlate with the development of FT (Guy, C. L. (1990) Annu. Rev. Plant Physiol. Plant Mol. Biol. 41: 187-223; Thomashow, M. F. (1990) Adv. Genet. 28: 99-131). A cryoprotective function has been proposed for several low temperature-responsive genes (Houde, M., et al. (1995) Plant J. 8: 583-593; Kurkela, S., et al. (1990) Plant Mol. Biol. 15: 137-144; Sieg, F., et al. (1996) Plant. Physiol. 111:215-221).
The molecular mechanisms governing gene expression at low temperature are not well understood. Recent reports suggest a role for calcium as second messenger in the early events following exposure to chilling temperatures (Knight, H., et al. (1996) Plant Cell 8:489-503; Monroy, A. F., et al. (1995) Plant Cell 7: 321-331). The Ca
2+
signal is likely transduced in a series of phosphorylation events which may involve Ca
2+
-dependent protein kinases. However, there is little information regarding the downstream signalling components leading to the activation of specific sets of genes in response to LT.
Few cis-acting elements responsive to low temperature have been identified so far. In the case of the cor15a gene of
Arabidopsis thaliana,
Baker, S. S., et al. ((1994) Plant Mol. Biol. 24:701-713) suggested a potential role of G-box-like elements in ABA (abscissic acid) and drought responsiveness. However it is unclear whether these elements also play a role in LT responsiveness. The DR1 core motif (TACCGACAT) in the promoter of the
A. thaliana
rd29A gene is a cis-element implicated in the response to dehydration, high salt and low temperature (Yamaguchi-Shinozaki. K., et al. (1994) Plant Cell 6:251-264). A similar low-temperature regulatory element (LTRE)(TGGCCGAC), found in the promoter of the
Brassica napus
BN115 gene, contains the pentamer CCGAC motif which imparts low-temperature responsiveness (White, T. C., et al. (1994b) Plant Physiol. 106:917-928; Jiang, C., et al. (1996) Plant Mol. Biol. 30: 679-684). This motif or similar variants is also present in the promoters of cor15a (Baker, S. S., supra), rd29A (Yamaguchi-Shinozaki, K., supra), Iti78 and Iti65 Arabidopsis genes (Nordin K., et al. (1993) Plant Mol. Biol. 21:641-653) and blt4.6 and blt4.9 barley genes (White A. J, et al. (1994a) J. Exp. Bot. 45:1885-1892).
Several LT-responsive cDNA clones from wheat have been characterized. Among these, the Wcs120 gene is specifically regulated by low temperature (Houde M., et al. (1992) Plant Physiol. 99:1381-1387). The encoded 50 kDa protein is the major member of the WCS120 protein family (Houde, M. (1995), supra). Southern analysis indicates that the gene copy number and gene organization are identical in both freezing tolerant and sensitive wheat cultivars. On the other hand, the accumulation of both Wcs120 mRNA and encoded protein is shown to correlate closely with the differential capacity of wheat cultivars to develop FT (Limin, A. E., et al. (1995) Genome 38:1023-1031). Homologs of Wcs120 and other cold-regulated genes are present in chilling sensitive Gramineae species such as rice and corn, but they are not induced by low temperature (Danyluk, J., et al. (1994) FEBS Lett. 344:20-24). It appears that the expression of the wcs120 gene is regulated mainly at the transcriptional level. It is possible that the inability of some species to CA and to develop FT is due to inefficient cis-acting elements in the promoter or the absence of LT-specific transcription factors.
There thus remains a need to identify the nuclear events regulating the cold-specific expression of the Wcs120 gene. It therefore appeared of great interest to characterize the promoter region of the wheat wcs120 gene and to analyse the role of the promoter in the development of FT in plants.
The present invention seeks to meet these and other needs.
SUMMARY OF THE INVENTION
The invention concerns the identification of nuclear events regulating cold-specific gene expression involved in CA of plants in the development of FT. Specifically, it is the characterization of a gene promoter in plants that is induced in response to low temperature to express genes involved in the CA of plants. The promoter of this invention was first isolated from the 5′ start site of the wheat wcs120 gene, sequenced and analysed for specific elements or motifs. A comparison of homologous function in promoters in other genes that are expressed in response to adverse environmental conditions, in the same and different species has shown conservation of some of these motifs. Further, by deletion analyses of the promoter, the functions of specific promoter regions are characterized and their importance in the response to the development of FT is identified. The invention additionally teaches the ability of the promoter to be used as a universal promoter in driving genes in response to low temperature involved in CA in dicots and monocot plant species for improvement of FT.
The invention additionally relates to the nuclear factors and events involved in the expression of the wheat low-temperature responsive gene wcs120. The results demonstrate that these nuclear factors regulate the expression of wcs120 at the level of the promoter by differential binding of these factors to the promoter under different environmental conditions.
The inventors are the first to isolate, identify, sequence and characterize the promoter of the wheat wcs120 gene. The inventors are also the first to characterize the promoter and functional variants, fragments and derivatives thereof by deletion analyses.
The inventors are the first to demonstrate that this promoter can be induced by low-temperatures in all plants, those that are cold-sensitive and cold-tolerant, and, in monocot and dicots plant species, thus demonstrating the universality of the promoter in inducing homologous and heterologous gene expression in response to low temperature.
The inventors are also the first to demonstrate the role of nuclear factors in regulating the transcription of the wheat wcs120 gene in response to different temperatures.
Before the present invention, it was believed that sensitivity to low temperature and inability to CA in plants was due to genetic variability in the low temperature responsive genes. The inventors are the first to demonstrate that the level of repression of the cold-induced genes, specifically wcs 120, is not due to variations in the gene itself or its promoter but rather to nuclear factors acting at the level of the promoter.
The promoter of the present invention and any derivatives or fragments thereof, can therefore be used in the design of transgenic plants in need of improving resistance to low temperatures. This can be accomplished by replacing the homologous promoter with the promoter of the present invention or any derivatives or fragments thereof, to drive the expression of the genes needed to cold-acclimate a plant and in this manner improve the FT of the plant.
In accordance with the present invention, there is therefore provided, a sequence of the promoter region of the wheat wcs120 gene.
In accordance with another aspect of the present invention, there is also provided the promoter, derivatives or fragments thereof being universal, such that the promoter, derivatives or fragments thereof are capable of driving the

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