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Molecular Genetics of Environmental Stress Tolerance

Abiotic stresses including extremes in temperature and water availability are major factors that determine the natural geographical distribution of plants and limit the agricultural production of crops. Our overarching interest is to understand the molecular genetic mechanisms that plants have evolved to tolerate environmental stresses. Most of our effort focuses on the cold acclimation response, the process whereby certain plants increase in freezing tolerance upon exposure to low nonfreezing temperatures. However, as one of the cold response pathways that we are studying includes genes that impart drought tolerance, we are also interested in how plants sense and respond to water deficit.

Our work on cold acclimation has centered on genes that are induced in response to low temperature. Our working hypothesis was that freezing tolerance involved, at least in part, genes that were induced with cold acclimation.  Indeed, the recent discovery of the Arabidopsis CBF cold-response pathway (Figure 1) has proven this to be true.  We have shown that Arabidopsis encodes a small family of cold-responsive transcriptional activators known either as CBF1, CBF2, and CBF3 or DREB1b, DREB1c and DREB1a, respectively.  The CBF transcription factors recognize the cold- and dehydration-responsive DNA regulatory element designated the CRT (C-repeat)/DRE (dehydration responsive element) that is present in the promoter regions of many cold and dehydration responsive genes of Arabidopsis.  The CBF genes are induced within 15 min of plants being exposed to low, nonfreezing temperatures followed at about 2 h by induction of cold-regulated genes that contain the CRT/DRE-regulatory element; i.e., the “CBF regulon”.  Over the next few days, expression of the CBF regulon leads to an increase in plant freezing tolerance.  In addition, expression of the regulon increases tolerance to both drought and high salinity stress.

Figure 1. Model of the Arabidopsis CBF Cold-Response Pathway. Low temperature leads to rapid induction of the CBF genes (CBF1, 2, and 3; see text), which in turn results in expression of the CBF regulon of the CRT/DRE-regulated genes. Action of the CBF regulon, which includes COR, ERD, and presumably yet to be discovered ("XYZ") cold-regulated genes, results in an increase in plant freezing tolerance.

A major aim now is to develop a detailed low temperature "wiring diagram" for the Arabidopsis genome. At completion, the diagram will include the identification of genes that are responsive to low temperature, an organization of the cold-responsive genes into regulons, the identification of transcription factors that have key roles in controlling expression of the regulons, and an identification of the low temperature "thermometers" that regulate the activities of the transcription factors. To date, we have used microarrays to identify 512 COS (cold-standard) genes that are robustly cold-responsive, 302 of which are up-regulated and 212 down-regulated. Further, we have assigned 93 of the COS genes to the CBF regulon, 85 of which are cold-induced, and 24 of the COS genes to the ZAT12 regulon, 9 of which are cold-induced. Significantly, 7 of the ZAT12 regulon genes are also members of the CBF regulon, indicating an overlap in these two regulatory networks. In addition, ZAT12 down-regulates expression of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that dampens expression of the CBF cold response pathway. Finally, expression of the ZAT12 regulon results in a detectible increase in plant freezing tolerance, though the increase is far less than that brought about by expression of the CBF regulon.

By what mechanisms are the CBF1-3 and ZAT12 genes induced in response to low temperature? Toward answering this fundamental question, we have identified (1) a 155-bp region of the CBF2 promoter that is sufficient to impart cold-regulated gene expression and (2) two regulatory elements within this region, ICEr1 and ICEr2 (induction of CBF expression region 1 and 2), that are involved in this response. We are now pursuing multiple approaches to identify the transcription factors that presumably bind to these regulatory elements. Once identified, these transcription factors will serve as entry points to step our way back into the inner workings of the low temperature "thermometer."

One additional interest of ours is to determine how highly conserved the CBF cold response pathway is among plant species. In our initial experiments, we established that the oilseed crop canola (Brassica napus), and the cereals wheat and barely, encode CBF-like genes that are induced in response to low temperature. Moreover, we showed that constitutive overexpression of the Arabidopsis CBF genes in canola increases the freezing tolerance of the transgenic plants. Thus, it would appear that the CBF cold response pathway is conserved in plants that cold acclimate. The question then is whether the CBF cold response pathway functions in plants that do not cold acclimate, such as tomato and other plants from tropical and subtropical regions. To date, we have found that tomato has three CBF genes, LeCBF1-3, and have demonstrated that one of these, LeCBF1, is quickly induced in response to low temperature and encodes a functional protein. However, whereas we have assigned 85 cold-induced genes to the CBF regulon of Arabidopsis, transcriptome analysis using a cDNA microarray surveying approximately 25% of the tomato genome resulted in only 3 genes being assigned to the tomato CBF regulon. Also, activation of the tomato CBF regulon, by constitutive expression of either LeCBF1 or AtCBF3, did not result in an increase in freezing tolerance. The difference in CBF regulon composition between Arabidopsis and tomato could signify that tomato has few genes with functional CRT/DRE elements in their promoters. Alternatively, tomato might have a regulatory mutation that results in poor expression of the CBF cold response pathway. A major goal now is to determine which of these alternative models is correct and whether the CBF cold response pathway of tomato can be altered to improve the cold tolerance of this crop species.

Fowler SG, Cook D, Thomashow MF (2005) Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol 137: 961-968. Abstract

Vogel JT, Zarka DG, Van Buskirk HA, Fowler SG, Thomashow MF (2005) Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis. Plant J 41: 195-211. Abstract

Gilmour S.J., S.G. Fowler and M.F. Thomashow (2004) Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities. Plant Mol Biol 54: 767-781. Abstract

Zhang X, Fowler S, Cheng H, Lou Y, Rhee SY, Stockinger EJ, Thomashow MF (2004) Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. Plant J 39: 905-919. Abstract

Cook D, Fowler S, Fiehn O, Thomashow MF (2004) A prominent role for the CBF cold response pathway in configuring the low temperature metabolome of Arabidopsis. Proc Natl Acad Sci USA 101: 15243-15248. Abstract

Vlachonasios K.E., M.F. Thomashow and S.J. Triezenberg (2003) Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expression. Plant Cell 15: 626-638. Abstract

Zarka, D.G., J.T. Vogel, D. Cook, M.F. Thomashow (2003) Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiol 133: 910-918. Abstract

Fowler  S, Thomashow MF (2002) Arabidopsis transcriptome profiling indicates multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold-response pathway.  Plant Cell 14: 1675-1690. Abstract

Haake V, Cook D, Riechmann JL, Pineda O, Thomashow MF, Zhang JZ (2002) Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis thaliana. Plant Physiol 130: 709-783 Abstract

Jaglo KR, Kleff S, Amundsen K, Zhang X, Haake V, Zhang J, Deits T, Thomashow MF (2001) Components of the Arabidopsis CBF cold-response pathway are conserved in Brassica napus and other plant species.  Plant Physiol 127: 910-917. Abstract

Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation.  Plant Physiol 124: 1854-1865. Abstract

Gilmour, SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression.  Plant J 16: 433-443. Abstract

Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF (1998)  Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance.  Science 280: 104-106. Abstract

Reviews on Cold Acclimation

Thomashow MF (2001) So what's new in the field of plant cold acclimation? Lots! Plant Physiol 125: 89-93 Abstract

Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50: 571-599  Abstract

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