Michael F. Thomashow

Arabidopsis Transcriptome Profiling Indicates That Multiple Regulatory Pathways Are Activated during Cold Acclimation in Addition to the CBF Cold Response Pathway

Many plants, including Arabidopsis, increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Previous studies established that cold acclimation involves rapid expression of the CBF transcriptional activators (also known as DREB1 proteins) in response to low temperature followed by induction of the CBF regulon (CBF-targeted genes), which contributes to an increase in freezing tolerance. Here, we present the results of transcriptome-profiling experiments indicating the existence of multiple low-temperature regulatory pathways in addition to the CBF cold response pathway. The transcript levels of ∼8000 genes were determined at multiple times after plants were transferred from warm to cold temperature and in warm-grown plants that constitutively expressed CBF1, CBF2, or CBF3. A total of 306 genes were identified as being cold responsive, with transcripts for 218 genes increasing and those for 88 genes decreasing threefold or more at one or more time points during the 7-day experiment. These results indicate that extensive downregulation of gene expression occurs during cold acclimation. Of the cold-responsive genes, 48 encode known or putative transcription factors. Two of these, RAP2.1 and RAP2.6, were activated by CBF expression and thus presumably control subregulons of the CBF regulon. Transcriptome comparisons indicated that only 12% of the cold-responsive genes are certain members of the CBF regulon. Moreover, at least 28% of the cold-responsive genes were not regulated by the CBF transcription factors, including 15 encoding known or putative transcription factors, indicating that these cold-responsive genes are members of different low-temperature regulons. Significantly, CBF expression at warm temperatures repressed the expression of eight genes that also were downregulated by low temperature, indicating that in addition to gene induction, gene repression is likely to play an integral role in cold acclimation.

The 5'-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression.

Previous nuclear run-on experiments indicated that the cor15a (cold-regulated) gene of Arabidopsis thaliana L. (Heyn) has a cold-inducible promoter (Hajela et al., Plant Physiol 93: 1246–1252, 1990). The data presented here indicate that the 5′ region of cor15a between nucleotides −305 and +78 (relative to the start of transcription) contains a cis-acting element(s) that can impart cold-regulated gene expression. Histochemical staining experiments indicated that the cor15a promoter is inactive, or very weakly active, in most of the tissues and organs of plants grown at normal temperature and that it becomes activated throughout most of the plant in response to low temperature. Notable exceptions to this general pattern include constitutive activity of the promoter in anthers of control grown plants and apparent inactivity of the promoter in the roots and ovaries of cold-treated plants. Histochemical staining experiments also indicated that low temperature regulation of cor15a does not involve the synthesis of a regulatory molecule that can spread throughout the plant and induce cor gene expression at normal growth temperature. Finally, gene fusion experiments indicated that the 5′ region of cor15a between nucleotides −305 and +78, in addition to imparting cold-regulated gene expression, can impart ABA- and drought-regulated gene expression.

Genes Galore: A Summary of Methods for Accessing Results from Large-Scale Partial Sequencing of Anonymous Arabidopsis cDNA Clones

High-throughput automated partial sequencing of anonymous cDNA clones provides a method to survey the repertoire of expressed genes from an organism. Comparison of the coding capacity of these expressed sequence tags (ESTs) with the sequences in the public data bases results in assignment of putative function to a significant proportion of the ESTs. Thus, the more than 13,400 plant ESTs that are currently available provide a new resource that will facilitate progress in many areas of plant biology. These opportunities are illustrated by a description of the results obtained from analysis of 1500 Arabidopsis ESTs from a cDNA library prepared from equal portions of poly(A+) mRNA from etiolated seedlings, roots, leaves, and flowering inflorescences. More than 900 different sequences were represented, 32% of which showed significant nucleotide or deduced amino acid sequence similarity to previously characterized genes or proteins from a wide range of organisms. At least 165 of the clones had significant deduced amino acid sequence homology to proteins or gene products that have not been previously characterized from higher plants. A summary of methods for accessing the information and materials generated by the Arabidopsis cDNA sequencing projects is provided.

Transcription Factor CBF4 Is a Regulator of Drought Adaptation in Arabidopsis

In plants, low temperature and dehydration activate a set of genes containing C-repeat/dehydration-responsive elements in their promoter. It has been shown previously that the Arabidopsis CBF/DREB1 transcription activators are critical regulators of gene expression in the signal transduction of cold acclimation. Here, we report the isolation of an apparent homolog of the CBF/DREB1 proteins (CBF4) that plays the equivalent role during drought adaptation. In contrast to the three already identified CBF/DREB1 homologs, which are induced under cold stress, CBF4 gene expression is up-regulated by drought stress, but not by low temperature. Overexpression of CBF4 in transgenic Arabidopsis plants results in the activation of C-repeat/dehydration-responsive element containing downstream genes that are involved in cold acclimation and drought adaptation. As a result, the transgenic plants are more tolerant to freezing and drought stress. Because of the physiological similarity between freezing and drought stress, and the sequence and structural similarity of the CBF/DREB1 and the CBF4 proteins, we propose that the plants response to cold and drought evolved from a common CBF-like transcription factor, first through gene duplication and then through promoter evolution.

Arabidopsis transcriptional activators CBF1, CBF2, and CBF3 have matching functional activities.

When Arabidopsis is exposed to low temperature a small gene family encoding transcription factors known as CBF1, CBF2, and CBF3 (also referred to as DREB1b, DREB1c, and DREB1a, respectively) is rapidly induced followed by expression of CBF-targeted genes, the CBF regulon, which act to bring about an increase in freezing tolerance. The CBF1, 2 and 3 proteins, though highly similar in amino acid sequence, are not identical, raising the question of whether the proteins have the same functions. Here we explored this issue by comparing the effects that overexpression of each CBF gene had on Arabidopsis growth and development, proline and sugar composition, freezing tolerance and gene expression. Taken together, the results support the conclusion that the CBF1, 2 and 3 transcriptional activators have redundant functional activities.

Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade

Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1–JAZ–DELLA–PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated.

Roles for Arabidopsis CAMTA Transcription Factors in Cold-Regulated Gene Expression and Freezing Tolerance

The Arabidopsis thaliana CBF cold response pathway plays a central role in cold acclimation. It is characterized by rapid cold induction of genes encoding the CBF1-3 transcription factors, followed by expression of the CBF gene regulon, which imparts freezing tolerance. Our goal was to further the understanding of the cis-acting elements and trans-acting factors involved in expression of CBF2. We identified seven conserved DNA motifs (CM), CM1 to 7, that are present in the promoters of CBF2 and another rapidly cold-induced gene encoding a transcription factor, ZAT12. The results presented indicate that in the CBF2 promoter, CM4 and CM6 have negative regulatory activity and that CM2 has both negative and positive activity. A Myc binding site in the CBF2 promoter was also found to have positive regulatory effects. Moreover, our results indicate that members of the calmodulin binding transcription activator (CAMTA) family of transcription factors bind to the CM2 motif, that CAMTA3 is a positive regulator of CBF2 expression, and that double camta1 camta3 mutant plants are impaired in freezing tolerance. These results establish a role for CAMTA proteins in cold acclimation and provide a possible point of integrating low-temperature calcium and calmodulin signaling with cold-regulated gene expression.

Molecular Basis of Plant Cold Acclimation: Insights Gained from Studying the CBF Cold Response Pathway

For the 75th Anniversary Issue of Plant Physiology , I contributed an article ([Thomashow, 2001][1]) in which I highlighted recent advances in the identification of genes with roles in cold acclimation: the process whereby certain plants increase in freezing tolerance in response to low nonfreezing

Low Temperature Induction of Arabidopsis CBF1, 2, and 3 Is Gated by the Circadian Clock

Exposing Arabidopsis (Arabidopsis thaliana) plants to low temperature results in rapid induction of CBF1, 2, and 3 (CBF1-3; also known as DREB1B, C, and A, respectively), which encode transcriptional activators that induce expression of a battery of genes that increase plant freezing and chilling tolerance. Recently, it has been shown that basal levels of CBF3 transcripts and those of certain CBF-regulated genes exhibit circadian cycling. Here, we further explored the regulation of CBF1-3 by the circadian clock. The results indicated that the extent to which CBF1-3 transcripts accumulated in response to low temperature was dependent on the time of day that the plants were exposed to low temperature and that this was regulated by the circadian clock. The highest and lowest levels of cold-induced CBF1-3 transcript accumulation occurred at 4 and 16 h after subjective dawn, respectively. An analysis of CBF2 promoter-reporter gene fusions indicated that this control included transcriptional regulation. In addition, the cold responsiveness of RAV1 and ZAT12, genes that are cold induced in parallel with CBF1-3, was also subject to circadian regulation. However, whereas the maximum level of cold-induced RAV1 transcript accumulation occurred at the same time of day as did CBF1-3 transcripts, that of ZAT12 was in reverse phase, i.e. the highest level of cold-induced ZAT12 transcript accumulation occurred 16 h after subjective dawn. These results indicate that cold-induced expression of CBF1-3, RAV1, and ZAT12 is gated by the circadian clock and suggest that this regulation likely occurs through at least two nonidentical (though potentially overlapping) signaling pathways.

cDNA sequence analysis and expression of two cold-regulated genes of Arabidopsis thaliana

The DNA sequences of cDNAs for twocor (cold-regulated) genes ofArabidopsis thaliana L. (Heyn) were determined. One cDNA (approximately 70% full-length) corresponds to acor gene, designatedcor47, that encodes a 47 kDa hydrophilic polypeptide. The data indicate that COR47 has amino acid sequence homology with Group II LEA (lateembryogenesisabundant) proteins, a class of proteins that accumulate late in embryo development. DNA sequence analysis of a second cDNA (containing the complete protein coding sequence) indicates that it represents acor gene, designatedcor6.6, that encodes an alanine-rich 6.6 kDa hydrophilic polypeptide. COR6.6 is almost identical to KIN1, a cold-regulatedArabidopsis gene that has been suggested to have amino acid sequence similarities with type I fish antifreeze proteins (S. Kurkela, M. Franck, Plant Mol Biol 15: 137–144, 1990). Northern analysis indicated that transcripts forcor47 andcor6.6 do not accumulate to high levels in late-developing embryos or fresh mature seeds as is typical oflea gene transcripts. The similarities and differences between COR and LEA proteins are discussed as are their possible roles in freezing and drought tolerance.