Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity and oxidative stress are serious threats to agriculture and the natural status of the environment. Increased salinization of arable land is expected to have devastating global effects, resulting in 30% land loss within the next 25 years, and up to 50% by the year 2050. Therefore, breeding for drought and salinity stress tolerance in crop plants (for food supply) and in forest trees (a central component of the global ecosystem) should be given high research priority in plant biotechnology programs. Molecular control mechanisms for abiotic stress tolerance are based on the activation and regulation of specific stress-related genes. These genes are involved in the whole sequence of stress responses, such as signaling, transcriptional control, protection of membranes and proteins, and free-radical and toxic-compound scavenging. Recently, research into the molecular mechanisms of stress responses has started to bear fruit and, in parallel, genetic modification of stress tolerance has also shown promising results that may ultimately apply to agriculturally and ecologically important plants. The present review summarizes the recent advances in elucidating stress-response mechanisms and their biotechnological applications. Emphasis is placed on transgenic plants that have been engineered based on different stress-response mechanisms. The review examines the following aspects: regulatory controls, metabolite engineering, ion transport, antioxidants and detoxification, late embryogenesis abundant (LEA) and heat-shock proteins.

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Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance

Plant growth and productivity are greatly affected by environmental stresses such as drought, high salinity, and low temperature. Expression of a variety of genes is induced by these stresses in various plants. The products of these genes function not only in stress tolerance but also in stress response. In the signal transduction network from perception of stress signals to stress-responsive gene expression, various transcription factors and cis-acting elements in the stress-responsive promoters function for plant adaptation to environmental stresses. Recent progress has been made in analyzing the complex cascades of gene expression in drought and cold stress responses, especially in identifying specificity and cross talk in stress signaling. In this review article, we highlight transcriptional regulation of gene expression in response to drought and cold stresses, with particular emphasis on the role of transcription factors and cis-acting elements in stress-inducible promoters.

Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses.

Abiotic stress, the field environment and stress combination

Plants respond to survive under water-deficit conditions via a series of physiological, cellular, and molecular processes culminating in stress tolerance. Many drought-inducible genes with various functions have been identified by molecular and genomic analyses in Arabidopsis, rice, and other plants, including a number of transcription factors that regulate stress-inducible gene expression. The products of stress-inducible genes function both in the initial stress response and in establishing plant stress tolerance. In this short review, recent progress resulting from analysis of gene expression during the drought-stress response in plants as well as in elucidating the functions of genes implicated in the stress response and/or stress tolerance are summarized. A description is also provided of how various genes involved in stress tolerance were applied in genetic engineering of dehydration stress tolerance in transgenic Arabidopsis plants.

Gene networks involved in drought stress response and tolerance

Plants respond to survive under water-deficit conditions via a series of physiological, cellular, and molecular processes culminating in stress tolerance. Many drought-inducible genes with various functions have been identified by molecular and genomic analyses in Arabidopsis, rice, and other plants, including a number of transcription factors that regulate stress-inducible gene expression. The products of stress-inducible genes function both in the initial stress response and in establishing plant stress tolerance. In this short review, recent progress resulting from analysis of gene expression during the drought-stress response in plants as well as in elucidating the functions of genes implicated in the stress response and/or stress toler ance are summarized. A description is also provided of how various genes involved in stress tolerance were applied in genetic engineering of dehydration stress tolerance in transgenic Arabidopsis plants.

Gene networks involved in drought stress response and

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.

https://bioinformatics.cs.vt.edu/~easychair/FowlerThomashow_PlantCell_2002.pdf

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

Summary The CBF cold response pathway has a prominent role in cold acclimation. The pathway includes action of three transcription factors, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), that are rapidly induced in response to low temperature followed by expression of the CBF-targeted genes (the CBF regulon) that act in concert to increase plant-freezing tolerance. The results of transcriptome profiling and mutagenesis experiments, however, indicate that additional cold response pathways exist and may have important roles in life at low temperature. To further understand the roles that the CBF proteins play in configuring the low temperature transcriptome and to identify additional transcription factors with roles in cold acclimation, we used the Affymetrix GeneChip containing probe sets for approximately 24,000 Arabidopsis genes to define a core set of cold-responsive genes and to determine which genes were targets of CBF2 and 6 other transcription factors that appeared to be coordinately regulated with CBF2. A total of 514 genes were placed in the core set of cold-responsive genes, 302 of which were upregulated and 212 downregulated. Hierarchical clustering and bioinformatic analysis indicated that the 514 cold-responsive transcripts could be assigned to one of seven distinct expression classes and identified multiple potential novel cis-acting cold-regulatory elements. Eighty-five cold-induced genes and eight cold-repressed genes were assigned to the CBF2 regulon. An additional nine cold-induced genes and 15 cold-repressed genes were assigned to a regulon controlled by ZAT12. Of the 25 core cold-induced genes that were most highly upregulated (induced over 15-fold), 19 genes (84%) were induced by CBF2 and another two genes (8%) were regulated by both CBF2 and ZAT12. Thus, the large majority (92%) of the most highly induced genes belong to the CBF and ZAT12 regulons. Constitutive expression of ZAT12 in Arabidopsis caused a small, but reproducible, increase in freezing tolerance, indicating a role for the ZAT12 regulon in cold acclimation. In addition, ZAT12 downregulated the expression of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that dampens expression of the CBF cold response pathway.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2004.02288.x

Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis.

The Arabidopsis CBF cold response pathway has a central role in cold acclimation, the process whereby plants increase in freezing tolerance in response to low nonfreezing temperatures. Here we examined the changes that occur in the Arabidopsis metabolome in response to low temperature and assessed the role of the CBF cold response pathway in bringing about these modifications. Of 434 metabolites monitored by GC-time-of-flight MS, 325 (75%) were found to increase in Arabidopsis Wassilewskija-2 (Ws-2) plants in response to low temperature. Of these 325 metabolites, 256 (79%) also increased in nonacclimated Ws-2 plants in response to overexpression of C-repeat/dehydration responsive element-binding factor (CBF)3. Extensive cold-induced changes also occurred in the metabolome of Arabidopsis Cape Verde Islands-1 (Cvi-1) plants, which were found to be less freezing tolerant than Ws-2 plants. However, low-temperature-induced expression of CBF1, CBF2, CBF3, and CBF-targeted genes was much lower in Cvi-1 than in Ws-2 plants, and the low-temperature metabolome of Cvi-1 plants was depleted in metabolites affected by CBF3 overexpression. Taken together, the results indicate that the metabolome of Arabidopsis is extensively reconfigured in response to low temperature, and that the CBF cold response pathway has a prominent role in this process.

https://www.pnas.org/content/pnas/101/42/15243.full.pdf

A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis

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.

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

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.

Sarah G. Fowler

Papers

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

Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis.

A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis

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

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