▪ Abstract Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. In this review, recent advances in determining the nature and function of genes with roles in freezing tolerance and the mechanisms involved in low temperature gene regulation and signal transduction are described. One of the important conclusions to emerge from these studies is that cold acclimation includes the expression of certain cold-induced genes that function to stabilize membranes against freeze-induced injury. In addition, a family of Arabidopsis transcription factors, the CBF/DREB1 proteins, have been identified that control the expression of a regulon of cold-induced genes that increase plant freezing tolerance. These results along with many of the others summarized here further our understanding of the basic mechanisms that plants have evolved to survive freezing temperatures. In addition, the findings have potential practical applications as freezing te...

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PLANT COLD ACCLIMATION: Freezing Tolerance Genes and Regulatory Mechanisms

Plant growth is greatly affected by drought and low temperature. Expression of a number of genes is induced by both drought and low temperature, although these stresses are quite different. Previous experiments have established that a cis-acting element named DRE (for dehydration-responsive element) plays an important role in both dehydration- and low-temperature-induced gene expression in Arabidopsis. Two cDNA clones that encode DRE binding proteins, DREB1A and DREB2A, were isolated by using the yeast one-hybrid screening technique. The two cDNA libraries were prepared from dehydrated and cold-treated rosette plants, respectively. The deduced amino acid sequences of DREB1A and DREB2A showed no significant sequence similarity, except in the conserved DNA binding domains found in the EREBP and APETALA2 proteins that function in ethylene-responsive expression and floral morphogenesis, respectively. Both the DREB1A and DREB2A proteins specifically bound to the DRE sequence in vitro and activated the transcription of the b-glucuronidase reporter gene driven by the DRE sequence in Arabidopsis leaf protoplasts. Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration. Overexpression of the DREB1A cDNA in transgenic Arabidopsis plants not only induced strong expression of the target genes under unstressed conditions but also caused dwarfed phenotypes in the transgenic plants. These transgenic plants also revealed freezing and dehydration tolerance. In contrast, overexpression of the DREB2A cDNA induced weak expression of the target genes under unstressed conditions and caused growth retardation of the transgenic plants. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.

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Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis.

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.

Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor

MicroRNAs (miRNAs) are ∼21-nucleotide noncoding RNAs that have been identified in both animals and plants. Although in animals there is direct evidence implicating particular miRNAs in the control of developmental timing, to date it is not known whether plant miRNAs also play a role in regulating temporal transitions. Through an activation-tagging approach, we demonstrate that miRNA 172 (miR172) causes early flowering and disrupts the specification of floral organ identity when overexpressed in Arabidopsis. miR172 normally is expressed in a temporal manner, consistent with its proposed role in flowering time control. The regulatory target of miR172 is a subfamily of APETALA2 (AP2) transcription factor genes. We present evidence that miR172 downregulates these target genes by a translational mechanism rather than by RNA cleavage. Gain-of-function and loss-of-function analyses indicate that two of the AP2-like target genes normally act as floral repressors, supporting the notion that miR172 regulates flowering time by downregulating AP2-like target genes.

Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes

Genes in the ERF family encode transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. In this study, a comprehensive computational analysis identified 122 and 139 ERF family genes in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L. subsp. japonica), respectively. A complete overview of this gene family in Arabidopsis is presented, including the gene structures, phylogeny, chromosome locations, and conserved motifs. In addition, a comparative analysis between these genes in Arabidopsis and rice was performed. As a result of these analyses, the ERF families in Arabidopsis and rice were divided into 12 and 15 groups, respectively, and several of these groups were further divided into subgroups. Based on the observation that 11 of these groups were present in both Arabidopsis and rice, it was concluded that the major functional diversification within the ERF family predated the monocot/dicot divergence. In contrast, some groups/subgroups are species specific. We discuss the relationship between the structure and function of the ERF family proteins based on these results and published information. It was further concluded that the expansion of the ERF family in plants might have been due to chromosomal/segmental duplication and tandem duplication, as well as more ancient transposition and homing. These results will be useful for future functional analyses of the ERF family genes.

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Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice

APETALA2 (AP2) plays a central role in the establishment of the floral meristem, the specification of floral organ identity, and the regulation of floral homeotic gene expression in Arabidopsis. We show here that in addition to its functions during flower development, AP2 activity is also required during seed development. We isolated the AP2 gene and found that it encodes a putative nuclear protein that is distinguished by an essential 68-amino acid repeated motif, the AP2 domain. Consistent with its genetic functions, we determined that AP2 is expressed at the RNA level in all four types of floral organs--sepals, petals, stamens, and carpels--and in developing ovules. Thus, AP2 gene transcription does not appear to be spatially restricted by the floral homeotic gene AGAMOUS as predicted by previous studies. We also found that AP2 is expressed at the RNA level in the inflorescence meristem and in nonfloral organs, including leaf and stem. Taken together, our results suggest that AP2 represents a new class of plant regulatory proteins that may play a general role in the control of Arabidopsis development.

Control of Arabidopsis flower and seed development by the homeotic gene APETALA2.

APETALA2 (AP2) plays an important role in the control of Arabidopsis flower and seed development and encodes a putative transcription factor that is distinguished by a novel DNA binding motif referred to as the AP2 domain. In this study we show that the AP2 domain containing or RAP2 (related to AP2) family of proteins is encoded by a minimum of 12 genes in Arabidopsis. The RAP2 genes encode two classes of proteins, AP2-like and EREBP-like, that are defined by the number of AP2 domains in each polypeptide as well as by two sequence motifs referred to as the YRG and RAYD elements that are located within each AP2 domain. RAP2 genes are differentially expressed in flower, leaf, inflorescence stem, and root. Moreover, the expression of at least three RAP2 genes in vegetative tissues are controlled by AP2. Thus, unlike other floral homeotic genes, AP2 is active during both reproductive and vegetative development.

https://www.pnas.org/content/pnas/94/13/7076.full.pdf

The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis

Plant development is governed by intrinsic and environmen- tal factors that regulate the identity and activity of meristems, organized tissues of pluripotent "stem" cells, that together de- termine plant form and architecture. However, little is known about how these factors act at the molecular leve1 to affect meristem identity and function. Genetic studies in Arabidop- sis and other plant species such as snapdragon, petunia, and maize have revealed a hierarchy of regulatory genes that func- tion together to promote the formation of the floral meristem and to regulate floral organogenesis. Mutations in these genes result in dramatic defects in flower development that can af- fect both meristem identity and organ development. Table 1 lists the Arabidopsis genes that are known to control meristem and organ identity and their snapdragon equivalents. One class of regulatory genes, the homeotic organ identity genes, have provided important insights into the genetic and molecular mechanisms that govern floral organ identity and development (Bowman et al., 1991; Coen and Meyerowitz, 1991; Coen and Carpenter, 1993, this issue; van der Krol and Chua, 1993, this issue). In Arabidopsis, these genes include

Regulation of Arabidopsis flower development.

We have analyzed the contributions of phytochrome and gibberellin signal transduction to the control of flower meristem identity in the Arabidopsis mutants apetala1 (ap1) and apetala2 (ap2). ap1 flowers are partially defective for the establishment of flower meristem identity and are characterized by the production of ectopic secondary or axillary flowers and by branching. Axillary flower production is also induced in ap2-1 flowers by short-day photoperiod and is suppressed by hy1, a mutation blocking phytochrome activity. The production of axillary flower by ap2-1 is also suppressed by exogenous gibberellins and by spindly (spy), a mutation that activates basal gibberellin signal transduction in hormone-independent manner. Ectopic axillary flower production and floral branching by ap1 flowers are also suppressed by spy. We conclude that gibberellins promote flower meristem identity and that the inflorescence-like traits of ap2-1 and ap1-1 flowers are due in part to SPY gene activity.

K D Jofuku

Papers

Control of Arabidopsis flower and seed development by the homeotic gene APETALA2.

The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis

Regulation of Arabidopsis flower development.

Photo and hormonal control of meristem identity in the Arabidopsis flower mutants apetala2 and apetala1.