Junya Mizoi

AP2/ERF family transcription factors in plant abiotic stress responses ☆

In terrestrial environments, temperature and water conditions are highly variable, and extreme temperatures and water conditions affect the survival, growth and reproduction of plants. To protect cells and sustain growth under such conditions of abiotic stress, plants respond to unfavourable changes in their environments in developmental, physiological and biochemical ways. These responses require expression of stress-responsive genes, which are regulated by a network of transcription factors. The AP2/ERF family is a large family of plant-specific transcription factors that share a well-conserved DNA-binding domain. This transcription factor family includes DRE-binding proteins (DREBs), which activate the expression of abiotic stress-responsive genes via specific binding to the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element in their promoters. In this review, we discuss the functions of the AP2/ERF-type transcription factors in plant abiotic stress responses, with special emphasis on the regulations and functions of two major types of DREBs, DREB1/CBF and DREB2. In addition, we summarise the involvement of other AP2/ERF-type transcription factors in abiotic stress responses, which has recently become clear. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.

AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation.

A myriad of drought stress-inducible genes have been reported, and many of these are activated by abscisic acid (ABA). In the promoter regions of such ABA-regulated genes, conserved cis-elements, designated ABA-responsive elements (ABREs), control gene expression via bZIP-type AREB/ABF transcription factors. Although all three members of the AREB/ABF subfamily, AREB1, AREB2, and ABF3, are upregulated by ABA and water stress, it remains unclear whether these are functional homologs. Here, we report that all three AREB/ABF transcription factors require ABA for full activation, can form hetero- or homodimers to function in nuclei, and can interact with SRK2D/SnRK2.2, an SnRK2 protein kinase that was identified as a regulator of AREB1. Along with the tissue-specific expression patterns of these genes and the subcellular localization of their encoded proteins, these findings clearly indicate that AREB1, AREB2, and ABF3 have largely overlapping functions. To elucidate the role of these AREB/ABF transcription factors, we generated an areb1 areb2 abf3 triple mutant. Large-scale transcriptome analysis, which showed that stress-responsive gene expression is remarkably impaired in the triple mutant, revealed novel AREB/ABF downstream genes in response to water stress, including many LEA class and group-Ab PP2C genes and transcription factors. The areb1 areb2 abf3 triple mutant is more resistant to ABA than are the other single and double mutants with respect to primary root growth, and it displays reduced drought tolerance. Thus, these results indicate that AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent gene expression for ABA signaling under conditions of water stress.

Comprehensive analysis of rice DREB2-type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes

DREB2s (dehydration-responsive element-binding protein 2s) are transcription factors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in water- and heat-shock stress responses and tolerance in Arabidopsis thaliana. In this study, we performed a comprehensive analysis of all five DREB2-type genes in rice (OsDREB2s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E and OsABI4) to determine which of them contribute to plant stress responses. We analysed the expression patterns of these genes under abiotic stress conditions, and we examined the subcellular localisation and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear specific localisation and the highest transactivation activity. OsDREB2B has functional and non-functional forms of its transcript similar to its orthologues in the grass family, and the functional form of its transcript was markedly increased during stress conditions. We analysed the splicing mechanism of OsDREB2B with transgenic rice that express the non-functional transcript and we found that the non-functional form is not a precursor of the functional form; thus, stress-inducible alternative splicing of pre-mRNA is an important mechanism for the regulation of OsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showed enhanced expression of DREB2A target genes and improved drought and heat-shock stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice.

Identification of cis-acting promoter elements in cold- and dehydration-induced transcriptional pathways in Arabidopsis, rice, and soybean.

The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (46 = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species.

The Phytochrome-Interacting Factor PIF7 Negatively Regulates DREB1 Expression under Circadian Control in Arabidopsis

Transcription factors of the DRE-Binding1 (DREB1)/C-repeat binding factor family specifically interact with a cis-acting dehydration-responsive element/C-repeat involved in low-temperature stress-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Expression of DREB1s is induced by low temperatures and is regulated by the circadian clock under unstressed conditions. Promoter sequences of DREB1s contain six conserved motifs, boxes I to VI. We analyzed the promoter region of DREB1C using transgenic plants and found that box V with the G-box sequence negatively regulates DREB1C expression under circadian control. The region around box VI contains positive regulatory elements for low-temperature-induced expression of DREB1C. Using yeast one-hybrid screens, we isolated cDNA encoding the transcriptional factor Phytochrome-Interacting Factor7 (PIF7), which specifically binds to the G-box of the DREB1C promoter. The PIF7 gene was expressed in rosette leaves, and the PIF7 protein was localized in the nuclei of the cells. Transactivation experiments using Arabidopsis protoplasts indicated that PIF7 functions as a transcriptional repressor for DREB1C expression and that its activity is regulated by PIF7-interacting factors TIMING OF CAB EXPRESSION1 and Phytochrome B, which are components of the circadian oscillator and the red light photoreceptor, respectively. Moreover, in the pif7 mutant, expression of DREB1B and DREB1C was not repressed under light conditions, indicating that PIF7 functions as a transcriptional repressor for the expression of DREB1B and DREB1C under circadian control. This negative regulation of DREB1 expression may be important for avoiding plant growth retardation by the accumulation of DREB1 proteins under unstressed conditions.

Arabidopsis HsfA1 transcription factors function as the main positive regulators in heat shock-responsive gene expression

Arabidopsis DREB2A is a key transcription factor of heat- and drought-responsive gene expression, and DREB2A expression is induced by these stresses. We analyzed the DREB2A promoter and found a heat shock element that functions as a cis-acting element in the heat shock (HS)-responsive expression of DREB2A. Among the 21 Arabidopsis heat shock factors, we chose 4 HsfA1-type proteins as candidate transcriptional activators (HsfA1a, HsfA1b, HsfA1d, and HsfA1e) based on transactivation activity and expression patterns. We generated multiple mutants and found that the HS-responsive expression of DREB2A disappeared in hsfa1a/b/d triple and hsfa1a/b/d/e quadruple mutants. Moreover, HS-responsive gene expression, including that of molecular chaperones and transcription factors, was globally and drastically impaired in the hsfa1a/b/d triple mutant, which exhibited greatly reduced tolerance to HS stress. HsfA1 protein accumulation in the nucleus was negatively regulated by their interactions with HSP90, and other factors potentially strongly activate the HsfA1 proteins under HS stress. The hsfa1a/b/d/e quadruple mutant showed severe growth retardation, and many genes were downregulated in this mutant even under non-stress conditions. Our study indicates that HsfA1a, HsfA1b, and HsfA1d function as main positive regulators in HS-responsive gene expression and four HsfA1-type proteins are important in gene expression for normal plant growth.

Functional analysis of an Arabidopsis heat-shock transcription factor HsfA3 in the transcriptional cascade downstream of the DREB2A stress-regulatory system

A transcription factor DREB2A functions as a key regulator not only in drought stress responses but also in heat stress (HS) responses, and activates expression of many abiotic stress-responsive-genes involved in drought and HS tolerance. HsfA3 is one of the most up-regulated heat-inducible genes in transgenic plants overexpressing DREB2A. In this study, the analyses of HsfA3 expression profile and the transactivation analysis of HsfA3 showed that the expression of HsfA3 was directly regulated by DREB2A under HS. Microarray analysis using transgenic plants overexpressing HsfA3 also showed that overexpression of HsfA3 induces many heat-inducible genes. Furthermore, we showed that thermotolerance of the HsfA3 overexpressors was increased, and that of the hsfA3 T-DNA tagged mutants was decreased. These results indicate that HsfA3 regulates expression of many heat-inducible genes in the transcriptional cascade downstream of the DREB2A stress-regulatory system and functions in acquisition of thermotolerance under the control of the DREB2A cascade.

An ABRE Promoter Sequence is Involved in Osmotic Stress-Responsive Expression of the DREB2A Gene, Which Encodes a Transcription Factor Regulating Drought-Inducible Genes in Arabidopsis

In plants, osmotic stress-responsive transcriptional regulation depends mainly on two major classes of cis-acting elements found in the promoter regions of stress-inducible genes: ABA-responsive elements (ABREs) and dehydration-responsive elements (DREs). ABRE has been shown to perceive ABA-mediated osmotic stress signals, whereas DRE is known to be involved in an ABA-independent pathway. Previously, we reported that the transcription factor DRE-BINDING PROTEIN 2A (DREB2A) regulates DRE-mediated transcription of target genes under osmotic stress conditions in Arabidopsis (Arabidopsis thaliana). However, the transcriptional regulation of DREB2A itself remains largely uncharacterized. To elucidate the transcriptional mechanism associated with the DREB2A gene under osmotic stress conditions, we generated a series of truncated and base-substituted variants of the DREB2A promoter and evaluated their transcriptional activities individually. We found that both ABRE and coupling element 3 (CE3)-like sequences located approximately -100 bp from the transcriptional initiation site are necessary for the dehydration-responsive expression of DREB2A. Coupling our transient expression analyses with yeast one-hybrid and chromatin immunoprecipitation (ChIP) assays indicated that the ABRE-BINDING PROTEIN 1 (AREB1), AREB2 and ABRE-BINDING FACTOR 3 (ABF3) bZIP transcription factors can bind to and activate the DREB2A promoter in an ABRE-dependent manner. Exogenous ABA application induced only a modest accumulation of the DREB2A transcript when compared with the osmotic stress treatment. However, the osmotic stress-induced DREB2A expression was found to be markedly impaired in several ABA-deficient and ABA-insensitive mutants. These results suggest that in addition to an ABA-independent pathway, the ABA-dependent pathway plays a positive role in the osmotic stress-responsive expression of DREB2A.

Abiotic stress‐inducible receptor‐like kinases negatively control ABA signaling in Arabidopsis

Membrane-anchored receptor-like protein kinases (RLKs) recognize extracellular signals at the cell surface and activate the downstream signaling pathway by phosphorylating specific target proteins. We analyzed a receptor-like cytosolic kinase (RLCK) gene, ARCK1, whose expression was induced by abiotic stress. ARCK1 belongs to the cysteine-rich repeat (CRR) RLK sub-family and encodes a cytosolic protein kinase. The arck1 mutant showed higher sensitivity than the wild-type to ABA and osmotic stress during the post-germinative growth phase. CRK36, an abiotic stress-inducible RLK belonging to the CRR RLK sub-family, was screened as a potential interacting factor with ARCK1 by co-expression analyses and a yeast two-hybrid system. CRK36 physically interacted with ARCK1 in plant cells, and the kinase domain of CRK36 phosphorylated ARCK1 in vitro. We generated CRK36 RNAi transgenic plants, and found that transgenic plants with suppressed CRK36 expression showed higher sensitivity than arck1-2 to ABA and osmotic stress during the post-germinative growth phase. Microarray analysis using CRK36 RNAi plants revealed that suppression of CRK36 up-regulates several ABA-responsive genes, such as LEA genes, oleosin, ABI4 and ABI5. These results suggest that CRK36 and ARCK1 form a complex and negatively control ABA and osmotic stress signal transduction.

Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using β-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of Km for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.