APETALA2/ethylene-responsive Element Binding Protein Research Articles

Rice transcription factor OsNAC2 maintains the homeostasis of immune responses to bacterial blight.

Rice (Oryza sativa) bacterial blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo), threatens plant growth and yield. However, the molecular mechanisms underlying rice immunity against Xoo remain elusive. Here, we identified a NAC (NAM-ATAF-CUC) transcription factor OsNAC2 as a negative regulator in the resistance to bacterial blight disease in rice. Constitutive overexpression of OsNAC2 inhibited the expression of salicylic acid (SA) biosynthesis-related genes (i.e. isochorismate synthase 1 (OsICS1), phenylalanine ammonia lyase 3 (OsPAL3), etc.) with adverse impacts on the pathogenesis-related proteins (PRs) responses and compromised blight resistance. Moreover, OsNAC2 interacted with APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factor OsEREBP1 and possibly threatened its protein stability, destroying the favorable interaction of OsEREBP1-Xa21-binding protein OsXb22a in the cytoplasm during Xoo-induced infection. On the contrary, downregulation of OsNAC2 resulted in enhanced resistance to bacterial blight in rice without any growth or yield penalties. Our results demonstrated that OsNAC2 inhibits SA signaling and stably interacted with OsEREBP1 to impair disease resistance. This OsNAC2-OsEREBP1-based homeostatic mechanism provided insights into the competition between rice and bacterial pathogens, and it will be useful to improve the disease resistance of important crops through breeding.

ZmEREB46, a maize ortholog of Arabidopsis WAX INDUCER1/SHINE1, is involved in the biosynthesis of leaf epicuticular very-long-chain waxes and drought tolerance

The aerial surfaces of plants are covered by a layer of cuticular wax that is composed of long-chain hydrocarbon compounds for protection against adverse environmental conditions. The current study identified a maize (Zea mays L.) APETALA2/ethylene-responsive element-binding protein (AP2/EREBP)-type transcription factor, ZmEREB46. Ectopic expression of ZmEREB46 in Arabidopsis increased the accumulation of epicuticular wax on the leaves and enhanced the drought tolerance of plants. The amounts of C24/C32 fatty acids, C32/C34 aldehydes, C32/C34 1-alcohols and C31 alkanes in zmereb46 (ZmEREB46 knockout mutant) leaves were reduced. The amount of leaf total epicuticular wax decreased approximately 50% in zmereb46. Compared to wild-type LH244 leaves, the cuticle permeability of zmereb46 leaves was increased, which resulted from decreased epicuticular wax load and a thinner cuticle layer. ZmEREB46 had transcriptional activation activity and directly bound to promoter regions of ZmCER2, ZmCER3.2 and ZmKCS1. The zmereb46 seedlings also exhibited reduced drought tolerance. These results, and the observations in ZmEREB46-overexpressing lines, suggest that ZmEREB46 is involved in cuticular metabolism by influencing the biosynthesis of very-long-chain waxes and participates in the cutin biosynthesis pathway. These results are helpful to further analyze the regulatory network of wax accumulation in maize.

Overexpression of Soybean GmWRI1a Stably Increases the Seed Oil Content in Soybean

WRINKLED1 (WRI1), an APETALA2/ethylene-responsive-element-binding protein (AP2/EREBP) subfamily transcription factor, plays a crucial role in the transcriptional regulation of plant fatty acid biosynthesis. In this study, GmWRI1a was overexpressed in the soybean cultivar ‘Dongnong 50’ using Agrobacterium-mediated transformation to generate three transgenic lines with high seed oil contents. PCR and Southern blotting analysis showed that the T-DNA was inserted into the genome at precise insertion sites and was stably inherited by the progeny. Expression analysis using qRT-PCR and Western blotting indicated that GmWRI1a and bar driven by the CaMV 35S promoter were significantly upregulated in the transgenic plants at different developmental stages. Transcriptome sequencing results showed there were obvious differences in gene expression between transgenic line and transgenic receptor during seed developmental stages. KEGG analysis found that the differentially expressed genes mainly annotated to metabolic pathways, such as carbohydrated metabolism and lipid metabolism. A 2-year single-location field trial revealed that three transgenic lines overexpressing GmWRI1a (GmWRI1a-OE) showed a stable increase in seed oil content of 4.97–10.35%. Importantly, no significant effect on protein content and yield was observed. Overexpression of GmWRI1a changed the fatty acid composition by increasing the linoleic acid (C18:2) content and decreasing the palmitic acid (C16:0) content in the seed. The three GmWRI1a-OE lines showed no significant changes in agronomic traits. The results demonstrated that the three GmWRI1a overexpression lines exhibited consistent increases in seed oil content compared with that of the wild type and did not significantly affect the seed yield and agronomic traits. The genetic engineering of GmWRI1a will be an effective strategy for the improvement of seed oil content and value in soybean.

OMICS approaches towards understanding plant's responses to counterattack heavy metal stress: An insight into molecular mechanisms of plant defense

Under constant climatic change and the existing environment, plants are exposed to several adverse factors such as extreme temperatures, high salt, heavy metals (HMs), drought, and pathogens. Prolonged exposure to such adverse conditions results in phytotoxicity and overall retardation of growth and development. Excess of HMs is one of the significant factors that affects plant growth significantly. Plants develop several defense mechanisms at the cellular and physiological levels to survive HMs stress. However, these internal defense mechanisms are not enough to overcome excessive HMs stress. Plants generate various secondary messengers to activate cell signaling, which results in the activation of several transcriptional responses associated with plant defense. Different transcription factors (TFs) gene families such as WRKY, my elob lastosis (MYB), basic leucine zipper domain (bZIP), basic helix-loop-helix (bHLH), APETALA2/ethylene-responsive element binding protein (AP2/EREBP), Cys2-His2 (C2H2), receptor genes/proteins, and kinases, like mitogen-activated protein kinase (MAPK), play an essential role in the regulation of stress using hormone-mediated pathways under HMs stress. Additionally, phytohormones and their signaling network also contribute to regulating cellular functions at the molecular level to combat HMs stress. Several mechanisms have been explored to understand the defense mechanism of plants under HMs stress. However, many more advanced molecular approaches are required to explore these defense mechanisms. Herein, we have described OMICS’ approaches to improve the plants' internal defense system to counteract HMs stress.

Genome-wide identification of AP2/EREBP in Fragaria vesca and expression pattern analysis of the FvDREB subfamily under drought stress

BackgroundDrought is a common phenomenon worldwide. It is also one of the main abiotic factors that affect the growth and quality of strawberry. The dehydration-responsive element binding protein (DREB) members that belong to the APETALA2/ethylene-responsive element binding protein (AP2/EREBP) superfamily are unique transcription factors in plants that play important roles in the abiotic stress response.ResultsHere, a total of 119 AP2/EREBP genes were identified in Fragaria vesca, and the AP2/EREBP superfamily was divided into AP2, RAV, ERF, DREB, and soloist subfamilies, containing 18, 7, 61, 32, and one member(s), respectively. The DREB subfamily was further divided into six subgroups (A-1 to A-6) based on phylogenetic analysis. Gene structure, conserved motifs, chromosomal location, and synteny analysis were conducted to comprehensively investigate the characteristics of FvDREBs. Furthermore, transcriptome analysis revealed distinctive expression patterns among the FvDREB genes in strawberry plants exposed to drought stress. The expression of FvDREB6 of the A-2 subgroup was down-regulated in old leaves and up-regulated in young leaves in response to drought. Furthermore, qRT-PCR analysis found that FvDREB8 from the A-2 subgroup had the highest expression level under drought stress. Together, analyses with the expression pattern, phylogenetic relationship, motif, and promoter suggest that FvDREB18 may play a critical role in the regulation of FvDREB1 and FvDREB2 expression.ConclusionsOur findings provide new insights into the characteristics and potential functions of FvDREBs. These FvDREB genes should be further studied as they appear to be excellent candidates for drought tolerance improvement of strawberry.

AtMYB92 enhances fatty acid synthesis and suberin deposition in leaves of Nicotiana benthamiana.

Acyl lipids are important constituents of the plant cell. Depending on the cell type, requirements in acyl lipids vary greatly, implying a tight regulation of fatty acid and lipid metabolism. The discovery of the WRINKLED1 (WRI1) transcription factors, members of the AP2-EREBP (APETALA2-ethylene-responsive element binding protein) family, has emphasized the importance of transcriptional regulation for adapting the rate of acyl chain production to cell requirements. Here, we describe the identification of another activator of the fatty acid biosynthetic pathway, the Arabidopsis MYB92 transcription factor. This MYB andall themembers of the subgroups S10 and S24 of MYB transcription factors can directly activate the promoter of BCCP2 that encodes a component of the fatty acid biosynthetic pathway. Two adjacent MYB cis-regulatory elements are essential for the binding and activation of the BCCP2 promoter by MYB92. Overexpression of MYB92 or WRI1 in Nicotiana benthamiana induces the expression of fatty acid biosynthetic genes but results in the accumulation of different types of acyl lipids. In the presence of WRI1, triacylglycerol biosynthetic enzymes coded by constitutively expressed genes efficiently channel the excess fatty acids toward reserve lipid accumulation. By contrast, MYB92 activates both fatty acid and suberin biosynthetic genes; hence, the remarkable increase in suberin monomers measured in leaves expressing MYB92. These results provide additional insight into the molecular mechanisms that control the biosynthesis of an important cell wall-associated acylglycerol polymer playing critical roles in plants.

The Maize AP2/EREBP Transcription Factor ZmEREB160 Enhances Drought Tolerance in Arabidopsis

Drought is a main factor affecting plant growth and crop production. APETALA2/Ethylene Responsive Element Binding Protein (AP2/EREBP) transcription factors (TFs) are involved in the response to abiotic stress and their functions in ABA signaling and the regulation of drought response have been intensively studied. However, AP2/EREBP TFs have been limited in the maize. The objective of our study was to identify the function of the novel maize AP2/EREBP gene ZmEREB160. Expression levels analysis in maize revealed that ZmEREB160 is significantly induced by PEG6000, NaCl and ABA treatment. ZmEREB160 localized to the nucleus when transiently expressed in Arabidopsis leaf protoplasts. ZmEREB160 activated the reporter gene and exhibited transcriptional activation activity in yeast cells. When overexpression of ZmEREB160 in Arabidopsis significantly enhanced tolerance to osmotic and ABA stress, overexpressed seedlings were longer roots under ABA and mannitol treatments compared with wild type. In addition, overexpression of ZmEREB160 Arabidopsis seedlings was found to elevate survival rate compared with wild type plants under drought stress. During the drought treatment, qRT-PCR assays showed that the expression levels of ABA/drought stress-related genes, ABI2, ABI5, COR15, DREB2A and RD29B were up-regulated in ZmEREB160 transgenic plants, transgenic plants accumulated more proline content than wild type plants. These results indicate that ZmEREB160 functions in response to drought and ABA stresses, and participates in ABA signaling pathway and may enhance drought tolerance.

OsERF101, an ERF family transcription factor, regulates drought stress response in reproductive tissues.

An ERF transcription factor OsERF101 is predominantly expressed in rice reproductive tissues and plays an important role in improving rice seed setting rate under drought stress. Drought reduces grain yield due to the cumulative damage effects to plant vegetative and reproductive developmental processes. However, the genes involved in these processes are still not completely understood. In this study, we identified a gene named OsERF101 as an important positive regulator in the adaptive responses to dehydration stress during the reproductive and vegetative stages. This gene encodes a member of APETALA2/Ethylene-Responsive Element Binding Protein (AP2/EREBP) family. OsERF101 was predominantly expressed in flowers, particularly in the tapetum and microspores under normal growth conditions. It was induced by drought, PEG6000 and abscisic acid (ABA) in leaves. During the vegetative stage, OsERF101-overexpression plants were more resistant to osmotic stress caused by PEG6000 compared to the control plants. They also had higher survival and seed setting rates than wild type when subjected to reproductive-stage drought stress. Further physiological analysis revealed that the pollen fertility was improved in the overexpression lines, while the knockout mutant and RNAi lines showed reduced pollen fertility and compromised drought tolerance during the reproductive stage. The increased proline content and peroxidase activity in OsERF101-overexpression plants might contribute to the improved drought-tolerance of plants. In addition, OsERF101-overexpression plants displayed ABA susceptible phenotype, in which the expression levels of ABA-responsive genes RD22, LEA3, and PODs were up-regulated. Taken together, our results indicate that OsERF101 is a gene that regulates dehydration responses during the vegetative and reproductive stages.

Molecular cloning and expression analysis of three ThERFs involved in the response to waterlogging stress of Taxodium 'Zhongshanshan406', and subcellular localization of the gene products.

As a subfamily of the APETALA 2/ethylene response element binding protein (AP2/EREBP) transcription factor superfamily, the ethylene response factor (ERF) is widely involved in the regulation of growth and response to various abiotic stresses in plants, and has been shown to be the main transcription factor regulating transcription of the genes related to hypoxia and waterlogging stress. In this study, three ThERF genes, with significant differences in expression profile in response to flooding stress, were identified from the transcriptomics data acquired from Taxodium hybrid ‘Zhongshanshan 406’ (T. mucronatum Tenore × T. distichum (L.) Rich) under waterlogging stress: ThERF15, ThERF39 and ThRAP2.3 (GenBank ID: KY463467, KY463468 and KY463470, respectively).The full-length cDNA of each of the three ERFs was obtained using the RACE (rapid amplification cDNA ends) method, and all three were intron-free. Multiple protein sequence alignments indicated that ThERF15, ThERF39 and ThRAP2.3 proteins all had only one AP2-ERF domain and belonged to the ERF subfamily. A transient gene expression assay demonstrated that ThERF15, ThERF39 and ThRAP2.3 were all localized to the nucleus. Real-time quantitative PCR (qPCR) revealed that the expression of ThERF15, ThERF39 and ThRAP2.3 exhibited significant differences, compared with the control, in response to two levels of flooding treatment (half-flooding or total-submergence) of ‘Zhongshanshan 406’. Quantification of ethylene concentration revealed that ethylene was more relevant to the level of expression than the period of flooding treatment. Based on the experimental results above, ThERF15, ThERF39 and ThRAP2.3 were identified as being related to the regulation of downstream flooding- responsive gene expression in ‘Zhongshanshan 406’. ThRAP2.3 is most likely to be a key downstream-response ERF gene to respond to the output of the ethylene signal generated by flooding stress.

A compendium of transcription factor and Transcriptionally active protein coding gene families in cowpea (Vigna unguiculata L.)

BackgroundCowpea (Vigna unguiculata (L.) Walp.) is the most important food and forage legume in the semi-arid tropics of sub-Saharan Africa where approximately 80% of worldwide production takes place primarily on low-input, subsistence farm sites. Among the major goals of cowpea breeding and improvement programs are the rapid manipulation of agronomic traits for seed size and quality and improved resistance to abiotic and biotic stresses to enhance productivity. Knowing the suite of transcription factors (TFs) and transcriptionally active proteins (TAPs) that control various critical plant cellular processes would contribute tremendously to these improvement aims.ResultsWe used a computational approach that employed three different predictive pipelines to data mine the cowpea genome and identified over 4400 genes representing 136 different TF and TAP families. We compare the information content of cowpea to two evolutionarily close species common bean (Phaseolus vulgaris), and soybean (Glycine max) to gauge the relative informational content. Our data indicate that correcting for genome size cowpea has fewer TF and TAP genes than common bean (4408 / 5291) and soybean (4408/ 11,065). Members of the GROWTH-REGULATING FACTOR (GRF) and Auxin/indole-3-acetic acid (Aux/IAA) gene families appear to be over-represented in the genome relative to common bean and soybean, whereas members of the MADS (Minichromosome maintenance deficient 1 (MCM1), AGAMOUS, DEFICIENS, and serum response factor (SRF)) and C2C2-YABBY appear to be under-represented. Analysis of the AP2-EREBP APETALA2-Ethylene Responsive Element Binding Protein (AP2-EREBP), NAC (NAM (no apical meristem), ATAF1, 2 (Arabidopsis transcription activation factor), CUC (cup-shaped cotyledon)), and WRKY families, known to be important in defense signaling, revealed changes and phylogenetic rearrangements relative to common bean and soybean that suggest these groups may have evolved different functions.ConclusionsThe availability of detailed information on the coding capacity of the cowpea genome and in particular the various TF and TAP gene families will facilitate future comparative analysis and development of strategies for controlling growth, differentiation, and abiotic and biotic stress resistances of cowpea.

Soybean (Glycine max) WRINKLED1 transcription factor, GmWRI1a, positively regulates seed oil accumulation.

Soybean is the world's most important leguminous crop producing high-quality protein and oil. Elevating oil accumulation in soybean seed is always many researchers' goal. WRINKLED1 (WRI1) encodes a transcription factor of the APETALA2/ethylene responsive element-binding protein (AP2/EREBP) family that plays important roles during plant seed oil accumulation. In this study, we isolated and characterized three distinct orthologues of WRI1 in soybean (Glycine max) that display different organ-specific expression patterns, among which GmWRI1a was highly expressed in maturing soybean seed. Electrophoretic mobility shift assays and yeast one-hybrid experiments demonstrated that the GmWRI1a protein was capable of binding to AW-box, a conserved sequence in the proximal upstream regions of many genes involved in various steps of oil biosynthesis. Transgenic soybean seeds overexpressing GmWRI1a under the control of the seed-specific napin promoter showed the increased total oil and fatty acid content and the changed fatty acid composition. Furthermore, basing on the activated expressions in transgenic soybean seeds and existence of AW-box element in the promoter regions, direct downstream genes of GmWRI1a were identified, and their products were responsible for fatty acid production, elongation, desaturation and export from plastid. We conclude that GmWRI1a transcription factor can positively regulate oil accumulation in soybean seed by a complex gene expression network related to fatty acid biosynthesis.

Complementary RNA-Sequencing Based Transcriptomics and iTRAQ Proteomics Reveal the Mechanism of the Alleviation of Quinclorac Stress by Salicylic Acid in Oryza sativa ssp. japonica

To uncover the alleviation mechanism of quinclorac stress by salicylic acid (SA), leaf samples of Oryza sativa ssp. Japonica under quinclorac stress with and without SA pre-treatment were analyzed for transcriptional and proteomic profiling to determine the differentially expressed genes (DEGs) and proteins (DEPs), respectively. Results showed that quinclorac stress altered the expression of 2207 DEGs (1427 up-regulated, 780 down-regulated) and 147 DEPs (98 down-regulated, 49 up-regulated). These genes and proteins were enriched in glutathione (GSH) metabolism, porphyrin and chlorophyll metabolism, the biosynthesis of secondary metabolites, glyoxylate and dicarboxylate metabolism, and so on. It also influenced apetala2- ethylene-responsive element binding protein (AP2-EREBP) family, myeloblastosis (MYB) family and WRKY family transcription factors. After SA pre-treatment, 697 genes and 124 proteins were differentially expressed. Pathway analysis showed similar enrichments in GSH, glyoxylate and dicarboxylate metabolism. Transcription factors were distributed in basic helix-loop-helix (bHLH), MYB, Tify and WRKY families. Quantitative real-time PCR results revealed that quinclorac stress induced the expression of glutathion reductase (GR) genes (OsGR2, OsGR3), which was further pronounced by SA pre-treatment. Quinclorac stress further mediated the accumulation of acetaldehyde in rice, while SA enhanced the expression of OsALDH2B5 and OsALDH7 to accelerate the metabolism of herbicide quinclorac for the protection of rice. Correlation analysis between transcriptome and proteomics demonstrated that, under quinclorac stress, correlated proteins/genes were mainly involved in the inhibition of intermediate steps in the biosynthesis of chlorophyll. Other interesting proteins/genes and pathways regulated by herbicide quinclorac and modulated by SA pre-treatment were also discussed, based on the transcriptome and proteomics results.

Expansion and stress responses of the AP2/EREBP superfamily in cotton

BackgroundThe allotetraploid cotton originated from one hybridization event between an extant progenitor of Gosssypium herbaceum (A1) or G. arboreum (A2) and another progenitor, G. raimondii Ulbrich (D5) 1–1.5 million years ago (Mya). The APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factors constitute one of the largest and most conserved gene families in plants. They are characterized by their AP2 domain, which comprises 60–70 amino acids, and are classified into four main subfamilies: the APETALA2 (AP2), Related to ABI3/VP1 (RAV), Dehydration-Responsive Element Binding protein (DREB) and Ethylene-Responsive Factor (ERF) subfamilies. The AP2/EREBP genes play crucial roles in plant growth, development and biotic and abiotic stress responses. Hence, understanding the molecular characteristics of cotton stress tolerance and gene family expansion would undoubtedly facilitate cotton resistance breeding and evolution research.ResultsA total of 269 AP2/EREBP genes were identified in the G. raimondii (D5) cotton genome. The protein domain architecture and intron/exon structure are simple and relatively conserved within each subfamily. They are distributed throughout all chromosomes but are clustered on various chromosomes due to genomic tandem duplication. We identified 73 tandem duplicated genes and 221 segmental duplicated gene pairs which contributed to the expansion of AP2/EREBP superfamily. Of them, tandem duplication was the most important force of the expansion of the B3 group. Transcriptome analysis showed that 504 AP2/EREBP genes were expressed in at least one tested G. hirsutum TM-1 tissues. In G. hirsutum, 151 non-repeated genes of the DREB and ERF subfamily genes were responsive to different stresses: 132 genes were induced by cold, 63 genes by drought and 94 genes by heat. qRT-PCR confirmed that 13 GhDREB and 15 GhERF genes were induced by cold and/or drought. No transcripts detected for 53 of the 111 tandem duplicated genes in TM-1. In addition, some homoeologous genes showed biased expression toward either A-or D-subgenome.ConclusionsThe AP2/EREBP genes were obviously expanded in Gossypium. The GhDREB and GhERF genes play crucial roles in cotton stress responses. Our genome-wide analysis of AP2/EREBP genes in cotton provides valuable information for characterizing the molecular functions of AP2/EREBP genes and reveals insights into their evolution in polyploid plants.

A transcriptome approach towards understanding the development of ripening capacity in 'Bartlett' pears (Pyrus communis L.).

BackgroundThe capacity of European pear fruit (Pyrus communis L.) to ripen after harvest develops during the final stages of growth on the tree. The objective of this study was to characterize changes in ‘Bartlett’ pear fruit physico-chemical properties and transcription profiles during fruit maturation leading to attainment of ripening capacity.ResultsThe softening response of pear fruit held for 14 days at 20 °C after harvest depended on their maturity. We identified four maturity stages: S1-failed to soften and S2- displayed partial softening (with or without ET-ethylene treatment); S3 - able to soften following ET; and S4 - able to soften without ET. Illumina sequencing and Trinity assembly generated 68,010 unigenes (mean length of 911 bp), of which 32.8 % were annotated to the RefSeq plant database. Higher numbers of differentially expressed transcripts were recorded in the S3-S4 and S1-S2 transitions (2805 and 2505 unigenes, respectively) than in the S2-S3 transition (2037 unigenes). High expression of genes putatively encoding pectin degradation enzymes in the S1-S2 transition suggests pectic oligomers may be involved as early signals triggering the transition to responsiveness to ethylene in pear fruit. Moreover, the co-expression of these genes with Exps (Expansins) suggests their collaboration in modifying cell wall polysaccharide networks that are required for fruit growth. K-means cluster analysis revealed that auxin signaling associated transcripts were enriched in cluster K6 that showed the highest gene expression at S3. AP2/EREBP (APETALA 2/ethylene response element binding protein) and bHLH (basic helix-loop-helix) transcripts were enriched in all three transition S1-S2, S2-S3, and S3-S4. Several members of Aux/IAA (Auxin/indole-3-acetic acid), ARF (Auxin response factors), and WRKY appeared to play an important role in orchestrating the S2-S3 transition.ConclusionsWe identified maturity stages associated with the development of ripening capacity in ‘Bartlett’ pear, and described the transcription profile of fruit at these stages. Our findings suggest that auxin is essential in regulating the transition of pear fruit from being ethylene-unresponsive (S2) to ethylene-responsive (S3), resulting in fruit softening. The transcriptome will be helpful for future studies about specific developmental pathways regulating the transition to ripening.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1939-9) contains supplementary material, which is available to authorized users.

Plant acclimation to environmental stress: a critical appraisal

Plant acclimation to environmental stress: a critical appraisal

A novel DREB transcription factor from Halimodendron halodendron leads to enhance drought and salt tolerance in Arabidopsis

A new member of the APETALA2/ethylene responsive element binding protein (AP2/EREBP) transcription factor family, HhDREB2, was isolated from Halimodendron halodendron. Based on the similarity of the AP2/ERF domain, HhDREB2 was classified into A-5 group of the DREB subfamily. The expression of HhDREB2 gene was induced by drought, high salinity, and low temperature, but not by exogenous plant hormones. Trans-activity assay demonstrated that HhDREB2 gene encodes a transcription activator. Furthermore, over-expression of HhDREB2 gene under the stress-inducible rd29A promotor in Arabidopsis resulted in enhanced tolerance to salt and drought stresses. The overall results reveal that HhDREB2 functioned as important transcription factor in regulation of stress-responsive signaling in plants and may be used for improving plant tolerance to abiotic stresses.

Ectopic expression of Dendrobium EREB5 gene in Arabidopsis influences leaf morphology

The Dendrobium genus belongs to the Orchidaceae family and includes a broad range of terrestrial, epiphytic, and lithophytic plants. The genus includes many well-loved ornamental plants and valuable sources of traditional Chinese herbal medicines. Although popular and useful, their slow growth and vulnerability to environmental stress has strongly limited their more widespread adoption. The aim of our research is to expand the understanding of the regulation of Dendrobium growth and development. In this study, the gene encoding EREB5, a member of the APETALA2/ethylene-responsive element-binding protein (AP2/EREBP) family of transcription factors, was cloned from Dendrobium officinale. The full-length ORF of 558 bp, is predicted to code for a protein of 185 amino acids. The putative protein contains one copy of the well-conserved AP2 domain and a typical EAR motif, present in members of the ethylene response factor (ERF) subfamily. Arabidopsis thaliana plants ectopically expressing the DoEREB5 gene showed several phenotypic differences from wild-type plants, including altered leaf shape and petiole size. Expression of a DoEREB5 RNAi construct in Arabidopsis led to reduced levels of the putative Arabidopsis ortholog, AtERF9. Plants with reduced levels of AtERF9 showed an increase in chlorophyll content and photosynthetic rates compared to wild-type and heterologous ectopic DoEREB5 expression plants. Photosynthetic rates in the RNAi plants were higher than those in WT plants under both high and low light. Our results have provided clues as to the in vivo role of the Dendrobium gene, DoEREB5, suggesting that it is involved in the important processes of leaf development and function.

The Arabidopsis thaliana At4g13040 gene, a unique member of the AP2/EREBP family, is a positive regulator for salicylic acid accumulation and basal defense against bacterial pathogens

The Arabidopsis genome contains a large number of putative transcription factors, containing a DNA binding domain similar to APETALA2/ethylene response element binding protein (AP2/EREBP), for most of which a function is not known. Phylogenetic analysis divides the Apetala 2 (AP2) super-family into 5 major groups: AP2, RAV, ethylene response factor (ERF), dehydration response element binding protein (DREB) and At4g13040. Similar to ERF and DREB, the At4g13040 protein contains only one AP2 domain; however, its structural uniqueness places it into a distinct group. In this article, we report that At4g13040 (referred herein as Apetala 2 family protein involved in SA mediated disease defense 1 - APD1) is an important regulator for SA mediated plant defense. The APD1 gene is upregulated upon pathogen inoculation, exogenous SA application and in the mutant that constitutively activates SA signaling. The T-DNA insertion lines (inserted in the APD1 promoter), which fail to induce expression upon pathogen inoculation, are compromised for resistance against virulent bacterial pathogens and show reduced induction of pathogenesis related 1 gene. Our results suggest that APD1 functions downstream of PAD4 in Arabidopsis and promotes pathogen-induced SA accumulation. Exogenous SA application completely restores the loss-of-resistance phenotype of the apd1 mutant. Thus, APD1 is a positive regulator of disease defense that functions upstream of SA accumulation.

Overexpression of <i>CnANT</i>, Coconut <i>BABYBOOM</i> Homologue Alters Plant Growth and Morphology in Transgenic <i>Arabidopsis</i> Plants

Identification of molecular markers for the recognition of tissues competent for in vitro regeneration has a considerable potential for increasing the efficiency of coconut somatic embryogenesis protocol. BABYBOOM (BBM), APETALA2/Ethylene Responsive Element Binding Protein (AP2/EREBP) family gene has shown a role during embryogenesis. To gain insight into the functions of CnANT, a BABYBOOM gene homologue of coconut was cloned under the cauliflower mosaic virus-35s promoter and the plant growth and developmental changes that occur in transgenic <em>Arabidopsis</em> plants were determined and compared with the data obtained for other BBM genes originated from other plant species. Construction of vectors for <em>Arabidopsis</em> transformation was based on pGREEN plasmid. Transformed plants were screened by spraying herbicide Harvest which contains the active ingredient gluphosinate ammonium. Five independent transgenic lines were developed. All lines showed the typical 3:1 segregation ratio suggesting that the bar genes had integrated into the <em>Arabidopsis</em> as a single segregating locus. In comparison to wild type plants, overexpression of CnANT resulted in pleiotropic phenotypes including compact rosettes, backward curly-leaves with serrated margins, larger flowers and smaller siliques. There were also modifications in plant growth such as delay in flowering. Similar phenotypic effects observed for CnANT along with BBM and other related genes from eudicots suggest similar biochemical targets governed by these proteins. Similarity of their expression, thus suggest that CnANT gene also has a potential to increase in vitro regeneration response. <em>Tropical Agricultural Research Vol. 23 (3): 249-260 (2012)</em> DOI: <a href="http://dx.doi.org/10.4038/tar.v23i3.4662">http://dx.doi.org/10.4038/tar.v23i3.4662</a>

Gene Structures, Classification and Expression Models of the AP2/EREBP Transcription Factor Family in Rice

We identified 163 AP2/EREBP (APETALA2/ethylene-responsive element-binding protein) genes in rice. We analyzed gene structures, phylogenies, domain duplication, genome localizations and expression profiles. Conserved amino acid residues and phylogeny construction using the AP2/ERF conserved domain sequence suggest that in rice the OsAP2/EREBP gene family can be classified broadly into four subfamilies [AP2, RAV (related to ABI3/VP1), DREB (dehydration-responsive element-binding protein) and ERF (ethylene-responsive factor)]. The chromosomal localizations of the OsAP2/EREBP genes indicated 20 segmental duplication events involving 40 genes; 58 redundant OsAP2/EREBP genes were involved in tandem duplication events. There were fewer introns after segmental duplication. We investigated expression profiles of this gene family under biotic stresses [infection with rice viruses such as rice stripe virus (RSV), rice tungro spherical virus (RTSV) and rice dwarf virus (RDV, three virus strains S, O and D84)], and various abiotic stresses. Symptoms of virus infection were more severe in RSV infection than in RTSV and RDV infection. Responses to biotic stresses are novel findings and these stresses enhance the ability to identify the best candidate genes for further functional analysis. The genes of subgroup B-5 were not induced under abiotic treatments whereas they were activated by the three RDV strains. None of the genes of subgroups A-3 were differentially expressed by any of the biotic stresses. Our 44K and 22K microarray results suggest that 53 and 52 non-redundant genes in this family were up-regulated in response to biotic and abiotic stresses, respectively. We further examined the stress responsiveness of most genes by reverse transcription-PCR. The study results should be useful in selecting candidate genes from specific subgroups for functional analysis.