APETALA2/ethylene-responsive Element Binding Factor Research Articles

Genome-Wide Exploration of the Ethylene-Responsive Element-Binding Factor Gene Family in Sweet Cherry (Prunus avium L.): Preliminarily Unveiling Insights into Normal Development and Fruit Cracking

The ERF subfamily, a significant part of the APETALA2/ethylene-responsive element-binding factor (AP2/ERF) transcription family, plays a crucial role in plant growth, development, and stress responses. Despite its importance, research on this gene family in sweet cherry (Prunus avium L.) is limited. This study identified and analyzed the sweet cherry ERF subfamily in terms of classification, physicochemical properties, structural characteristics, chromosome distribution, gene replication and collinearity, Cis-acting elements, and potential protein interactions. Preliminary investigations of transcription during fruit cracking and normal development were also conducted. Fifty ERFs (PatiERF1~50) were identified, distributed unevenly across eight chromosomes and classified into ten groups with nineteen conserved motifs. Collinearity analysis with other plant species revealed homology, with the highest number of ERF orthologous genes found in apple (Malus domestica L.). Cis-acting elements, particularly abscisic acid response factor, were abundant in PatiERF promoters. Weighted gene co-expression network analysis (WGCNA) and quantitative real-time PCR (RT-qPCR) analysis indicated the involvement of PatiERFs in sweet cherry fruit development and cracking, and nine and four significant candidates related to these processes were speculated, respectively. Furthermore, four other classes of transcription factors (TFs), namely MYB, GRAS, BHLH, and BZIP, as well as 23 structure genes, were predicted to have co-expression and interaction relationships with PatiERFs during fruit development. This suggests their potential synergistic regulation with ERFs in the cherry fruit development process. Our study represents the first comprehensive genome-wide analysis of the ERF subfamily in sweet cherry, laying a crucial foundation for a deeper understanding of the molecular mechanisms correlated with fruit growth, development, and cracking mediated by ERF genes.

PagERF81 regulates lignin biosynthesis and xylem cell differentiation in poplar.

Lignin is a major component of plant cell walls and is essential for plant growth and development. Lignin biosynthesis is controlled by a hierarchical regulatory network involving multiple transcription factors. In this study, we showed that the gene encoding an APETALA 2/ethylene-responsive element binding factor (AP2/ERF) transcription factor, PagERF81, from poplar 84 K (Populus alba × P. glandulosa) is highly expressed in expanding secondary xylem cells. Two independent homozygous Pagerf81 mutant lines created by gene editing, produced significantly more but smaller vessel cells and longer fiber cells with more lignin in cell walls, while PagERF81 overexpression lines had less lignin, compared to non-transgenic controls. Transcriptome and reverse transcription quantitative PCR data revealed that multiple lignin biosynthesis genes including Cinnamoyl CoA reductase 1 (PagCCR1), Cinnamyl alcohol dehydrogenase 6 (PagCAD6), and 4-Coumarate-CoA ligase-like 9 (Pag4CLL9) were up-regulated in Pagerf81 mutants, but down-regulated in PagERF81 overexpression lines. In addition, a transient transactivation assay revealed that PagERF81 repressed the transcription of these three genes. Furthermore, yeast one hybrid and electrophoretic mobility shift assays showed that PagERF81 directly bound to a GCC sequence in the PagCCR1 promoter. No known vessel or fiber cell differentiation related genes were differentially expressed, so the smaller vessel cells and longer fiber cells observed in the Pagerf81 lines might be caused by abnormal lignin deposition in the secondary cell walls. This study provides insight into the regulation of lignin biosynthesis, and a molecular tool to engineer wood with high lignin content, which would contribute to the lignin-related chemical industry and carbon sequestration.

Genome-Wide Analysis of the AP2/ERF Family in Oily Persimmon (Diospyros oleifera) and Their Preliminary Roles Exploration in Response to Polyamines for Adventitious Root Formation in Cultivated Persimmon (D. kaki)

The AP2/ERF (APETALA2/Ethylene−Responsive element binding factor) family genes play crucial roles in plant growth and development, and responses to environmental factors; however, this family has not been characterized in Diospyros species. In Diospyros, the diploid Oily persimmon (D. oleifera, 2n = 2x = 30) has been released with complete genome assembly, which makes it possible for genome-wide gene family identification and exploration of molecular function in cultivated persimmon (D. kaki, 2n = 6x = 90). Here, we identified the AP2/ERF family in Oily persimmon for the first time and investigated its classification, main physicochemical properties, structural characteristic, chromosome distribution, gene replication and collinearity, cis-factor binding sites deduction, GO term annotation, and PPI interaction, as well as its expression profiles in different tissue and under the treatment of polyamines. A total of 157 AP2/ERF genes, including four subfamilies (AP2, RAV, Soloist, and ERF), were identified with distribution on all 15 chromosomes. DkAP2/ERF gene expression patterns were extensive and diverse. They were detected expression in every examined tissue, with the highest number of DkAP2/ERF genes expressed in the root. DkAP2/ERF gene expression analysis in adventitious root generation and elongation of polyamines showed their different responses to the action of polyamines, and more pairs of DkAP2/ERF genes with high correlation in gene expression were obtained. In addition, some DkAP2/ERF genes were detected remarkably correlated with genes related to polyamine synthesis and cell metabolism, including S-adenosyl-L-methionine Decarboxyla2 (SAMDC2), D-type cyclin1 (CYCD1), and D-type cyclin2 (CYCD2) genes,. indicating that DkAP2/ERF genes may play a synergistic role in adventitious root development This study was the first to analyze the AP2/ERF gene comprehensively in Diospyros on a genome−wide scale and will provide insights into the application of adventitious root formation in cultivated persimmon.

Full-length fruit transcriptomes of southern highbush (Vaccinium sp.) and rabbiteye (V. virgatum Ait.) blueberry

BackgroundBlueberries (Vaccinium sp.) are native to North America and breeding efforts to improve blueberry fruit quality are focused on improving traits such as increased firmness, enhanced flavor and greater shelf-life. Such efforts require additional genomic resources, especially in southern highbush and rabbiteye blueberries.ResultsWe generated the first full-length fruit transcriptome for the southern highbush and rabbiteye blueberry using the cultivars, Suziblue and Powderblue, respectively. The transcriptome was generated using the Pacific Biosciences single-molecule long-read isoform sequencing platform with cDNA pooled from seven stages during fruit development and postharvest storage. Raw reads were processed through the Isoseq pipeline and full-length transcripts were mapped to the ‘Draper’ genome with unmapped reads collapsed using Cogent. Finally, we identified 16,299 and 15,882 non-redundant transcripts in ‘Suziblue’ and ‘Powderblue’ respectively by combining the reads mapped to Northern Highbush blueberry ‘Draper’ genome and Cogent analysis. In both cultivars, > 80% of sequences were longer than 1,000 nt, with the median transcript length around 1,700 nt. Functionally annotated transcripts using Blast2GO were > 92% in both ‘Suziblue’ and ‘Powderblue’ with overall equal distribution of gene ontology (GO) terms in the two cultivars. Analyses of alternative splicing events indicated that around 40% non-redundant sequences exhibited more than one isoform. Additionally, long non-coding RNAs were predicted to represent 5.6% and 7% of the transcriptomes in ‘Suziblue’ and ‘Powderblue’, respectively. Fruit ripening is regulated by several hormone-related genes and transcription factors. Among transcripts associated with phytohormone metabolism/signaling, the highest number of transcripts were related to abscisic acid (ABA) and auxin metabolism followed by those for brassinosteroid, jasmonic acid and ethylene metabolism. Among transcription factor-associated transcripts, those belonging to ripening-related APETALA2/ethylene-responsive element-binding factor (AP2/ERF), NAC (NAM, ATAF1/2 and CUC2), leucine zipper (HB-zip), basic helix-loop-helix (bHLH), MYB (v-MYB, discovered in avian myeloblastosis virus genome) and MADS-Box gene families, were abundant.Further we measured three fruit ripening quality traits and indicators [ABA, and anthocyanin concentration, and texture] during fruit development and ripening. ABA concentration increased during the initial stages of fruit ripening and then declined at the Ripe stage, whereas anthocyanin content increased during the final stages of fruit ripening in both cultivars. Fruit firmness declined during ripening in ‘Powderblue’. Genes associated with the above parameters were identified using the full-length transcriptome. Transcript abundance patterns of these genes were consistent with changes in the fruit ripening and quality-related characteristics.ConclusionsA full-length, well-annotated fruit transcriptome was generated for two blueberry species commonly cultivated in the southeastern United States. The robustness of the transcriptome was verified by the identification and expression analyses of multiple fruit ripening and quality–regulating genes. The full-length transcriptome is a valuable addition to the blueberry genomic resources and will aid in further improving the annotation. It will also provide a useful resource for the investigation of molecular aspects of ripening and postharvest processes.

Herbaceous peony AP2/ERF transcription factor binds the promoter of the tryptophan decarboxylase gene to enhance high-temperature stress tolerance.

Global warming has multifarious adverse effects on plant growth and productivity. Nonetheless, the effects of endogenous phytomelatonin on the high-temperature resistance of plants and the underlying genetic mechanisms remain unclear. Here, herbaceous peony (Paeonia lactiflora Pall.) tryptophan decarboxylase (TDC) gene involved in phytomelatonin biosynthesis was shown to respond to high-temperature stress at the transcriptional level, and its transcript level was positively correlated with phytomelatonin production. Moreover, overexpression of PlTDC enhanced phytomelatonin production and high-temperature stress tolerance in transgenic tobacco, while silencing PlTDC expression decreased these parameters in P. lactiflora. In addition, a 2402 bp promoter fragment of PlTDC was isolated, and DNA pull-down assay revealed that one APETALA2/ethylene-responsive element-binding factor (AP2/ERF) transcription factor, PlTOE3, could specifically activate the PlTDC promoter, which was further verified by yeast one-hybrid assay and luciferase reporter assay. PlTOE3 was a nucleus-localized protein, and its transcript level responded to high-temperature stress. Additionally, transgenic tobacco overexpressing PlTOE3 showed enhanced phytomelatonin production and high-temperature stress tolerance, while silencing PlTDC expression obtained the opposite results. These results illustrated that PlTOE3 bound the PlTDC promoter to enhance high-temperature stress tolerance by increasing phytomelatonin production in P. lactiflora.

Genome-Wide Screening of AP2 Transcription Factors Involving in Fruit Color and Aroma Regulation of Cultivated Strawberry.

Fragaria × ananassa Duch, which among the youngest fruit crops, comprises many popular cultivars that are famous for their favored color and aroma. The regulation roles of AP2/ERF (APETALA2/ethylene-responsive element-binding factor) transcription factors in fruit flavor and color regulation have been studied in several fruit crops. The AP2 family of strawberry, which was ignored in recent AP2/ERF identification studies, was explored in this study. A total of 64 FaAP2 (Fragaria × ananassa AP2) transcription factors belonging to the euAP2, euANT (AINTEGUMENTA), and baselANT groups were identified with canonical insertion motifs in two AP2 domains. The motif identification illustrated that motifs 1, 5, and 2 indicated a corresponding AP2 domain repeat 1 with a linker region, and motifs 6, 4, 3 indicated a corresponding AP2 domain repeat 2, all of which were highly conserved. By synteny analysis, FaAP2 paralogs were identified in each sub-genome, and FaAP2 gene duplication and loss explained the unequal AP2 loci of sub-genomes. The expression profile in three cultivars indicated that six FaAP2 paralogs—four WRI (WRINKLED) gene homologs and two AP2 gene homologs—were candidate regulators of red fruit color and/or special fruit aroma. All these finds provide a basis for further investigations into role of AP2 in fruit color and aroma and would be helpful in the targeted selection of strawberry fruit quality to improve breeding.

Expression of Three Related to ABI3/VP1 Genes in Medicago truncatula Caused Increased Stress Resistance and Branch Increase in Arabidopsis thaliana.

Related to ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1(VP1)(RAV) transcription factors, which encode a B3 domain and an APETALA2(AP2) domain, belong to the APETALA2/ethylene-responsive element binding factor(AP2/ERF) or B3 superfamily and play an important role in regulating plant growth and development and responding to abiotic stress. Although there have been many functional studies on RAV, the functional differences between RAVs are not clear. Therefore, in this study, the functional differences of RAVs of Medicago truncatula were analyzed. Based on sequence data from the plant transcription factor database and the M. truncatula genome database, we cloned three RAV genes from M. truncatula, named MtRAV1, MtRAV2, and MtRAV3. The cis-acting elements of these genes promoters were predicted, and the expression patterns of MtRAVs under exogenous conditions (4°C, NaCl, Polyethylene Glycol, Abscisic acid) were analyzed. MtRAVs transgenic Arabidopsis thaliana were obtained and subjected to adversity treatment. Subcellular localization results indicated that MtRAVs were located in the nucleus. A much lower expression level was observed for MtRAV3 than the levels of MtRAV1 and MtRAV2 in M. truncatula for growth in normal conditions, but under 4°C or PEG and NaCl treatment, the expression level of MtRAV3 was significantly increased. Only the MtRAV3 overexpression transgenic plants showed strong cold resistance, but the overexpressed MtRAV1 and MtRAV2 transgenic plants showed no difference from wild type plants. MtRAV transgenic plants exhibited similar response to exogenous mannitol, NaCl, and ABA, and the expression of some adverse-related marker genes were up-regulated, such as COLD REGULATED 414 THYLAKOID MEMBRANE 1 (COR414-TM1), Arabidopsis thaliana drought-induced 21 (AtDI21), and Arabidopsis thaliana phosphatidylinositol-specific phospholipase C (ATPLC). MtRAVs transgenic Arabidopsis thaliana exhibited increasing of branch number. These results indicated that there was some function redundancy during MtRAVs proteins of M. truncatula, and MtRAV3 has increased function compared to the other two genes. The results of this study should provide the foundation for future application of MtRAVs in legumes.

Overexpression of Malus hupehensis MhSHN1 Gene Enhances Salt and Osmotic Stress Tolerance in Transgenic Tobacco Plants

Plant APETALA2/Ethylene-responsive element binding factor (AP2/ERF) transcription factor involved in various biological functions such as plant development, flower development, fruit and seed maturation, wounding, pathogen defense, high salty, drought, and so on. Here, we reported the function analysis of an AP2/ERF member, MhSHN1, from Malus hupehensis (Pamp.) Rehder. The MhSHN1 gene has a total of 1171 bp and consists of two exons of 81 and 531 bp, one intron of 559 bp. Phylogenetic analysis indicated MhSHN1 belongs to group V. The expression of MhSHN1 was highest in flower, then in seed and fruit, with low expression in root, leaves and stems. The MhSHN1 transcripts were barely affected by jasmonic acid (JA) and 1-aminocyclopropane- 1-carboxylic acid (ACC) within the first 48 h of treatment. Salicylic acid (SA) and abscisic acid (ABA) induce significantly the expression of MhSHN1. MhSHN1 mRNA levels were increased in response to mannitol and salt treatments. However, low temperature could not induce the expression of MhSHN1 gene. Overexpression of the MhSHN1 gene does not regulate seed germination but does positively regulate plant tolerance to salt and osmotic stresses at the stage of seed germination. These results showed the MhSHN1 gene play key role in resistance to mannitol and salt stresses in M. hupehensis.

Overexpression of ERF1-V from Haynaldia villosa Can Enhance the Resistance of Wheat to Powdery Mildew and Increase the Tolerance to Salt and Drought Stresses.

The APETALA 2/Ethylene-responsive element binding factor (AP2/ERF) transcription factor gene family is widely involved in the biotic and abiotic stress regulation. Haynaldia villosa (VV, 2n = 14), a wild species of wheat, is a potential gene pool for wheat improvement. H. villosa confers high resistance to several wheat diseases and high tolerance to some abiotic stress. In this study, ERF1-V, an ethylene-responsive element-binding factor gene of the AP2/ERF transcription factor gene family from wild H. villosa, was cloned and characterized. Sequence and phylogenetic analysis showed that ERF1-V is a deduced B2 type ERF gene. ERF1-V was first identified as a Blumeria graminis f. sp. tritici (Bgt) up-regulated gene, and later found to be induced by drought, salt and cold stresses. In responses to hormones, ERF1-V was up-regulated by ethylene and abscisic acid, but down-regulated by salicylic acid and jasmonic acid. Over expression of ERF1-V in wheat could improve resistance to powdery mildew, salt and drought stress. Chlorophyll content, malondialdehyde content, superoxide dismutase and peroxidase activity were significantly differences between the recipient Yangmai158 and the transgenic plants following salt treatment. Furthermore, the expression levels of some stress responsive genes were differences after drought or salt treatments. Although ERF1-V was activated by the constitutive promoter, the agronomic traits, including flowering time, plant height, effective tiller number, spikelet number per spike and grain size, did not changed significantly. ERF1-V is a valuable gene for wheat improvement by genetic engineering.

Divergent Expression Patterns in Two Vernicia Species Revealed the Potential Role of the Hub Gene VmAP2/ERF036 in Resistance to Fusarium oxysporum in Vernicia montana.

Tung oil tree (Vernicia fordii) is a promising industrial oil crop; however, this tree is highly susceptible to Fusarium wilt disease. Conversely, Vernicia montana is resistant to the pathogen. The APETALA2/ethylene-responsive element binding factor (AP2/ERF) transcription factor superfamily has been reported to play a significant role in resistance to Fusarium oxysporum. In this study, comprehensive analysis identified 75 and 81 putative Vf/VmAP2/ERF transcription factor-encoding genes in V. fordii and V. montana, respectively, which were divided into AP2, ERF, related to ABI3 and VP1 (RAV) and Soloist families. After F. oxysporum infection, a majority of AP2/ERF superfamily genes showed strong patterns of repression in both V. fordii and V. montana. We then identified 53 pairs of one-to-one orthologs in V. fordii and V. montana, with most pairs of orthologous genes exhibiting similar expression in response to the pathogen. Further investigation of Vf/VmAP2/ERF gene expression in plant tissues indicated that the pairs of genes with different expression patterns in response to F. oxysporum tended to exhibit different tissue profiles in the two species. In addition, VmAP2/ERF036, showing the strongest interactions with 666 genes, was identified as a core hub gene mediating resistance. Moreover, qRT-PCR results indicated VmAP2/ERF036 showed repressed expression while its orthologous gene VfAP2/ERF036 had the opposite expression pattern during pathogen infection. Overall, comparative analysis of the Vf/VmAP2/ERF superfamily and indication of a potential hub resistance gene in resistant and susceptible Vernicia species provides valuable information for understanding the molecular basis and selection of essential functional genes for V. fordii genetic engineering to control Fusarium wilt disease.

Overexpression of the Transcription Factors GmSHN1 and GmSHN9 Differentially Regulates Wax and Cutin Biosynthesis, Alters Cuticle Properties, and Changes Leaf Phenotypes in Arabidopsis.

SHINE (SHN/WIN) clade proteins, transcription factors of the plant-specific APETALA 2/ethylene-responsive element binding factor (AP2/ERF) family, have been proven to be involved in wax and cutin biosynthesis. Glycine max is an important economic crop, but its molecular mechanism of wax biosynthesis is rarely characterized. In this study, 10 homologs of Arabidopsis SHN genes were identified from soybean. These homologs were different in gene structures and organ expression patterns. Constitutive expression of each of the soybean SHN genes in Arabidopsis led to different leaf phenotypes, as well as different levels of glossiness on leaf surfaces. Overexpression of GmSHN1 and GmSHN9 in Arabidopsis exhibited 7.8-fold and 9.9-fold up-regulation of leaf cuticle wax productions, respectively. C31 and C29 alkanes contributed most to the increased wax contents. Total cutin contents of leaves were increased 11.4-fold in GmSHN1 overexpressors and 5.7-fold in GmSHN9 overexpressors, mainly through increasing C16:0 di-OH and dioic acids. GmSHN1 and GmSHN9 also altered leaf cuticle membrane ultrastructure and increased water loss rate in transgenic Arabidopsis plants. Transcript levels of many wax and cutin biosynthesis and leaf development related genes were altered in GmSHN1 and GmSHN9 overexpressors. Overall, these results suggest that GmSHN1 and GmSHN9 may differentially regulate the leaf development process as well as wax and cutin biosynthesis.

Evolution of the APETALA2 Gene Lineage in Seed Plants.

Gene duplication is a fundamental source of functional evolutionary change and has been associated with organismal diversification and the acquisition of novel features. The APETALA2/ETHYLENE RESPONSIVE ELEMENT-BINDING FACTOR (AP2/ERF) genes are exclusive to vascular plants and have been classified into the AP2-like and ERF-like clades. The AP2-like clade includes the AINTEGUMENTA (ANT) and the euAPETALA2 (euAP2) genes, both regulated by miR172 Arabidopsis has two paralogs in the euAP2 clade, namely APETALA2 (AP2) and TARGET OF EAT3 (TOE3) that control flowering time, meristem determinacy, sepal and petal identity and fruit development. euAP2 genes are likely functionally divergent outside Brassicaceae, as they control fruit development in tomato, and regulate inflorescence meristematic activity in maize. We studied the evolution and expression patterns of euAP2/TOE3 genes to assess large scale and local duplications and evaluate protein motifs likely related with functional changes across seed plants. We sampled euAP2/TOE3 genes from vascular plants and have found three major duplications and a few taxon-specific duplications. Here, we report conserved and new motifs across euAP2/TOE3 proteins and conclude that proteins predating the Brassicaceae duplication are more similar to AP2 than TOE3. Expression data show a shift from restricted expression in leaves, carpels, and fruits in non-core eudicots and asterids to a broader expression of euAP2 genes in leaves, all floral organs and fruits in rosids. Altogether, our data show a functional trend where the canonical A-function (sepal and petal identity) is exclusive to Brassicaceae and it is likely not maintained outside of rosids.

Genome-wide investigation and expression analysis of AP2-ERF gene family in salt tolerant common bean.

Apetala2-ethylene-responsive element binding factor (AP2-ERF) superfamily with common AP2-DNA binding domain have developmentally and physiologically important roles in plants. Since common bean genome project has been completed recently, it is possible to identify all of the AP2-ERF genes in the common bean genome. In this study, a comprehensive genome-wide in silico analysis identified 180 AP2-ERF superfamily genes in common bean (Phaseolus vulgaris). Based on the amino acid alignment and phylogenetic analyses, superfamily members were classified into four subfamilies: DREB (54), ERF (95), AP2 (27) and RAV (3), as well as one soloist. The physical and chemical characteristics of amino acids, interaction between AP2-ERF proteins, cis elements of promoter region of AP2-ERF genes and phylogenetic trees were predicted and analyzed. Additionally, expression levels of AP2-ERF genes were evaluated by in silico and qRT-PCR analyses. In silico micro-RNA target transcript analyses identified nearly all PvAP2-ERF genes as targets of by 44 different plant species' miRNAs were identified in this study. The most abundant target genes were PvAP2/ERF-20-25-62-78-113-173. miR156, miR172 and miR838 were the most important miRNAs found in targeting and BLAST analyses. Interactome analysis revealed that the transcription factor PvAP2-ERF78, an ortholog of Arabidopsis At2G28550, was potentially interacted with at least 15 proteins, indicating that it was very important in transcriptional regulation. Here we present the first study to identify and characterize the AP2-ERF transcription factors in common bean using whole-genome analysis, and the findings may serve as a references for future functional research on the transcription factors in common bean.

Genome-Wide Investigation and Expression Profiling of AP2/ERF Transcription Factor Superfamily in Foxtail Millet (Setaria italica L.)

The APETALA2/ethylene-responsive element binding factor (AP2/ERF) family is one of the largest transcription factor (TF) families in plants that includes four major sub-families, namely AP2, DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and RAV (Related to ABI3/VP). AP2/ERFs are known to play significant roles in various plant processes including growth and development and biotic and abiotic stress responses. Considering this, a comprehensive genome-wide study was conducted in foxtail millet (Setaria italica L.). A total of 171 AP2/ERF genes were identified by systematic sequence analysis and were physically mapped onto nine chromosomes. Phylogenetic analysis grouped AP2/ERF genes into six classes (I to VI). Duplication analysis revealed that 12 (∼7%) SiAP2/ERF genes were tandem repeated and 22 (∼13%) were segmentally duplicated. Comparative physical mapping between foxtail millet AP2/ERF genes and its orthologs of sorghum (18 genes), maize (14 genes), rice (9 genes) and Brachypodium (6 genes) showed the evolutionary insights of AP2/ERF gene family and also the decrease in orthology with increase in phylogenetic distance. The evolutionary significance in terms of gene-duplication and divergence was analyzed by estimating synonymous and non-synonymous substitution rates. Expression profiling of candidate AP2/ERF genes against drought, salt and phytohormones revealed insights into their precise and/or overlapping expression patterns which could be responsible for their functional divergence in foxtail millet. The study showed that the genes SiAP2/ERF-069, SiAP2/ERF-103 and SiAP2/ERF-120 may be considered as potential candidate genes for further functional validation as well for utilization in crop improvement programs for stress resistance since these genes were up-regulated under drought and salinity stresses in ABA dependent manner. Altogether the present study provides new insights into evolution, divergence and systematic functional analysis of AP2/ERF gene family at genome level in foxtail millet which may be utilized for improving stress adaptation and tolerance in millets, cereals and bioenergy grasses.

Genome-Wide Analysis of the AP2/ERF Gene Family in Prunus mume

The APETALA 2/ethylene-responsive element binding factor (AP2/ERF) transcription factors play important roles in plant development and responses to stress. Although the entire genomes of four Rosaceae species have been fully sequenced, no genome-wide analysis of AP2/ERF gene family has yet been reported in the Rosaceae family. In this study, 116 AP2/ERF genes were identified from Chinese plum (Prunus mume). Based on the number of AP2/ERF domains, these PmAP2/ERF genes were classified into three families (AP2, ERF, and RAV) along with a single member. The ERF family was subdivided into 11 groups. Of those, 22 and 41 PmAP2/ERF genes were involved in segmental and tandem duplications, respectively. Putative orthologs in Arabidopsis were identified for 73 PmAP2/ERF genes following synteny analysis. Transcriptome sequencing analysis showed that expression of PmAP2/ERF genes was widely variable.

A Proximal Promoter Region of Arabidopsis DREB2C Confers Tissue‐specific Expression under Heat Stress

The dehydration-responsive element-binding factor 2C (DREB2C) is a member of the CBF/DREB subfamily of proteins, which contains a single APETALA2/Ethylene responsive element-binding factor (AP2/ERF) domain. To identify the expression pattern of the DREB2C gene, which contains multiple transcription cis-regulatory elements in its promoter, an approximately 1.4 kb upstream DREB2C sequence was fused to the β-glucuronidase reporter gene (GUS) and the recombinant p1244 construct was transformed into Arabidopsis thaliana (L.) Heynh. The promoter of the gene directed prominent GUS activity in the vasculature in diverse young dividing tissues. Upon applying heat stress (HS), GUS staining was also enhanced in the vasculature of the growing tissues. Analysis of a series of 5'-deletions of the DREB2C promoter revealed that a proximal upstream sequence sufficient for the tissue-specific spatial and temporal induction of GUS expression by HS is localized in the promoter region between -204 and -34 bps relative to the transcriptional start site. Furthermore, electrophoretic mobility shift assay (EMSA) demonstrated that nuclear protein binding activities specific to a -120 to -32 bp promoter fragment increased after HS. These results indicate that the TATA-proximal region and some latent trans-acting factors may cooperate in HS-induced activation of the Arabidopsis DREB2C promoter.

Over-expression of the Arabidopsis DRE/CRT-binding transcription factor DREB2C enhances thermotolerance

The dehydration responsive element binding protein 2 (DREB2) subgroup belongs to the plant-specific APETALA2/ethylene-responsive element binding factor (AP2/ERF) family of transcription factors. We have characterized cDNA encoding Arabidopsis thaliana DREB2C, which is induced by mild heat stress. Both an electrophoretic mobility shift assay (EMSA) and a yeast one-hybrid assay revealed that DREB2C 145–528 was able to form a complex with the dehydration responsive element/C-repeat (DRE/CRT; A/GCCGAC) motif. A trans-activating ability test in yeast demonstrated that DREB2C could effectively function as a trans-activator. Constitutive expression of DREB2C under the control of the cauliflower mosaic virus (CaMV) 35S promoter led to enhanced thermotolerance in transgenic lines of Arabidopsis. Microarray and RT-PCR analyses of transgenic plants revealed that DREB2C regulates expression of several heat stress-inducible genes that contain DRE/CRT elements in their promoters. From these data, we deduced that DREB2C is a regulator of heat stress tolerance in Arabidopsis.