Transcriptome-Based Identification and Expression Profiling of AP2/ERF Members in Caragana Intermedia and Functional Analysis of CiDREB3

Caragana. intermedia Kuang et H.C.Fu., a xerophytic deciduous shrub that widely distributed in arid and semi-arid desert areas of North-west China, is highly tolerant to cold, drought, salt-alkali and barren. In this study, the transcriptome C. intermedia under drought treatment was screened for the genes encoding the AP2/ERF family of transcription factors. As a result, 22 sequences with complete open reading frames (ORFs) were obtained. All sequences were divided into 13 groups, including the DREB A1 to A6, ERF B1 to B6, and RAV groups. The results of multiple sequence alignment (MSA) analysis, domain distribution analysis, conservative motif analysis and tertiary structure prediction showed that all the AP2/ERF transcription factors contained the AP2 domain. The AP2 domain contained the YRG and RAYD elements. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to analyze the expression patterns of the 22 AP2/ERF transcription factors in C. intermedia. Most of the transcription factors exhibited tissue-specific expression. The expression of most transcription factors was induced to varying degrees by cold, heat, salt and mannitol stress. Furthermore, Heterologous expression of CiDREB3 in transgenic Arabidopsis L. Heynh decreased root length and fresh weight, decreased rosette leaf diameter and number of leaf, and improved drought tolerance during seedlings development. Taken together, the results of the present study help to better understand the functions of the AP2/ERF family of transcription factors in plant responses to multiple abiotic stresses and deeply insight the roles of CiDREB3 in Arabidopsis morphology and conferring abiotic stress tolerance.

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.

Characteristics and phylogeny of DREB gene subfamily in soybeans [Glycine max (L.) Meril]

Dehydration responsive element binding proteins (DREB) are transcription factors linked to cis-acting elements of the promoter region, which regulate plant gene expression in response to abiotic stress. In this study, 69 DREB gene sequences of soybean from NCBI belonging to 18 GmDREB (Glycine max DREB) genes of 1 to 8 copies distributed on 17 chromosomes were identified in which GmDREB3 has 8 copies and the rest consisted of 1 to 4 ones. The motif PTPEMAARAYDVAALALKGPSARLNFPEL containing 11 points associated with the promoter of functional genes existed in 4 main types with the most popular one RGRRWKERRWT found in 13/18 DREB proteins was regarded as the popular AP2 domain of DREB protein. The phylogenetic tree based on the nucleotide sequences of GmDREB genes and the amino acid sequences of the AP2 domain expresses the evolution and relationship of the DREB subfamily in soybean. This study provides comprehensive information about the DREB subfamily, which formed the basis for experimental analyses to clarify the function of some members of this subfamily.

Molecular cloning and characterization of GhERF105, a gene contributing to the regulation of gland formation in upland cotton (Gossypium hirsutum L.)

Background Gossypium hirsutum L. (cotton) is one of the most economically important crops in the world due to its significant source of fiber, feed, foodstuff, oil and biofuel products. However, the utilization of cottonseed was limited due to the presence of small and darkly pigmented glands that contain large amounts of gossypol, which is toxic to human beings and non-ruminant animals. To date, some progress has been made in the pigment gland formation, but the underlying molecular mechanism of its formation was still unclear. Results In this study, we identified an AP2/ERF transcription factor named GhERF105 (GH_A12G2166), which was involved in the regulation of gland pigmentation by the comparative transcriptome analysis of the leaf of glanded and glandless plants. It encoded an ERF protein containing a converved AP2 domain which was localized in the nucleus with transcriptional activity, and showed the high expression in glanded cotton accessions that contained much gossypol. Virus-induced gene silencing (VIGS) against GhERF105 caused the dramatic reduction in the number of glands and significantly lowered levels of gossypol in cotton leaves. GhERF105 showed the patterns of spatiotemporal and inducible expression in the glanded plants. Conclusions These results suggest that GhERF105 contributes to the pigment gland formation and gossypol biosynthesis in partial organs of glanded plant. It also provides a potential molecular basis to generate ‘glandless-seed’ and ‘glanded-plant’ cotton cultivar.

Molecular cloning and characterization of GhERF105, a gene contributing to the regulation of gland formation in upland cotton (Gossypium hirsutum L.)

Background: Gossypium hirsutum L. ( cotton) is one of the most economically important crops in the world due to its significant source of fiber, feed, foodstuff, oil and biofuel products. However, the utilization of cottonseed was limited due to the presence of small and darkly pigmented glands that contain large amounts of gossypol, which is toxic to human beings and non-ruminant animals. To date, some progress has been made in the pigment gland formation, but the underlying molecular mechanism of its formation was still unclear.Results: In this study, we identified an AP2/ERF transcription factor named GhERF105 (GH_A12G2166), which was involved in the regulation of gland pigmentation by the comparative transcriptome analysis of the leaf of glanded and glandless plants. It encoded an ERF protein containing a converved AP2 domain which was localized in the nucleus with transcriptional activity, and showed the high expression in glanded cotton accessions that contained much gossypol. Virus-induced gene silencing(VIGS) against GhERF105 caused the dramatic reduction in the number of glands and significantly lowered levels of gossypol in cotton leaves. GhERF105 showed the patterns of spatiotemporal and inducible expression in the glanded plants. Conclusions: These results suggest that GhERF105 contributes to the pigment gland formation and gossypol biosynthesis in partial organs of glanded plant. It also provides a potential molecular basis to generate ‘glandless-seed’ and ‘glanded-plant’ cotton cultivar.

Molecular cloning and characterization of GhERF105, a gene contributing to the regulation of gland formation in upland cotton (Gossypium hirsutum L.)

Background: Gossypium hirsutum L. ( cotton) is one of the most economically important crops in the world due to its significant source of fiber, feed, foodstuff, oil and biofuel products. However, the utilization of cottonseed was limited due to the presence of small and darkly pigmented glands that contain large amounts of gossypol, which is toxic to human beings and non-ruminant animals. To date, some progress has been made in the pigment gland formation, but the underlying molecular mechanism of its formation was still unclear.Results: In this study, we identified an AP2/ERF transcription factor named GhERF105 (GH_A12G2166), which was involved in the regulation of gland pigmentation by the comparative transcriptome analysis of the leaf of glanded and glandless plants. It encoded an ERF protein containing a converved AP2 domain which was localized in the nucleus with transcriptional activity, and showed the high expression in glanded cotton accessions that contained much gossypol. Virus-induced gene silencing(VIGS) against GhERF105 caused the dramatic reduction in the number of glands and significantly lowered levels of gossypol in cotton leaves. GhERF105 showed the patterns of spatiotemporal and inducible expression in the glanded plants. Conclusions: These results suggest that GhERF105 contributes to the pigment gland formation and gossypol biosynthesis in partial organs of glanded plant. It also provides a potential molecular basis to generate ‘glandless-seed’ and ‘glanded-plant’ cotton cultivar.

Genome Wide Characterization, Identification And Expression Analysis Of Erf Gene Family In Cotton

ERF is a subfamily of AP2/EREBP superfamily, contained single AP2 domain. The overexpression of ERF genes steered to abiotic stress tolerance and pathogen resistance in transgenic plants. Here, a genome-wide analysis of ERF gene family within two diploid species (G. arboreum & G. raimondii) and two tetraploid species (G. barbadense, G. hirsutum) was performed. A total of 118, 120, 213, 220 genes contained sequence single AP2 domain were identified in G. arboreum, G. raimondii, G. barbadense and G. hirsutum respectively. The identified genes were unevenly distributed across 13/26 chromosomes of A and D genomes of cotton. Genome comparison revealed that segmental duplication may have played crucial roles in the expansion of the cotton ERF gene family, and tandem duplication also played a minor role. Analysis of RNA-Seq data indicated that cotton ERF gene expression levels varied across different tissues and in response to different abiotic stress. Overall, our results could provide valuable information for better understanding the evolution of cotton ERF genes and lays a foundation for future investigation in cotton.

Genome-Wide Identification, Expression and Potential Function Analysis of the ERF and DREB Subfamily Members in Tomato

Background: APETALA2/ethylene responsive factors (AP2/ERFs) are unique regulators found in the plant kingdom that are involved in all life activity processes, including flowering, fruit ripening, floral meristem growth, and defense responses. In tomato (Solanum lycopersicum), there are 60 DREB and 80 ERF subfamily members, however, their functionality remains poorly understood.Results: In this work, the AP2 domain conserved amino acid sequences of 68 ERF proteins from 20 plant species were compared and a Multiple Em for Motif Elicitation (MEME) analysis was conducted. Results revealed that the 9th amino acid of the AP2 domain exhibited marked characteristics during the selection of DRE/CRT and/or GCC boxes as protein binding sites. Moreover, motifs near the AP2 domain may be involved in protein binding to DNA, whereas motifs far away from the AP2 domain may function as a part of the transactivation domain. Furthermore, we compared the expression levels of all ERF genes in 30 tomato organs and under biotic and abiotic stresses. Results indicated that most of 17 ERF and DREB repressor genes were highly expressed in almost all tomato organs and under some biotic and abiotic stress. The transcripts per million (TPM) value ratios of all repressor genes exceeded that of all activator genes in 16 tomato organs. Thus, it can be inferred that these repressor genes play vital roles in balancing the regulatory functions of activator genes and activator genes may also conversely compete with repressor genes to ensure normal growth, development, and defense responses in tomato.Conclusions: This work uncovered the potential functions of all ERF and DREB genes that regulate tomato growth, development, and defense responses, and considers the binding ability of the AP2 domain unique sequences with DRE/CRT and GCC boxes, as well as the relationship of unique motifs with the transactivation domain. These findings will expand upon our understanding of the functions of ERF and DREB genes in tomato.

A functional genomics approach to dissect spotted alfalfa aphid resistance in Medicago truncatula

Aphids are virus-spreading insect pests affecting crops worldwide and their fast population build-up and insecticide resistance make them problematic to control. Here, we aim to understand the molecular basis of spotted alfalfa aphid (SAA) or Therioaphis trifolii f. maculata resistance in Medicago truncatula, a model organism for legume species. We compared susceptible and resistant near isogenic Medicago lines upon SAA feeding via transcriptome sequencing. Expression of genes involved in defense and stress responses, protein kinase activity and DNA binding were enriched in the resistant line. Potentially underlying some of these changes in gene expression was the finding that members of the MYB, NAC, AP2 domain and ERF transcription factor gene families were differentially expressed in the resistant versus susceptible lines. A TILLING population created in the resistant cultivar was screened using exome capture sequencing and served as a reverse genetics tool to functionally characterise genes involved in the aphid resistance response. This screening revealed three transcription factors (a NAC, AP2 domain and ERF) as important regulators in the defence response, as a premature stop-codon in the resistant background led to a delay in aphid mortality and enhanced plant susceptibility. This combined functional genomics approach will facilitate the future development of pest resistant crops by uncovering candidate target genes that can convey enhanced aphid resistance.

Characterization and expression QTL analysis of TaABI4, a pre-harvest sprouting related gene in wheat

Pre-harvest sprouting (PHS) induced by the decline of seed dormancy causes a severe reduction in crop yield and flour quality. In this study, we isolated and characterized TaABI4, an ABA-responsive transcription factor that participates in regulating seed germination in wheat. Sequence analysis revealed that TaABI4 has three homologues, located on chromosomes 1A/1B/1D. TaABI4 contains a conserved AP2 domain, and AP2-associated, LRP, and potential PEST motifs. Putative cis-acting regulatory elements (CE1-like box, W-box, ABRE elements, and RY-elements) were identified in the TaABI4 promoter region that showed high conservation in 17 wheat cultivars and wheat-related species. Expression profiling of TaABI4 indicated that it is a seed-specific gene accumulating during the middle stages of seed development. Transcript accumulation of TaABI4 in wheat cultivar Chuanmai 32 (CM32, PHS susceptible) was 5.07-fold and 1.39-fold higher than that in synthetic hexaploidy wheat SHW-L1 (PHS resistant) at 15DPA and 20DPA, respectively. Six expression quantitative trait loci (eQTL) of TaABI4 on chromosome 2A, 2D, 3B, and 4A were characterized based on the accumulated transcripts of TaABI4 in SHW-L1 and CM32 derived recombinant inbred lines. These QTLs explained from 10.7% to 46.1% of the trait variation with 4.53~10.59 of LOD scores, which contain genes that may affect the expression of TaABI4.