APETALA1 Research Articles

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.

Promoter Trapping and Deletion Analysis Show Arabidopsis thaliana APETALA2 Gene Promoter Is Bidirectional and Functions as a Pollen- and Ovule-Specific Promoter in the Reverse Orientation.

The Arabidopsis thaliana promoter trap mutant Bitrap-112 expressing green fluorescent protein (GFP) gene in the ovules was found to carry transferred DNA (T-DNA) insertion at -309 position of the APETALA2 (AP2) gene. Bitrap-112 line did not show phenotype associated with the AP2 mutation, suggesting that T-DNA insertion did not interrupt the AP2 promoter. Further, head-to-head orientation of GFP and AP2 genes indicated that the AP2 promoter could be bidirectional. A detailed deletion analysis of the upstream sequences of the AP2 gene was done to identify the promoter. GUS assay of transgenic A. thaliana plants carrying various AP2 upstream fragments fused to the uidA gene showed that ~200-bp 5' UTR sequences are capable of driving gene expression at low levels in vegetative tissues whereas inclusion of further upstream sequences (~300bp) enhanced uidA expression comparable to native AP2 expression levels in various tissues including ovules. In the reverse orientation, the 519-bp AP2 upstream fragment was found to drive gene expression in immature ovules and pollen. Absence of antisense transcripts corresponding to the sequences upstream of AP2 gene in wild-type A. thaliana plants suggests that promoter trapping has uncovered a cryptic promoter, which in reverse orientation is capable of driving gene expression in ovules and anthers.

ABI4 represses the expression of type-A ARRs to inhibit seed germination in Arabidopsis.

The plant hormone abscisic acid (ABA) plays a crucial role in regulating seed germination and post-germination growth. ABSCISIC ACID INSENSITIVE4 (ABI4), an APETALA2 (AP2)-type transcription factor, is required for the ABA-mediated inhibition of seed germination. Cytokinins promote seed germination and seedling growth by antagonizing ABA signaling. However, the interaction between ABA and cytokinin signaling during seed germination remains unclear. Here, we report that ABA signaling downregulates Arabidopsis response regulators (ARRs), a class of cytokinin-inducible genes, during seed germination and cotyledon greening. We found that the application of exogenous ABA repressed the expression of type-A ARRs in Arabidopsis seeds and seedlings. Among the type-A ARR family members, the expression of ARR6, ARR7 and ARR15 was upregulated in ABA-deficient mutants, indicating that the transcriptional inhibition of type-A ARRs requires the ABA signaling pathway. Single and multiple mutations of these ARRs resulted in increased ABA sensitivity during germination and cotyledon greening; overexpression of ARR7 or ARR15 led to an ABA-insensitive phenotype. These observations suggest that type-A ARRs inhibit the ABA response during seed germination and cotyledon greening. Further analysis showed that ABI4 negatively regulated the transcription of ARR6, ARR7 and ARR15 by directly binding to their promoters. Genetic analysis showed that loss-of-function mutations of ARR7 and ARR15 partially rescued the ABA insensitivity of abi4-1. Thus, this study revealed that ABI4 plays a key role in ABA and cytokinin signaling by inhibiting the transcription of type-A ARRs to inhibit seed germination and cotyledon greening.

In silico Functional Analysis of FLC and FT-Genes Responsible for Postponingand Accelerating the Onset of Flowering

In silico functional analyses of FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT) genes were investigated herein. The recognition of cis-regulatory modules and their organization are a prerequisite to information on the regulation of gene expression. Accordingly, this study carried out a promoter analysis on FT and FLC genes in Arabidopsis and its orthologs in Prunus persica by PlantPan and PLANCARE servers in which the integrating transcription factor binding site (TFBs) was involved in hormonal and stress signals. Light responsive elements were also studied. Among the responsive elements identified in FT and FLC, Arabidopsis had the highest number of responsive elements. This is partly because P. persica is day-neutral, whereas Arabidopsis is a facultative long day plant. The results of motif recognition in FT and FLC proteins with the MEME server showed that FT is highly conserved, but the number of motifs suggests that the FLC protein had far more numerous motifs, which is likely due to its higher amino acid length. Some specified TFs like AGAMOUS (AG), AGAMOUS LIKE (AGL), LEAFY (LFY) and APETALA1 (AP1) were found by the PlantPan web server in FT and FLC promoters’ analysis. They were also present in the protein-protein interaction network analysis which raises the question whether or not FT or FLC can be positive or negative regulators of their own expression in a feedback loop.

Duplication and Whorl-Specific Down-Regulation of the Obligate AP3-PI Heterodimer Genes Explain the Origin of Paeonia lactiflora Plants with Spontaneous Corolla Mutation.

Herbaceous peony (Paeonia lactiflora) is a globally important ornamental plant. Spontaneous floral mutations occur frequently during cultivation, and are selected as a way to release new cultivars, but the underlying evolutionary developmental genetics remain largely elusive. Here, we investigated a collection of spontaneous corolla mutational plants (SCMPs) whose other floral organs were virtually unaffected. Unlike the corolla in normal plants (NPs) that withered soon after fertilization, the transformed corolla (petals) in SCMPs was greenish and persistent similar to the calyx (sepals). Epidermal cellular morphology of the SCMP corolla was also similar to that of calyx cells, further suggesting a sepaloid corolla in SCMPs. Ten floral MADS-box genes from these Paeonia plants were comparatively characterized with respect to sequence and expression. Codogenic sequence variation of these MADS-box genes was not linked to corolla changes in SCMPs. However, we found that both APETALA3 (AP3) and PISTILLATA (PI) lineages of B-class MADS-box genes were duplicated, and subsequent selective expression alterations of these genes were closely associated with the origin of SCMPs. AP3-PI obligate heterodimerization, essential for organ identity of corolla and stamens, was robustly detected. However, selective down-regulation of these duplicated genes might result in a reduction of this obligate heterodimer concentration in a corolla-specific manner, leading to the sepaloid corolla in SCMPs, thus representing a new sepaloid corolla model taking advantage of gene duplication. Our work suggests that modifying floral MADS-box genes could facilitate the breeding of novel cultivars with distinct floral morphology in ornamental plants, and also provides new insights into the functional evolution of the MADS-box genes in plants.

OsMADS1 Represses microRNA172 in Elongation of Palea/Lemma Development in Rice.

Specification of floral organ identity is critical for the establishment of floral morphology and inflorescence architecture. Although multiple genes are involved in the regulation of floral organogenesis, our understanding of the underlying regulating network is still fragmentary. MADs-box genes are principle members in the ABCDE model that characterized floral organs. OsMADS1 specifies the determinacy of spikelet meristem and lemma/palea identity in rice. However, the pathway through which OsMADS1 regulates floral organs remains elusive; here, we identified the microRNA172 (miR172) family as possible regulators downstream of OsMADS1. Genetic study revealed that overexpression of each miR172 gene resulted in elongated lemma/palea and indeterminacy of the floret, which resemble the phenotype of osmads1 mutant. On the contrary, overexpression of each target APETALA2 (AP2) genes resulted in shortened palea/lemma. Expression level and specificity of miR172 was greatly influenced by OsMADS1, as revealed by Northern blot analysis and In situ hybridization. Genetically, AP2-3 and AP2-2 over expression rescued the elongation and inconsistent development of the lemma/palea in OsMADS1RNAi transgenic plants. Our results suggested that in rice, OsMADS1 and miR172s/AP2s formed a regulatory network involved in floral organ development, particularly the elongation of the lemma and the palea.

Functional conservation and divergence of five SEPALLATA-like genes from a basal eudicot tree, Platanus acerifolia.

Five SEP -like genes were cloned and identified from Platanus acerifolia through the analysis of expression profiles, protein-protein interaction patterns, and transgenic phenotypes, which suggested that they play conservative and diverse functions in floral initiation and development, fruit development, bud growth, and dormancy. SEPALLATA (SEP) genes have been well characterized in core eudicots and some monocots, and they play important and diverse roles in plant development, including flower meristem initiation, floral organ identity, and fruit development and ripening. However, the knowledge on the function and evolution of SEP-like genes in basal eudicot species is very limited. Here, we cloned and identified five SEP-like genes from London plane (Platanus acerifolia), a basal eudicot tree that is widely used for landscaping in cities. Sequence alignment and phylogenetic analysis indicated that three genes (PlacSEP1.1, PlacSEP1.2, and PlacSEP1.3) belong to the SEP1/2/4 clade, while the other two genes (PlacSEP3.1 and PlacSEP3.2) are grouped into the SEP3 clade. Quantitative real-time PCR (qRT-PCR) analysis showed that all PlacSEPs, except PlacSEP1.1 and PlacSEP1.2, were expressed during the male and female inflorescence initiation, and throughout the flower and fruit development process. PlacSEP1.2 gene expression was only detected clearly in female inflorescence at April. PlacSEP1.3 and PlacSEP3.1 were also expressed, although relatively weak, in vegetative buds of adult trees. No evident PlacSEPs transcripts were detected in various organs of juvenile trees. Overexpression of PlacSEPs in Arabidopsis and tobacco plants resulted in different phenotypic alterations. 35S:PlacSEP1.1, 35S:PlacSEP1.3, and 35S:PlacSEP3.2 transgenic Arabidopsis plants showed evident early flowering, with less rosette leaves but more cauline leaves, while 35S:PlacSEP1.2 and PlacSEP3.1 transgenic plants showed no visible phenotypic changes. 35S:PlacSEP1.1 and 35S:PlacSEP3.2 transgenic Arabidopsis plants also produced smaller and curled leaves. Overexpression of PlacSEP1.1 and PlacSEP3.1 in tobacco resulted in the early flowering and producing more lateral branches. Yeast two-hybrid analysis indicated that PlacSEPs proteins can form homo- or hetero-dimers with the Platanus APETALA1 (AP1)/FRUITFULL (FUL), B-, C-, and D-class MADS-box proteins in different interacting patterns and intensities. Our results suggest that the five PlacSEP genes may play important and divergent roles during floral initiation and development, as well as fruit development, by collaborating with FUL, B-, C-, and D-class MADS-box genes in London plane; PlacSEP1.3 and PlacSEP3.1 genes might also involve in vegetative bud growth and dormancy. The results provide valuable data for us to understand the functional evolution of SEP-like genes in basal eudicot species.

RAP2.4a Is Transported through the Phloem to Regulate Cold and Heat Tolerance in Papaya Tree (Carica papaya cv. Maradol): Implications for Protection Against Abiotic Stress.

Plants respond to stress through metabolic and morphological changes that increase their ability to survive and grow. To this end, several transcription factor families are responsible for transmitting the signals that are required for these changes. Here, we studied the transcription factor superfamily AP2/ERF, particularly, RAP2.4 from Carica papaya cv. Maradol. We isolated four genes (CpRap2.4a, CpRAap2.4b, CpRap2.1 and CpRap2.10), and an in silico analysis showed that the four genes encode proteins that contain a conserved APETALA2 (AP2) domain located within group I and II transcription factors of the AP2/ERF superfamily. Semiquantitative PCR experiments indicated that each CpRap2 gene is differentially expressed under stress conditions, such as extreme temperatures. Moreover, genetic transformants of tobacco plants overexpressing CpRap2.4a and CpRap2.4b genes show a high level of tolerance to cold and heat stress compared to non-transformed plants. Confocal microscopy analysis of tobacco transgenic plants showed that CpRAP2.4a and CpRAP2.4b proteins were mainly localized to the nuclei of cells from the leaves and roots and also in the sieve elements. Moreover, the movement of CpRap2.4a RNA in tobacco grafting was analyzed. Our results indicate that CpRap2.4a and CpRap2.4b RNA in the papaya tree have a functional role in the response to stress conditions such as exposure to extreme temperatures via direct translation outside the parental RNA cell.

Profiling of microRNAs in wild type and early flowering transgenic Chrysanthemum morifolium by deep sequencing

Here, we performed comparative miRNA profiling in wild type and early flowering transgenic Chrysanthemum morifolium with constitutive expression of APETALA1 (AP1)-like gene, HAM92 (Helianthus annuus). Six sRNA libraries constructed from leaves and shoot apexes after the short day photoperiod initiation, as well as from opened inflorescence after anthesis were sequenced and analyzed. A total of 324 members (163 families) of putative conserved miRNAs and 30 candidate novel miRNAs specific for C. morifolium (cmo-miRNAs) were identified. Bioinformatic analysis revealed 427 and 138 potential mRNA targets for conserved and novel cmo-miRNAs, respectively. These genes were described in Gene Ontology terms and found to be implicated in a broad range of signaling pathways. Plant- and tissue-specific expression of 9 highly conserved cmo-miRNAs was compared between wild type and transgenic chrysanthemum lines with ectopic expression of AP1-like genes HAM92 and CDM111 (C. morifolium), using RT-qPCR and cmo-miR162a as a reference miRNA. The results of our study provide a framework for further investigation of miRNA evolution and functions in higher plants, as well as their roles in flowering control.

Differential isoform expression and protein localization from alternatively spliced Apetala2 in peanut under drought stress

APETALA2 (AP2) belongs to the AP2/Ethylene Responsive Factor (ERF) family and regulates expression levels of downstream stress responsive genes as a transcription factor. In this study, we cloned six different isoforms of AhAP2 from peanut (Arachis hypogaea). Four isoforms (AhAP2.1, AhAP2.2, AhAP2.3 and AhAP2.4) had both AP2/ERF DNA binding domains and ERF-associated amphiphilic repression (EAR) motifs. Two isoforms (AhAP2.5 and AhAP2.6) only had an EAR suppressor domain. After agroinfiltration, AhAP2.1, AhAP2.3, and AhAP2.4 fused to yellow fluorescent protein (YFP) showed localization to the nucleolus, which is the site of transcription and ribosome biogenesis. AhAP2.2-YFP showed a dispersed signal in the nucleus. AhAP2.5 and AhAP2.6 fused to YFP localized to both the nucleus and cytoplasm. In addition, increased levels of AhAP2.1 and AhAP2.2 transcripts were observed in drought-treated peanut leaves, suggesting differential transcriptional regulation under drought stress conditions.

Characterization and functional analysis of a MADS-box transcription factor gene (GbMADS9) from Ginkgo biloba

The MADS-box transcription factors play diverse roles in plant development, but little is known about the role of MADS-box genes in Ginkgo biloba. Here a MADS-box transcription factor gene, designated as GbMADS9, was cloned and functionally characterized from G. biloba. The open reading frame of GbMADS9 comprised 684bp encoding a polypeptide of 227 amino acids. Phylogenetic analysis showed that GbMADS9 belonged to the GGM13 clade of Bsister-class MADS-box proteins. Real-time PCR results revealed that GbMADS9 was preferentially expressed in strobili and ovules. In addition, its expression was up-regulated with the increase in the size of ginkgo pulp, indicating that GbMADS9 had a positive function in ginkgo ovule development. The expression of GbMADS9 in ginkgo was up-regulated in response to salinity, drought, and cold stress, as well as to phytohormones gibberellic acid and abscisic acid. GbMADS9 transgenic Arabidopsis plants presented early flowering phenotypes compared with wild-type plants. The expression of the FLOWERING LOCUS T (FT), APETALA1 (AP1), LEAFY (LFY), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) genes involved in flowering was up-regulated, whereas the expression of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) was down-regulated in the GbMADS9 transgenic lines. The GbMADS9 transgenic plants exhibited better growth than wild-type plants under high osmotic stress. Enhanced tolerance of transgenic Arabidopsis plants to osmotic stress was confirmed by changes in the chlorophyll, proline, and malondialdehyde contents. Moreover, increased tolerance to osmotic stress in GbMADS9 transgenic lines was associated with improved antioxidant enzymatic activities. These findings suggested that GbMADS9 might be a promising gene to manipulate flowering time and improve abiotic stress tolerance in G. biloba.

Fine mapping and candidate gene analysis of qHD5, a novel major QTL with pleiotropism for yield-related traits in rice (Oryza sativa L.).

A major QTL for heading date, qHD5, was fine-mapped to a 52.59-kb region on the short arm of rice chromosome 5. Heading date (HD) is one of the most important traits that enables rice to adapt to seasonal differences and specific growth conditions in diverse growing regions. In this study, a major-effect quantitative trait locus (QTL), qHD5, was resolved as a single Medelian factor that causes NIL(BG1) and NIL(XLJ) (two near-isogenic lines (NILs) used in our study) to have at a minimum of 10-day difference in HD under both long-day and short-day conditions in rice. qHD5 was initially mapped to a 309.52-kb genomic region in our previous study. Here, using an advanced BC4F3 population and map-based cloning, we further narrowed the location of qHD5 to a 52.59-kb region between the H71 and RD502 markers. Sequence analysis revealed that Os05g03040, which putatively encodes an AP2 (APETALA2) transcription factor, has six single nucleotide polymorphisms (SNPs) between NIL(BG1) and NIL(XLJ). On this basis, this gene was concluded to be the most probable candidate gene for qHD5. Our results also showed that Hd3a, RFT1, Hd1, Ehd1, and Ghd7 were differentially expressed in the two NILs. Moreover, qHD5 was found to affect yield-related traits such as flag leaf width, flag leaf length, branch number, and 1000-grain weight.

Expansion and Functional Divergence of AP2 Group Genes in Spermatophytes Determined by Molecular Evolution and Arabidopsis Mutant Analysis.

The APETALA2 (AP2) genes represent the AP2 group within a large group of DNA-binding proteins called AP2/EREBP. The AP2 gene is functional and necessary for flower development, stem cell maintenance, and seed development, whereas the other members of AP2 group redundantly affect flowering time. Here we study the phylogeny of AP2 group genes in spermatophytes. Spermatophyte AP2 group genes can be classified into AP2 and TOE types, six clades, and we found that the AP2 group homologs in gymnosperms belong to the AP2 type, whereas TOE types are absent, which indicates the AP2 type gene are more ancient and TOE type was split out of AP2 type and losing the major function. In Brassicaceae, the expansion of AP2 and TOE type lead to the gene number of AP2 group were up to six. Purifying selection appears to have been the primary driving force of spermatophyte AP2 group evolution, although positive selection occurred in the AP2 clade. The transition from exon to intron of AtAP2 in Arabidopsis mutant leads to the loss of gene function and the same situation was found in AtTOE2. Combining this evolutionary analysis and published research, the results suggest that typical AP2 group genes may first appear in gymnosperms and diverged in angiosperms, following expansion of group members and functional differentiation. In angiosperms, AP2 genes (AP2 clade) inherited key functions from ancestors and other genes of AP2 group lost most function but just remained flowering time controlling in gene formation. In this study, the phylogenies of AP2 group genes in spermatophytes was analyzed, which supported the evidence for the research of gene functional evolution of AP2 group.

APETALA2 antagonizes the transcriptional activity of AGAMOUS in regulating floral stem cells in Arabidopsis thaliana.

Summary APETALA2 (AP2) is best known for its function in the outer two floral whorls, where it specifies the identities of sepals and petals by restricting the expression of AGAMOUS (AG) to the inner two whorls in Arabidopsis thaliana. Here, we describe a role of AP2 in promoting the maintenance of floral stem cell fate, not by repressing AG transcription, but by antagonizing AG activity in the center of the flower.We performed a genetic screen with ag‐10 plants, which exhibit a weak floral determinacy defect, and isolated a mutant with a strong floral determinacy defect. This mutant was found to harbor another mutation in AG and was named ag‐11. We performed a genetic screen in the ag‐11 background to isolate mutations that suppress the floral determinacy defect. Two suppressor mutants were found to harbor mutations in AP2.While AG is known to shut down the expression of the stem cell maintenance gene WUSCHEL (WUS) to terminate floral stem cell fate, AP2 promotes the expression of WUS. AP2 does not repress the transcription of AG in the inner two whorls, but instead counteracts AG activity.

Genome-Wide Analysis of APETALA2/Ethylene-Responsive Factor (AP2/ERF) Gene Family in Barley (Hordeum vulgare L.).

APETALA2/Ethylene-Responsive Factor (AP2/ERF) gene family is plant specific transcription factor. It plays critical roles in development process, tolerance to biotic and abiotic stresses, and responses to plant hormones. However, limited data are available on the contributions of AP2/ERF gene family in barley (Hordeum vulgare L.). In the present study, 121 HvAP2/ERF genes in barley were identified by using bioinformatics methods. A total of 118 HvAP2/ERF (97.5%) genes were located on seven chromosomes. According to phylogenetic classification of AP2/ERF family in Arabidopsis, HvAP2/ERF proteins were divided into AP2 (APETALA2), RAV (Related to ABI3/VP), DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and soloist sub families. The analysis of duplication events indicated that tandem repeat and segmental duplication contributed to the expansion of the AP2/ERF family in barley. HvDREB1s/2s genes displayed various expression patterns under abiotic stress and phytohormone. Taken together, the data generated in this study will be useful for genome-wide analysis to determine the precise role of the HvAP2/ERF gene during barley development, abiotic stress and phytohormone responses with the ultimate goal of improving crop production.

Characterizing Two Inter-specific Bin Maps for the Exploration of the QTLs/Genes that Confer Three Soybean Evolutionary Traits.

Annual wild soybean (Glycine soja Sieb. and Zucc.), the wild progenitor of the cultivated soybean [Glycine max (L.) Merr.], is valuable for improving the later. The construction of a linkage map is crucial for studying the genetic differentiation between these species, but marker density is the main factor limiting the accuracy of such a map. Recent advances in next-generation sequencing technologies allow for the generation of high-density linkage maps. Here, two sets of inter-specific recombinant inbred line populations, named NJIRNP and NJIR4P, composed of 284 and 161 lines, respectively, were generated from the same wild male parent, PI 342618B, and genotyped by restriction-site-associated DNA sequencing. Two linkage maps containing 5,728 and 4,354 bins were constructed based on 89,680 and 80,995 single nucleotide polymorphisms, spanning a total genetic distance of 2204.6 and 2136.7 cM, with an average distance of 0.4 and 0.5 cM between neighboring bins in NJRINP and NJRI4P, respectively. With the two maps, seven well-studied loci, B1 for seed bloom; G and I for seed coat color; E2, E3, qDTF16.1 and two linked FLOWERING LOCUS T for days to flowering, were detected. In addition, two SB and two DTF loci were newly identified in wild soybean. Using two high-density maps, the mapping resolution was enhanced, e.g., G was narrowed to a region of 0.4 Mb on chromosome 1, encompassing 54 gene models, among which only Glyma01g40590 was predicted to be involved in anthocyanin accumulation, and its interaction with I was verified in both populations. In addition, five genes, Glyma16g03030, orthologous to Arabidopsis Phytochrome A (PHYA); Glyma13g28810, Glyma13g29920, and Glyma13g30710 predicted to encode the APETALA 2 (AP2) domain; and Glyma02g00300, involved in response to red or far red light, might be candidate DTF genes. Our results demonstrate that RAD-seq is a cost-effective approach for constructing high-density and high-quality bin maps that can be used to map QTLs/genes into such small enough regions that their candidate genes can be predicted.

High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis).

BackgroundG1 + HBP is a male sterile cybrid line with nuclear genome from Hirado Buntan pummelo (C. grandis Osbeck) (HBP) and mitochondrial genome from “Guoqing No.1” (G1, Satsuma mandarin), which provides a good opportunity to study male sterility and nuclear-cytoplasmic cross talk in citrus. High-throughput sRNA and degradome sequencing were applied to identify miRNAs and their targets in G1 + HBP and its fertile type HBP during reproductive development.ResultsA total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2) and transport inhibitor response 1 (TIR1). Eight target genes were confirmed to be sliced by corresponding miRNAs using 5’ RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between sterile line G1 + HBP and fertile line HBP were identified. Differential expression of miRNAs and their target genes between two lines was validated by quantitative RT-PCR, and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulatory mechanism of miR167a was investigated by yeast one-hybrid and dual-luciferase assays that one dehydrate responsive element binding (DREB) transcription factor binds to miR167a promoter and transcriptionally repress miR167 expression.ConclusionOur study reveals the altered expression of miRNAs and their target genes in a male sterile line of pummelo and highlights that miRNA regulatory network may be involved in floral bud development and cytoplasmic male sterility in citrus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2882-0) contains supplementary material, which is available to authorized users.

Upregulation of LINC-AP2 is negatively correlated with AP2 gene expression with Turnip crinkle virus infection in Arabidopsis thaliana.

A long intergenic noncoding RNA LINC - AP2 is upregulated and negatively correlated with AP2 gene expression with Turnip crinkle virus infection in Arabidopsis. Plant vegetative growth and floral reproductive structure were severely retarded and distorted in Turnip crinkle virus (TCV)-infected Arabidopsis thaliana. Compared to mock-inoculated plants, the stamen filaments were shorter in flowers of TCV-infected plants. However, TCV-infected plants can still produce normal seeds through artificial pollination, indicating both its pollen and stigma were biologically functional. From our high-throughput RNA-Seq transcriptome analysis, a floral structure-related APETALA2 (AP2) gene was found to be downregulated and its neighboring long intergenic noncoding RNAs (lincRNA), At4NC069370 (named LINC-AP2 in this study), were upregulated significantly in TCV-infected plants. This LINC-AP2 was further confirmed for its existence using 5'RACE technology. LINC-AP2 overexpression (LINC-AP2 OE) transgenic Arabidopsis plants were generated to compare with TCV-infected WT plants. TCV-infected LINC-AP2 OE plants which contained lower AP2 gene expression displayed more severe symptoms (including floral structure distortion) and higher TCV-CP gene transcript and coat protein levels. Furthermore, compared to TCV-infected WT plants, TCV-infected ap2 mutant plants failed to open their flower buds and displayed more severe viral symptoms. In conclusion, upregulation of LINC-AP2 is negatively correlated with AP2 gene expression with TCV infection in Arabidopsis.

Isolation and Functional Characterization of SOC1-like Genes in Prunus mume

The MADS-box gene SOC1/TM3 (suppressor of overexpression of constans 1/tomato MADS-box gene 3) integrates multiple flowering signals to regulate the transition from vegetative to reproductive development in arabidopsis (Arabidopsis thaliana). Although SOC1-like genes have been isolated from a wide range of plant species, their orthologs are not well characterized in mei (Prunus mume), an important ornamental and fruit plant in east Asia. To better understand the molecular regulation of flower development in mei, we isolated and characterized three putative orthologs of arabidopsis SOC1, including PmSOC1-1, PmSOC1-2, and PmSOC1-3. The phylogenetic tree revealed that these genes fall into different subgroups within the SOC1-like gene group, suggesting distinct functions. PmSOC1-1 and PmSOC1-3 were mainly expressed in vegetative organs and at low expression levels in floral parts of the plants, whereas PmSOC1-2 was expressed only in vegetative organs. Furthermore, the expression level decreased significantly during flower bud differentiation development, suggesting a role for these genes in the transition from the vegetative to the reproductive phase. Overexpression of PmSOC1-1, PmSOC1-2, and PmSOC1-3 in arabidopsis caused early flowering. Early flowering also increased expression levels of four other flowering promoters, agamous-like 24 (AGL24), leafy (LFY), apetala 1 (AP1), and fruitfull (FUL). Moreover, the overexpression of PmSOC1-1 and PmSOC1-2 resulted in a range of floral phenotype changes such as sepals into leaf-like structures, petal color into green, and petal into filament-like structures. These results suggested that the genes PmSOC1-1, PmSOC1-2, and PmSOC1-3 play an evolutionarily conserved role in promoting flowering in mei, and may have distinct roles during flower development. Our findings will help elucidate the molecular mechanisms involved in the transition from vegetative to reproductive development in mei.

AP2/ERF Transcription Factors Involved in Response to Tomato Yellow Leaf Curly Virus in Tomato.

Tomato yellow leaf curly virus (TYLCV), transmitted by the whitefly (), causes leaf curling and yellowing, plant dwarfism, and growth inhibition in tomato ( L.). The APETALA2 (AP2) and ethylene response factor (ERF) transcription factor (TF) family, the largest plant-specific TF family, was identified to function in plant development and pathogen defense. Our study aimed to analyze the mechanism underlying the function of ERF (SlERF) TFs in response to TYLCV infection and improve useful information to increase the resistance to TYLCV in tomato. A total of 22 tomato AP2/ERF TFs in response to TYLCV were identified according to transcriptome database. Five ERF-B3 TFs were identified in cultivars Hongbeibei (highly resistant), Zheza-301, Zhefen-702 (both resistant), Jinpeng-1, and Xianke-6 (both susceptible). Interaction network indicated that SlERF TFs could interact with mitogen-activated protein kinase (MAPK). Expression profiles of five ERF-B3 genes (, , , , and ) were detected by quantitative real-time-polymerase chain reaction (qRT-PCR) after TYLCV infection in five tomato cultivars. expression was upregulated in five tomato cultivars. The expressions of three genes (, , and ) were upregulated in Zheza-301 and Zhefen-702. and expressions were downregulated in Hongbeibei and Xianke-6, respectively. Yeast one-hybrid showed that the GCC-box binding ability of ERF-B3 TFs differed in resistant and susceptible tomato cultivars. Expression profiles were related to the GCC-box binding ability of SlERF TFs in resistant and susceptible tomato cultivars. The defense mechanism underlying the tomato's response to TYLCV involved a complicated network, which provided important information for us in breeding and genetic analysis.