Basic helix-loop-helix (bHLH) transcription factors are involved in many abiotic stress responses as well as flavonol and anthocyanin biosynthesis. In grapes (Vitis vinifera L.), flavonols including anthocyanins and condensed tannins are most abundant in the skins of the berries. Flavonols are important phytochemicals for viticulture and enology, but grape bHLH genes have rarely been examined. We identified 94 grape bHLH genes in a genome-wide analysis and performed Nr and GO function analyses for these genes. Phylogenetic analyses placed the genes into 15 clades, with some remaining orphans. 41 duplicate gene pairs were found in the grape bHLH gene family, and all of these duplicate gene pairs underwent purifying selection. Nine triplicate gene groups were found in the grape bHLH gene family and all of these triplicate gene groups underwent purifying selection. Twenty-two grape bHLH genes could be induced by PEG treatment and 17 grape bHLH genes could be induced by cold stress treatment including a homologous form of MYC2, VvbHLH007. Based on the GO or Nr function annotations, we found three other genes that are potentially related to anthocyanin or flavonol biosynthesis: VvbHLH003, VvbHLH007, and VvbHLH010. We also performed a cis-acting regulatory element analysis on some genes involved in flavonoid or anthocyanin biosynthesis and our results showed that most of these gene promoters contained G-box or E-box elements that could be recognized by bHLH family members.

Plant long non-coding RNA (lncRNA) undergoes dynamic regulation and acts in developmental and stress regulation. In this study, we surveyed the expression dynamics of lncRNAs in grapevine (Vitis vinifera L.) under cold stress using high-throughput sequencing. Two-hundred and three known lncRNAs were significantly up-regulated and 144 known lncRNAs were significantly down-regulated in cold-treated grapevine. In addition, 2 088 novel lncRNA transcripts were identified in this study, with 284 novel lncRNAs significantly up-regulated and 182 novel lncRNAs significantly down-regulated in cold-treated grapevine. Two-hundred and forty-two differentially expressed grapevine lncRNAs were predicted to target 326 protein-coding genes in a cis-regulatory relationship. Many differentially expressed grapevine lncRNAs targeted stress response-related genes, such as CBF4 transcription factor genes, late embryogenesis abundant protein genes, peroxisome biogenesis protein genes, and WRKY transcription factor genes. Sixty-two differentially expressed grapevine lncRNAs were predicted to target 100 protein-coding genes in a trans-regulatory relationship. The expression of overall target genes in both cis and trans-regulatory relationships were positively related to the expression of lncRNAs in grapevines under cold stress. We identified 31 known lncRNAs as 34 grapevine micro RNA (miRNA) precursors and some miRNAs may be derived from multiple lncRNAs. We found 212 lncRNAs acting as targets of miRNAs in grapevines, involving 150 miRNAs; additionally, 120 grapevine genes were predicted as targets of grapevine miRNAs and lncRNAs. We found one gene cluster that was up-regulated and showed the same expression trend. In this cluster, many genes may be involved in abiotic stress response such as WRKY, Hsf, and NAC transcription factor genes.

Epigenetic modifications play important roles in plant and animal development. DNA methylation impacts the transposable element (TE) silencing, gene imprinting and expression regulation. Through a genome-wide analysis, DNA methylation peaks were characterized and mapped in maize embryo and endosperm genome, respectively. Distinct methylation level was observed across maize embryo and endosperm. The maize embryo genome contained more DNA methylation than endosperm. Totally, 985,478 CG islands (CGIs) were identified and most of them were unmethylated. More CGI shores were methylated than CGIs in maize suggested that DNA methylation level was not positively correlated with CpG density. The promoter sequence and transcriptional termination region (TTR) were more methylated than the gene body (intron and exon) region based on peak number and methylated depth. Result showed that 99% TEs were methylated in maize embryo, but a large portion of them (34.8%) were not methylated in endosperm. Maize embryo and endosperm exhibit distinct pattern/level of methylation. The most differentially methylated region between embryo and endosperm are CGI shores. Our results indicated that DNA methylation is associated with both gene silencing and gene activation in maize. Many genes involved in embryogenesis and seed development were found differentially methylated in embryo and endosperm. We found 41.5% imprinting genes were similarly methylated and 58.5% imprinting genes were differentially methylated between embryo and endosperm. Methylation level was associated with allelic silencing of only a small number of imprinting genes. The expression of maize DEMETER-like (DME-like) gene and MBD101 gene (MBD4 homolog) were higher in endosperm than in embryo. These two genes may be associated with distinct methylation levels across maize embryo and endosperm. Through MeDIP-seq we systematically analyzed the methylomes of maize embryo and endosperm and results indicated that the global methylation status of embryo was more than that of the endosperm. Differences could be observed at the total number of methylation peaks, DMRs and specific methylated genes which were tightly associated with development of embryo and endosperm. Our results also revealed that many DNA methylation regions didn’t affect transcription of the corresponding genes.

Heat shock transcription factors (Hsfs) are important transcription factors (TFs) in protecting plants from damages caused by various stresses. The released whole genome sequences of wild peanuts make it possible for genome-wide analysis of Hsfs in peanut. In this study, a total of 16 and 17 Hsf genes were identified from Arachis duranensis and A. ipaensis, respectively. We identified 16 orthologous Hsf gene pairs in both peanut species; however HsfXs was only identified from A. ipaensis. Orthologous pairs between two wild peanut species were highly syntenic. Based on phylogenetic relationship, peanut Hsfs were divided into groups A, B, and C. Selection pressure analysis showed that group B Hsf genes mainly underwent positive selection and group A Hsfs were affected by purifying selection. Small scale segmental and tandem duplication may play important roles in the evolution of these genes. Cis-elements, such as ABRE, DRE, and HSE, were found in the promoters of most Arachis Hsf genes. Five AdHsfs and two AiHsfs contained fungal elicitor responsive elements suggesting their involvement in response to fungi infection. These genes were differentially expressed in cultivated peanut under abiotic stress and Aspergillus flavus infection. AhHsf2 and AhHsf14 were significantly up-regulated after inoculation with A. flavus suggesting their possible role in fungal resistance.

As a typical geocarpic plant, peanut embryogenesis and pod development are complex processes involving many gene regulatory pathways and controlled by appropriate hormone level. MicroRNAs (miRNAs) are small non-coding RNAs that play indispensable roles in post-transcriptional gene regulation. Recently, identification and characterization of peanut miRNAs has been described. However, whether miRNAs participate in the regulation of peanut embryogenesis and pod development has yet to be explored. In this study, small RNA and degradome libraries from peanut early pod of different developmental stages were constructed and sequenced. A total of 70 known and 24 novel miRNA families were discovered. Among them, 16 miRNA families were legume-specific and 12 families were peanut-specific. 30 known and 10 novel miRNA families were differentially expressed during pod development. In addition, 115 target genes were identified for 47 miRNA families by degradome sequencing. Several new targets that might be specific to peanut were found and further validated by RNA ligase-mediated rapid amplification of 5′ cDNA ends (RLM 5′-RACE). Furthermore, we performed profiling analysis of intact and total transcripts of several target genes, demonstrating that SPL (miR156/157), NAC (miR164), PPRP (miR167 and miR1088), AP2 (miR172) and GRF (miR396) are actively modulated during early pod development, respectively. Large numbers of miRNAs and their related target genes were identified through deep sequencing. These findings provided new information on miRNA-mediated regulatory pathways in peanut pod, which will contribute to the comprehensive understanding of the molecular mechanisms that governing peanut embryo and early pod development.

Peanut zygotes typically divide a few times to form a pre-embryo before further embryonic development halts under normal day/night photoperiods. Ovary elongation, however, continuesforming a downward growing peg-like structure. When the peg is buried in the soil, embryo development resumes in the darkness. The embryo-located region (ER) of the peg begins to enlarge and form a pod, while the basal region (BR) of the peg has a distinct fate. The molecular mechanisms governing these unique embryo development processes are unknown. In this study, histological analysis demonstrated that from 4 days after pollination to 3 days after soil penetration, the peanut pre-embryo remained morphologically similar. By 9 days after soil penetration, the embryo had changed to a globular embryo. Transcriptome analysis revealed differentially expressed genes in the ER and BR before and after peg soil penetration. In addition to light signaling and plant hormone metabolism genes, we identified differentially expressed genes in the ER that contribute to embryo development and pod formation processes, including MADS-box transcription factors, xyloglucan endotransglucosylase/hydrolase protein, cellulose synthase, homeobox-leucine zipper (HD-Zip) protein family genes, amino acid permease, and seed growth and embryo morphogenesis regulators (DA1, TCP3, and YABBY). A large number of genes were found to be differentially expressed in the ER and BR across three developmental peg stages. Exact changes in gene expression were also identified in the ER during early embryo and pod development. This information provides an expanded knowledgebase for understanding the mechanisms of early peanut pod formation.