MrERF039 transcription factor plays an active role in the cold response of Medicago ruthenica as a sugar molecular switch.

Cold stress severely restricts plant development, causing significant agricultural losses. We found a critical transcription factor network in Medicago ruthenica was involved in plant adaptation to low-temperature. APETALA2/ethylene responsive factor (AP2/ERF) transcription factor MrERF039 was transcriptionally induced by cold stress in M. ruthenica. Overexpression of MrERF039 significantly increased the glucose and maltose content, thereby improving the tolerance of M. ruthenica. MrERF039 could bind to the DRE cis-acting element in the MrCAS15A promoter. Additionally, the methyl group of the 14th amino acid in MrERF039 was required for binding. Transcriptome analysis showed that MrERF039 acted as a sugar molecular switch, regulating numerous sugar transporters and sugar metabolism-related genes. In addition, we found that MrERF039 could directly regulate ß-amylase gene, UDP glycosyltransferase gene, and C2H2 zinc finger protein gene expression. In conclusion, these findings suggest that high expression of MrERF039 can significantly improve the cold tolerance of M. ruthenica root tissues during cold acclimation. Our results provide a new theoretical basis and candidate genes for breeding new legume forage varieties with high resistance.

Identification and characterization analysis of the HDM gene family in melon (Cucumis melo L.).

Histone demethylase (HDM) play crucial roles in regulating plant growth and environmental adaptation. In this study, the HDM gene family in melon was identified by bioinformatics methods and the expression patterns of the CmHDM family members in different melon tissues were analyzed using transcriptome data. The results showed that 20 CmHDM genes were identified in the melon genome, which were unevenly distributed across each chromosome. These members fall into two major categories: LSD1 and JmjC. The JmjC group could be further divided into five subgroups with different numbers. The results of collinearity analysis of intraspecific and interspecific relationships showed that there were only one pair of segmental duplication in melon HDM genes, and more collinearity in genetic relationship of HDM genes between melon and tomato. The numbers of conserved domains, exons and introns in each member vary and various cis-acting elements responding to hormones and environmental signals existed in the respective promoter regions. Expression analysis showed that the respective gene members were expressed at different levels in male flowers, female flowers, roots, stems, leaves, ovary, and mature fruits of melon. These results will contribute to the understanding on the potential functions of the HDM genes and their potential functions in regulating melon growth and environmental adaptation.

Genome-wide identification of microRNAs involved in the regulation of fruit ripening and climacteric stages in melon (Cucumis melo).

Fruit ripening is influenced by multiple plant hormones and the regulation of genes. However, studies on posttranscriptional regulators (e.g., miRNAs) of fruit growth and ripening are limited. We used miRNA sequencing and degradome methods to identify miRNAs and their target genes in melon (Cucumis melo cv. Hetao melon). A total of 61 conserved miRNAs and 36 novel miRNAs were identified from fruit growth, ripening, climacteric, and postclimacteric developmental stage samples, of which 32 conserved miRNAs were differentially expressed between developmental stage samples. Sixty-two target genes of 43 conserved miRNAs and 1 novel miRNA were identified from degradome sequencing. To further investigate miRNA influencing fruit ripening, transgenic melon plants overexpressing pre-cme-miR393 (cme-miR393-OE) were generated and characterized. The results showed that fruit ripening was delayed in cme-miR393-OE transgenic lines compared to nontransgenic fruits. The target of cme-miR393 was also identified, and the expression of CmAFB2 was repressed in transgenic plants. These results provide evidence that miRNA regulates melon fruit ripening and provide potential targets to improve the horticultural traits of melon fruit.

Genome-wide identification and characterization of long non-coding RNAs involved in fruit ripening and the climacteric in Cucumis melo.

BACKGROUND: Cucumis melo is a suitable study material for investigation of fruit ripening owing to its climacteric nature. Long non-coding RNAs have been linked to many important biological processes, such as fruit ripening, flowering time regulation, and abiotic stress responses in plants. However, knowledge of the regulatory roles of lncRNAs underlying the ripening process in C. melo are largely unknown. In this study the complete transcriptome of Cucumis melo L. cv. Hetao fruit at four developmental stages was sequenced and analyzed. The potential role of lncRNAs was predicted based on the function of differentially expressed target genes and correlated genes. RESULTS: In total, 3857 lncRNAs were assembled and annotated, of which 1601 were differentially expressed between developmental stages. The target genes of these lncRNAs and the regulatory relationship (cis- or trans-acting) were predicted. The target genes were enriched with GO terms for biological process, such as response to auxin stimulus and hormone biosynthetic process. Enriched KEGG pathways included plant hormone signal transduction and carotenoid biosynthesis. Co-expression network construction showed that LNC_002345 and LNC_000154, which were highly expressed, might co-regulate with mutiple genes associated with auxin signal transduction and acted in the same pathways. We identified lncRNAs (LNC_000987, LNC_000693, LNC_001323, LNC_003610, LNC_001263 and LNC_003380) that were correlated with fruit ripening and the climacteric, and may participate in the regulation of ethylene biosynthesis and metabolism and the ABA signaling pathway. A number of crucial transcription factors, such as ERFs, WRKY70, NAC56, and NAC72, may also play important roles in the regulation of fruit ripening in C. melo. CONCLUSIONS: Our results predict the regulatory functions of the lncRNAs during melon fruit development and ripening, and 142 highly expressed lncRNAs (average FPKM > 100) were identified. These lncRNAs participate in the regulation of auxin signal transduction, ethylene, sucrose biosynthesis and metabolism, the ABA signaling pathway, and transcription factors, thus regulating fruit development and ripening.

Molecular Phylogenetic Analysis of the Orthoptera (Arthropoda, Insecta) based on Hexamerin Sequences.

The higher taxa classification and phylogeny of the insect order Orthoptera have long been controversial. Hexamerin, as a member of the highly conserved arthropod hemocyanin superfamily, has been shown to be a good marker for the phylogenetic study of insects. However, few studies have used hexamerins on the phylogeny of Orthoptera. In this study, we determined twenty-seven different hexamerin subunit type sequences in seventeen speices of Orthoptera. In order to infer the phylogenetic relationships among the superfamilies within Orthoptera and test the monophyly of Orthoptera, phylogenic trees were reconstructed using Neighbor-Joining (NJ) and Bayesian inference (BI) methods with two dipluran and three hymenopteran hexamerin sequences as outgroups. The result supported the monophyly of Orthoptera, which includes two monophyletic suborders Caelifera and Ensifera. The Caelifera includes Acridoidea, Eumastacoidea, Tetrigoidea and Tridactyloidea, and the Ensifera includes Tettigonioidea, Grylloidea and Gryllotalpoidea. Our study is basically consistent with the study of morphological classification. In addition, our study indicates that a relatively comprehensive taxa sampling is essential to solve some problems in phylogenetic reconstruction.

Cloning and characterization of a Ca(2+)/H(+) exchanger from the halophyte Salicornia europaea L.

The calcium ion (Ca(2+)), which functions as a second messenger, plays an important role in plants' responses to various abiotic stresses, and Ca(2+)/H(+) exchangers (CAXs) are an important part of this process. In this study, we isolated and characterized a putative Ca(2+)/H(+) exchanger gene (SeCAX3) from Salicornia europaea L., a succulent, leafless euhalophyte. The SeCAX3 open reading frame was 1368 bp long and encoded a 455-amino-acid polypeptide that showed 67.9% similarity to AtCAX3. SeCAX3 was expressed in the shoots and roots of S. europaea. Expression of SeCAX3 was up-regulated by Ca(2+), Na(+), sorbitol, Li(+), abscisic acid, and cold treatments in shoots, but down-regulated by Ca(2+), sorbitol, abscisic acid, and cold treatments in roots. When SeCAX3 was transformed into a Ca-sensitive yeast strain, the transformed cells were able to grow in the presence of 200 mM Ca(2+). Furthermore, SeCAX3 conferred drought, salt, and cold tolerance in yeast. Compared with the control strains, the yeast transformants expressing SeCAX3 were able to grow well in the presence of 30 mM Li(+), 150 mM Mg(2+), or 6 mM Ba(2+). These results showed that the expression of SeCAX3 in yeast suppressed its Ca(2+) hypersensitivity and conferred tolerance to Mg(2+) and Ba(2+). Together, these findings suggest that SeCAX3 might be a Ca(2+) transporter that plays a role in regulating cation tolerance and the responses of S. europaea to various abiotic stresses.

Efficient one-pot cyclization/folding of Rhesus θ-defensin-1 (RTD-1).

We report an efficient approach for the chemical synthesis of Rhesus θ-defensin-1 (RTD-1) using Fmoc-based solid-phase peptide synthesis in combination with an intramolecular version of native chemical ligation. The corresponding linear thioester precursor was cyclized and folded in a one-pot reaction using reduced glutathione. The reaction was extremely efficiently yielding natively folded RTD-1 with minimal or no purification at all. This approach is fully compatible with the high throughput production of chemical libraries using this peptide scaffold.

Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway.

PURPOSE OF WORK: Melons have short shelf-lives due to fruit ripening caused by ethylene production. The 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene is essential for ethylene biosynthesis. As fruit ripening in other fruit crops can be deterred by down-regulation of ACC oxidase expression, we have carried out similar work to improve fruit quality and shelf-life of the melon Cucumis melo. A marker-free and vector-free antisense 1-aminocyclopropane-1-carboxylic acid oxidase construct was transformed into melon via the pollen-tube pathway. Based on phenotype analysis together with RT-PCR data, a transformation frequency of 0.7% was achieved. The transgenic fruits showed respiration rate and endogenous ethylene production level at approx. 15 and 6% of those of wild-type fruits, respectively. These fruits also demonstrated improved flesh firmness and exhibited extended shelf-life of 30 days compared to less than 12 days for the wild type fruits.