Genome-wide characterization and expression of DELLA genes in Cucurbita moschata reveal their potential roles under development and abiotic stress.

DELLA gene family plays a key role in regulating plant development and responding to stress. Currently, many DELLA family members have been identified in plants, however, information on DELLA genes in pumpkin (Cucurbita moschata) is scarce. In this study, physical and chemical properties, gene structure cis-regulatory elements and expression of CmoDELLA genes were examined in pumpkin. We found that seven CmoDELLA genes were identified in pumpkin, and they were unevenly classified into five chromosomes. CmoDELLA proteins were relatively unstable and their secondary structures were mainly made up α-helix and random coil. All seven CmoDELLA proteins contained typical DELLA domain and GRAS domain, however, motif numbers between CmoDELLA proteins were unevenly distributed, implying the complex evolution and functional diversification of CmoDELLA proteins. Cis-regulatory elements analysis revealed that CmoDELLA genes might play an essential role in regulating plant growth and development, and response to stress in pumpkin. Transcriptome data in the roots, stems, leaves and fruits demonstrated that CmoDELLA2, CmoDELLA3 and CmoDELLA7 were related to the stems development, CmoDELLA1, CmoDELLA4, CmoDELLA5 and CmoDELLA6 were associated with the fruits development. Furthermore, we found that CmoDELLA1 and CmoDELLA5 were up-regulated under NaCl stress. CmoDELLA1, CmoDELLA2, CmoDELLA3, CmoDELLA5, CmoDELLA6 and CmoDELLA7 were remarkably induced under waterlogging stress. While, all of the 7 CmoDELLA genes showed significantly induced expression under cold stress. The expression patterns under abiotic stress suggested that CmoDELLA genes might mediate the stress response of pumpkin to NaCl, waterlogging and cold, however, the functions of different CmoDELLA genes varied under different stress. Overall, our study provides valuable information for further research about the potential functions and regulatory networks of CmoDELLA genes in pumpkin.

Identification and Characterization of Csa-miR395s Reveal Their Involvements in Fruit Expansion and Abiotic Stresses in Cucumber.

The miR395 plays an indispensable role in biochemical processes by regulating their target genes. However, little is known about the roles of miR395 in cucumber fruit expansion and response to abiotic stresses. Here, 4 Csa-miR395s and 8 corresponding target genes were identified in the cucumber genome. Csa-miR395s were all located on the same chromosome (Chr 5). Csa-miR395a/b/c and Csa-miR395d were distributed in different branches without a closer genetic relationship. Massive cis-acting elements, including light, phytohormone, and stress response elements, were detected in the promoter regions of Csa-MIR395s, indicating that Csa-miR395s might be involved in complex regulatory networks to control cucumber growth and development and stress response. In addition, Csa-miR395a/b/c shared the same target genes, and Csa-miR395d had its specific target genes. Tissue-specific expression analysis showed that Csa-miR395a/b/c were all expressed in the leaf, root, ovary, and expanded fruit of cucumber and highly expressed in the expanded fruits compared to the ovary, while Csa2G215520 and Csa1G502860 (target genes of Csa-miR395a/b/c) presented a downregulated trend in the expanded fruit compared to the ovary. Meanwhile, the protein co-expression network revealed that these target genes had interactions in sulfur metabolism. These results suggested that Csa-miR395a/b/c targeting Csa2G215520 and Csa1G502860 might promote cucumber fruit expansion by affecting sulfur metabolism. Additionally, Quantitative Real-time PCR analysis validated that Csa-miR395s could be regulated by NaCl stress, and Csa-miR395a/b/c could respond to PEG stress, which further confirmed the reliability of cis-acting elements data. Taken together, our results could be helpful for further exploration of the functions of miR395s in cucumber fruit expansion and response to abiotic stresses.

Identification and Expression Analysis of miR160 and Their Target Genes in Cucumber.

miR160 plays a crucial role in various biological processes by regulating their target gene auxin response factor (ARF) in plants. However, little is known about miR160 and ARF in cucumber fruit expansion. Here, 4 Csa-MIR160 family members and 17 CsARFs were identified through a genome-wide search. Csa-miR160 showed a closer relationship with those in melon. Phylogenetic analysis revealed that CsARFs were divided into four classes and most of CsARFs presented a closer evolutionary relationship with those from tomato. Putative cis-elements analysis predicted that Csa-MIR160 and CsARFs were involved in light, phytohormone and stress response, which proved that they might take part in light, phytohormone and stress signaling pathway by the miR160-ARF module. In addition, CsARF5, CsARF11, CsARF13 and CsARF14 were predicted as the target genes of Csa-miR160. qRT-PCR revealed that Csa-miR160 and their target gene CsARFs were differentially expressed in differential cucumber tissues and developmental stages. Csa-miR160d was only expressed in the expanded cucumber fruit. CsARF5, CsARF11 and CsARF13 exhibited the lower expression in the expanded fruit than those in the ovary, while, CsARF14 showed the reverse trend. Our results suggested that Csa-miR160d might play a crucial role in cucumber fruit expansion by negatively targeting CsARF5, CsARF11 and CsARF13. This is the first genome-wide analysis of miR160 in cucumber. These findings provide useful information and resources for further studying the role of miR160 and ARF in cucumber fruit expansion.

Identification of miRNAs and Their Target Genes Involved in Cucumber Fruit Expansion Using Small RNA and Degradome Sequencing.

Fruit expansion is an essential and very complex biological process. Regulatory roles of microRNAs (miRNAs) and miRNA-mRNA modules in the cucumber fruit expansion are not yet to be investigated. In this work, 1253 known and 1269 novel miRNAs were identified from nine cucumber fruit small RNA (sRNA) libraries through high-throughput sequencing. A total of 105 highly differentially expressed miRNAs were recognized in the fruit on five days post anthesis with pollination (EXP_5d) sRNA library. Further, expression patterns of 11 differentially expressed miRNAs were validated by quantitative real-time PCR (qRT-PCR). The expression patterns were similar to sRNAs sequencing data. Transcripts of 1155 sequences were predicted as target genes of differentially expressed miRNAs by degradome sequencing. Gene Ontology (GO) enrichment showed that these target genes were involved in 24 biological processes, 15 cell components and nine molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that these target genes were significantly enriched in 19 pathways and the enriched KEGG pathways were associated with environmental adaptation, signal transduction and translation. Based on the functional prediction of miRNAs and target genes, our findings suggest that miRNAs have a potential regulatory role in cucumber fruit expansion by targeting their target genes, which provide important data for understanding the miRNA-mediated regulatory networks controlling fruit expansion in cucumber. Specific miRNAs could be selected for further functional research and molecular breeding in cucumber.