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Conserved miR396b-GRF Regulation Is Involved in Abiotic Stress Responses in Pitaya (Hylocereus polyrhizus).

ABSTRACT: MicroRNA396 (miR396) is a conserved microRNA family that targets growth-regulating factors (GRFs), which play significant roles in plant growth and stress responses. Available evidence justifies the idea that miR396-targeted GRFs have important functions in many plant species; however, no genome-wide analysis of the pitaya (Hylocereus polyrhizus) miR396 gene has yet been reported. Further, its biological functions remain elusive. To uncover the regulatory roles of miR396 and its targets, the hairpin sequence of pitaya miR396b and the open reading frame (ORF) of its target, HpGRF6, were isolated from pitaya. Phylogenetic analysis showed that the precursor miR396b (MIR396b) gene of plants might be clustered into three major groups, and, generally, a more recent evolutionary relationship in the intra-family has been demonstrated. The sequence analysis indicated that the binding site of hpo-miR396b in HpGRF6 is located at the conserved motif which codes the conserved "RSRKPVE" amino acid in the Trp-Arg-Cys (WRC) region. In addition, degradome sequencing analysis confirmed that four GRFs (GRF1, c56908.graph_c0; GRF4, c52862.graph_c0; GRF6, c39378.graph_c0 and GRF9, c54658.graph_c0) are hpo-miR396b targets that are regulated by specific cleavage at the binding site between the 10th and 11th nucleotides from the 5' terminus of hpo-miR396b. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that hpo-miR396b is down-regulated when confronted with drought stress (15% polyethylene glycol, PEG), and its expression fluctuates under other abiotic stresses, i.e., low temperature (4 ± 1 °C), high temperature (42 ± 1 °C), NaCl (100 mM), and abscisic acid (ABA; 0.38 mM). Conversely, the expression of HpGRF6 showed the opposite trend to exposure to these abiotic stresses. Taken together, hpo-miR396b plays a regulatory role in the control of HpGRF6, which might influence the abiotic stress response of pitaya. This is the first documentation of this role in pitaya and improves the understanding of the molecular mechanisms underlying the tolerance to drought stress in this fruit.

SUBMITTER: Li AL

PROVIDER: S-EPMC6566180 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Conserved miR396b-GRF Regulation Is Involved in Abiotic Stress Responses in Pitaya (Hylocereus polyrhizus).

Li A-Li AL Wen Zhuang Z Yang Kun K Wen Xiao-Peng XP

International journal of molecular sciences 20190521 10

MicroRNA396 (miR396) is a conserved microRNA family that targets growth-regulating factors (GRFs), which play significant roles in plant growth and stress responses. Available evidence justifies the idea that miR396-targeted GRFs have important functions in many plant species; however, no genome-wide analysis of the pitaya (Hylocereus polyrhizus) miR396 gene has yet been reported. Further, its biological functions remain elusive. To uncover the regulatory roles ...[more]

PMID: 31117184

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Project description:BackgroundCanker disease caused by Neoscytalidium dimidiatum is the most serious disease that attacks the pitaya industry. One pathogenic fungus, referred to as ND8, was isolated from the wild-type red-fleshed pitaya (Hylocereus polyrhizus) of Hainan Province. In the early stages of this disease, stems show little spots and a loss of green color. These spots then gradually spread until the stems became rotten due to infection by various strains. Canker disease caused by Neoscytalidium dimidiatum poses a significant threat to pitaya commercial plantations with the growth of stems and the yields, quality of pitaya fruits. However, a lack of transcriptomic and genomic information hinders our understanding of the molecular mechanisms underlying the pitaya defense response.ResultsWe investigated the host responses of red-fleshed pitaya (H. polyrhizus) cultivars against N. dimidiatum using Illumina RNA-Seq technology. Significant expression profiles of 23 defense-related genes were further analyzed by qRT-PCR. The total read length based on RNA-Seq was 25,010,007; mean length was 744, the N50 was 1206, and the guanine-cytosine content was 44.48%. Our investigation evaluated 33,584 unigenes, of which 6209 (18.49%) and 27,375 (81.51%) were contigs and singlets, respectively. These unigenes shared a similarity of 16.62% with Vitis vinifera, 7.48% with Theobroma cacao, 6.6% with Nelumbo nucifera and 5.35% with Jatropha curcas. The assembled unigenes were annotated into non-redundant (NR, 25161 unigenes), Kyoto Encyclopedia of Genes and Genomes (KEGG, 17895 unigenes), Clusters of Orthologous Groups (COG, 10475 unigenes), InterPro (19,045 unigenes), and Swiss-Prot public protein databases (16,458 unigenes). In addition, 24 differentially expressed genes, which were mainly associated with plant pathology pathways, were analyzed in-depth.ConclusionsThis study provides a basis for further in-depth research on the protein function of the annotated unigene assembly with cDNA sequences.

Project description:Cross-pollination can improve the percentage of fruit set and fruit weight for most red flesh varieties in pitaya. The technology of pollen storage was very important for successful cross-pollination. However, till present, the technology of pollen storage is unsatisfactory in pitaya production. In this study, pitaya pollen stored at low temperature was taken as the research object, and its physicochemical indexes, metabolomics, and transcriptomics were studied. The results showed that in vitro pollen germination rate decreased significantly with the increase in storage time. Soluble sugar and soluble protein content of pollen peaked on the first day of storage, whereas its relative conductivity, and manlondialdehyde (MDA) and proline contents increased gradually during storage. At the same time, the antioxidant enzyme system of pollen was also affected. Superoxide dismutase (SOD) activity decreased, while the activities of catalase (CAT) and peroxidase (POD) increased and superoxide anion generation rate increased gradually during storage. According to the metabolomics results, amino acid, peptide, nucleotide, plant hormone, terpene, alcohol, phenol, flavonoid, sterol, vitamin, ester, sphingolipid, and ketone contents increased significantly during storage, whereas flavonoid and pigment contents declined gradually. During pollen storage, the gene expressions related to carbohydrate metabolism, protein metabolism, acid and lipid metabolism, sterol metabolism, plant hormone metabolism, and signal transductions were significantly downregulated. With KEGG pathway analysis, isoquinoline alkaloid biosynthesis, tyrosine metabolism, alanine, aspartate, and glutamate metabolism of pollen were affected significantly during low-temperature storage. Correlation analysis showed that the gene expression patterns of HuRP2, HuUPL1, and HuAAT2 had significant effects on pollen germination. D-arabinose 5-phosphate and myricetin were positively correlated with pollen germination rate, which was valuable for studying preservation agents. In this study, the changes in pollen during low-temperature storage were described from the level of metabolites and genes, which could provide theoretical support for the research and development of pollen long-term storage technology in pitaya.

Dataset Information

Conserved miR396b-GRF Regulation Is Involved in Abiotic Stress Responses in Pitaya (Hylocereus polyrhizus).

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Conserved <i>miR396b-GRF</i> Regulation Is Involved in Abiotic Stress Responses in Pitaya (<i>Hylocereus polyrhizus</i>).

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