A near-isogenic line (NIL) DC90 which was generated by introgressing a wild rice (Oryza rufipogon Griff.) locus CTS-12 into the 9311(Oryza sativa L. ssp. indica) background confers chilling tolerance phenotype. Here, our pilot trials showed that chilling tolerance was positively correlated with abscisic acid (ABA) biosynthesis. To understand how CTS-12 mediated the ABA-dependent multi-levels of regulation, the integration of transcriptomic and metabolomic profiling using the two-way orthogonal projections to latent structures (O2PLS) and discriminant analysis (OPLS-DA) modeling was performed to investigate the mechanisms underlying chilling tolerance. Our results revealed that metabolic shifts, including the activation of stachyose biosynthesis, amino acid metabolism pathways, phenylpropanoid/flavonoid biosynthesis, ABA biosynthesis, and perturbation of glycolysis, occurred under chilling treatment; in the recovery period, glutamate-related pathways, β-alanine biosynthesis and degradation, and serotonin biosynthesis pathways were differentiated between 9311 and DC90. Particularly, the differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs), including galactinol, β-alanine, glutamate, naringenin, serotonin, ABA, and LOC_Os03g44380 (9-cis-epoxycarotenoid dioxygenase 3, OsNCED3), might be involved in the chilling tolerance variation of 9311 and DC90. CRISPR/Cas9-edited OsNCED3 resulted in chilling sensitive of japonica rice ZH11, demonstrating the involvement of ABA pathway in chilling stress response. In addition, chilling tolerance of rice was associated with the balance of water uptake and loss that was modulated by stomatal movement under chilling stress. Therefore, we speculated that the CTS-12mediated ABA signaling pathway leads to transcriptional regulation of chilling-responsive genes and, in turn, triggers metabolic shifts to coordinately regulate the stomatal movement of guard cells. The results of this study improve our understanding of the multilevel regulation of wild rice in response to chilling stress.
A chromosome single segment substitution line (CSSL) DC90, which was generated by introgressing CTS-12, a locus derived from common wild rice (Oryza rufipogon Griff.), into the 9311 (Oryza sativa L. ssp. indica) background, exhibits a chilling tolerance phenotype under chilling stress. Here, an integration of microRNA (miRNA) deep sequencing and transcriptomic sequencing analysis was performed to explore the expression profiles of miRNAs and their target genes mediated by CTS-12 under chilling stress, and to reveal the possible regulatory mechanisms of miRNAs that are involved in chilling tolerance. Integration analysis revealed that a number of differentially expressed miRNAs (DEMs) and putative target genes with different expression patterns and levels were identified in 9311 and DC90 under chilling stress. KEGG enrichment analysis revealed that the target genes that are regulated by chilling-induced miRNAs are involved in the regulation of various biological processes/pathways, including protein biosynthesis, redox process, photosynthetic process, and chloroplast development in two genotypes. CRISPR/Cas9 editing of the target genes of the key DEMs in a chilling tolerant rice variety Zhonghua 11 (ZH11) found that LOC_Os11g48020 (OsGL1-11), one of the putative target genes of osa-miR1846a/b-5p and encoding a wax synthesis protein, is correlated with a chilling stress tolerance phenotype, implying osa-miR1846a/b-5p/OsGL1-11 plays an important role in CTS-12-mediated chilling stress tolerance regulatory pathway(s). Therefore, we speculate that the CTS-12 may regulate the key miRNA target genes in response to chilling stress by differential regulation of miRNAs in wild rice, thereby resulting in the variation of chilling tolerance phenotype between 9311 and DC90.
Background The testis has been reported to be a naturally O 2 -deprived organ, dimethyloxaloylglycine (DMOG) can inhibit hypoxia inducible factor-1alpha (HIF-1α) subject to degradation under normal oxygen condition in cells. Objectives The objective of this study is to detect the effects of DMOG on the proliferation and differentiation of spermatogonial stem cells (SSCs) in Bama minipigs. Methods Gradient concentrations of DMOG were added into the culture medium, HIF-1α protein in SSCs was detected by western blot analysis, the relative transcription levels of the SSC-specific genes were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Six days post-induction, the genes related to spermatogenesis were detected by qRT-PCR, and the DNA content was determined by flow cytometry. Results Results revealed that the levels of HIF-1α protein increased in SSCs with the DMOG treatment in a dose-dependent manner. The relative transcription levels of SSC-specific genes were significantly upregulated ( p < 0.05) by activating HIF-1α expression. The induction results showed that DMOG significantly increased ( p < 0.05) the spermatogenesis capability of SSCs, and the populations of haploid cells significantly increased ( p < 0.05) in DMOG-treated SSCs when compared to those in DMOG-untreated SSCs. Conclusion We demonstrate that DMOG can promote the spermatogenesis activity of SSCs.
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