| Larch(Larix leptolepis)is an important and fast-growing afforetation and timber species in the alpine and subapline regions of northern China.Larch somatic embryogenesis system,as a suitable and valuable system,was used for large-scale breeding in cell engineering,and also provided excellent materials for the functional studies of trees genes.Our laboratory has investigated the molecular mechanism of embryogenic maintenance and high rate of abnormal embryos formation during somatic embryogenesis at the morphological,physiological,biochemical and molecular levels.It has been shown that the molecule hydrogen as an antioxidant or signal molecule,can promote cell proliferation,regulate cell cycle and apoptosis,relieve oxidative damage,and delay cell senescence in plants and animals.However,the biological effects of exogenous hydrogen on plant somatic embryos have not been documented to date.Thus,the aim of this study was to optimize of larch somatic embryogenesis system through exogenous hydrogen treatment(hydrogen-rich culture)in the process,and examine the influences of hydrogen-rich culture on the development of pro-embryogenic masses(PEMs)and high-quality cotyledonary embryos,and further explore miRNAs and mRNAs regulatory mechanism underlying the effects.The main results are as follows:1.We analyzed the effects of hydrogen-rich culture on the PEMs embryogenic maintenance,the synchronization of PEMIII development and the quantity and quality of cotyledonary embryos in the larch S287 cells line.Compared with the control,the fresh weight of PEMs proliferation increased by 18.9%,the PEMIII synchronization rate raised by 25.4%,and the survival rates of active PEMIII improved by 12.9% after hydrogen-rich culture during the PEMs phase;the formation rate of early cotyledonary embryos increased by 10.3%,and the formation rate of normal mature cotyledon embryos elevated by 19.4% after continual hydrogen-rich culture during PEMs and somatic embryo(SEs)phases.Moreover,the level of ROS decreased by 53.6%,and the antioxidant enzyme activities of SOD,CAT and POD were enhenced by 89.2%,76%,and 21.4%,respectively.These results indicate that hydrogen is involved in the regulation of PEMs and cotyledonary embryos development.2.Twenty-two cDNA libraries from larch PEMs sub-cultured on hydrogen-rich and control media at six time points(0 h,12 h,48 h,7 d,21 d,and 36 d)were sequenced by RNA-seq.A total of 572,103,351 clean reads were obtained.After de novo assembly of the clean reads,we obtained 62,729 transcripts and 45,393 unigenes.Further,we identified 4253 differentially expressed genes(DEGs)by comparing the hydrogen-treatment and control samples;2379 were up-regulated and 1874 were down-regulated.By Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses,many of these DEGs were found to be involved in ROS homeostasis,programmed cell death process,cell cycle regulation,and plant hormones signal transduction pathways.3.Twenty-two miRNA libraries were sequenced by miRNA-seq.A total of 315 miRNAs were identified.The 315 miRNAs were compared to known miRNAs;49 of them matched 40 known miRNAs families.A further 266 miRNAs were identified as novel mi RNAs,having a corresponding miRNA* and the characteristic hairpin structure.23 conserved miRNAs family members were expressed in the PEMs phase and the SEs phase,but the expression levels were obviously different;17 and 16 conserved miRNAs family members were specifically expressed at the PEMs phase and the SEs phase,respectively.These results suggest that more unknown mi RNAs are involved in the regulation of larch PEMs development and there are different mi RNAs expression profiles in the PEMs phase and the SEs phase during somatic embryogenesis.4.96 differentially expressed miRNAs were obtained after hydrogen treatment compared with untreated controls;49 were up-regulated and 47 were down-regulated.Then,we identified 4399 potential target genes for 285 of the miRNAs.After 12 h,48 h,7 d,21 d,and 36 d of hydrogen-rich culture,we found 8,13,5,7,and 8 differently expressed miRNAs that putatively regulated 8,40,5,9,and 10 DEGs targets,respectively.Among them,42 miRNAs-targets pairs displayed negative correlation.miRNAs-mRNAs interaction network was further constructed.These results suggest that miRNAs and mRNAs coordinately participate in the response pathways of larch PEMs to hydrogen-rich culture.5.We cloned and identified the LlCuZnSOD gene encoding a copper and zinc superoxide dismutase.Bioinformatics,sequences alignment and molecular phylogenetic tree analyses revealed that highly conserved Cu ZnSOD were found in bacteria,plant and animal species.qRTPCR results showed that the expression was active in the PEMs phase and reached the highest level in the early SEs phase,and then the expression declined sharply in the late SEs phase.Moreover,protoplasts transient transfection assays displayed the encoded protein was localized in cytoplasm.Taken together,hydrogen-rich culture can promote the proliferation and synchronization of PEMs,delayed the senescence of PEMs,and improved the survival rates of active embryogenic cells at the PEMs phase;promoted the muration of SEs and increased the proportions of normal mature cotyledon embryos at the SEs phase.Therefore,hydrogen-rich culture can be used to optimize the larch somatic embryogenesis system.The transcriptome data in this study also suggest that the responses of larch PEMs to the exogenous hydrogen involve multiple biological pathways and processes.The differential expression data of mRNAs and miRNAs together with the complex mi RNAs-targets interaction network described here will contribute to a more comprehensive understanding of the molecular mechanism underlying how hydrogen affects the development of larch PEMs.Moreover,these analyses provide new insights into and a resource for the futher dissection of the regulatory network during somatic embryogenesis,also provide a feasible method for the larch large-scale breeding in cell engineering. |
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