| Somatic embryogenesis(SE)is a perfect system for deciphering dedifferentiation and redifferentiation mechanisms of differentiated tissues and organs and also necessary for creating transgenic plants in numerous species,which makes the mechanism researches in SE of great value for both theoretic study and application.micro RNAs(miRNAs)are a class of endogenous ~19-25 nucleotide non-coding small RNAs,which play important roles in plant development,hormone signal transduction,and stress responses.However,very few studies about miRNA functions were reported in cotton.Here,comprehensive exploration of dynamic regulation of miRNAs during cotton SE was conducted by identifying miRNAs and targets through small RNA and degradome sequencing using an elite variety YZ1,charicterized by perfect SE.In addition,the biological functions of GhmiR157 a and its target GhSPL10 in dedifferentiation has been analyzed.The main results are as follows: 1.Identification of mi RNAs and targets during cotton SERNA-SEQ libraries of EC and CK were constructed in this study,and 36 differentially expressed conserved miRNA family members were identified,in which 29 precusors were found belonging to 19 miRNA families,as well as 25 novel miRNAs and their precusors.The length of these miRNAs varying from 19-24 nt,of which 21 nt miRNAs represented the most abundant.qRT-PCR revealed that several miRNAs showed dynamic expression patterns during cotton SE.By degradome sequencing,234 transcripts in EC and 322 transcripts in CK were identified cutting by 23 and 30 conserved miRNA families respectively,and also 16 transcripts were found cutting by 8 novel miRNAs.Targets of conserved miRNAs contain transcription factors and genes involved in hormone signaling and stress responses.Interestingly,four tasiRNAs and their targets AFR3/4 were also identified.Cutting sites of several conserved targets were confirmed using 5’ RACE,which further validated the fidelity of the degradome sequencing.Expression patterns between miR169 and NF-YA3,or miR396 and GRF8,showed perfect negative relationships during cotton SE.These results suggested that cotton SE were controled by diverse downstream signaling pathways mediated by diverse miRNA regulated target degradation.2.GhmiR157a/GhSPL10 module was involved in regulation of cotton SEqRT-PCR indicated that Ghmi R157 a and GhSPL10 showed opposite expression pattern in both the initial dedifferentiation stage and embryonic redifferention process during cotton SE.In situ hybridization further showed that GhSPL10 transcripts accumulated in cambium of explants and prevascular system in torpedo-stage embryos,implying that GhmiR157 a and GhSPL10 might control SE progress through modulating cell divison and differentiation.Although no significant phenotype during cotton SE was observed by Ghmi R157 overexpression,GhSPL10 overexpression conferred faster callus proliferation rate(CPR)and embryonic redifferentiation.Paraffin sections showed that vascular tissue pattern was also changed in 35S:r SPL10-7 line which exibited more and smaller xylem cells with continous distribution pattern in hypocotyls,and more cambium cells in explants induced for 3 d,coupled with upregulated expression of cell cycle related genes during initial cellular dedifferention.These results indicated that GhSPL10 overexpression promote dedifferentiation rate in cotton through enhancing cambium cell division in explants.3.Increased ethylene signaling and flavonoid biosynthesis pathways might be the reasons for the phenotypes in GhSPL10 overexpression lines To further determine the mechanisms of GhSPL10 overexpression in regulating dedifferentiation,RNA-SEQ was conducted using hypocotyls of 35S:rSPL10-7 and a segregated negative control-Null,and the results showed that: 1)35S:rSPL10-7 line occupied 1600 differentially expressed genes(DEGs)compared with control,among which 1005 were upregulated and 633 were downregulated.2)GO and KEGG analysis both pointed out that flavonoid biosynthesis and hormonal signaling pathways were significantly enriched.3)qRT-PCR confirmed that ethylene biosynthesis genes ACOs and flavonoid biosynthesis related genes were upregulated in 35S:rSPL10-7 compared to control;Flavonoid content measurement confirmed the overaccumulation of several flavonols in GhSPL10 overexpression lines.F1 hybridiztion line constructed using 35S:rSPL10-7 as maternal line and a F3 H RNAi line-Ri3 as paternal line,can reverse the higher CPR observed in GhSPL10 overexpression lines.Besides,flavonol application to wild type explants also promotes CPR and cell cycle related gene expression.These results indicated that excessive flavonoid accumulation largely contributed to the higher CPR observed in GhSPL10 overexpression lines.4.Activation of ethylene signaling pathway in GhSPL10 overexpression lines partially contributed to the overrepresentation of flavonoid biosynthesisEthylene biosynthesis inhitor AVG treatment completely inhibited callus initiation in 35S:rSPL10-7,and the ethylene precursor ACC treatement led to higher CPR in wild type,while ethylene biosynthesis inhitor AVG and Co Cl2 or response inhibitor Ag2S2SO3 treatment redarded callus proliferation severely,indicating that elevated ethylene signaling positively contributed to the higher CPR in GhSPL10 overexpression lines.qRT-PCR revealed that expression of F3 H can be further elevated in 35S:rSPL10-7 post ACC treatment compared to the level in 35S:rSPL10-7 without ACC treatment,which was inhibited in rSPL10-7/F3H-Ri3 under normal culture condition and cannot be further promoted post ACC treatment,confirming that an ethylene mediated flavonoid biosynthesis pathway was changed by GhSPL10 overexpression.Moreover,expression of flavonoid biosynthesis related genes and content of flavonoids can both be elevated by either ACC treatment in wild type or in EIN2 overexpression or CTR1 RNAi,and Ag2S2O3 treatment caused the opposite effect to some extent during callus initiation stage in wild type,indicating ethylene was a key factor contributed to overaccumulation of flavonoids in GhSPL10 overexpression lines.Except for ethylene,IAA treatment also promoted relative expression of flavonoid biosynthesis related genes in wild type during callus initiation stage,suggesting auxin might be another factor resulting into the overaccumulation of flavonoids in GhSPL10 overexpression lines. |
PDF Full Text Request