- Integrated mRNA and miRNA transcriptome analysis reveals a regulatory network for tuber expansion in Chinese yam ( Dioscorea opposita. - However, little is known about the regulatory mechanism of tuber expansion. - In order to identify the genes and miRNAs involved in tuber expansion, we examined the mRNAs and small RNAs in Dioscorea opposita (Chinese yam) cv. - Results: A total of 14,238 differentially expressed genes in yam tuber at its expansion stage were identified by using RNA sequencing technology. - Functional analysis revealed the coordination of tuber plant involved in processes of cell events, metabolism, biosynthesis, and signal transduction pathways at transcriptional level, suggesting that these differentially expressed genes are somehow involved in response to tuber expansion, including CDPK, CaM, CDL, SAUR, DELLA, SuSy, and expansin. - transcription factor genes showed differential expression during the expansion stage at transcriptional level. - MADS, bHLH, and GRAS were involved in cell differentiation, division, and expansion, which may relate to tuber expansion. - The integrated analysis of miRNA-mRNA showed that 4 known miRNAs and 11 genes formed 14 miRNA-target mRNA pairs were co-expressed in expansion stage. - miRNA160, miRNA396, miRNA535 and miRNA5021 may be involved in complex network to regulate cell division and differentiation in yam during its expansion stage.. - The expansion stage can be divided into three periods: early expansion stage, middle expansion stage, late expansion stage [2, 3]. - However, fundamental knowledges of endogenous metabolic networks are poor in tuber expansion.. - Although many DNA molecular markers have been used to uncover the genetic diversity and rela- tionship among yam germplasms [16–18], little is known about specific genes involved in tuber morphogenesis.. - PE2.1 and PE53 are the members of pectinesterase (PE) super- family, which may be involved in the regulation of starch and sucrose metabolism and signaling pathways. - Tuber morphogenesis is a complex biological process involving many specific genes and proteins, espe- cially at yam tuber expansion stage. - Former tran- scriptomic study revealed that some putative genes were involved in dioscin biosynthesis [25], along with this chal- cone isomerase (CHS), flavanone 3-hydroxylase (F3H),. - miRNA172 and miR156 were involved in tuberization process, either as a compo- nent or a regulator of long-distance gibberellin signaling pathways [31, 32]. - However many studies have found that miRNAs are involved in tuber and root development, the miRNA- mediated regulatory network during tuber expansion is still unclear.. - rotundata) had been per- formed for sex determination [33], a detailed comparative mRNA and miRNA analysis during yam tuber expansion stage need to be detected. - Among them, 5723 genes were up- regulated, 8515 genes were down-regulated, respectively, and it were differentially expressed in expansion stage (GH16_E), compared to initiation stage (GH16_I) (Add- itional file 2: Table S1).. - and biological regulation (9%) were enriched during expansion stage (Fig. - genes were involved in hemicellulose. - Besides, the results also revealed several sig- nificant expression genes involved in tissue development, root morphogenesis, root system development, and root development (Table 4).. - KEGG is a signal pathway database with vibrant signal pathway map, 20 pathways were identified during yam tuber expansion stage. - Interestingly, KEGG pathway ana- lysis showed that plant hormone signal transduction (ko04075), biosynthesis of amino acids (ko01230) were enriched with DEGs during expansion stage (Fig. - Comprehensive analysis of differentially expressed genes in expansion stage. - Compared with initiation stage, there were a large num- ber of DEGs in tuber expansion stage using NR, GO, and KEGG annotation. - A total of 242 DEGs were identified to be highly similar to many plant hormone signal pathways, including 131 down-regulated and 111 up-regulated DEGs in expansion stage (Additional file 2: Table S1). - Interestingly, most plant hormone-related genes in GA, IAA, and ABA signal pathways were discovered during expansion stage.. - Total_1) were low expression level during expansion stage (Additional file 3: Table S2).. - In contrast, DELLA proteins were highly expressed during the expansion stage. - Meanwhile, pro- tein phosphatase 2C(PP2C) was highly expressed during expansion stage.. - Regulation of genes related to MAPK and calcium signal- ing during the expansion stage were also investigated. - Table 4 Functional classification and pathway assignment of differentially expressed DEG by GO in expansion stage. - It is worth noticing that CBLs were down-regulated during expansion stage (Additional file 3: Table S2).. - A total of 98 transcripts homologous to the genes associ- ated with cell wall and cell cycle were observed as differ- entially regulated during expansion stage (Additional file 2: Table S1), including xyloglucan endotransglucosylase/. - All of the expansin, extension, cell wall synthesis, and CKS genes were down- regulated during expansion stage. - Meanwhile, most of the cell cytoskeleton and XTH were down-regulated during expansion stage in yam (Additional file 3: Table S2).. - The major constituents of starch and sucrose metabolism during expansion stage are sucrose synthase genes (SuSy), sucrose phosphate synthase genes (SPS), starch synthase (SS), and invertase genes (INV) (Additional file 2: Table S1). - Interestingly, dioscorins, the major storage proteins in yam tubers, were significantly up-regulated during the expansion stage (Additional file 3: Table S2).. - These results indicated that many functional genes were involved in expansion stage of yam tuber.. - The most abundant TF gene families with the highest number of expressions during expansion stage were depicted by heat map (Fig. - More- over, these genes were involved in circadian rhythm path- way, starch and sucrose metabolism pathway, and GA pathway by KEGG analysis respectively.. - 4 The numbers of up-regulated and down-regulated transcription factors during expansion stage. - it showed that 40 miRNAs were down-regulated, and 4 miRNAs were up-regulated during expansion stage (Table 5). - Notably, 4 known miRNAs and 11genes formed 14 miRNA-target mRNA pairs with co- expressed expression in expansion stage (Fig. - Table 5 Differentially expressed miRNAs involved in expansion stage. - involved in multiple pathways. - On the other hand, some target genes were revealed to be involved in transport and catabolism processes, such as autophagy-related protein 13b (ATG13, targeted by miRNA160), IST1-like protein (IST1, targeted by miR396a-. - Yam tuber expansion stage is an attractive theoretical model for studying the develop- ment of underground organs because it is the primary storage organ as nutrients reservoir. - Therefore, the under- standing of candidate genes involved in tuber expansion stage is supreme importance. - In this study, 14,238 genes were identified that have shown significant differential ex- pression during expansion stage.. - KEGG enrichment analysis revealed that a large number of genes were involved in pathways related to MAPK signaling pathway, plant hormone signal transduction, starch and su- crose metabolism, and carbon metabolism (Fig. - These genes are involved in cell wall biosynthesis, cell prolifera- tion, cell expansion, nutrient accumulation, primary metab- olism, and hormone signal transduction . - Cellular processes are triggered by specific stimuli and hor- mones that are involved in a series of signaling pathways.. - A potato Ca 2+ -dependent protein kinase, StCDPK1, which was reported to be expressed during tuberization, possibly be involved in potato tuberization through transcriptional ac- tivation of certain genes [40, 43]. - These re- sults demonstrated that calcium signal is regulated during tuber expansion stage. - Besides, MAPK signal pathways are known to play a central role in cell proliferation, differenti- ation and hormones, but it is not known to be involved in tuber or root formation [49]. - In this study, MAPK signal- related genes were up-regulated during expansion stage, and it means that MAPK signal pathways take part in yam tuber expansion. - Cell number and cell size are two key de- terminants of plant and organ size, while involved in cell division, cell expansion and cell cycle [50, 51]. - In this study, XTH, expansin, extension, CKS, ftsHs, CDC5, CDC, CDKs, and CDKIs involved in cell division, cell expansion and cell cycles were differentially regulated during expansion stage.. - These differentially regulated genes involved in cell wall and cell cycle metabolism were identified in Arabidopsis, Rehmannia glutinosa, and radish . - In this study, 12 MADS, 52 bHLH, and 17 GRAS were found to be expressed differently during expansion stage at transcript level, which plays key regulatory functions in cell differ- entiation, division and expansion (Fig. - All data suggest that these transcription factors may be potentially involved in the expansion stage.. - A set of transcriptome evidence suggests that hormone sig- nal pathway-related genes involved in regulating of storage organ formation have been identified in sweet potato, potato, radish, lotus, and carrot, while may be potentially involved in cell division, differentiation, and expansion . - In radish, most Aux/IAA, ARFs, and SAUR genes were abundantly expressed in the root cortex splitting and expansion stage, implying that these transcripts may be involved in cambium cell expansion [35]. - Among auxin signal-related genes, Aux/IAA, ARFs were highly expressed in expansion stage in this study, and auxin has been identified to regulate cell division and expansion by altering genes expression [1, 38], suggesting that Aux/IAA, ARFs may offer excellent candi- dates during expansion stage.. - Interestingly, in this study, DELLA genes were up- regulated, while GIDI, like GID2, were down-regulated during expansion stage (Additional file 3: Table S2).. - Overall, these results in- dicate that hormone signaling-related genes have a com- plex regulatory network involved in tuber expansion.. - The SuSy gene was down-regulated during expansion stage. - The results were similar to the taproot expansion stage in radish [35], implying that they may play a signifi- cant role in tuber expansion stage of yam. - Therefore, these starch and sucrose metabol- ism genes are necessary for tuber expansion.. - miRNAs regulation by targeting the potential genes miRNA mediated gene regulation has been extensively studied in root and tuber development through transcrip- tional and post-transcriptional levels, which provides a bet- ter understanding of molecular regulatory network during tuber expansion stage. - In this study, most miRNAs were down-regulated during expansion stage (Table 5). - However, miR160 was down-regulated during yam tuber expansion stage (Table 5). - In this study, some miRNA-mRNA pairs were observed during expansion stage (Fig. - 6), miR160 was involved in root cell division and differentiation by regulat- ing auxin response factors (ARFs) affecting root develop- ment in Arabidopsis thaliana [30]. - miRNA160 was down- regulated during expansion stage, auxin response factor ARF18 and ARF17 were up-regulated during expansion stage (Fig. - Previous studies have shown that the miRNA160-targeted ARF has been identified to be involved in cell expansion and cell differentiation in radish root and potato tuber [28].. - miRNA160, miRNA396, and miR535 may be involved in complex a network of regulating cell division and differenti- ation during expansion stage.. - miRNA and target genes have been extensively studied in tuber development [21, 28], which significantly advanced our understanding of molecular regulatory networks underlying during yam tuber expansion. - In this study, based on our RNA-Seq and small RNA analysis, previous transcriptomics analysis and results of other tuber crops, a putative model of regulatory network associated with yam tuber expansion was proposed (Fig. - Some specific genes pro- mote cell differentiation division and expansion at tuber expansion. - XTH, expansin, extension, CKS, ftsHs, CDC5, CDC, CDKs, and CDKIs involved in cell division, cell expansion and cell cycles were differentially regulated during expan- sion stage. - A finding similar to these differences in cell wall and cell cycle regulatory genes are involved in cell wall metabolism in Arabidopsis, Rehmannia glutinosa, radish . - Most Aux/IAA, ARFs and SAUR genes were enriched and expressed in the root cortex during splitting and expand- ing stage in radish, suggesting that these transcripts may be involved in cell expansion in the cambium [35]. - In this study, some miRNA and target genes were observed during yam tuber expansion stage, bHLH (target by miR5021), SBP (target by miR535a), and ARFs (tar- geted by miRNA160 and miR396), were identified to be involved in cell expansion and cell differentiation in radish root and potato tuber [28, 35]. - Taken together, the result suggested that these DEGs may play a vital role in the regulatory network of tuber expansion.. - Fig.8 A proposed model of genetic and molecular interactions in the regulatory network during tuber expansion in yam. - Arrows represents genes were involved in signal transduction pathways or carbohydrate metabolism, red circle represents target genes of miRNA. - A total of 14,238 genes expressed differentially during yam tuber expansion stage were identified first time using RNA-Seq technology. - These results reveal the co- ordination of tuber involved in the processes of cellular events, metabolism, biosynthesis, and signal transduction pathways at transcriptional levels. - It is worth noticing that the integrated analysis of miRNA-mRNA was iden- tified to be differentially expressed in tuber expansion.. - After stringent quality filering, a mixed sample of initi- ation and expansion stage library and six RNA-Seq li- braries clean reads were used to map the two reference genome sequences (D. - Differentially expressed genes (DEGs) in expansion stage compared with initiation stage.. - The differentially expressed genes involved in signal transduction pathways in expansion stage (FPKM>. - The differentially expressed transcription factors involved in expansion stage.. - Identification of miRNAs target genes in expansion stage.. - Genome-wide transcriptome analysis reveals small RNA profiles involved in early stages of stolon-to-tuber transitions in potato under photoperiodic conditions. - Transcriptome analysis reveals novel genes potentially involved in photoperiodic tuberization in potato. - Comparative transcriptome analysis identifies putative genes involved in dioscin biosynthesis in Dioscorea zingiberensis
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