- Global identification of long non-coding RNAs involved in the induction of spinach flowering. - Results: In the present study, we identified a total of 1141 lncRNAs, of which 111 were differentially expressed between vegetative and reproductive stages. - The GO and KEGG analyses carried out on the cis target gene of lncRNAs showed that the lncRNAs play an important role in the regulation of flowering spinach. - Conclusions: This study is the first report on identifying bolting and flowering-related lncRNAs based on transcriptome sequencing in spinach, which provides a useful resource for future functional genomics studies, genes expression researches, evaluating genes regulatory networks and molecular breeding programs in the regulation of the genetic mechanisms related to bolting in spinach.. - Spinach (Spinacia oleracea L.) from the Amaranthaceae family is a beneficial annual vegetable species that is widely cultivated in different parts of the world and used in the human diet. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article Ghorbani et al. - Indeed, lncRNAs can promote gene expression by competing with miRNAs for specific binding sites in the non-coding regions of mRNAs and preventing the transcriptional repression caused by miRNAs [22, 23].. - According to the expression profile, 186 known lncRNAs, and 89 novel lncRNAs were found associated with pistil development in Prunus mume, which could provide new indications to elucidate how lncRNAs and their targets play role in pistil differentiation and flower development [36], highlighting the potential contribu- tions of lncRNA in the flowering process. - According to the results of previous research, the vegetative characteristics of 44 spinach accessions [46], two accessions Viroflay and Kashan were placed in the group of late and early flowering spinach, respect- ively. - Using a rigorous filtering pipeline to remove transcripts with any known protein domains docu- mented in the Pfam database, housekeeping RNAs such as tRNAs, rRNAs, snRNAs, and snoRNAs in the Rfam database, and encoding any conserved protein, 186 tran- scripts were detected and filtered out from the further analysis. - In total, 1141 transcripts were identified as lncRNAs in the spinach transcriptome. - Ghorbani et al. - The comparison result between spinach lncRNAs and lncRNAs available in the above-mentioned databases in- dicated that more spinach lncRNAs appear to be exten- sively conserved. - Among unique DE- lncRNAs, 8 and 67 genes were up-regulated in the re- productive stage of Kashan and Viroflay. - By comparison of log fold change (logFC) of DE-mRNAs and DE- lncRNAs in both accessions, we found that lncRNAs were expressed at different levels in the Kashan and Vir- oflay (Fig. - Since lncRNAs located upstream and downstream of protein-coding genes may be involved in regulatory activ- ities (cis and trans roles), we searched protein-coding genes from 100 kb upstream and downstream of potential lncRNAs to explore their function in the cis role. - According to the details of the GO analysis, of the 2715 target genes located upstream of lncRNAs and 1280 were successfully annotated with GO as- signments in the three main categories, including bio- logical process (BP), molecular function (MF), and cellular component (CC), respectively. - 0009790), post-embryonic development (GO and pollination (GO which are well known to be involved in the transition from the vegetative stage to the flowering stage. - Assessment of conservation and expression of identified lncRNAs in the spinach flower tissues. - The expression profile of these lncRNAs across all the flower-related samples along with the young leaf-related ones was depicted in the heatmap plot (Fig. - The majority of 64 DE-lncRNAs displayed over- expression in the “FM” stage which among them vMSTRG.8252.1, vMSTRG4066.1, and vMSTRG4448.1 indicated the highest level of expression.. - The significant down expression of this lncRNA was observed in the late stages (“ODVO” and “OM”) of flower development. - Another flower-related lncRNA, vMSTRG.15493.1, appeared with a positive regulatory role in the development of spinach flower because of its significant overexpression in each of “FM”, “FO”, and. - Additionally, its significant overexpression in the latest stage (“OM”) in comparison with “FO” and “ODVO” was observed.. - According to the results, in order, 7 and 8 upstream and downstream DEGs were identified as potential targets of 10 and 11 DE-lncRNAs in Kashan respectively. - 5 The heatmap of DE-lncRNAs identified in flower tissues. - Regarding the annotation of trans target genes located in downstream of DE-lncRNAs in Viroflay, Serotonin N-acetyltransferase (SNAT2:. - In the present study, 66 DE-lncRNAs were identified as eTMs of 65 miRNAs (Table S2). - Through KEGG functional enrichment analysis, we found only genes in the turquoise and blue modules were significantly enriched in 14 and 11 pathways (Fig. - flowering and inflorescence development, we found sev- eral carbohydrate-related pathways, including “starch and sucrose metabolism” and “galactose metabolism” in the turquoise module and “fructose and mannose me- tabolism” in the blue module. - rhythm” in its correspondent module, suggesting that they are engaged in the flowering pathway. - Table.2 The number of genes per module and the percentage of DE-lncRNAs in each module. - Module name Number of mRNAs Number of DE-lncRNAs % of DE-lncRNAs. - Thus, most of these genes should be up-regulated in the transition from the vegetative stage to the flowering stage. - To further understand each co-expressed module’s particularity concerning their expression patterns in the different datasets, we plotted eigengene expression values of the genes belonging to each module along with each sample. - According to these results, we observed that in all detected modules except the blue module and some replicates in the brown (KV_2 and KV_3), the gene expression levels were higher in the reproductive stage. - of both accessions than in the vegetative stage (Fig. - 9B, C) genes displayed a similar trend with more induction in the reproductive. - 9D) appeared to be modulated in the vegetative stage of both accessions through down-regulation, whereas in the black module (Fig. - 9G) genes were positively regulated in the reproductive stage of Viroflay, suggesting genes in this module play important central and key roles in regulating the flowering time of Viroflay as a late-bolting accession.. - To identify the central and highly connected genes in the modules, we selected 50 top genes in the four main modules and visualized these genes by Cytoscape (Fig. - In the turquoise module, we identified 47 protein-coding genes and 3 lncRNAs. - In this module, several well-known TFs, including MADS-box, GATA, and BZIP, were found in the list of the protein-coding genes. - expression, was found in the turquoise module with a higher expression in the vegetative stage of accession Viroflay. - Strikingly, we found lncRNAs in the turquoise module closely linked to UGT1 and TFs, indicating that these lncRNAs may function as regulators of these genes in spinach.. - In the green module, we identified 36 protein-coding genes and 14 lncRNAs. - UGT and Early light-induced protein were identified as putative genes related to the flowering in the blue. - Spo20470, ERF5:Spo25961, ERF510:Spo06220), Zinc- finger, GRAS family, and Heat shock in the yellow mod- ule that could be important regulators of bolting in spinach.. - To validate the expression of DE-lncRNAs obtained through RNA-Seq, we selected six hub DE-lncRNAs identified in the four main modules. - The qPCR results showed that the expression of the lncRNAs, including vMSTRG.13194.1, vMSTRG.2206.1, and vMSTRG.3613.1 were significantly up-regulated only in the vegetative stage of accession Viroflay compared to the reproductive stage (Fig. - 11), whereas kvMSTRG.6436.1 was significantly up-regulated in the vegetative stage of both accessions. - On the con- trary, the expression of the lncRNA kvMSTRG.1075.1 was significantly down-regulated in the vegetative stage of both accessions compared to the reproductive stage.. - vMSTRG.11356.1 was found to be up-regulated only in the reproductive stage of accession Viroflay. - One way to extract this information could be facilitated through sub-categorizing of protein-coding genes and lncRNAs in gene co-expression networks because it has been demonstrated that genes in the same module are. - Hence, lncRNAs with different expressions in the accession with different bolting times may be in- volved in the flowering time control. - Notably, no homologous lncRNA was iden- tified in spinach compared with Arabidopsis in the NONCODE database [62]. - Among the main regulatory pathways for flowering control, including the photo- period, vernalization, autonomous, age, and gibberellin pathways, vernalization is well-known to involve lncRNAs in the regulation of FLC expression. - Recent studies have demonstrated that at least two types of lncRNAs, including COOLAIR (located at the 3’ end of the FLC locus) and COLDAIR, are involved in the modulation of FLC expression . - The GO term classified in the biological process category was mainly derived from target genes located upstream of all lncRNAs and mostly associated with the accession. - Viroflay for DE-lncRNAs. - In the present study, the upregulation of PIF4 was observed in vegetative stage of Kashan compared to the reproductive stage. - The key role of this gene has been detected in the GA-related flowering pathway and sup- pression of FT gene expression as well [71]. - Lee et al.. - They attributed delay of flowering time to decrement in the expression levels of gibberellin biosynthetic genes like ent-kaurene synthase (KS) and reduction in FT ex- pression levels. - According to this study, the upregulation of SNAT2 in the Kashan vegetative stage presumably re- vealed its positive regulatory role on early floral transi- tion of this accession in comparison with the Viroflay, which exhibited the downregulation of SNAT2. - Additionally, ELF6 was predicted as another downstream-trans target of DE-lncRNAs in Viroflay. - This gene is identified as a leading gene negatively involved in the regulation of flowering time through repressing the photoperiodic floral regulatory pathway [74]. - Jeong et al. - PKL is well-known as a CHD3 chromatin- remodeling factor that promotes flowering initiation (Henderson et al., 2004). - Fu et al. - Kraft et al.. - Since this ob- served expression pattern in the vegetative stage might prolong flowering initiation, we can conclude the prob- able pivotal role of this gene in induction of late bolting phenotype of Viroflay. - Similar to PHL, GASA5 emerged as the overexpressed gene in the vegetative stage. - These results in- dicate that the lncRNA-miRNA (MSTRG.16566.1- mir172) pairs might be important novel regulatory com- ponents in the flowering/bolting of spinach.. - In the turquoise model, TFs such as ERF, BHLH, AP2/B3-like, BZIP, MYB, WRKY, GATA, NAC domain, Zinc finger, MADS-box, and CCAAT-binding factor were also iden- tified as the paramount regulators of flowering . - TFs in the blue module included GRAS, GATA zinc finger, MYB, ERF, and NAC domain. - In the black module, five DE-lncRNAs were recognized as the flowering- associated lncRNA, together with four well-known TFs including zinc finger, F-box, ERF, WRKYand other neighboring co-expressed mRNAs such as sugar transporter and UDP-glycosyltransferase. - importantly ELF3 and COL1, were also identified in the other modules.. - Furthermore, the KEGG analysis of genes from two turquoise and blue modules supports that the candidate target genes of lncRNAs existing in these modules may be involved in the regulation of circadian rhythm.. - Altogether, we found that novel identified lncRNAs in this study may be involved in the regulation of key flowering-related genes and related pathways like circa- dian rhythm.. - In the current study, one dataset included six samples of accession Kashan repre- senting three biological replicates at each developmental stage. - babraham.ac.uk/projects/fastqc/) and Trimmomatic v0.30 program [107](Bolger et al., 2014), respectively.. - Tracking of identified lncRNAs in the spinach flower tissues. - To investigate whether lncRNAs identified in the leaf samples are also conserved and expressed in flower tis- sues, we employed the transcriptome dataset of young leaf (YL) and female flower samples of s. - oleracea in five different developmental stages including, the formation of ovary (FO), sepal primordia and carpel primordium (SPCP), floral meristem (FM), ovule differentiates within the ovary (ODVO), and ovule matures (OM), available in the European Nucleotide Archive (ENA, https://www.. - Identification of DE-lncRNAs that act as eTMs. - Finally, to determine the genes that are highly connected in the modules, we selected the 50 top hub genes through the Cyto-Hubba plugin [127] and vi- sualized these genes by Cytoscape [128] as an unsigned network.. - The formal identification of the plant material was undertaken by the herbarium of Agricultural and Natural Resources College, University of Tehran, and no voucher specimens were collected and deposited in the collection (it is not necessary as we don ’ t describe a novel species). - We also stated that the field studies were in compliance with local legislation of Iran in the experimental greenhouse and growth chamber of Isfahan University of Technology, Isfahan, and no specific licenses were required.. - Use of spinach powder as functional ingredient in the manufacture of UF-Soft cheese. - Homeologous regulation of Frigida-like genes provides insights on reproductive development and somatic embryogenesis in the allotetraploid Coffea arabica. - Genome-wide screening and functional analysis identify a large number of long noncoding RNAs involved in the sexual reproduction of rice. - Yang Z, Yang Z, Yang C, Wang Z, Chen D, Xie Y, et al. - Zhao X, Gan L, Yan C, Li C, Sun Q, Wang J, et al. - Identification and characterization of regulatory pathways involved in early flowering in the new leaves of alfalfa (Medicago sativa L.) by transcriptome analysis. - Li N, Meng Z, Tao M, Wang Y, Zhang Y, Li S, et al. - Divergent roles of a pair of homologous jumonji/zinc-finger-class transcription factor proteins in the regulation of Arabidopsis flowering time. - Cross-Repressive Interactions between SOC1 and the GATAs GNC and GNL/CGA1 in the Control of Greening, Cold Tolerance, and Flowering Time in Arabidopsis
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