A New Insight into Flowering Regulation: Molecular Basis of Flowering Initiation in Magnolia × soulangeana 'Changchun'.
ABSTRACT: Magnolia × soulangeana 'Changchun' are trees that bloom in spring and summer respectively after flower bud differentiation. Here, we use phenological and morphological observation and RNA-seq technology to study the molecular basis of flowering initiation in 'Changchun'. During the process of flowering initiation in spring and summer, the growth of expanded flower buds increased significantly, and their shape was obviously enlarged, which indicated that flowering was initiated. A total of 168,120 expressed genes were identified in spring and summer dormant and expanded flower buds, of which 11,687 genes showed significantly differential expression between spring and summer dormant and expanded flower buds. These differentially expressed genes (DEGs) were mainly involved in plant hormone signal transduction, metabolic processes, cellular components, binding, and catalytic activity. Analysis of differential gene expression patterns revealed that gibberellin signaling, and some transcription factors were closely involved in the regulation of spring and summer flowering initiation in 'Changchun'. A qRT-PCR (quantitative Real Time Polymerase Chain Reaction) analysis showed that BGISEQ-500 sequencing platform could truly reflect gene expression patterns. It also verified that GID1B (GIBBERELLIN INSENSITIVE DWARF1 B), GID1C, SPL8 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8), and GASA (GIBBERELLIC ACID-STIMULATED ARABIDOPSIS) family genes were expressed at high levels, while the expression of SPY (SPINDLY) was low during spring and summer flowering initiation. Meanwhile, the up- and down-regulated expression of, respectively, AGL6 (AGAMOUS-LIKE 6) and DREB3 (DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 3), AG15, and CDF1 (CYCLIC DOF FACTOR 1) might also be involved in the specific regulation of spring and summer flowering initiation. Obviously, flowering initiation is an important stage of the flowering process in woody plants, involving the specific regulation of relevant genes and transcription factors. This study provides a new perspective for the regulation of the flowering process in perennial woody plants.
Project description:Flowering is an integral part of the life cycle of flowering plants, which is essential for plant survival and crop production. Most woody fruit trees such as apples and pears bloom in spring, but loquat blooms in autumn and winter. Gibberellin (GA) plays a key role in the regulation of plant flower formation. In this study, we sprayed loquat plants with exogenous GA<sub>3</sub>, which resulted in vigorous vegetative growth rather than floral bud formation. We then performed a comprehensive RNA-seq analysis on GA<sub>3</sub>-treated and control-treated leaves and buds over three time periods to observe the effects of exogenous GA<sub>3</sub> application on floral initiation and development. The results showed that 111 differentially expressed genes (DEGs) and 563 DEGs were down-regulated, and 151 DEGs and 506 DEGs were up-regulated in buds and leaves, respectively, upon treatment with GA<sub>3</sub>. Among those that are homologs of the DELLA-mediated GA signal pathway genes, some may be involved in the positive regulation of flower development, including <i>EjWRKY75, EjFT, EjSOC1, EjAGL24, EjSPL, EjLFY, EjFUL</i>, and <i>EjAP1</i>; while some may be involved in the negative regulation of flower development, including <i>EjDELLA, EjMYC3, EjWRKY12</i>, and <i>EjWRKY13</i>. Finally, by analyzing the co-expression of DEGs and key floral genes <i>EjSOC1s, EjLFYs, EjFULs, EjAP1s</i>, 330 candidate genes that may be involved in the regulation of loquat flowering were screened. These genes belong to 74 gene families, including Cyclin_C, Histone, Kinesin, Lipase_GDSL, MYB, P450, Pkinase, Tubulin, and ZF-HD_dimer gene families. These findings provide new insights into the regulation mechanism of loquat flowering.
Project description:The molecular mechanism underlying dormancy release and the induction of flowering remains poorly understood in woody plants. Mu-legacy is a valuable blueberry mutant, in which a transgene insertion caused increased expression of a RESPONSE REGULATOR 2-like gene (VcRR2). Mu-legacy plants, compared with nontransgenic 'Legacy' plants, show dwarfing, promotion of flower bud formation, and can flower under nonchilling conditions. We conducted transcriptomic comparisons in leaves, chilled and nonchilled flowering buds, and late-pink buds, and analyzed a total of 41 metabolites of six groups of hormones in leaf tissues of both Mu-legacy and 'Legacy' plants. These analyses uncovered that increased VcRR2 expression promotes the expression of a homolog of Arabidopsis thaliana ENT-COPALYL DIPHOSPHATE SYNTHETASE 1 (VcGA1), which induces new homeostasis of hormones, including increased gibberellin 4 (GA4) levels in Mu-legacy leaves. Consequently, increased expression of VcRR2 and VcGA1, which function in cytokinin responses and gibberellin synthesis, respectively, initiated the reduction in plant height and the enhancement of flower bud formation of the Mu-legacy plants through interactions of multiple approaches. In nonchilled flower buds, 29 differentially expressed transcripts of 17 genes of five groups of hormones were identified in transcriptome comparisons between Mu-legacy and 'Legacy' plants, of which 22 were chilling responsive. Thus, these analyses suggest that increased expression of VcRR2 was collectively responsible for promoting flower bud formation in highbush blueberry under nonchilling conditions. We report here for the first time the importance of VcRR2 to induce a suite of downstream hormones that promote flowering in woody plants.
Project description:The role of gibberellins (GAs) during floral induction has been widely studied in the annual plant Arabidopsis thaliana. Less is known about this control in perennials. It is thought that GA is a major regulator of flowering in rose. In spring, low GA content may be necessary for floral initiation. GA inhibited flowering in once-flowering roses, whereas GA did not block blooming in continuous-flowering roses. Recently, RoKSN, a homologue of TFL1, was shown to control continuous flowering. The loss of RoKSN function led to continuous flowering behaviour. The objective of this study was to understand the molecular control of flowering by GA and the involvement of RoKSN in this inhibition. In once-flowering rose, the exogenous application of GA(3) in spring inhibited floral initiation. Application of GA(3) during a short period of 1 month, corresponding to the floral transition, was sufficient to inhibit flowering. At the molecular level, RoKSN transcripts were accumulated after GA(3) treatment. In spring, this accumulation is correlated with floral inhibition. Other floral genes such as RoFT, RoSOC1, and RoAP1 were repressed in a RoKSN-dependent pathway, whereas RoLFY and RoFD repression was RoKSN independent. The RoKSN promoter contained GA-responsive cis-elements, whose deletion suppressed the response to GA in a heterologous system. In summer, once-flowering roses did not flower even after exogenous application of a GA synthesis inhibitor that failed to repress RoKSN. A model is presented for the GA inhibition of flowering in spring mediated by the induction of RoKSN. In summer, factors other than GA may control RoKSN.
Project description:Flower bud formation and flowering in chrysanthemums occur under short day conditions (SD), but the molecular basis for the switch to reproductive growth is less well understood than in model plants. Here, a spontaneous mutant able to flower under long days is described. In an attempt to reveal the pathway(s) involved in the formation of flower buds under contrasting daylengths, transcriptome sequencing was carried out in plants grown both under SD and long day conditions (LD). A number of differentially transcribed genes involved in the various known flowering pathways were identified. Both circadian clock genes and Chrysanthemum FLOWERING LOCUS T Like3 (CmFTL3) were up-regulated under SD, thereby inducing floral bud formation and flowering. The gibberellin (GA) signaling pathway-related genes Gibberellin 20-oxidase (GA20ox) and Gibberellin receptor (GID1) were up-regulated in the mutant under LD, while the catabolic genes Gibberellin 2-oxidase (GA2ox) and GA-INSENSITIVE (GAI) were down-regulated, thereby inducing the transcription of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and LEAFY (LFY). The GA content of the leaf was higher in the mutant than in the wild type (WT) under LD and SD, and the mutant has more branching than WT plants under LD or SD. When treated with GA, the mutant flowered earlier under both SD and LD relative to WT, but there was no detectable phenotype difference between the two lines. The indication was that the photoperiod pathway majorly regulates flower bud formation and flowering time in chrysanthemums under SD. The GA signaling pathway only plays a subsidiary role for flowering. However, the GA signaling pathway predominated for flowering under LD.
Project description:BACKGROUND:Blueberry cultivars require a fixed quantity of chilling hours during winter endo-dormancy for vernalization. In this study, transcriptome analysis using RNA sequencing data from nonchilled, chilled, and late pink buds of southern highbush blueberry 'Legacy' was performed to reveal genes associated with chilling accumulation and bud break. RESULTS:Fully chilled 'Legacy' plants flowered normally whereas nonchilled plants could not flower. Compared to nonchilled flower buds, chilled flower buds showed differential expression of 89% of flowering pathway genes, 86% of MADS-box genes, and 84% of cold-regulated genes. Blueberry orthologues of FLOWERING LOCUS T (FT) did not show a differential expression in chilled flower buds (compared to nonchilled flower bud) but were up-regulated in late-pink buds (compared to chilled flower bud). Orthologoues of major MADS-box genes were significantly up-regulated in chilled flower buds and down-regulated in late-pink buds. Functional orthologues of FLOWERING LOCUS C (FLC) were not found in blueberry. Orthologues of Protein FD (FD), TERMINAL FLOWER 1 (TFL1), and LEAFY (LFY) were down-regulated in chilled flower buds and in late-pink buds compared to nonchilled flower bud. CONCLUSIONS:The changes from nonchilled to chilled and chilled to late-pink buds are associated with transcriptional changes in a large number of differentially expressed (DE) phytohormone-related genes and DE flowering pathway genes. The profile of DE genes suggests that orthologues of FT, FD, TFL1, LFY, and MADS-box genes are the major genes involved in chilling-mediated blueberry bud-break. The results contribute to the comprehensive investigation of the vernalization-mediated flowering mechanism in woody plants.
Project description:BACKGROUND:Floral induction is an important stage in the apple tree life cycle. In 'Nagafu No. 2', which was derived from a 'Fuji' bud sport, flower bud formation is associated with serious problems, such as fewer and inferior flower buds, a long juvenile phase, and an alternate bearing phenotype. Moreover, the molecular regulatory mechanisms underlying apple floral induction remain unknown. To characterize these mechanisms, we compared the RNA-sequencing-based transcriptome profiles of buds during floral induction in profusely flowering 'Qinguan' and weakly flowering 'Nagafu No. 2' apple varieties. RESULTS:Genes differentially expressed between the buds of the two varieties were mainly related to carbohydrate, fatty acid, and lipid pathways. Additionally, the steady up-regulated expression of genes related to the fatty acid and lipid pathways and the down-regulated expression of starch synthesis-related genes in the carbon metabolic pathway of 'Qinguan' relative to 'Nagafu No. 2' were observed to contribute to the higher flowering rate of 'Qinguan'. Additionally, global gene expression profiling revealed that genes related to cytokinin, indole-3-acetic acid, and gibberellin synthesis, signalling, and responses (i.e., factors contributing to cell division and differentiation and bud growth) were significantly differentially expressed between the two varieties. The up-regulated expression of genes involved in abscisic acid and salicylic acid biosynthesis via shikimate pathways as well as jasmonic acid production through fatty acid pathways in 'Qinguan' buds were also revealed to contribute to the floral induction and relatively high flowering rate of this variety. The differential expression of transcription factor genes (i.e., SPL, bZIP, IDD, and MYB genes) involved in multiple biological processes was also observed to play key roles in floral induction. Finally, important flowering genes (i.e., FT, FD, and AFL) were significantly more highly expressed in 'Qinguan' buds than in 'Nagafu No. 2' buds during floral induction. CONCLUSIONS:A complex genetic network of regulatory mechanisms involving carbohydrate, fatty acid, lipid, and hormone pathways may mediate the induction of apple tree flowering.
Project description:Chrysanthemum is a leading cut flower species. Most conventional cultivars flower during the fall, but the Chrysanthemum morifolium 'Yuuka' flowers during the summer, thereby filling a gap in the market. To date, investigations of flowering time determination have largely focused on fall-flowering types. Little is known about molecular basis of flowering time in the summer-flowering chrysanthemum. Here, the genome-wide transcriptome of 'Yuuka' was acquired using RNA-Seq technology, with a view to shedding light on the molecular basis of the shift to reproductive growth as induced by variation in the photoperiod.Two sequencing libraries were prepared from the apical meristem and leaves of plants exposed to short days, three from plants exposed to long days and one from plants sampled before any photoperiod treatment was imposed. From the ~316 million clean reads obtained, 115,300 Unigenes were assembled. In total 70,860 annotated sequences were identified by reference to various databases. A number of transcription factors and genes involved in flowering pathways were found to be differentially transcribed. Under short days, genes acting in the photoperiod and gibberellin pathways might accelerate flowering, while under long days, the trehalose-6-phosphate and sugar signaling pathways might be promoted, while the phytochrome B pathway might block flowering. The differential transcription of eight of the differentially transcribed genes was successfully validated using quantitative real time PCR.A transcriptome analysis of the summer-flowering cultivar 'Yuuka' has been described, along with a global analysis of floral transition under various daylengths. The large number of differentially transcribed genes identified confirmed the complexity of the regulatory machinery underlying floral transition.
Project description:BACKGROUND:Tree peony (Paeonia suffruticosa Andrews) is a globally famous ornamental flower, with large and colorful flowers and abundant flower types. However, a relatively short and uniform flowering period hinders the applications and production of ornamental tree peony. Unfortunately, the molecular mechanism of regulating flowering time and floral organ development in tree peony has yet to be elucidated. Because of the absence of genomic information, 454-based transcriptome sequence technology for de novo transcriptomics was used to identify the critical flowering genes using re-blooming, non-re-blooming, and wild species of tree peonies. RESULTS:A total of 29,275 unigenes were obtained from the bud transcriptome, with an N50 of 776?bp. The average length of unigenes was 677.18?bp, and the longest sequence was 5815?bp. Functional annotation showed that 22,823, 17,321, 13,312, 20,041, and 9940 unigenes were annotated by NCBI-NR, Swiss-Prot, COG, GO, and KEGG, respectively. Within the differentially expressed genes (DEGs) 64 flowering-related genes were identified and some important flowering genes were also characterized by bioinformatics methods, reverse transcript polymerase chain reaction (RT-PCR), and rapid-amplification of cDNA ends (RACE). Then, the putative genetic network of flowering induction pathways and a floral organ development model were put forward, according to the comparisons of DEGs in any two samples and expression levels of the important flowering genes in differentiated buds, buds from different developmental stages, and with GA or vernalization treated. In tree peony, five pathways (long day, vernalization, autonomous, age, and gibberellin) regulated flowering, and the floral organ development followed an ABCE model. Moreover, it was also found that the genes PsAP1, PsCOL1, PsCRY1, PsCRY2, PsFT, PsLFY, PsLHY, PsGI, PsSOC1, and PsVIN3 probably regulated re-blooming of tree peony. CONCLUSION:This study provides a comprehensive report on the flowering-related genes in tree peony for the first time and investigated the expression levels of the critical flowering related genes in buds of different cultivars, developmental stages, differentiated primordium, and flower parts. These results could provide valuable insights into the molecular mechanisms of flowering time regulation and floral organ development.
Project description:Members of the genus <i>Paphiopedilum</i> are world-famous for their large, colourful flowers, unique floral morphology and long floral lifespan. Most <i>Paphiopedilum</i> species bloom in spring or autumn. The control of flowering time is of great significance to the commercial production of floral crops, because it affects the sales and prices of flowers. However, the mechanism that regulates when <i>Paphiopedilum</i> species bloom is unclear. In the present study, floral bud initiation and development of <i>P. micranthum</i> (spring-flowering species with one flower per stalk), <i>P. dianthum</i> (autumn-flowering species with multiple flowers per stalk) and <i>P. henryanum</i> (autumn-flowering species with one flower per stalk) were investigated by morphological and anatomical methods. We divided <i>Paphiopedilum</i> floral bud differentiation into six phases: the initiation of differentiation, inflorescence primordium differentiation, flower primordium differentiation, sepal primordium differentiation, petal primordium differentiation and column primordium differentiation. We found that the timing of floral bud differentiation for the three species was synchronized when experiencing the same environment, while the period from initiation to flowering largely differed. In addition, initiation of floral bud differentiation in <i>P. dianthum</i> was earlier at a warmer environment. The difference in flowering time of three species was mainly caused by the duration of floral bud development, rather than the initiation time. The findings were of great significance for the cultivation and flowering regulation of <i>Paphiopedilum</i> species.
Project description:To investigate the role of lncRNAs in citrus flowering, paired-end strand-specific RNA sequencing was performed between precocious trifoliate orange and its wild-type. Total 6,584 potential lncRNAs were identified, 51.6% of which came from intergenic regions. It was also observed that 555 lncRNAs were significantly up-regulated, and 276 lncRNAs were down-regulated in precocious trifoliate orange, indicating that lncRNAs could be involved in the regulation of citrus flowering. Overall design: Two samples, the terminal bud and the five following buds (the major node position for flower formation) from spring flushes of the MT and the WT 2-year-old trees were collected at flower initiation (after self-pruning). And then subjected to RNA-seq.