Transcriptome Profiling and Gene Network Analysis Revealed Regulatory Mechanisms of Bract Development in Bougainvillea glabra

Transcriptome Profiling and Gene Network Analysis Revealed Regulatory Mechanisms of Bract Development in Bougainvillea glabra | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptome Profiling and Gene Network Analysis Revealed Regulatory Mechanisms of Bract Development in Bougainvillea glabra xiangdong liu, Yaonan Peng, Qinghui Zeng, Yuwan Ma, Jin Liu, Yaqi Huang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4275941/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: Bracts are important in ornamental plants and their developmental regulation is complex, but relatively little research has been done on them. In this study, physiological, biochemical and morphological changes in Bougainvillea glabra leaves, leaf buds and bracts during seven developmental periods were systematically investigated in B. glabra bracts. Meanwhile, transcriptome data of B. glabra bracts were obtained using PacBio and Illumina sequencing technologies, and key genes regulating their development were screened. Results: Scanning electron microscopy revealed that the bracts develop with a process of regression of hairs, changing colour from green to white; Transcriptome sequencing yielded 79,130,973 bp of transcript sequences, totalling 45,788 transcripts; Differential gene analysis revealed 50 expression patterns across seven developmental periods, with significant variability in transcription factors such as BgAP1 , BgFULL , BgCMB1, BgSPL16 , BgEIL1, and BgBH305 KEGG and GO analyses of growth and development concerning chlorophyll metabolism and hormone-conducting metabolic pathways; Key genes for chlorophyll metabolism include PORA , SGR , PPH , PAO and RCCR ; The growth hormone and abscisic acid signalling pathways involve 44 and 23 homologous genes, and co-expression network analyses revealed that the screened genes BgAPRR5 and BgEXLA1 are involved in the regulation of bract development. Conclusions: These findings promote the understanding of the molecular mechanism of plant bract development, as well as provide important guidance for the molecular regulation and genetic improvement of the growth and development of ornamental plants, mainly ornamental bracts. Bract development Bougainvillea glabra MADS-box Squamosa promoter binding protein (SBP) Plant hormone signal transduction Chlorophyll Metabolic Pathways Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Bract is the metamorphosed leaf that appears accompanying inflorescence. In general, any leaf associated with inflorescence can be defined as a bract [1,2] . Strictly speaking, bract does not belong to the inflorescence structure, while it can be considered as an extension of floral organs. The bract primordia originated from the stem meristem usually generated at the very early stage of reproductive development [3] . However, a “bract suppression system” has been observed in many, but not all, angiosperm species. This system will cease the development of the bract and eventually subsume the bract primordia into the floral meristem. Therefore, bract is absent in many model plants, such as Arabidopsis , maize ( Zea mays ) and rice ( Oryza sativa ). The bract development system has been suppressed rather than removed in these higher plants. Moreover, a study in rice indicated that bract suppression is not only necessary for floral development but also critical for the transition from vegetative to reproductive branching [4,5] . Although bract suppression is a conserved mechanism in most angiosperm lineages, some plants are retaining the trait of natural bract development, such as species belonging to the Cornaceae , Nyssaceae, Nyctaginaceae and Araceae families . In many ornamental plants, the bract is an important organ undertaking reproductive events, as well as an ornamental character [6] . The bract with ornamental value is called a petaloid bract, which results from the ectopic expression of genes determining the petal identity in the leaf. This process is supposed to involve the developmental signals transduction and the activation or suppression of related genes. Ectopic petalization (petal-like characteristics in non-petal organs) contributed to the diversity of flower morphology in the process of angiosperm evolution. The MADS-box gene family belonging to the B-class of the ABC model has been verified to play a key role in ectopic petalization. A MADS-box gene, AGL6 , was identified to regulate floral organ development and flowering time in Arabidopsis [7] . Overexpression of the AGL6 gene in Arabidopsis promoted the growth of petal-like bracts. Similarly, ectopic expression of the MADS-box genes was observed in Cornus officinalis . Significant upregulation of the CorPI-B , CorPI-A and CorAP3 genes was detected in the C. officinalis bracts along with the developmental stages [8,9] . In Aristolochia (Aristolochiaceae, basal angiosperm), expression of an AP3 -like gene was detected only during the late stages of petaloid perianth development and expression of B-class paralogs was detected in late development of Aquilegia petaloid sepal [10] . The JAGGED gene is the only gene that has been reported to positively regulate bract development in Arabidopsis . Overexpression of the JAGGED gene promoted bract development in Arabidopsis , and bract development was inhibited in the APETALA1 ( AP1 ) 、 jagged double mutant [11] . A recent study has identified a bract-specific gene, DiASR1 (abscisic acid, stress and ripening protein), from the dove tree ( Davidia involucrata ). Overexpression of the DiASR1 gene induced bract-like leaves in Arabidopsis [ 2] . Although there is a large body of literature on bract development, most of it focuses on bract inhibitory systems and is carried out in model plants, whereas the mechanism of natural bract development remains largely unknown. More genes regulating bract development are expected to be explored in those naturally bracted species. Ornamental plant bracts are currently more studied mainly in pigments, For example, Bougainvillea is a woody plant of the Nyctaginaceae [12] , rich in colour, such as chalcone--flavonone isomerase 1( CHI1 ), 4,5-DOPA dioxygenase extradiol ( DOD ) and flavanone 3-hydroxylase( F3H ), have all been shown to be involved in bract colour change [13,14] . But there is very little research on how bracts develop. However, the molecular mechanism underlying the key events of bract development, including organogenesis, chloroplast degeneration, petal identity determination, rapid growth and abscission needs integrative investigation. B. glabra 'Mrs. Eva White' bracts have significant ornamental value. Its bracts are large and thin, making it ideal for studying bract development. To unravel the developmental mechanisms of bracts in ornamental plants. We conducted a comprehensive analysis of B. glabra 'Mrs. Eva White' at the morphological level and transcriptome to identify key genes involved in these processes. Results Morphological and photosynthetic pigment changes during bract development in B.glabra The development of B. glabra buds was found to go through five main periods as observed by resin sections. At the initial stage, it contains only bracts and growth points (Fig. S1A-a). The primordium forms the outermost whorl of the inflorescence, around which the primordium of the bracts appears in rapid succession and consists of outer and inner cells, the former being larger and looser, the latter smaller and more compact, followed by a gradual growth of the flower primordium (Fig. 1B-a). The floret primordium rapidly differentiates ((Fig. S1A-b) and the perianth whorl begins to appear around the primordium (Fig. 1A, S1A-b). The floral organs develop further, the buds expand, the sepals and petals gradually elongate, the growth cone broadens, the outer petals differentiate into small projections, and the stamen primordia begin to form (Fig. 1B-c). During the LB period（Late Bud）, the stage of bract protocorm differentiation, the androecium has not yet completed its differentiation and is still in the protocorm stage (Fig. S1B-a). Subsequently, androgynous differentiation is completed, and this process is known as the stage of floral primordium differentiation (Fig. S1B-b). Scanning electron microscopy showed that bract villi gradually degenerated during development(Fig. 1C). During the BR1 period, the villi were dense and gradually decreased as the bracts developed (Fig. S1C). The development of B. glabra bracts shows a slow-fast-slow pattern. The bracts were initially green and then showed a change in chlorophyll degradation. From the onset of bract primordium, growth is slow for the first 5 days, accelerates on day 6, and enters a period of rapid growth on day 17, when the bracts gradually change from green to white (Fig. 1D). Chlorophyll content tended to decrease during development, especially significantly from bract period 2, indicating that bract development was accompanied by a process of chlorophyll regreening. However, the chlorophyll content of bracts increased slightly during senescence. Overall, the chlorophyll and carotene content of B. glabra bracts was significantly lower than that of the leaf blade (Fig. 1E). Transcriptome sequencing and sequence analysis of leaf buds, leaves and bracts at different developmental periods In this study, the samples were subjected to transcriptome sequencing using the Circular Consensus Sequence Technique, which yielded a total length of 1041,643,869bp of the circular consensus sequence. The homologues and polyA tails were removed by Isoseq processing and 463,177 FLNC reads were obtained. Subsequently, the FLNC sequences were clustered and de-redundant using the ICE tool of SMRTlink software, resulting in a non-redundant transcript sequence of 79,134,466bp. Thereafter, to improve sequence accuracy, the transcript sequence was further corrected using LoRDEC error correction software, resulting in a corrected transcript sequence of 79,130,973bp. After obtaining the transcript sequences, they were clustered and de-redundant using cd-hit software to create the final full-length transcript sequences, which were used as the reference transcript sequences for second-generation data comparison. The comparison results showed that a total of 45,788 transcripts were detected in the samples, with a total base of 706,647,796 bp and an average length of 1,544 bp. These findings provide an important database for subsequent transcriptome analyses. Transcriptome differential gene analysis Principal Component Analysis (PCA) of the gene expression levels (FPKM) of all the samples revealed that the samples from the LB, FB and BR1 periods clustered together, whereas the BR2 period began to show a trend of dispersion. Over time, samples from the BR3, BR4 and BR5 periods showed more similar gene expression patterns. However, samples from the LE and BR6 periods showed a clear trend of separation from the other periods (Fig. 2A). Analyses for differentially expressed genes between LB/FB, FB/BR2, BR2/BR5 and BR5/LE revealed 245 differentially expressed genes between LB/FB and 2,039 differentially expressed genes between FB/BR2. 3,769 differentially expressed genes were found between BR2/BR5 (Fig. 2B). Subsequently, these 37,842 differentially expressed genes were analysed by clustering expression trends over time using STEM software. The results showed that these differentially expressed genes exhibited 50 different expression patterns within 7 periods. Among them, 20709 differentially expressed genes showed 8 significantly clustered expression patterns ( P < 0.05). The more critical of these patterns include expression pattern I, which is down-regulated, and expression pattern II, which is down-regulated and then up-regulated. In addition, differential genes in expression pattern 22 were up-regulated from BR1 to BR2, then down-regulated from BR2 to BR3, and then up-regulated from BR3 to BR4 and BR5 to BR6, while expression pattern 8 showed a trend of down-regulation followed by up-regulation (Fig. 2C). Analysis by GO enrichment showed that these differentially expressed genes were involved in important pathways such as reproduction, reproductive processes and growth (Fig. 2D). The SBP and MADS-box families were mainly involved. Among them, members of the MADS-box family include BgAP1 , BgFULL , and BgCMB1 , etc., while members of the SBP family include SPL16 , SPL8 , and SPL14 , etc. (Fig. 2E, F, G). These results further revealed the important role of gene expression regulation in plant bract development. The metabolic pathways related to bract development were found to be mainly porphyrin and chlorophyll metabolism, Plant hormone signal transduction and other metabolic pathways by KEGG enrichment analysis (Fig. 2H, Fig. S2). Chlorophyll Metabolic Pathways During Bract development B. glabra 'Mrs. Eva White' bracts evolve in colour from green to white during their development, a transition that is regulated by chlorophyll metabolism. Chlorophyll metabolism consists of three main processes: synthesis, recycling and degradation. As can be seen in Figure 3, in the synthetic pathway, hydroxymethyl chlorophyll was gradually reduced to Chlorophyllide by the catalysis of NADPH - POR . PORA was highly expressed in the early stage of bract development and gradually decreased to very low levels as development progressed, suggesting that the ternary complex formed by hydroxymethyl chlorophyll a, NADPH , and POR is the key to the green colour of bracts in the early stage of bract development. The genes found to interact with PORA by differential protein network analysis were PPOC , PAO , and BgCHLH (Fig. S3). The expression of SGR , PPH , PAO and RCCR genes was low at the early stage of bract development, and then gradually increased, especially at the BR3 and BR4 stages. Therefore, it can be hypothesized that this period is the critical period for bracts to change from green to white. Metabolism of auxin and Abscisic Acid during bract development Endogenous plant hormones play an important role in leaf development and have a regulatory role in the development of B. glabra bracts. Further in-depth analysis of the plant hormone signalling pathways led to the detection of a large number of differential genes involved in hormone signalling response and transduction at key sites of bract formation. These genes are involved in a variety of hormones such as auxin, Abscisic Acid, cytokinin, gibberellins, ethylene, and jasmonic acid. Indole-3-acetic acid (IAA) synthesis can be divided into the tryptophan-dependent pathway and the tryptophan-independent pathway. In the growth hormone signal transduction pathway, we further identified 44 homologous genes that encode 12 enzymes in the growth hormone synthesis pathway. The tryptophan-independent pathway includes two encoding ASB1 and TRPX , five encoding PAT1 , two encoding PAI1 , and six encoding TRPC genes. The TRPX , ASB1 , PAT1 , PAI1 , PRPC , and TRA2 genes in this pathway are all highly expressed in the early stages of bract development and decrease in the later stages. The tryptophan-dependent pathway includes 15 coding TRPB , 4 coding TIR1 , 3 coding AMI1 , and 3 coding ALDO2 , TAR1 , YUC , and TRPA2 all with only 1 code. The expression of the YUC and AMI1 genes of this pathway was higher in the early stage, The YUC gene started to decline gradually in the BR2 period, while the AMI1 gene started to decline gradually in the BR3 period, and the expression of the ALDO2 gene increased with bract development. These results suggest that they may play an important regulatory role in bract development (Fig. 4). In the abscisic acid signalling pathway, we further identified 23 homologous genes encoding four enzymes based on the growth hormone synthesis pathway. These include 2 encoding ECED2 , 3 encoding ECED1 , 5 encoding ALDO3 genes, and 12 encoding ABA2 . NCED2 was up-regulated during BR5 NCED1 and ALDO3 genes were up-regulated during BR6.The ABA2 base was up-regulated during early development. Therefore, it is inferred that these genes play an important regulatory role in bract senescence (Fig. 5) Weighted correlation network analysis （ WGCNA analysis ） A systems biology approach using weighted gene WGCNAs was used to construct WGCNAs, which included 12,370 genes (FPKM ≥ 10) and 1,506 transcription factors, as a way to reveal the function of the network. In this network, the blue and brown modules showed a significant negative correlation with bract area and chlorophyll content, while the variability was particularly significant for the red and brown modules (Fig. 6A, Fig. S4). Several key HUB genes were identified, including Bg_3140 , Bg_11315 , and Bg_23959 in MEblue (Fig. 6B, Fig. S5), and transcription factor HUB genes Bg_26128 and Bg_10088 in MEbrown (Fig. 6C,Fig. S6), with structural genes with HUBs mainly Bg_23913 and Bg_37656 (Fig. 6D). Notably, the HUB genes BgPORA and BgEXLA1 showed significant expression differences during BR1. Among them, the HUB gene EXLA1 encodes an expansion protein that plays an important role in plant cell wall relaxation and regulates the expansion of B. glabra bract cells. The HUB gene POR A, on the other hand, is a key gene in the process of chlorophyll metabolism and is involved in the biosynthesis of light-sensitive pigments by regulating the expression of POR A, which in turn affects chlorophyll biosynthesis, which is consistent with chlorophyll degradation during bract development in Trigonella foetidum(Fig. 6D). In addition, we also found that the brown module HUB genes mainly included BgBZP44, Bg APRR5 and BgIAA7 , which were related to the regulation of phy B photoreceptors and the regulation of plant growth hormones. During the development of B. glabra bracts, APRR5 showed a gradually increasing trend, especially with high expression levels during the BR6 period (Fig. 6E). BgTLP8 , BgAPRR2 , BgBZP44 , BgEXLA1 , and BgPORA are highly expressed in the blue module during the early stages of bract development, which plays an important role in the regulation of B. glabra bract development and senescence and other processes. qRT-PCR of bract development-related candidate genes Based on the above analysis, we performed qRT-PCR on one structural gene and eight transcription factors of the screened chlorophyll metabolic pathway. qRT-PCR results were also compared with RNA-seq (FPKM) results to verify the consistency of the gene expression pattern with the sequencing results. The results showed that the results were similar to the expression trends obtained by RNA-Seq(Fig. 7). Discussion Involvement of MADS-box in B. glabra bract development There are more studies on flower development, but relatively few studies on bract development in ornamental plants, and the molecular mechanism of their bract development is still unclear. MADS-box proteins are a large family of transcription factors that play key roles in eukaryotic development, regulating the intensity of target gene expression in a given time and space by binding to cis-acting elements that interact with the target gene [15] .In this study, we found that the differentially expressed genes BgAP1 , BgCMB1 , BgDEFA , and BgFULL were all in the MADS-box family during the development of B. glabra bracts and that the expression of BgAP1 was largely inversely related to that of BgFULL . It has been suggested that FRUITFULL ( FULL) is negatively regulated by AP1 during the early stages of Arabidopsis thaliana development. FULL is hardly expressed in early trophic organs until after the onset of inflorescence development [16] . AP1 represses FULL expression during sepal and petal development . AP3, on the other hand, inhibits the expression of FULL in stamen development [17] . Although it is unclear whether there is negative regulation of FULL by AP1 during bract development, it is hypothesised that there may be mutual regulation of BgAP1 and BgFULL based on their expression patterns at different developmental periods. Specifically, BgAP1 was expressed mainly in the later stages of bract development, whereas BgFULL was expressed in the early stages of bract development. It is more consistent with the results of previous studies. While most of the MADS-box genes are expressed only in flowers, FULL is expressed at different specific stages of bract development. Investigations in a variety of plant species suggest that the requirement for B-class gene function during heterotopic petaloidy varies dramatically, and may function early, late, or not at all during the development of petaloid organs [18,19] . In Aristolochia ( Aristolochiaceae , basal angiosperm), expression of an AP3 -like gene was detected only during the late stages of petaloid perianth development and expression of B-class paralogs was detected in late development of Aquilegia petaloid sepals, suggesting that B-class homologs may only be required relatively late in the development of petaloid organs in these species [20] . CMB1 is a gene of the MADS-box family, be involved in the regulation of pistil and sepal development in some plants. It has been suggested that PpCMB1 regulates pistil and sepal development in Prunus persica [21] . CMB1 is involved in fruit ripening and sepal development [22] ; In Solanum lycopersicum, it was studied that SlCMB1 is a SEP-like MADS-box gene involved in inflorescence and sepal development by interacting with other genes [23] . In this study, the expression of BgCMB1 was found to trend upward with bract development and was positively correlated with BgAP1 expression. BgAP1, BgFULL, BgCMB1, and BgDEFA were barely expressed in leaves but were highly expressed during bract development. These results suggest a potential role for BgCMB1 in bract development and may be related to the fact that BgCMB1 interacts with BgAP1 and BgFULL to regulate bract development. SBP family involved in B. glabra bract development SBP is a plant-specific class of transcription factors that are closely related to plant inflorescence development and yield [24] .SBP proteins have a zinc finger structure that recognises and binds to the promoter of the MADS-box gene SQUAMOSA (SQUA), which is involved in plant growth and development as well as a variety of physiological and biochemical processes. In addition, SBP a class of transcription factors can regulate flower and fruit development [25 ] . In A. thaliana , SBP homologues SPL3 , SPL4 and SPL5 regulate A. thaliana flower development, whereas the SPL8 and SPL14 genes regulate pollen development [ 26] , which in turn affects yield. The MADS-box transcription factor FULL , together with the SBP transcription factor, plays a role in reproductive transformation and meristem identity transformation [ 27 ] . AtSPL3 in A. thaliana was identified as a direct upstream activator of FULL and the activation of FULL by SPL3 was very strong [ 28 ] . In A. thaliana Yamasaki also demonstrated that At AP1 is downstream of the AtSPL3 gene [ 29 ] . Further studies showed that after silencing and overexpression of the Pp SPL16 gene in P. persica , the expression of the PpFULL and PpAP2a genes were down-regulated and up-regulated, respectively, which further regulated fruit ripening in P. persica through the regulation of the PpFULL and PpAP2a genes [30] . Whether SPL16 interacts with FULL during bract development is unknown, but the analyses in this study revealed that both BgSPL16 and BgAP1 as well as BgSPL16 have a crucial role in B. glabra bract development. In this study, the BgSPL genes showed different temporal and spatial expression patterns. The high expression levels of BgSPL8 , BgSPL13 and BgSPL16 at the stage of floral differentiation further confirmed the role of SPLs in regulating the asexual to reproductive transition through different regulatory roles during flower bud differentiation. SPL3 does not play a major role in asexual changes or at anthesis, but it promotes a shift in floral phloem identity by activating floral phloem-specific genes [3 1 ] .In addition, SPL7 and SPL8 induced phase transition and flowering in Gramineae by directly upregulating SEPALLATA3 ( SEP3 ) and MADS32 [ 32 ] . It was also found that SPL8 was involved in GA signalling and positively regulated trichome formation on sepals and stamen filament elongation [ 33 ] . In this study, we found that BgSPL8 was expressed in bracts but not in leaves during bract development, and BgSPL16 was highly expressed during the FB period, with lower expression at other periods. Therefore, the present study hypothesized that BgSPL8 and SPL16 are associated with bract development, which provides important clues for further studies on plant growth and development. Chlorophyll metabolism during bract development in B. glabra In this study, it was found that B. glabra 'Snow White' bract pigmentation consisted of chlorophyll, carotenoids, and Flavonoids, and that at the beginning of the developmental stage B. glabra bracts had a high content of chlorophyll, but as the bracts developed, the colour gradually changed to white. Currently, phytochrome-mediated light signalling has been reported to induce PORA gene expression in Monocotyledons such as Hordeum vulgare and similarly in dicotyledons such as A. thaliana [ 34,35 ] . Meanwhile, it has been suggested that PORA is abundant in yellowing plants, is active mainly during seedling de-yellowing, and acts synergistically with the light-trapping complex [ 36 ] . Identified a mutant type in A. thaliana with yellow leaves that do not turn green when grown in white light, confirming that the presence of PORA confers a certain degree of photoprotection to the plant. The PORA of Pchlide is the only step of light-requiring enzyme mediating the biosynthesis of chlorophyll in higher plants [ 37 ] , and its expression is regulated by photosensitive pigments, thus affecting chlorophyll biosynthesis. This study further reveals that BgPORA may be a key gene for chlorophyll formation during bract development. In addition, STAY-GREEN Proteins( SGR ), play key regulatory roles in chlorophyll degradation and function independently of PAO enzymes. Meanwhile, chlorophyll degrading enzymes including PPH , PAO and RCCR , the genes for which have been identified in model crops such as Oryza sativa , A. thaliana , etc., and the alteration of their transcriptional expression levels affect the process of chlorophyll degradation, and consequently the yellowing or senescence phenotypes of the leaves. It has been suggested that knockdown or inhibition of SGR expression delays chlorophyll degradation, leading to a stagnant green phenotype in plants during natural development or dark-induced senescence [ 38 ] . In this study, B. glabra bracts were found to be up-regulated during development with SGR in the later stages of B.glabra bract development, which may be a key gene regulating bract greening to whitening. Effects of auxin and Abscisic Acid during bract development Auxin is a Phytohormone that plays an important role in plant growth and development. It has an unsaturated aromatic ring and an acetic acid side chain, in which indole-3-acetaldoxime can be converted to indole-3-acetaldehyde, and can be catalytically generated into IAA by ALDO2 enzyme. This pathway is currently relatively understudied, especially concerning bract development. The indole-3-pyruvic acid pathway(IPA) is the main pathway for IAA biosynthesis in plants and is widely present in most plant bodies [39] . It has been shown that YUC CA ( YUC ) is involved in the IPA pathway in synergy with tryptophan aminotransferase of Arabidopsis (TAA), in which TAA is involved in catalysing the conversion of tryptophan to IPA, and subsequently, YUC is involved in catalysing the production of IAA from IPA [ 40,41 ] . The YUC and TAA families mainly regulate embryonic development after the spherical embryo stage [ 42 ] .In this study, we found that the Bg YUC gene is highly expressed mainly in the prophase of the growth hormone synthesis pathway and plays a regulatory role in the prophase of bract development. In this study, we hypothesised that YUC expression in this pathway is high in the early stages of bract development and low in the later stages, and its molecular mechanism with bract development remains to be investigated. Abscisic Acid metabolic pathway mainly includes the Terpenoid pathway and the Carotenoid pathway [43] . The terpenoid pathway, also known as the C15 direct pathway, consists of the direct formation of 15-carbon ABA from FARNESYLPYROPHOSPHATE (FPP) via cyclisation and oxidation whereas the carotenoid pathway, also known as the C40 indirect pathway, is the main pathway for major ABA synthesis in higher plants. In this pathway, the conversion of zeaxanthin to violaxanthin is catalysed by zeaxanthin epoxidase(ZEP)which is followed by 9-cis-epoxy carotenoid dioxygenase(NCED)which catalyzes the production of xanthoxin from 9-cis-neoxanthin. The short-chain alcohol dehydrogenase ABA2 converts to ABA-aldehyde, which is ultimately converted to ABA by ALDO3 [ 44 ] .In this study, we found that Bg ALDO3 was highly expressed during the BR6 period at the end of bract development, suggesting that bract senescence is associated with Bg ALDO3 .It has been suggested that the ABA signalling pathway is associated with PSEUDO-RESPONSE REGULATOR5 ( APRR5 ) and APRR7 and that ABI5 , a key regulator in the ABA signalling pathway, specifically interacts with APRR5 and APRR7 [45] .In this study, BgAPRR5 was found to be highly expressed during the BR6 period, and it was hypothesised that this gene is associated with the Abscisic Acid metabolic pathway, which provides important clues for understanding the molecular mechanisms of plant Auxin and Abscisic Acid in bract development. Conclusion In conclusion, our investigation into the development of Bougainvillea glabra bracts has identified critical physiological and genetic mechanisms. We observed key changes in bract morphology, including increased area and chlorophyll content, accompanied by a reduction in hairiness (glabrescence). These morphological changes correlate strongly with the dynamics of hormone transduction pathways and chlorophyll metabolism. Importantly, genes within the MADS-box and SBP families, such as BgAP1 , BgFULL , BgSPL16 , BgCMB1 , and BgDEFA , play pivotal roles in these developmental processes. Our study also highlights the significance of BgPORA and BgSGR genes in the biosynthesis and breakdown of chlorophyll during bract development. The expression patterns of BgPRPC and BgTRA2 suggest their involvement in early developmental stages through both tryptophan-dependent and independent growth hormone pathways. Furthermore, the up-regulation of BgALDO3 during the BR6 period underscores its potential importance in later developmental stages, particularly in the abscisic acid pathway. These findings not only enhance our understanding of bract development in B. glabra but also suggest avenues for future research to explore the regulatory mechanisms of floral architecture in ornamental plants. Materials and methods Plant materia l , sample collection B.glabra 'Mrs. Eva White' planted in the flower base of Hunan Agricultural University was selected as the plant material for this study in August 2021, and the different developmental stages of the bracts were divided into eight stages of bract sample collection (Fig. 1A). Three plants were set up as 1 biological replicate, and 3 biological replicates were obtained at each developmental stage. Twenty representative samples were collected from each biological replicate and mixed. These samples were immediately placed in liquid nitrogen and stored in a refrigerator at -80°C for RNA extraction and then sent to Fraser Bioinformatics for sequencing using a PacBio sequencer. Microscopic observation, paraffin sections, scanning electron microscopy methods Dissection of delphiniums at the time of flower bud point to observe their internal structure. Buds from LB and FB periods were selected for paraffin sectioning, Samples from the LB and FB periods were taken and soaked in FAA fixative, and the follow-up experiments were entrusted to Wuhan Safeway Biotechnology Co. To understand the flower bud development for the resin section, collect buds in different periods, immediately soak in FAA fixative (70% ethanol: glacial acetic acid: formaldehyde: 90:5:5) fixed, dehydration after completing using Technovit 7100 resin embedding kit for embedding, embedding after using Leica EM UC6 section, using 0.1% toluidine blue dye solution for staining with the microscope. To observe the phenotypic changes of bract development, samples were taken from BR1, BR3, BR5, LE period for electron microscopy scanning, and the subsequent experiments were entrusted to Wuhan Sevier Biotechnology Co. Bract area, chlorophyll measurement The rate of bract development was observed daily, and the area of delphinium bracts was measured using a leaf area meter. The chlorophyll and carotenoid contents of leaf buds, leaf blades and bracts at different periods of development were determined by Ultraviolet-visible Spectrophotometer, respectively. Several samples were harvested, washed and cut into strips of about 2 mm, weighed 0.2 g, and extracted in 95% ethanol for 24 hours, and three replicates were set up for each species in each treatment. After 24 hours, the absorbance at 470 nm, 665 nm and 649 nm was measured by Ultraviolet-visible Spectrophotometer, and the content of chlorophyll a, chlorophyll b and carotenoids were calculated. Iso-Seq analysis method Total RNA was extracted from the tissue samples, the concentration and purity of the extracted RNA were tested by Nanodrop 2000, genomic contamination, purity and RNA integrity of the samples were detected by agarose gel electrophoresis, and the RIN values were determined by Agilent 2100. High-quality RNA is the basis for successful sequencing. To ensure the accuracy of the sequencing data, the samples were tested and the results met the requirements before library construction. After library testing, full-length transcriptome sequencing was performed using a PacBio sequencer according to the target downstream data volume. Raw sequencing data were preprocessed using SMRTlink software and full-length transcript sequences were obtained using the Iso-Seq analysis process. Identification of Coexpression Modules and Visualization of Hub Genes The weighted gene correlation network analysis (WGCNA) package in R was used to construct the gene co-expression network analysis for Bougainvillea based on the gene-level FPKM data from differentially expressed genes during the 8 bract developmental stages. Module detection was performed using the TOM-based similarity measure and the dynamic tree-cutting algorithm to cut the hierarchal clustering tree and define modules as branches from the tree cutting. The minimum number of genes per module was set as 30 genes by default, and the threshold of module merging correlation for eigengene similarity was 0.9 (Fig. S7). module-tissue association analysis, the eigengene value was calculated for each module and used to test the association with each tissue type. The total connectivity and intramodular connectivity, kME, and kME-p-value were calculated and represent the Pearson correlation between the expression level of that gene and the ME. Additionally, genes with the highest degree of intramodular connectivity within a module are referred to as hub genes. The networks were visualized using Cytoscape_v.3.8.0. Quantitative real-time PCR The BgActin gene was used as the internal control. Primers used in the study are listed in Supplemental Table S1. Real-time fluorescence quantitative qPCR was performed using (the ChamQ Universal SYBR qPCR Master Mix) kit. The relative gene expression levels were normalized using the 2 -ΔΔCt method. Data analysis The experimental results were expressed as mean ± standard error and analyzed using Excel 2010 and SPSS 22.0. The significance of differences among different data sets was analyzed using Duncan's multiple range test at a significance level of p < 0.05. Abbreviations LB: Late Bud; FB: Floral Bud; BR: Bract; LE: Leaf; WGCNA: Weighted correlation network analysis; IAA: Indole-3-acetic acid ; IPA: indole-3-pyruvic acid ; SBP: Squamosa promoter binding protein; PCA: Principal Component Analysis; AP1 : APETALA1; FULL : FRUITFULL; HEM11 : glutamyl-tRNA reductase 1; GSA : glutamate-1-semialdehyde 2,1-aminomutase; HEM2: delta-aminolevulinic acid dehydratase; DCUP : Uroporphyrinogen decarboxylase; HEM4: uroporphyrinogen-III synthase; HEM3 : porphobilinogen deaminase; HEM6 : oxygen-dependent coproporphyrinogen-III oxidase; POOC : protoporphyrinogen oxidase 1; CHLH : Magnesium-chelatase subunit ; CHLD : magnesium-chelatase subunit ; CHLI : magnesium-chelatase subunitI; CHLM : magnesium protoporphyrin IX methyltransferase; CRD : dicarboxylate diiron protein; PORA : protochlorophyllide oxidoreductase A; DCVR : Divinyl chlorophyllide a 8-vinyl-reductase; CHLG : chlorophyll synthase; CHLP : geranylgeranyl reductase; CAO : chlorophyllide a oxygenase; SGR : STAY-GREEN protein ; HCAR : coenzyme F420 hydrogenase family / dehydrogenase; NYC1 : NAD:P-binding Rossmann-fold superfamily protein; PPH: pheophytinase; CHLH: pheophorbide a oxygenase, chloroplastic; RCCR: accelerated cell death ; TRPX : anthranilate synthase alpha subunit 2; ASB1 : anthranilate synthase beta subunit 1; PAT1 : scarecrow-like protein 21 isoform X2 ; PAI1: phosphoribosylanthranilate isomerase 1; TRPC : Indole-3-glycerol phosphate synthase; TRPA2 : tryptophan synthase alpha chain isoform X1; TRPB: hypothetical protein; TAR1 : tryptophan aminotransferase related 1; TIR1 : TRANSPORT INHIBITOR RESPONSE 1-like ; YUC : Probable indole-3-pyruvate monooxygenase ; AMI1 : amidase 1; ALDO2 :fructose-bisphosphate aldolase; NCED2 : nine-cis-epoxycarotenoid dioxygenase 2; NCED1 : 9-cis-epoxycarotenoid dioxygenase; ABA2 : NAD(P)-binding Rossmann-fold superfamily protein; ALDO3 : aldolase C. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material All data generated/analyzed during this study are included in this article and its supplementary files. The sequencing data associated with transcription profiles in this study have been deposited in the China National Center for Bioinformation database with accession number GSA :CRA006581( https://bigd.big.ac.cn/ GSA /browse/CRA006581 ). Competing interests The authors declare that there are no competing interests Funding This study was conducted by Key project of Hunan Provincial Department of Education (22A0155); the Forest Bureau of Hunan Provence (XLKY2024); Hunan province education department project(22C1207, 22C1212). Authors' contributions X.D.L., Y.N.P., and Q.H.Z collected the sample and conceived and designed the study X.D.L., Y.W.M., Y.Q.H and J.L the Transcriptome analysis. 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Forests, 2024, 15(3): 547. Shkryl Y N, Vasyutkina E A, Gorpenchenko T V, et al. Salicylic acid and jasmonic acid biosynthetic pathways are simultaneously activated in transgenic Arabidopsis expressing the rolB/C gene from Ipomoea batatas[J]. Plant Physiology and Biochemistry, 2024: 108521. Yanru H, Xiao H, Milian Y, et al. The Transcription Factor INDUCER OF CBF EXPRESSION1 Interacts with ABSCISIC ACID INSENSITIVE5 and DELLA Proteins to Fine-Tune Abscisic Acid Signaling during Seed Germination in Arabidopsis[J]. The Plant Cell, (7):7,2024-04-11. Additional Declarations No competing interests reported. Supplementary Files attachment.docx Additional file1:Table S1. Primers used in this article Additional file2: Fig. S1. A Resin section for B. glabra bud development process, (a) undifferentiated period. (b) floret primordial differentiation period.(c) whorls around the primordial perianth. B Paraffin sections of leaf buds and flower buds. (a) paraffin section at LB period. (b) paraffin section at FB period. C electron microscope scan of the development of B. glabra bracts. (a) B.glabra bracts at the BR1 period. (b) B. glabra bracts at the BR3 period. (c) B. glabra bract BR5 period. Additional file3: Fig. S2. Significantly enriched KEGG metabolic pathways at developmental stages are represented by bubble plots. A FB-VS-BR1 period. B BR2-VS-BR3 period. C BR1-VS-BR5 Additional file4: Fig. S3. Differential protein network interaction map. Additional file5: Fig. S4. Gene module partition map. Additional file6: Fig. S5.Heatmap of gene expression pattern and bar plot of eigengen expression of genes in blue module. Additional file7: Fig. S6. Heatmap of gene expression pattern and bar plot of eigengen expression of genes in brown module. Additional file8: Fig. S7. Gene scale independence and average connectivity of different powers under the assumption of scaleless networks. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 May, 2024 Reviews received at journal 03 May, 2024 Reviews received at journal 01 May, 2024 Reviewers agreed at journal 01 May, 2024 Reviews received at journal 29 Apr, 2024 Reviewers agreed at journal 24 Apr, 2024 Reviewers agreed at journal 23 Apr, 2024 Reviewers invited by journal 23 Apr, 2024 Editor assigned by journal 23 Apr, 2024 Submission checks completed at journal 23 Apr, 2024 First submitted to journal 16 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4275941","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":295493433,"identity":"2a5df0d1-80c6-4fa8-af25-acde3555d0ed","order_by":0,"name":"xiangdong liu","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"xiangdong","middleName":"","lastName":"liu","suffix":""},{"id":295493434,"identity":"254a3bf0-e3a6-45c3-92d2-b86e18e11e99","order_by":1,"name":"Yaonan Peng","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yaonan","middleName":"","lastName":"Peng","suffix":""},{"id":295493435,"identity":"56a2a2d3-f5c5-4a85-a241-aea4057f59a7","order_by":2,"name":"Qinghui Zeng","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qinghui","middleName":"","lastName":"Zeng","suffix":""},{"id":295493436,"identity":"b8ac1839-ec9d-4984-a986-28c7efac0e06","order_by":3,"name":"Yuwan Ma","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuwan","middleName":"","lastName":"Ma","suffix":""},{"id":295493437,"identity":"3bbecb97-5452-4e06-af99-a8d4f28159ad","order_by":4,"name":"Jin Liu","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Liu","suffix":""},{"id":295493438,"identity":"85935dc2-5993-451a-9eb4-65104e359cbd","order_by":5,"name":"Yaqi Huang","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yaqi","middleName":"","lastName":"Huang","suffix":""},{"id":295493439,"identity":"545dbcb7-6e9e-4261-8cb0-da3f00ad89f0","order_by":6,"name":"Xiaoying Yu","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Yu","suffix":""},{"id":295493440,"identity":"dfb4f9f9-a516-447e-8e3a-beee61c12ed6","order_by":7,"name":"Jun Luo","email":"","orcid":"","institution":"Hunan Botanical Garden","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Luo","suffix":""},{"id":295493441,"identity":"0464a513-7b52-4ed7-8c4a-c666030cbd68","order_by":8,"name":"Yanlin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBACPmYQWSDBwC8BEzpAQAsbWIuBBIPkDKK1gEkDILpBtBZ2HsPPBQYWeca3e8we/GxjkOO7kcD4uQCvw3iMpWcYSBSb3TljbtjbxmAseSOBWXoGfi0G0jwGEonbbuSYSfC2MSRuuJEAFCRgy2+Qls0zcswk/7Yx1BOjxQxsywaJHDNpoC0JBoS1sJVZg7TMuJFWJi1zTsJw5pmHzdL4tPDzH958m6eiLrF/RvI2yTdlNvJ8x5MPfsanhYGBwwCZB0oDjA14NTAwsD8goGAUjIJRMApGPAAA7mk99nn8/qoAAAAASUVORK5CYII=","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yanlin","middleName":"","lastName":"Li","suffix":""},{"id":295493442,"identity":"40a0f8ae-1092-41c7-b8a1-81bc2a7d990d","order_by":9,"name":"Meng Li","email":"","orcid":"","institution":"Central South University of Forestry and Technology","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Li","suffix":""},{"id":295493443,"identity":"d2dd3c5c-371c-477c-95fa-e6329f7765a2","order_by":10,"name":"Fuxiang Cao","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fuxiang","middleName":"","lastName":"Cao","suffix":""}],"badges":[],"createdAt":"2024-04-16 12:13:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4275941/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4275941/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55362826,"identity":"9e0aef63-9bf8-432d-ba5a-7a1704bb072f","added_by":"auto","created_at":"2024-04-26 09:00:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6703900,"visible":true,"origin":"","legend":"\u003cp\u003eMorphostructural and chlorophyll changes during bract development in\u003cem\u003e B. glabra.\u003c/em\u003e \u003cstrong\u003eA\u003c/strong\u003e\u003cem\u003e B. glabra \u003c/em\u003eleaf buds, bracts 7 developmental periods, and leaf sampling periods; \u003cstrong\u003eB\u003c/strong\u003e Resin section of \u003cem\u003eB. glabra\u003c/em\u003e during bud development. (a) Inflorescence primordium differentiation stage. (b) Late stage of floral primordial differentiation. (c)The organs of the third chakra begin to develop consecutively; \u003cstrong\u003eC\u003c/strong\u003e Electron microscope scan of the bract development process of\u003cem\u003eB. glabra. \u003c/em\u003e(a) Electron microscope scan of bracts from the BR1 period. (b) Bract fluff shedding. (c) Electron microscope scan of bracts at the time of BR5. \u003cstrong\u003eD \u003c/strong\u003eChanges in bract area during bract development in \u003cem\u003eB. glabra\u003c/em\u003e. \u003cstrong\u003eE\u003c/strong\u003e Chlorophyll and carotene content of leaf buds, bracts and leaves at seven developmental stages of \u003cem\u003eB.glabra\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/04e59ed3e6beaac523de6263.jpg"},{"id":55362831,"identity":"8ec78cc6-bd51-48c6-925c-e6dcad7c14f9","added_by":"auto","created_at":"2024-04-26 09:00:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3228932,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of genetic differences. \u003cstrong\u003eA\u003c/strong\u003e Sample FPKM values PCA analysis plot. \u003cstrong\u003eB\u003c/strong\u003e Differential gene Wayne plots for LB/FB, FB/BR2, BR2/BR5, BR5/LE.\u003cstrong\u003eC\u003c/strong\u003e STEM trend analysis plot. \u003cstrong\u003eD\u003c/strong\u003e Differential transcription factor KEGG enrichment analysis plot. \u003cstrong\u003eE\u003c/strong\u003e Differential transcription factor GO enrichment analysis plot. \u003cstrong\u003eF \u003c/strong\u003eHeatmap of SPLS family members of differential genes in significant expression trends. \u003cstrong\u003eG\u003c/strong\u003eHeat map of differential gene MADS-box family members in significant expression trend. \u003cstrong\u003eH\u003c/strong\u003e Differential protein network interactions graph.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/e3f00d9cb119f4d4955b3ed3.jpg"},{"id":55362825,"identity":"5d930514-80d3-4865-adc6-dd83681fdc31","added_by":"auto","created_at":"2024-04-26 09:00:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4858201,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll metabolism pathway.\u003cem\u003eHEM11\u003c/em\u003e(glutamyl-tRNA reductase 1);\u003cem\u003eGSA\u003c/em\u003e(glutamate-1-semialdehyde 2,1-aminomutase); HEM2(delta-aminolevulinic acid dehydratase); \u003cem\u003eDCUP\u003c/em\u003e(Uroporphyrinogen decarboxylase); \u003cem\u003eHEM4\u003c/em\u003e(uroporphyrinogen-III synthase); \u003cem\u003eHEM3\u003c/em\u003e(porphobilinogen deaminase); \u003cem\u003eHEM6\u003c/em\u003e(oxygen-dependent coproporphyrinogen-III oxidase); \u003cem\u003eppoc\u003c/em\u003e(protoporphyrinogen oxidase 1); \u003cem\u003eCHLH\u003c/em\u003e(Magnesium-chelatase subunit \u003cem\u003eCHLH\u003c/em\u003e); \u003cem\u003eCHLD\u003c/em\u003e(magnesium-chelatase subunit \u003cem\u003eCHLD\u003c/em\u003e); \u003cem\u003eCHLI\u003c/em\u003e(magnesium-chelatase subunitI); \u003cem\u003eCHLM\u003c/em\u003e(magnesium protoporphyrin IX methyltransferase); \u003cem\u003eCRD\u003c/em\u003e(dicarboxylate diiron protein); \u003cem\u003ePORA\u003c/em\u003e(protochlorophyllide oxidoreductase A); \u003cem\u003eDCVR\u003c/em\u003e(Divinyl chlorophyllide a 8-vinyl-reductase); \u003cem\u003eCHLG\u003c/em\u003e(chlorophyll synthase); \u003cem\u003eCHLP\u003c/em\u003e(geranylgeranyl reductase); \u003cem\u003eCAO\u003c/em\u003e(chlorophyllide a oxygenase); \u003cem\u003eSGR\u003c/em\u003e(protein STAY-GREEN 1); \u003cem\u003eHCAR\u003c/em\u003e(coenzyme F420 hydrogenase family / dehydrogenase); \u003cem\u003eNYC1\u003c/em\u003e(NAD(P)-binding Rossmann-fold superfamily protein); PPH(pheophytinase); \u003cem\u003ePAO\u003c/em\u003e(pheophorbide a oxygenase, chloroplastic); RCCR(accelerated cell death )\u003c/p\u003e","description":"","filename":"Figure3..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/d825062d408d162524a5f66a.jpg"},{"id":55362827,"identity":"498add46-f3c3-4fde-8ae0-c190e2a052ba","added_by":"auto","created_at":"2024-04-26 09:00:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2472796,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analysis of plant growth hormone synthesis genes in \u003cem\u003eB. glabra \u003c/em\u003ebract development stages. \u003cem\u003eTRPX\u003c/em\u003e(anthranilate synthase alpha subunit 2); \u003cem\u003eASB1\u003c/em\u003e(anthranilate synthase beta subunit 1); \u003cem\u003ePAT1\u003c/em\u003e(scarecrow-like protein 21 isoform X2)\u003cem\u003e PAI1\u003c/em\u003e(phosphoribosylanthranilate isomerase 1);TRPC(Indole-3-glycerol phosphate synthase); \u003cem\u003eTRPA2\u003c/em\u003e(tryptophan synthase alpha chain isoform X1);\u003cem\u003e TRPB\u003c/em\u003e(hypothetical protein) ; \u003cem\u003eTAR1\u003c/em\u003e(tryptophan aminotransferase related 1); \u003cem\u003eTIR1\u003c/em\u003e(TRANSPORT INHIBITOR RESPONSE 1-like); \u003cem\u003eYUC\u003c/em\u003e(Probable indole-3-pyruvate monooxygenase ); \u003cem\u003eAMI1\u003c/em\u003e(amidase 1);\u003cem\u003eALDO2\u003c/em\u003e(fructose-bisphosphate aldolase)\u003c/p\u003e","description":"","filename":"Figure4..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/6d218bc7599713551f078ed5.jpg"},{"id":55362828,"identity":"dffaf5f7-c832-4b7a-94f6-3e7f6367ef58","added_by":"auto","created_at":"2024-04-26 09:00:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1187642,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analysis of plant abscisic acid synthesis genes at \u003cem\u003eB. glabra\u003c/em\u003e bract developmental stages. \u003cem\u003eNCED2 \u003c/em\u003e(nine-cis-epoxycarotenoid dioxygenase 2); \u003cem\u003eNCED1\u003c/em\u003e(9-cis-epoxycarotenoid dioxygenase); \u003cem\u003eABA2\u003c/em\u003e(NAD(P)-binding Rossmann-fold superfamily protein); \u003cem\u003eALDO3\u003c/em\u003e(aldolase C).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/37c5c2bf78efd45169080a6b.jpg"},{"id":55362833,"identity":"63a298a5-0ea2-4779-be01-8f5c833797b1","added_by":"auto","created_at":"2024-04-26 09:00:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2549940,"visible":true,"origin":"","legend":"\u003cp\u003eWeighted correlation network analysis of transcripts. \u003cstrong\u003eA\u003c/strong\u003e WGCNA analysis with module-physiological growth indicator associations. Each row corresponds to a module. The names of the modules are shown on the left. Each column corresponds to a specific physiological growth indicator. The colour of each cell at the row-column intersection indicates the correlation coefficient between the module and the sample. High correlations between specific modules and samples are indicated in red. \u003cstrong\u003eB\u003c/strong\u003e Interaction diagram of the gene network of the co-expressed gene ME blue module transcription factor HUB. \u003cstrong\u003eC\u003c/strong\u003e ME brown module transcription factor HUB gene network interaction plot. \u003cstrong\u003eD\u003c/strong\u003e ME brown module structural gene HUB gene network inter-operation diagram. \u003cstrong\u003eE\u003c/strong\u003e Heatmap of related HUB gene expression.\u003c/p\u003e","description":"","filename":"Figure6..jpg","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/83c285a29a6cc4e740cad787.jpg"},{"id":55363412,"identity":"897b7c93-5bf2-4ad3-9a42-caf437b33658","added_by":"auto","created_at":"2024-04-26 09:08:25","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":8849121,"visible":true,"origin":"","legend":"\u003cp\u003eCandidate gene qRT-PCR identification FPKM values were derived from\u003cem\u003e B. glabra \u003c/em\u003etranscriptome data. actin gene was used as an internal control. lb as a control was assigned an arbitrary value of 1.0. The data represent three biological replicates and their mean values, and the error line represents the standard deviation of the three biological replicates.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/a2602e48bbf1bf2fe77d101e.jpg"},{"id":55363972,"identity":"a0668410-d82f-4d2f-a3f1-301047c6b61c","added_by":"auto","created_at":"2024-04-26 09:16:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1568449,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/154481f6-82c9-4aa7-bbb5-5bbafd2b3b3d.pdf"},{"id":55362830,"identity":"c2254a32-cbf9-4ef1-8d61-f746df0353b0","added_by":"auto","created_at":"2024-04-26 09:00:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":567273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file1:\u003c/strong\u003eTable S1. Primers used in this article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file2: \u003c/strong\u003eFig. S1. \u003cstrong\u003eA\u003c/strong\u003e Resin section for\u003cem\u003e B. glabra\u003c/em\u003e bud development process, (a) undifferentiated period. (b) floret primordial differentiation period.(c) whorls around the primordial perianth. \u003cstrong\u003eB\u003c/strong\u003e Paraffin sections of leaf buds and flower buds. (a) paraffin section at LB period. (b) paraffin section at FB period. \u003cstrong\u003eC \u003c/strong\u003eelectron microscope scan of the development of\u003cem\u003eB. glabra \u003c/em\u003ebracts. (a) \u003cem\u003eB.glabra\u003c/em\u003e bracts at the BR1 period. (b)\u003cem\u003e B. glabra \u003c/em\u003ebracts at the BR3 period. (c) \u003cem\u003eB. glabra \u003c/em\u003ebract BR5 period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file3: \u003c/strong\u003eFig. S2. Significantly enriched KEGG metabolic pathways at developmental stages are represented by bubble plots. \u003cstrong\u003eA\u003c/strong\u003e FB-VS-BR1 period. \u003cstrong\u003eB\u003c/strong\u003e BR2-VS-BR3 period. \u003cstrong\u003eC \u003c/strong\u003eBR1-VS-BR5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file4: \u003c/strong\u003eFig. S3. Differential protein network interaction map.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file5: \u003c/strong\u003eFig. S4. Gene module partition map.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file6: \u003c/strong\u003eFig. S5.Heatmap of gene expression pattern and bar plot of eigengen expression of genes in blue module.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file7: \u003c/strong\u003eFig. S6. Heatmap of gene expression pattern and bar plot of eigengen expression of genes in brown module.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file8: \u003c/strong\u003eFig. S7. Gene scale independence and average connectivity of different powers under the assumption of scaleless networks.\u003c/p\u003e","description":"","filename":"attachment.docx","url":"https://assets-eu.researchsquare.com/files/rs-4275941/v1/06c46653937cd94dedf278d6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptome Profiling and Gene Network Analysis Revealed Regulatory Mechanisms of Bract Development in Bougainvillea glabra","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBract is the metamorphosed leaf that appears accompanying inflorescence. In general, any leaf associated with inflorescence can be defined as a bract\u003csup\u003e[1,2]\u003c/sup\u003e. Strictly speaking, bract does not belong to the inflorescence structure, while it can be considered as an extension of floral organs. The bract primordia originated from the stem meristem usually generated at the very early stage of reproductive development\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[3]\u003c/sup\u003e. However, a \u0026ldquo;bract suppression system\u0026rdquo; has been observed in many, but not all, angiosperm species. This system will cease the development of the bract and eventually subsume the bract primordia into the floral meristem. Therefore, bract is absent in many model plants, such as \u003cem\u003eArabidopsis\u003c/em\u003e, maize (\u003cem\u003eZea mays\u003c/em\u003e) and rice (\u003cem\u003eOryza sativa\u003c/em\u003e).\u0026nbsp;The bract development system has been suppressed rather than removed in these higher plants. Moreover, a study in rice indicated that bract suppression is not only necessary for floral development but also critical for the transition from vegetative to reproductive branching\u003csup\u003e[4,5]\u003c/sup\u003e.\u0026nbsp;Although bract suppression is a conserved mechanism in most angiosperm lineages, some plants are retaining the trait of natural bract development, such as species belonging to the\u003cem\u003e\u0026nbsp;Cornaceae\u003c/em\u003e, \u003cem\u003eNyssaceae,\u0026nbsp;\u003c/em\u003e\u003cem\u003eNyctaginaceae\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Araceae families\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn many ornamental plants, the bract is an important organ undertaking reproductive events, as well as an ornamental character\u003csup\u003e[6]\u003c/sup\u003e. The bract with ornamental value is called a petaloid bract, which results from the ectopic expression of genes determining the petal identity in the leaf. This process is supposed to involve the developmental signals transduction and the activation or suppression of related genes. Ectopic petalization (petal-like characteristics in non-petal organs) contributed to the diversity of flower morphology in the process of angiosperm evolution. The MADS-box gene family belonging to the B-class of the ABC model has been verified to play a key role in ectopic petalization. A MADS-box gene, \u003cem\u003eAGL6\u003c/em\u003e, was identified to regulate floral organ development and flowering time in \u003cem\u003eArabidopsis\u003c/em\u003e \u003csup\u003e[7]\u003c/sup\u003e. Overexpression of the \u003cem\u003eAGL6\u0026nbsp;\u003c/em\u003egene in \u003cem\u003eArabidopsis\u003c/em\u003e promoted the growth of petal-like bracts. Similarly, ectopic expression of the MADS-box genes was observed in \u003cem\u003eCornus officinalis\u003c/em\u003e. Significant upregulation of the \u003cem\u003eCorPI-B\u003c/em\u003e, \u003cem\u003eCorPI-A\u003c/em\u003e and \u003cem\u003eCorAP3\u003c/em\u003e genes was detected in the \u003cem\u003eC. officinalis\u003c/em\u003e bracts along with the developmental stages\u003csup\u003e[8,9]\u003c/sup\u003e.\u0026nbsp;In\u0026nbsp;\u003cem\u003eAristolochia\u003c/em\u003e (Aristolochiaceae, basal angiosperm), expression of an\u0026nbsp;\u003cem\u003eAP3\u003c/em\u003e\u003cem\u003e-like\u003c/em\u003e gene was detected only during the late stages of petaloid perianth development and expression of B-class paralogs was detected in late development of\u0026nbsp;Aquilegia\u0026nbsp;petaloid sepal\u003csup\u003e[10]\u003c/sup\u003e.\u0026nbsp;The \u003cem\u003eJAGGED\u003c/em\u003e gene is the only gene that has been reported to positively regulate bract development in \u003cem\u003eArabidopsis\u003c/em\u003e. Overexpression of the \u003cem\u003eJAGGED\u003c/em\u003e gene promoted bract development in \u003cem\u003eArabidopsis\u003c/em\u003e, and bract development was inhibited in the\u0026nbsp;\u003cem\u003eAPETALA1\u003c/em\u003e(\u003cem\u003eAP1\u003c/em\u003e)\u0026nbsp;、\u003cem\u003ejagged\u003c/em\u003e double mutant \u003csup\u003e[11]\u003c/sup\u003e. A recent study has identified a bract-specific gene, \u003cem\u003eDiASR1\u003c/em\u003e (abscisic acid, stress and ripening protein), from the dove tree (\u003cem\u003eDavidia involucrata\u003c/em\u003e). Overexpression of the \u003cem\u003eDiASR1\u003c/em\u003e gene induced bract-like leaves in \u003cem\u003eArabidopsis\u003c/em\u003e\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e2]\u003c/sup\u003e. Although there is a large body of literature on bract development, most of it focuses on bract inhibitory systems and is carried out in model plants, whereas the mechanism of natural bract development remains largely unknown. More genes regulating bract development are expected to be explored in those naturally bracted species.\u003c/p\u003e\n\u003cp\u003eOrnamental plant bracts are currently more studied mainly in pigments, For example, \u003cem\u003eBougainvillea\u003c/em\u003e is a woody plant of the\u003cem\u003e\u0026nbsp;Nyctaginaceae\u003c/em\u003e\u003csup\u003e[12]\u003c/sup\u003e, rich in colour, such as\u0026nbsp;chalcone--flavonone isomerase 1(\u003cem\u003eCHI1\u003c/em\u003e),\u0026nbsp;4,5-DOPA dioxygenase extradiol (\u003cem\u003eDOD\u003c/em\u003e) and\u0026nbsp;flavanone 3-hydroxylase(\u003cem\u003eF3H\u003c/em\u003e), have all been shown to be involved in bract colour change\u003csup\u003e[13,14]\u003c/sup\u003e. But there is very little research on how bracts develop. However, the molecular mechanism underlying the key events of bract development, including organogenesis, chloroplast degeneration, petal identity determination, rapid growth and abscission needs integrative investigation. \u003cem\u003eB. glabra\u003c/em\u003e \u0026apos;Mrs. Eva White\u0026apos; bracts have significant ornamental value. Its bracts are large and thin, making it ideal for studying bract development. To unravel the developmental mechanisms of bracts in ornamental plants. We conducted a comprehensive analysis of \u003cem\u003eB. glabra\u003c/em\u003e \u0026apos;Mrs. Eva White\u0026apos; at the morphological level and transcriptome to identify key genes involved in these processes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMorphological and photosynthetic pigment changes during bract development in B.glabra\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe development of\u0026nbsp;\u003cem\u003eB. glabra\u0026nbsp;\u003c/em\u003ebuds was found to go through five main periods as observed by resin sections. At the initial stage, it contains only bracts and growth points (Fig. S1A-a). The primordium forms the outermost whorl of the inflorescence, around which the primordium of the bracts appears in rapid succession and consists of outer and inner cells, the former being larger and looser, the latter smaller and more compact, followed by a gradual growth of the flower primordium (Fig. 1B-a). The floret primordium rapidly differentiates ((Fig. S1A-b) and the perianth whorl begins to appear around the primordium (Fig. 1A, S1A-b). The floral organs develop further, the buds expand, the sepals and petals gradually elongate, the growth cone broadens, the outer petals differentiate into small projections, and the stamen primordia begin to form (Fig. 1B-c). During the LB period（Late Bud）, the stage of bract protocorm differentiation, the androecium has not yet completed its differentiation and is still in the protocorm stage (Fig. S1B-a). Subsequently, androgynous differentiation is completed, and this process is known as the stage of floral primordium differentiation (Fig. S1B-b).\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy showed that bract villi gradually degenerated during development(Fig. 1C). During the BR1 period, the villi were dense and gradually decreased as the bracts developed (Fig. S1C). The development of \u003cem\u003eB. glabra\u0026nbsp;\u003c/em\u003ebracts shows a slow-fast-slow pattern. The bracts were initially green and then showed a change in chlorophyll degradation. From the onset of bract primordium, growth is slow for the first 5 days, accelerates on day 6, and enters a period of rapid growth on day 17, when the bracts gradually change from green to white (Fig. 1D). Chlorophyll content tended to decrease during development, especially significantly from bract period 2, indicating that bract development was accompanied by a process of chlorophyll regreening. However, the chlorophyll content of bracts increased slightly during senescence. Overall, the chlorophyll and carotene content of \u003cem\u003eB. glabra\u003c/em\u003e bracts was significantly lower than that of the leaf blade (Fig. 1E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing and sequence analysis of leaf buds, leaves and bracts at different developmental periods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the samples were subjected to transcriptome sequencing using the Circular Consensus Sequence Technique, which yielded a total length of 1041,643,869bp of the circular consensus sequence. The homologues and polyA tails were removed by Isoseq processing and 463,177 FLNC reads were obtained. Subsequently, the FLNC sequences were clustered and de-redundant using the ICE tool of SMRTlink software, resulting in a non-redundant transcript sequence of 79,134,466bp. Thereafter, to improve sequence accuracy, the transcript sequence was further corrected using LoRDEC error correction software, resulting in a corrected transcript sequence of 79,130,973bp.\u003c/p\u003e\n\u003cp\u003eAfter obtaining the transcript sequences, they were clustered and de-redundant using cd-hit software to create the final full-length transcript sequences, which were used as the reference transcript sequences for second-generation data comparison. The comparison results showed that a total of 45,788 transcripts were detected in the samples, with a total base of 706,647,796 bp and an average length of 1,544 bp. These findings provide an important database for subsequent transcriptome analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome differential gene analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrincipal Component Analysis (PCA) of the gene expression levels (FPKM) of all the samples revealed that the samples from the LB, FB and BR1 periods clustered together, whereas the BR2 period began to show a trend of dispersion. Over time, samples from the BR3, BR4 and BR5 periods showed more similar gene expression patterns. However, samples from the LE and BR6 periods showed a clear trend of separation from the other periods (Fig. 2A).\u003c/p\u003e\n\u003cp\u003eAnalyses for differentially expressed genes between LB/FB, FB/BR2, BR2/BR5 and BR5/LE revealed 245 differentially expressed genes between LB/FB and 2,039 differentially expressed genes between FB/BR2. 3,769 differentially expressed genes were found between BR2/BR5 (Fig. 2B). Subsequently, these 37,842 differentially expressed genes were analysed by clustering expression trends over time using STEM software. The results showed that these differentially expressed genes exhibited 50 different expression patterns within 7 periods. Among them, 20709 differentially expressed genes showed 8 significantly clustered expression patterns (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The more critical of these patterns include expression pattern I, which is down-regulated, and expression pattern II, which is down-regulated and then up-regulated. In addition, differential genes in expression pattern 22 were up-regulated from BR1 to BR2, then down-regulated from BR2 to BR3, and then up-regulated from BR3 to BR4 and BR5 to BR6, while expression pattern 8 showed a trend of down-regulation followed by up-regulation (Fig. 2C).\u003c/p\u003e\n\u003cp\u003eAnalysis by GO enrichment showed that these differentially expressed genes were involved in important pathways such as reproduction, reproductive processes and growth (Fig. 2D). The SBP and MADS-box families were mainly involved. Among them, members of the MADS-box family include \u003cem\u003eBgAP1\u003c/em\u003e, \u003cem\u003eBgFULL\u003c/em\u003e, and \u003cem\u003eBgCMB1\u003c/em\u003e, etc., while members of the SBP family include \u003cem\u003eSPL16\u003c/em\u003e, \u003cem\u003eSPL8\u003c/em\u003e, and \u003cem\u003eSPL14\u003c/em\u003e, etc. (Fig. 2E, F, G). These results further revealed the important role of gene expression regulation in plant bract development. The metabolic pathways related to bract development were found to be mainly porphyrin and chlorophyll metabolism, Plant hormone signal transduction and other metabolic pathways by KEGG enrichment analysis (Fig. 2H, Fig. S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChlorophyll Metabolic Pathways During Bract development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB. glabra\u003c/em\u003e \u0026apos;Mrs. Eva White\u0026apos; bracts evolve in colour from green to white during their development, a transition that is regulated by chlorophyll metabolism. Chlorophyll metabolism consists of three main processes: synthesis, recycling and degradation. As can be seen in Figure 3, in the synthetic pathway, hydroxymethyl chlorophyll was gradually reduced to Chlorophyllide by the catalysis of \u003cem\u003eNADPH\u003c/em\u003e-\u003cem\u003ePOR\u003c/em\u003e.\u003cem\u003ePORA\u003c/em\u003e was highly expressed in the early stage of bract development and gradually decreased to very low levels as development progressed, suggesting that the ternary complex formed by hydroxymethyl chlorophyll a, \u003cem\u003eNADPH\u003c/em\u003e, and \u003cem\u003ePOR\u003c/em\u003e is the key to the green colour of bracts in the early stage of bract development. The genes found to interact with \u003cem\u003ePORA\u003c/em\u003e by differential protein network analysis were \u003cem\u003ePPOC\u003c/em\u003e, \u003cem\u003ePAO\u003c/em\u003e, and \u003cem\u003eBgCHLH\u003c/em\u003e (Fig. S3). The expression of \u003cem\u003eSGR\u003c/em\u003e, \u003cem\u003ePPH\u003c/em\u003e, \u003cem\u003ePAO\u003c/em\u003e and \u003cem\u003eRCCR\u003c/em\u003e genes was low at the early stage of bract development, and then gradually increased, especially at the BR3 and BR4 stages. Therefore, it can be hypothesized that this period is the critical period for bracts to change from green to white.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolism of auxin and Abscisic Acid during bract development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndogenous plant hormones play an important role in leaf development and have a regulatory role in the development of \u003cem\u003eB. glabra\u003c/em\u003e bracts. Further in-depth analysis of the plant hormone signalling pathways led to the detection of a large number of differential genes involved in hormone signalling response and transduction at key sites of bract formation. These genes are involved in a variety of hormones such as auxin, Abscisic Acid, cytokinin, gibberellins, ethylene, and jasmonic acid.\u003c/p\u003e\n\u003cp\u003eIndole-3-acetic acid (IAA) synthesis can be divided into the tryptophan-dependent pathway and the tryptophan-independent pathway. In the growth hormone signal transduction pathway, we further identified 44 homologous genes that encode 12 enzymes in the growth hormone synthesis pathway. The tryptophan-independent pathway includes two encoding \u003cem\u003eASB1\u003c/em\u003e and \u003cem\u003eTRPX\u003c/em\u003e, five encoding \u003cem\u003ePAT1\u003c/em\u003e, two encoding\u003cem\u003e\u0026nbsp;PAI1\u003c/em\u003e, and six encoding \u003cem\u003eTRPC\u0026nbsp;\u003c/em\u003egenes. The \u003cem\u003eTRPX\u003c/em\u003e, \u003cem\u003eASB1\u003c/em\u003e, \u003cem\u003ePAT1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;PAI1\u003c/em\u003e, \u003cem\u003ePRPC\u003c/em\u003e, and \u003cem\u003eTRA2\u003c/em\u003e genes in this pathway are all highly expressed in the early stages of bract development and decrease in the later stages. The tryptophan-dependent pathway includes 15 coding\u003cem\u003e\u0026nbsp;TRPB\u003c/em\u003e, 4 coding \u003cem\u003eTIR1\u003c/em\u003e, 3 coding \u003cem\u003eAMI1\u003c/em\u003e, and 3 coding \u003cem\u003eALDO2\u003c/em\u003e, \u003cem\u003eTAR1\u003c/em\u003e, \u003cem\u003eYUC\u003c/em\u003e, and \u003cem\u003eTRPA2\u003c/em\u003e all with only 1 code. The expression of the \u003cem\u003eYUC\u003c/em\u003e and \u003cem\u003eAMI1\u003c/em\u003e genes of this pathway was higher in the early stage, The \u003cem\u003eYUC\u003c/em\u003e gene started to decline gradually in the BR2 period, while the \u003cem\u003eAMI1\u003c/em\u003e gene started to decline gradually in the BR3 period, and the expression of the \u003cem\u003eALDO2\u003c/em\u003e gene increased with bract development. These results suggest that they may play an important regulatory role in bract development (Fig. 4).\u003c/p\u003e\n\u003cp\u003eIn the abscisic acid signalling pathway, we further identified 23 homologous genes encoding four enzymes based on the growth hormone synthesis pathway. These include 2 encoding \u003cem\u003eECED2\u003c/em\u003e, 3 encoding \u003cem\u003eECED1\u003c/em\u003e, 5 encoding \u003cem\u003eALDO3\u003c/em\u003e genes, and 12 encoding \u003cem\u003eABA2\u003c/em\u003e. \u003cem\u003eNCED2 \u0026nbsp;\u0026nbsp;\u003c/em\u003ewas up-regulated during BR5\u003cem\u003e\u0026nbsp;NCED1\u0026nbsp;\u003c/em\u003eand \u003cem\u003eALDO3\u003c/em\u003e genes were up-regulated during BR6.The \u003cem\u003eABA2\u003c/em\u003e base was up-regulated during early development. Therefore, it is inferred that these genes play an important regulatory role in bract senescence (Fig. 5)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWeighted correlation network analysis\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eWGCNA analysis\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA systems biology approach using weighted gene WGCNAs was used to construct WGCNAs, which included 12,370 genes (FPKM \u0026ge; 10) and 1,506 transcription factors, as a way to reveal the function of the network. In this network, the blue and brown modules showed a significant negative correlation with bract area and chlorophyll content, while the variability was particularly significant for the red and brown modules (Fig. 6A, Fig. S4).\u003c/p\u003e\n\u003cp\u003eSeveral key HUB genes were identified, including \u003cem\u003eBg_3140\u003c/em\u003e, \u003cem\u003eBg_11315\u003c/em\u003e, and \u003cem\u003eBg_23959\u003c/em\u003e in MEblue (Fig. 6B, Fig. S5), and transcription factor HUB genes \u003cem\u003eBg_26128\u003c/em\u003e and \u003cem\u003eBg_10088\u003c/em\u003e in MEbrown (Fig. 6C,Fig. S6), with structural genes with HUBs mainly \u003cem\u003eBg_23913\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBg_37656\u003c/em\u003e (Fig. 6D). Notably, the HUB genes \u003cem\u003eBgPORA\u003c/em\u003e and\u003cem\u003e\u0026nbsp;BgEXLA1\u003c/em\u003e showed significant expression differences during BR1. Among them, the HUB gene\u003cem\u003e\u0026nbsp;EXLA1\u003c/em\u003e encodes an expansion protein that plays an important role in plant cell wall relaxation and regulates the expansion of \u003cem\u003eB. glabra\u003c/em\u003e bract cells. The HUB gene \u003cem\u003ePOR\u003c/em\u003eA, on the other hand, is a key gene in the process of chlorophyll metabolism and is involved in the biosynthesis of light-sensitive pigments by regulating the expression of \u003cem\u003ePOR\u003c/em\u003eA, which in turn affects chlorophyll biosynthesis, which is consistent with chlorophyll degradation during bract development in Trigonella foetidum(Fig. 6D).\u003c/p\u003e\n\u003cp\u003eIn addition, we also found that the brown module HUB genes mainly included \u003cem\u003eBgBZP44,\u003c/em\u003e \u003cem\u003eBg\u003c/em\u003e\u003cem\u003eAPRR5\u003c/em\u003e and \u003cem\u003eBgIAA7\u003c/em\u003e, which were related to the regulation of phy B photoreceptors and the regulation of plant growth hormones. During the development of \u003cem\u003eB. glabra\u003c/em\u003e bracts, \u003cem\u003eAPRR5\u0026nbsp;\u003c/em\u003eshowed a gradually increasing trend, especially with high expression levels during the BR6 period (Fig. 6E). \u003cem\u003eBgTLP8\u003c/em\u003e, \u003cem\u003eBgAPRR2\u003c/em\u003e, \u003cem\u003eBgBZP44\u003c/em\u003e, \u003cem\u003eBgEXLA1\u003c/em\u003e, and \u003cem\u003eBgPORA\u003c/em\u003e are highly expressed in the blue module during the early stages of bract development, which plays an important role in the regulation of \u003cem\u003eB. glabra\u003c/em\u003e bract development and senescence and other processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR of bract development-related candidate genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the above analysis, we performed qRT-PCR on one structural gene and eight transcription factors of the screened chlorophyll metabolic pathway. qRT-PCR results were also compared with RNA-seq (FPKM) results to verify the consistency of the gene expression pattern with the sequencing results. The results showed that the results were similar to the expression trends obtained by RNA-Seq(Fig. 7).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eInvolvement of MADS-box in\u003cem\u003e\u0026nbsp;B. glabra\u003c/em\u003e bract development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are more studies on flower development, but relatively few studies on bract development in ornamental plants, and the molecular mechanism of their bract development is still unclear. MADS-box proteins are a large family of transcription factors that play key roles in eukaryotic development, regulating the intensity of target gene expression in a given time and space by binding to cis-acting elements that interact with the target gene\u003csup\u003e[15]\u003c/sup\u003e.In this study, we found that the differentially expressed genes \u003cem\u003eBgAP1\u003c/em\u003e, \u003cem\u003eBgCMB1\u003c/em\u003e, \u003cem\u003eBgDEFA\u003c/em\u003e, and \u003cem\u003eBgFULL\u003c/em\u003e were all in the MADS-box family during the development of \u003cem\u003e\u0026nbsp;B. glabra\u003c/em\u003e bracts and that the expression of \u003cem\u003eBgAP1\u003c/em\u003e was largely inversely related to that of \u003cem\u003eBgFULL\u003c/em\u003e. It has been suggested that\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eFRUITFULL\u003c/em\u003e(\u003cem\u003eFULL)\u0026nbsp;\u003c/em\u003eis negatively regulated by \u003cem\u003eAP1\u003c/em\u003e during the early stages of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e development. \u003cem\u003eFULL\u003c/em\u003e is hardly expressed in early trophic organs until after the onset of inflorescence development \u003csup\u003e[16]\u003c/sup\u003e. \u003cem\u003eAP1\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003erepresses\u0026nbsp;\u003cem\u003eFULL\u003c/em\u003e expression during sepal and petal development\u003cem\u003e.\u003c/em\u003eAP3, on the other hand, inhibits the expression of \u003cem\u003eFULL\u003c/em\u003e in stamen development \u003csup\u003e[17]\u003c/sup\u003e. Although it is unclear whether there is negative regulation of \u003cem\u003eFULL\u003c/em\u003e by \u003cem\u003eAP1\u003c/em\u003e during bract development, it is hypothesised that there may be mutual regulation of \u003cem\u003eBgAP1\u003c/em\u003e and \u003cem\u003eBgFULL\u003c/em\u003e based on their expression patterns at different developmental periods. Specifically, \u003cem\u003eBgAP1\u003c/em\u003e was expressed mainly in the later stages of bract development, whereas \u003cem\u003eBgFULL\u003c/em\u003e was expressed in the early stages of bract development. It is more consistent with the results of previous studies. While most of the MADS-box genes are expressed only in flowers, \u003cem\u003eFULL\u003c/em\u003e is expressed at different specific stages of bract development.\u0026nbsp;Investigations in a variety of plant species suggest that the requirement for B-class gene function during heterotopic petaloidy varies\u0026nbsp;dramatically, and may function early, late, or not at all during the development of\u0026nbsp;petaloid organs\u003csup\u003e[18,19]\u003c/sup\u003e. In\u0026nbsp;\u003cem\u003eAristolochia\u003c/em\u003e (\u003cem\u003eAristolochiaceae\u003c/em\u003e, basal angiosperm), expression of an\u0026nbsp;\u003cem\u003eAP3\u003c/em\u003e\u003cem\u003e-like\u003c/em\u003e gene was detected only during the late stages of petaloid perianth development and expression of B-class paralogs was detected in late development of\u0026nbsp;\u003cem\u003eAquilegia\u003c/em\u003e petaloid sepals, suggesting that B-class homologs may only be required relatively late in the development of petaloid organs in these species\u003csup\u003e[20]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCMB1\u003c/em\u003e is a gene of the MADS-box family, be involved in the regulation of pistil and sepal development in some plants. It has been suggested that \u003cem\u003ePpCMB1\u0026nbsp;\u003c/em\u003eregulates pistil and sepal development in \u003cem\u003ePrunus persica\u003c/em\u003e\u003csup\u003e[21]\u003c/sup\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003eCMB1\u003c/em\u003e is involved in fruit ripening and sepal development\u003csup\u003e[22]\u003c/sup\u003e;\u003csup\u003e\u0026nbsp;\u003c/sup\u003eIn \u003cem\u003eSolanum lycopersicum,\u003c/em\u003e it was studied that \u003cem\u003eSlCMB1\u003c/em\u003e is a SEP-like MADS-box gene involved in inflorescence and sepal development by interacting with other genes \u003csup\u003e[23]\u003c/sup\u003e. In this study, the expression of \u003cem\u003eBgCMB1\u003c/em\u003e was found to trend upward with bract development and was positively correlated with \u003cem\u003eBgAP1\u003c/em\u003e expression. \u003cem\u003eBgAP1, BgFULL, BgCMB1, and BgDEFA\u0026nbsp;\u003c/em\u003ewere barely expressed in leaves but were highly expressed during bract development. These results suggest a potential role for \u003cem\u003eBgCMB1\u003c/em\u003e in bract development and may be related to the fact that \u003cem\u003eBgCMB1\u003c/em\u003e interacts with \u003cem\u003eBgAP1\u003c/em\u003e and \u003cem\u003eBgFULL\u003c/em\u003e to regulate bract development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSBP family involved in \u003cem\u003eB. glabra\u003c/em\u003e bract development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSBP is a plant-specific class of transcription factors that are closely related to plant inflorescence development and yield\u003csup\u003e[24]\u003c/sup\u003e.SBP proteins have a zinc finger structure that recognises and binds to the promoter of the MADS-box gene SQUAMOSA (SQUA), which is involved in plant growth and development as well as a variety of physiological and biochemical processes. In addition, SBP a class of transcription factors can regulate flower and fruit development\u0026nbsp;\u003csup\u003e[25\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. In \u003cem\u003eA. thaliana\u003c/em\u003e, SBP homologues \u003cem\u003eSPL3\u003c/em\u003e, \u003cem\u003eSPL4\u003c/em\u003e and \u003cem\u003eSPL5\u003c/em\u003e regulate \u003cem\u003eA. thaliana\u003c/em\u003e flower development, whereas the \u003cem\u003eSPL8\u003c/em\u003e and\u003cem\u003e\u0026nbsp;SPL14\u003c/em\u003e genes regulate pollen development \u003csup\u003e[\u003c/sup\u003e\u003csup\u003e26]\u003c/sup\u003e, which in turn affects yield.\u003c/p\u003e\n\u003cp\u003eThe MADS-box transcription factor \u003cem\u003eFULL\u003c/em\u003e, together with the SBP transcription factor, plays a role in reproductive transformation and meristem identity transformation\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e27\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. \u003cem\u003eAtSPL3\u003c/em\u003e in \u003cem\u003eA. thaliana\u003c/em\u003e was identified as a direct upstream activator of\u003cem\u003e\u0026nbsp;FULL\u003c/em\u003e and the activation of \u003cem\u003eFULL\u0026nbsp;\u003c/em\u003eby \u003cem\u003eSPL3\u003c/em\u003e was very strong \u003csup\u003e[\u003c/sup\u003e\u003csup\u003e28\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. In\u003cem\u003e\u0026nbsp;A. thaliana\u003c/em\u003e Yamasaki also demonstrated that \u003cem\u003eAt\u003c/em\u003e\u003cem\u003eAP1\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eis downstream of the \u003cem\u003eAtSPL3\u0026nbsp;\u003c/em\u003egene \u003csup\u003e[\u003c/sup\u003e\u003csup\u003e29\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. Further studies showed that after silencing and overexpression of the \u003cem\u003ePp\u003c/em\u003e\u003cem\u003eSPL16\u003c/em\u003e gene in \u003cem\u003eP. persica\u003c/em\u003e, the expression of the \u003cem\u003ePpFULL\u003c/em\u003e and \u003cem\u003ePpAP2a\u0026nbsp;\u003c/em\u003egenes were down-regulated and up-regulated, respectively, which further regulated fruit ripening in \u003cem\u003eP. persica\u003c/em\u003e through the regulation of the \u003cem\u003ePpFULL\u003c/em\u003e and \u003cem\u003ePpAP2a\u003c/em\u003e genes\u003csup\u003e\u0026nbsp;[30]\u003c/sup\u003e. Whether\u0026nbsp;\u003cem\u003eSPL16\u003c/em\u003e interacts with \u003cem\u003eFULL\u003c/em\u003e during bract development is unknown, but the analyses in this study revealed that both\u0026nbsp;\u003cem\u003eBgSPL16\u003c/em\u003e and\u0026nbsp;\u003cem\u003eBgAP1\u003c/em\u003e as well as\u0026nbsp;\u003cem\u003eBgSPL16\u003c/em\u003e have a crucial role in\u003cem\u003e\u0026nbsp;B. glabra\u003c/em\u003e bract development. In this study, the \u003cem\u003eBgSPL\u003c/em\u003e genes showed different temporal\u0026nbsp;and spatial expression patterns. The high expression levels of \u003cem\u003eBgSPL8\u003c/em\u003e, \u003cem\u003eBgSPL13\u003c/em\u003e and\u0026nbsp;\u003cem\u003eBgSPL16\u003c/em\u003e at the stage of floral differentiation further confirmed the role of SPLs in regulating the asexual to reproductive transition through different regulatory roles during flower bud differentiation.\u003cem\u003eSPL3\u0026nbsp;\u003c/em\u003edoes not play a major role in asexual changes or at anthesis, but it promotes a shift in floral phloem identity by activating floral phloem-specific genes\u003csup\u003e[3\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e.In addition, \u003cem\u003eSPL7\u003c/em\u003e and \u003cem\u003eSPL8\u003c/em\u003e induced phase transition and flowering in Gramineae\u0026nbsp;by directly upregulating \u003cem\u003eSEPALLATA3\u003c/em\u003e (\u003cem\u003eSEP3\u003c/em\u003e) and \u003cem\u003eMADS32\u003c/em\u003e\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e32\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e It was also found that\u0026nbsp;\u003cem\u003eSPL8\u003c/em\u003e was involved in GA signalling and positively regulated trichome formation on sepals and stamen filament elongation \u003csup\u003e[\u003c/sup\u003e\u003csup\u003e33\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. In this study, we found that \u003cem\u003eBgSPL8\u003c/em\u003e was expressed in bracts but not in leaves during bract development, and\u0026nbsp;\u003cem\u003eBgSPL16\u003c/em\u003e was highly expressed during the FB period, with lower expression at other periods. Therefore, the present study hypothesized that \u003cem\u003eBgSPL8\u003c/em\u003e and\u0026nbsp;\u003cem\u003eSPL16\u003c/em\u003e are associated with bract development, which provides important clues for further studies on plant growth and development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChlorophyll metabolism during bract development in\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eB. glabra\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, it was found that\u0026nbsp;\u003cem\u003eB. glabra\u003c/em\u003e \u0026apos;Snow White\u0026apos; bract pigmentation consisted of chlorophyll, carotenoids, and Flavonoids, and that at the beginning of the developmental stage\u0026nbsp;\u003cem\u003eB. glabra\u003c/em\u003e bracts had a high content of chlorophyll, but as the bracts developed,\u0026nbsp;the colour gradually changed to white.\u0026nbsp;Currently, phytochrome-mediated light signalling has been reported to induce\u0026nbsp;\u003cem\u003ePORA\u003c/em\u003e gene expression in \u003cem\u003eMonocotyledons\u003c/em\u003e such as \u003cem\u003eHordeum vulgare\u003c/em\u003e and similarly in dicotyledons such as \u003cem\u003eA. thaliana\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e34,35\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e.\u0026nbsp;Meanwhile, it has been suggested that\u0026nbsp;\u003cem\u003ePORA\u003c/em\u003e is abundant in yellowing\u0026nbsp;plants, is\u0026nbsp;active mainly during seedling de-yellowing, and\u0026nbsp;acts synergistically with the light-trapping complex\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e36\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e.\u0026nbsp;Identified a mutant type in \u003cem\u003eA. thaliana\u003c/em\u003e with yellow leaves that do not turn green when grown in white light, confirming that the presence of\u0026nbsp;\u003cem\u003ePORA\u003c/em\u003e confers a certain degree of photoprotection to the plant. The\u0026nbsp;\u003cem\u003ePORA\u003c/em\u003e of Pchlide is the only step of light-requiring enzyme mediating the biosynthesis of chlorophyll in higher plants\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e37\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e, and its expression is regulated by photosensitive pigments, thus affecting chlorophyll biosynthesis. This study further reveals that\u0026nbsp;\u003cem\u003eBgPORA\u003c/em\u003e may be a key gene for chlorophyll formation during bract development.\u003c/p\u003e\n\u003cp\u003eIn addition,\u0026nbsp;STAY-GREEN Proteins(\u003cem\u003eSGR\u003c/em\u003e), play key regulatory roles in chlorophyll degradation and function independently of\u0026nbsp;\u003cem\u003ePAO\u003c/em\u003e enzymes. Meanwhile, chlorophyll degrading enzymes including \u003cem\u003ePPH\u003c/em\u003e,\u0026nbsp;\u003cem\u003ePAO\u003c/em\u003e and\u0026nbsp;\u003cem\u003eRCCR\u003c/em\u003e, the genes for which have been identified in model crops such as \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eA. thaliana\u003c/em\u003e, etc., and the alteration of their transcriptional expression levels affect the process of chlorophyll degradation, and consequently the yellowing or senescence phenotypes of the leaves. It has been suggested that knockdown or inhibition of\u0026nbsp;\u003cem\u003eSGR\u003c/em\u003e expression delays chlorophyll degradation, leading to a stagnant green phenotype in plants during natural development or dark-induced senescence\u003csup\u003e\u0026nbsp;[\u003c/sup\u003e\u003csup\u003e38\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e.\u0026nbsp;In\u0026nbsp;this study,\u0026nbsp;\u003cem\u003eB. glabra\u003c/em\u003e bracts were found to be up-regulated during development with\u0026nbsp;\u003cem\u003eSGR\u003c/em\u003e in the later stages of\u0026nbsp;\u003cem\u003eB.glabra\u003c/em\u003e bract development, which may be a key gene regulating bract greening to whitening.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of auxin and Abscisic Acid during bract development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuxin is a Phytohormone that plays an important role in plant growth and development. It has an unsaturated aromatic ring and an acetic acid side chain, in which indole-3-acetaldoxime can be converted to indole-3-acetaldehyde, and can be catalytically generated into IAA by\u0026nbsp;\u003cem\u003eALDO2\u003c/em\u003e enzyme. This pathway is currently relatively understudied, especially concerning bract development.\u0026nbsp;The\u0026nbsp;indole-3-pyruvic acid\u0026nbsp;pathway(IPA)\u0026nbsp;is the main pathway for IAA biosynthesis in plants and is widely present in most plant bodies\u003csup\u003e[39]\u003c/sup\u003e. It has been shown that\u0026nbsp;\u003cem\u003eYUC\u003c/em\u003e\u003cem\u003eCA\u003c/em\u003e (\u003cem\u003eYUC\u003c/em\u003e) is involved in the IPA pathway in synergy with tryptophan aminotransferase of Arabidopsis (TAA), in which TAA is involved in catalysing the conversion of tryptophan to IPA, and subsequently,\u0026nbsp;\u003cem\u003eYUC\u003c/em\u003e is involved in catalysing the production of IAA from IPA\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e40,41\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. The\u0026nbsp;\u003cem\u003eYUC\u003c/em\u003e and TAA families mainly regulate embryonic development after the spherical embryo stage\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e42\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e.In this study, we found that the \u003cem\u003eBg\u003c/em\u003e\u003cem\u003eYUC\u003c/em\u003e gene is highly expressed mainly in the prophase of the growth hormone synthesis pathway and plays a regulatory role in the prophase of bract development. In this study, we hypothesised that\u0026nbsp;\u003cem\u003eYUC\u003c/em\u003e expression in this pathway is high in the early stages of bract development and low in the later stages, and its molecular mechanism with bract development remains to be investigated.\u003c/p\u003e\n\u003cp\u003eAbscisic Acid metabolic pathway mainly includes the Terpenoid pathway and the Carotenoid pathway\u003csup\u003e\u0026nbsp;[43]\u003c/sup\u003e. The terpenoid pathway, also known as the C15 direct pathway, consists of the direct formation\u0026nbsp;of 15-carbon ABA from FARNESYLPYROPHOSPHATE (FPP) via cyclisation and oxidation whereas the carotenoid pathway, also known as the C40 indirect pathway, is the main pathway for major ABA synthesis in higher plants. In this pathway, the conversion of zeaxanthin to violaxanthin is catalysed by zeaxanthin epoxidase(ZEP)which is followed by 9-cis-epoxy carotenoid dioxygenase(NCED)which catalyzes the production of xanthoxin from 9-cis-neoxanthin. The short-chain alcohol dehydrogenase\u0026nbsp;\u003cem\u003eABA2\u003c/em\u003e converts to ABA-aldehyde, which is ultimately converted to ABA by\u0026nbsp;\u003cem\u003eALDO3\u003c/em\u003e\u003csup\u003e\u0026nbsp;[\u003c/sup\u003e\u003csup\u003e44\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e.In this study, we found that\u0026nbsp;Bg\u003cem\u003eALDO3\u003c/em\u003e was highly expressed during the BR6 period at the end of bract development, suggesting that bract senescence is associated with\u0026nbsp;Bg\u003cem\u003eALDO3\u003c/em\u003e.It has been suggested that the ABA signalling pathway is associated with PSEUDO-RESPONSE REGULATOR5 (\u003cem\u003eAPRR5\u003c/em\u003e) and\u0026nbsp;\u003cem\u003eAPRR7\u003c/em\u003e and that \u003cem\u003eABI5\u003c/em\u003e, a key regulator in the ABA signalling pathway, specifically interacts with\u0026nbsp;\u003cem\u003eAPRR5\u003c/em\u003e and\u0026nbsp;\u003cem\u003eAPRR7\u003c/em\u003e\u003csup\u003e[45]\u003c/sup\u003e.In this study,\u0026nbsp;\u003cem\u003eBgAPRR5\u003c/em\u003e was found to be highly expressed during the BR6 period, and it was hypothesised that this gene is associated with the Abscisic Acid metabolic pathway, which provides important clues for understanding the molecular mechanisms of plant Auxin and Abscisic Acid in bract development.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our investigation into the development of \u003cem\u003eBougainvillea glabra\u003c/em\u003e bracts has identified critical physiological and genetic mechanisms. We observed key changes in bract morphology, including increased area and chlorophyll content, accompanied by a reduction in hairiness (glabrescence). These morphological changes correlate strongly with the dynamics of hormone transduction pathways and chlorophyll metabolism. Importantly, genes within the MADS-box and SBP families, such as \u003cem\u003eBgAP1\u003c/em\u003e, \u003cem\u003eBgFULL\u003c/em\u003e, \u003cem\u003eBgSPL16\u003c/em\u003e, \u003cem\u003eBgCMB1\u003c/em\u003e, and \u003cem\u003eBgDEFA\u003c/em\u003e, play pivotal roles in these developmental processes. Our study also highlights the significance of \u003cem\u003eBgPORA\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBgSGR\u003c/em\u003e genes in the biosynthesis and breakdown of chlorophyll during bract development. The expression patterns of \u003cem\u003eBgPRPC\u003c/em\u003e and \u003cem\u003eBgTRA2\u003c/em\u003e suggest their involvement in early developmental stages through both tryptophan-dependent and independent growth hormone pathways. Furthermore, the up-regulation of \u003cem\u003eBgALDO3\u003c/em\u003e during the BR6 period underscores its potential importance in later developmental stages, particularly in the abscisic acid pathway. These findings not only enhance our understanding of bract development in \u003cem\u003eB. glabra\u003c/em\u003e but also suggest avenues for future research to explore the regulatory mechanisms of floral architecture in ornamental plants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materia\u003c/strong\u003e\u003cstrong\u003el\u003c/strong\u003e\u003cstrong\u003e, sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB.glabra\u0026nbsp;\u003c/em\u003e\u0026apos;Mrs. Eva White\u0026apos;\u0026nbsp;planted in the flower base of Hunan Agricultural University was selected as the plant material for this study in August 2021, and the different developmental stages of the bracts were divided into eight stages of bract sample collection (Fig. 1A). Three plants were set up as 1 biological replicate, and 3 biological replicates were obtained at each developmental stage. Twenty representative samples were collected from each biological replicate and mixed. These samples were immediately placed in liquid nitrogen and stored in a refrigerator at -80\u0026deg;C for RNA extraction and then sent to Fraser Bioinformatics for sequencing using a PacBio sequencer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopic observation, paraffin sections, scanning electron microscopy methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDissection of delphiniums at the time of flower bud point to observe their internal structure. Buds from LB and FB periods were selected for paraffin sectioning, Samples from the LB and FB periods were taken and soaked in FAA fixative, and the follow-up experiments were entrusted to Wuhan Safeway Biotechnology Co. To understand the flower bud development for the resin section, collect buds in different periods, immediately soak in FAA fixative (70% ethanol: glacial acetic acid: formaldehyde: 90:5:5) fixed, dehydration after completing using Technovit 7100 resin embedding kit for embedding, embedding after using Leica EM UC6 section, using 0.1% toluidine blue dye solution for staining with the microscope. To observe the phenotypic changes of bract development, samples were taken from BR1, BR3, BR5, LE period for electron microscopy scanning, and the subsequent experiments were entrusted to Wuhan Sevier Biotechnology Co.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBract area, chlorophyll measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rate of bract development was observed daily, and the area of delphinium bracts was measured using a leaf area meter. The chlorophyll and carotenoid contents of leaf buds, leaf blades and bracts at different periods of development were determined by Ultraviolet-visible Spectrophotometer, respectively. Several samples were harvested, washed and cut into strips of about 2 mm, weighed 0.2 g, and extracted in 95% ethanol for 24 hours, and three replicates were set up for each species in each treatment. After 24 hours, the absorbance at 470 nm, 665 nm and 649 nm was measured by Ultraviolet-visible Spectrophotometer, and the content of chlorophyll a, chlorophyll b and carotenoids were calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIso-Seq analysis method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the tissue samples, the concentration and purity of the extracted RNA were tested by Nanodrop 2000, genomic contamination, purity and RNA integrity of the samples were detected by agarose gel electrophoresis, and the RIN values were determined by Agilent 2100. High-quality RNA is the basis for successful sequencing. To ensure the accuracy of the sequencing data, the samples were tested and the results met the requirements before library construction. After library testing, full-length transcriptome sequencing was performed using a PacBio sequencer according to the target downstream data volume. Raw sequencing data were preprocessed using SMRTlink software and full-length transcript sequences were obtained using the Iso-Seq analysis process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of Coexpression Modules and Visualization of Hub Genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe weighted gene correlation network analysis (WGCNA) package in R was used to construct the gene co-expression network analysis for\u0026nbsp;\u003cem\u003eBougainvillea\u003c/em\u003e based on the gene-level FPKM data from differentially expressed genes during the 8 bract developmental stages. Module detection was performed using the TOM-based similarity measure and the dynamic tree-cutting algorithm to cut the hierarchal clustering tree and define modules as branches from the tree cutting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe minimum number of genes per module was set as 30 genes by default, and the threshold of module merging correlation for eigengene similarity was 0.9 (Fig. S7). module-tissue association analysis, the eigengene value was calculated for each module and used to test the association with each tissue type. The total connectivity and\u0026nbsp;intramodular connectivity, kME, and kME-p-value were calculated and represent the Pearson correlation between the expression level of that gene and the ME. Additionally, genes with the highest degree of intramodular connectivity within a module are referred to as hub genes. The networks were visualized using Cytoscape_v.3.8.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003e\u0026nbsp;BgActin\u0026nbsp;\u003c/em\u003egene was used as the internal control. Primers used\u0026nbsp;in the study are listed in Supplemental Table S1. Real-time fluorescence quantitative qPCR was performed using (the ChamQ Universal SYBR qPCR Master Mix) kit. The relative\u0026nbsp;gene\u0026nbsp;expression\u0026nbsp;levels\u0026nbsp;were\u0026nbsp;normalized\u0026nbsp;using\u0026nbsp;the\u0026nbsp;2\u003csup\u003e\u0026nbsp;-\u0026Delta;\u0026Delta;Ct\u0026nbsp;\u003c/sup\u003emethod.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental results were expressed as mean \u0026plusmn; standard error and analyzed using Excel 2010 and SPSS 22.0. The significance of differences among different data sets was analyzed using Duncan\u0026apos;s multiple range test at a significance level of\u003cem\u003e\u0026nbsp;p\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLB:\u0026nbsp;Late Bud;\u003c/p\u003e\n\u003cp\u003eFB:\u0026nbsp;Floral Bud;\u003c/p\u003e\n\u003cp\u003eBR: Bract;\u003c/p\u003e\n\u003cp\u003eLE: Leaf;\u003c/p\u003e\n\u003cp\u003eWGCNA:\u0026nbsp;Weighted correlation network analysis;\u003c/p\u003e\n\u003cp\u003eIAA: Indole-3-acetic acid ;\u003c/p\u003e\n\u003cp\u003eIPA:\u0026nbsp;indole-3-pyruvic acid\u0026nbsp;;\u003c/p\u003e\n\u003cp\u003eSBP:\u0026nbsp;Squamosa promoter binding protein;\u003c/p\u003e\n\u003cp\u003ePCA: Principal Component Analysis;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAP1\u003c/em\u003e:\u0026nbsp;APETALA1;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFULL\u003c/em\u003e: FRUITFULL;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHEM11\u003c/em\u003e:\u0026nbsp;glutamyl-tRNA reductase 1;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGSA\u003c/em\u003e: glutamate-1-semialdehyde 2,1-aminomutase;\u003c/p\u003e\n\u003cp\u003eHEM2: delta-aminolevulinic acid dehydratase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDCUP\u003c/em\u003e: Uroporphyrinogen decarboxylase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHEM4:\u0026nbsp;\u003c/em\u003europorphyrinogen-III synthase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHEM3\u003c/em\u003e: porphobilinogen deaminase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHEM6\u003c/em\u003e: oxygen-dependent coproporphyrinogen-III oxidase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePOOC\u003c/em\u003e: protoporphyrinogen oxidase 1;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCHLH\u003c/em\u003e: Magnesium-chelatase subunit ;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCHLD\u003c/em\u003e: magnesium-chelatase subunit ;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCHLI\u003c/em\u003e: magnesium-chelatase subunitI;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCHLM\u003c/em\u003e: magnesium protoporphyrin IX methyltransferase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCRD\u003c/em\u003e: dicarboxylate diiron protein;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePORA\u003c/em\u003e: protochlorophyllide oxidoreductase A;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDCVR\u003c/em\u003e: Divinyl chlorophyllide a 8-vinyl-reductase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCHLG\u003c/em\u003e: chlorophyll synthase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCHLP\u003c/em\u003e: geranylgeranyl reductase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCAO\u003c/em\u003e: chlorophyllide a oxygenase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSGR\u003c/em\u003e: STAY-GREEN protein ;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHCAR\u003c/em\u003e: coenzyme F420 hydrogenase family / dehydrogenase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNYC1\u003c/em\u003e: NAD:P-binding Rossmann-fold superfamily protein;\u003c/p\u003e\n\u003cp\u003ePPH: pheophytinase;\u003c/p\u003e\n\u003cp\u003eCHLH: pheophorbide a oxygenase, chloroplastic;\u003c/p\u003e\n\u003cp\u003eRCCR: accelerated cell death ;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTRPX\u003c/em\u003e: anthranilate synthase alpha subunit 2;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eASB1\u003c/em\u003e: anthranilate synthase beta subunit 1;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePAT1\u003c/em\u003e: scarecrow-like protein 21 isoform X2 ;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePAI1:\u0026nbsp;phosphoribosylanthranilate isomerase 1;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTRPC\u003c/em\u003e: Indole-3-glycerol phosphate synthase;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTRPA2\u003c/em\u003e: tryptophan synthase alpha chain isoform X1;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTRPB: hypothetical protein;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTAR1\u003c/em\u003e:\u0026nbsp;tryptophan aminotransferase related 1;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTIR1\u003c/em\u003e: TRANSPORT INHIBITOR RESPONSE 1-like ;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eYUC\u003c/em\u003e: Probable indole-3-pyruvate monooxygenase ;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAMI1\u003c/em\u003e: amidase 1; \u003cem\u003eALDO2\u003c/em\u003e:fructose-bisphosphate aldolase;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNCED2\u003c/em\u003e:\u0026nbsp;nine-cis-epoxycarotenoid dioxygenase 2;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNCED1\u003c/em\u003e: 9-cis-epoxycarotenoid dioxygenase;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eABA2\u003c/em\u003e:\u0026nbsp;NAD(P)-binding Rossmann-fold superfamily protein;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eALDO3\u003c/em\u003e: aldolase C.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated/analyzed during this study are included in this article and its supplementary files. The sequencing data associated with transcription profiles in this study have been deposited in the China National Center for Bioinformation database with accession number\u0026nbsp;\u003cem\u003eGSA\u003c/em\u003e:CRA006581( https://bigd.big.ac.cn/\u003cem\u003eGSA\u003c/em\u003e/browse/CRA006581\u003cu\u003e).\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted by Key project of Hunan Provincial Department of Education (22A0155); the Forest Bureau of Hunan Provence (XLKY2024); Hunan province education department project(22C1207, 22C1212).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.D.L., Y.N.P.,\u0026nbsp;and Q.H.Z collected the sample and conceived and designed the study\u0026nbsp;X.D.L., Y.W.M., Y.Q.H and\u0026nbsp;J.L\u0026nbsp;the Transcriptome analysis.\u0026nbsp;X.D.L., J.L., M.L.,\u0026nbsp;X.Y.Y.,\u0026nbsp;Y.L.L., and\u0026nbsp;F.X.C\u0026nbsp;wrote the manuscript.\u0026nbsp;All the authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlsamadany H. 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The Plant Cell, (7):7,2024-04-11.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bract development, Bougainvillea glabra, MADS-box, Squamosa promoter binding protein (SBP), Plant hormone signal transduction, Chlorophyll Metabolic Pathways","lastPublishedDoi":"10.21203/rs.3.rs-4275941/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4275941/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Bracts are important in ornamental plants and their developmental regulation is complex, but relatively little research has been done on them. In this study, physiological, biochemical and morphological changes in \u003cem\u003eBougainvillea glabra\u003c/em\u003eleaves, leaf buds and bracts during seven developmental periods were systematically investigated in \u003cem\u003eB. glabra \u003c/em\u003ebracts. Meanwhile, transcriptome data of\u003cem\u003e B. glabra\u003c/em\u003e bracts were obtained using PacBio and Illumina sequencing technologies, and key genes regulating their development were screened.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eScanning electron microscopy revealed that the bracts develop with a process of regression of hairs, changing colour from green to white; Transcriptome sequencing yielded 79,130,973 bp of transcript sequences, totalling 45,788 transcripts; Differential gene analysis revealed 50 expression patterns across seven developmental periods, with significant variability in transcription factors such as\u003cem\u003e BgAP1\u003c/em\u003e, \u003cem\u003eBgFULL\u003c/em\u003e, \u003cem\u003eBgCMB1, BgSPL16\u003c/em\u003e, \u003cem\u003eBgEIL1,\u003c/em\u003e and \u003cem\u003eBgBH305\u003c/em\u003e KEGG and GO analyses of growth and development concerning chlorophyll metabolism and hormone-conducting metabolic pathways; Key genes for chlorophyll metabolism include \u003cem\u003ePORA\u003c/em\u003e, \u003cem\u003eSGR\u003c/em\u003e, \u003cem\u003ePPH\u003c/em\u003e, \u003cem\u003ePAO \u003c/em\u003eand \u003cem\u003eRCCR\u003c/em\u003e; The growth hormone and abscisic acid signalling pathways involve 44 and 23 homologous genes, and co-expression network analyses revealed that the screened genes \u003cem\u003eBgAPRR5\u003c/em\u003e and \u003cem\u003eBgEXLA1\u003c/em\u003e are involved in the regulation of bract development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e These findings promote the understanding of the molecular mechanism of plant bract development, as well as provide important guidance for the molecular regulation and genetic improvement of the growth and development of ornamental plants, mainly ornamental bracts.\u003c/p\u003e","manuscriptTitle":"Transcriptome Profiling and Gene Network Analysis Revealed Regulatory Mechanisms of Bract Development in Bougainvillea glabra","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-26 09:00:16","doi":"10.21203/rs.3.rs-4275941/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-06T07:34:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-03T09:28:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-01T09:15:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b68b649d-2413-4b99-b65b-6ffa83169f15","date":"2024-05-01T08:49:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-29T04:04:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223b09cb-83bd-4ba8-9782-42e2739e2575","date":"2024-04-24T10:27:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37bedcfb-4cf4-4534-89f5-daf8acada304","date":"2024-04-23T15:13:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-23T14:54:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-23T09:41:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-23T09:41:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-04-16T12:12:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5a973bfb-3e36-458e-8776-ce5254004f7e","owner":[],"postedDate":"April 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-04T09:11:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-26 09:00:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4275941","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4275941","identity":"rs-4275941","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}