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 PORA, 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 PORA, 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, BgAPRR5 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).