Morphology analysis of petal colour transitions
The petal colour of every single flower was transformed continuously from green to white and then to yellow during flower development in L. japonica. Early in the development of floral buds, primary buds with green petals grew to approximately 3.5 cm in length (Fig. 1a). At the early stage of anthesis, the petals turned from green to white (Fig. 1b). Then, the petals gradually transformed to yellow from white before the withering stage (Fig. 1c). During petal colour transitions, petals at the green bud, white flower and yellow flower stages were selected. The changes in the colour index of GB_Pe, WF_Pe and YF_Pe were significantly different (Table S2). The values of redness (a*) in GB_Pe, WF_Pe and YF_Pe were -12.36, -0.58 and 1.25, respectively. The parameter lightness (L*) in WF_Pe was 82.40, which was higher than that in GB_Pe and YF_Pe. The index of yellowness (b*) in YF_Pe was the highest (42.55).
Carotenoid and anthocyanin accumulation in L. japonica petals at various stages
To obtain an accurate understanding of carotenoid accumulation, carotenoid profiling was analysed in L. japonica petals using LC-MS/MS during petal colour transitions. A total of 13 carotenoids were detected from GB_Pe, WF_Pe and YF_Pe (Table 1). The major carotenoids of GB_Pe were lutein, violaxanthin, neoxanthin and zeaxanthin. The lutein content significantly decreased from 39.30 μg/g in GB_Pe to 1.46 μg/g in WF_Pe and slightly decreased to 1.09 μg/g in YF_Pe. The violaxanthin content first significantly decreased from 18.87 μg/g in GB_Pe to 0.60 μg/g in WF_Pe and then drastically increased to 43.81 μg/g in YF_Pe. The trends of neoxanthin and zeaxanthin were similar to those of violaxanthin. Compared with GB_Pe and WF_Pe, most carotenoids, including α-carotene, antheraxanthin, lycopene, zeaxanthin, violaxanthin, γ-carotene, neoxanthin, β-carotene, β-cryptoxanthin and apocarotenal, were significantly upregulated in YF_Pe. Among them, violaxanthin (43.81 μg/g), zeaxanthin (27.45 μg/g), α-carotene (20.84 μg/g) and γ-carotene (19.97 μg/g) were the major carotenoid compounds in YF_Pe. Except for zeaxanthin, the contents of the remaining 12 carotenoids were all detected at lower levels in WF_Pe.
To better understand the content changes of anthocyanins, quantitative analysis of anthocyanins was further performed by LC-MS/MS technology. A total of 10 anthocyanins were identified from GB_Pe, WF_Pe and YF_Pe (Table 2). In GB_Pe, delphinidin was the predominant component of anthocyanins. Specifically, delphinidin was reduced by 3.55- and 2.85-fold in WF_Pe and YF_Pe, respectively, compared with GB_Pe (Table S3). In contrast, pelargonidin was not detected in GB_Pe but at higher levels in WF_Pe and YF_Pe. In the YF_Pe vs GB_Pe comparison, pelargonidin and cyanidin O-syringic acid significantly increased, while delphinidin, cyanidin O-malonyl-malonylhexoside and delphin chloride significantly decreased. Compared with WF_Pe, the contents of pelargonidin and cyanidin were increased by 2.11- and 2.36-fold in YF_Pe, respectively. However, cyanidin O-malonyl-malonylhexoside and delphin chloride were not detected in YF_Pe.
Effects of endogenous hormones during petal color transitions
To obtain the changes in endogenous hormones, the concentrations of IAA, ZR, GA, BR, MeJA and ABA were analysed. During petal colour transitions, the concentrations of IAA, ZR, GA, BR and MeJA decreased, but the content of ABA increased (Fig. 2). The IAA concentration decreased significantly from 717.3 ng·g-1 FW (GB_Pe) to 191.0 ng·g-1 FW (WF_Pe) and then to 118.8 ng·g-1 FW (YF_Pe). The ZR and GA concentrations both first decreased significantly from GB_Pe to WF_Pe and then remained stable from WF_Pe to YF_Pe. The BR concentration was highest in GB_Pe. From GB_Pe to YF_Pe, the BR concentration decreased significantly from 9.2 ng·g-1 FW in GB_Pe to 7.3 ng·g-1 FW in WF_Pe and then increased slightly to 8.3 ng·g-1 FW in YF_Pe. The level of MeJA first decreased significantly from GB_Pe to WF_Pe, reaching the lowest level, and then slightly increased from WF_Pe to YF_Pe. However, the ABA concentration increased significantly from 98.0 ng·g-1 FW to 205.2 ng·g-1 FW from GB_Pe to YF_Pe (Fig. 2).
Sequencing, de novo assembly and annotation
To identify key candidate genes for petal colour transitions, RNA sequencing was carried out from GB_Pe, WF_Pe and YF_Pe. Nine cDNA libraries were sequenced and 448 565 884 raw reads were generated. After data filtering, 408 576 816 (91.1%) clean reads were produced, and the Q30 values were greater than 96.7%. For each sample, clean reads were obtained from 6.6 to 7.1 Gb (Table S4). A total of 69 946 unigenes were generated with an average length of 871 bp and an N50 of 1 636 bp (Table S5). Most unigenes (96.6%) were generated from 200 to 3 200 bp in length, and 2 383 (3.4%) unigenes were more than 3 200 bp in length (Fig. S1).
A total of 34 068 assembled unigenes were annotated (Table S6). Based on sequence similarity, 22 662 (32.4%) unigenes were enriched into three groups (biological process, cellular component and molecular function) based on GO term analysis (Fig. S2). The biological processes were mainly focused on ‘cellular process’ and ‘metabolic process’. The cellular components were mainly involved in ‘cell part’. The molecular functions were mainly classified into ‘binding’ and ‘catalytic activity’. KEGG term analysis was used to identify the functional classifications of the unigenes. A total of 9 309 (13.31%) unigenes were enriched in 32 KEGG pathway groups, of which ‘signal transduction’ represented the largest group, followed by ‘carbohydrate metabolism’, ‘translation’ and ‘folding, sorting and degradation’ (Fig. S3).
Identification and analysis of DEGs
To detect alterations in gene expression, transcriptomic analyses of WF_Pe vs GB_Pe, YF_Pe vs WF_Pe and YF_Pe vs GB_Pe were carried out to identify the key DEGs during petal colour transition in L. japonica (Fig. S4). A total of 29 679 DEGs were identified based on a 2-fold change at P < 0.05 (Fig. S4a). For each comparison, the numbers of total DEGs, upregulated DEGs and downregulated DEGs were counted, as shown in Fig. S4b.
All 29 679 identified DEGs were further classified into 8 clusters on the basis of expression alterations during petal colour transition (Fig. 3a). A total of 3 470 DEGs were classified into two profiles based on expression changes across the three developmental stages: expression stable and then increased (profile 4) and expression stable and then decreased (profile 3). The opposite change patterns of gene expression during the petal colour transition from white to yellow suggest a tight linkage of these genes with petal colour transition in L. japonica.
GO enrichment analysis was further performed to investigate the biological functions of these 1 897 DEGs (RPKM > 1 in at least one sample from the 3 470 DEGs) that showed higher or lower expression in YF_Pe. The hormone-mediated signalling pathway was significantly enriched in the biological process subcategory (Fig. 3b). DEGs involved in hormone-mediated signalling pathways, such as small auxin-up RNA (SAUR) and PYRABACTIN RESISTANCE1-like (PYL), were significantly differentially expressed between yellow petals and non-yellow petals and seemed relevant to the goal of this study.
Analysis of DEGs involved in hormone-mediated signalling pathways
GO enrichment analysis showed that DEGs were mainly enriched in hormone-mediated signalling pathways. To better investigate hormonal regulation in the colour transitions, we analysed the 67 DEGs (>1 RPKM) that were enriched in the signalling pathways of auxin, cytokinin, GA, BR, jasmonic acid (JA), ABA and ethylene in YF_Pe vs GB_Pe and YF_Pe vs WF_Pe (Fig. S5 and Table S7).
In the auxin signalling pathway, 15 DEGs were identified, of which the AUX1, TIR1, ARF and SAUR genes were significantly downregulated from GB_Pe to YF_Pe, while three IAAs were upregulated at WF_Pe (Fig. S5a). A total of 18 DEGs were enriched in the cytokine signalling pathway, including HKs, HPs, type-B RRs and type-A RRs. All of these DEGs were downregulated from GB_Pe to WF_Pe and YF_Pe (Fig. S5b). Meanwhile, in the GA signalling pathway, GID1, GID2 and DELLA genes were identified and significantly downregulated in YF_Pe (Fig. S5c). In the BR signalling pathway, 13 DEGs were identified, most of which were first downregulated and then upregulated in the transition. Specifically, the expression of BRI1, BSK, BZR1_2, CYCD3 and TCH4 was significantly higher in YF_Pe than in WF_Pe (Fig. S5d). Four DEGs were enriched in the JA signalling pathway, and their expression levels were higher in GB_Pe than in WF_Pe and YF_Pe (Fig. S5e). Furthermore, JAR1, COI-1 and MYC2 were expressed at higher levels in YF_Pe than in WF_Pe, while JAZ was expressed at lower levels in YF_Pe than in WF_Pe. However, seven DEGs were identified in the ABA signalling pathway, including PYL, PP2C, SNRK2 and ABF, of which PYLs and SNRK2 were significantly upregulated in YF_Pe (Fig. S5f). In the ethylene signalling pathway, five DEGs were identified, of which EIN3, ERS and ERF1 were significantly upregulated in YF_Pe (Fig. S5g).
Analysis of pigment-related DEGs during petal colour transitions
To investigate the pathways of pigment synthesis/degradation during the transitions, the expression levels of carotenoid, anthocyanin and chlorophyll metabolism-related genes were analysed. A total of 49 DEGs (>1 RPKM) regulating carotenoid, anthocyanin and chlorophyll metabolism were identified and significantly differentially expressed between yellow petals and non-yellow petals (GB_Pe or WF_Pe) (Fig. 4 and Table S7).
In the carotenoid biosynthesis pathway, PSY1, PDS1, ZDS1 and ZDS2 were significantly upregulated in YF_Pe. However, three carotenoid degradation-related genes, carotenoid cleavage dioxygenase 4 (CCD4), CCD7 and abscisic-aldehyde oxidase 3 (AAO3), were significantly downregulated in YF_Pe (Fig. 4a). Meanwhile, the expression levels of chlorophyll metabolism-related genes showed significant differences. Among these genes, biosynthesis-related genes, including glutamyl-tRNA synthetase (GltX), protoporphyrinogen IX oxidase (PPO) and chlorophyll synthase (CHLG), were significantly upregulated in GB_Pe. However, pheophytinase (PPH), pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR) were significantly downregulated in GB_Pe (Fig. 4b).
In the basic upstream pathway of flavonoid/anthocyanin biosynthesis, some DEGs were identified in WF_Pe vs GB_Pe, YF_Pe vs WF_Pe and/or YF_Pe vs GB_Pe comparisons, such as phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-monooxygenase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3β-hydroxylase (F3H), flavonoid 3'-monooxygenase (F3’H), dihydroflavonol 4-reductase (DFR) and ANS. In the specific anthocyanin downstream branch, one anthocyanidin 3-O-glucosyltransferase (BZ1) gene and one anthocyanidin 3-O-glucoside 5-O-glucosyltransferase (UGT75C1) gene were identified as DEGs. The expression levels of ANS and BZ1 were significantly upregulated in YF_Pe. In contrast, some DEGs, including CHS2, DFRs and UGT75C1 ,were upregulated in GB_Pe. Although the expression levels of four DFRs and one UGT75C1 first declined in white petals, they then slightly rose from WF_Pe to YF_Pe (Fig. 4c).
Validation of the expression analysis of key pigment-related genes
A total of ten pigment-related unigenes were randomly selected and identified by RT-qPCR. The expression patterns of these DEGs corresponded well with the RPKM values obtained by RNAseq (Fig. 5). Pearson correlation analysis showed high correlation coefficients between the RNA-seq and RT-qPCR data, suggesting that the sequencing data are reliable.