Flower bud differentiation of ‘Red Globe’ grape under red-blue light combination. Before the transcriptome study, we analyzed the floral bud development process in morphological analysis (Fig. 1). In the first stage (April 30), the budding body was flat green; the growth point was conical, the bud scale was not lignification, the T1 branch diameter was less than S1, two groups of flower buds were information of Anlagen or uncommitted primordia stage (Supplementary Figure S1). In the second stage (June 30), the buds expanded, green, and increased. The branches of S2 began to turn from green to brown. Some buds of T2 and S2 were at the stage of differentiation of Anlagen to form inflorescence primordia. In the third stage (July 30), the buds became plump and brown. 70% of the flower buds in T3 were at the stage of differentiation of Anlagen to form inflorescence primordia, while the buds in S3 reached 90%. S4 stage (September 15), bud hard, dark brown, scales semi-lignified, T4 and S4 bud all in the differentiation of Anlagen to form inflorescence primordia stage.
Changes of hormone content during flower bud development. Flower bud differentiation is closely related to hormones. Therefore, the contents of IAA, CKs, GAs, and ABA in flower buds of red-blue light combination and control at four stages were determined by HPLC. HPLC analysis showed that the contents of these hormones changed significantly in different flower bud differentiation stages. The IAA content in the control was the highest in the second stage (Fig. 2A) and then decreased with development. Interestingly, the IAA content in the red-blue combination light treatment was always higher than control, reaching the highest in the third and lowest in the fourth stages. As shown in Fig. 2B, the trend of CKs content in the two treatments was utterly different. The CKs content in the flower bud increased gradually under the control, and the highest in the second stage and the lowest in the fourth stage under the red-blue combination light treatment. GAs content fluctuated in the flower bud differentiation stage. The control was higher than the red-blue combination light in the first and the third stage of flower bud differentiation (Fig. 2C). The ABA content in flower bud differentiation under control was consistently lower than that under red-blue combination light treatment. In the third stage, the ABA content of control was the lowest, and that of red-blue combination light treatment was the highest, 24.6% higher than that of control (Fig. 2D).
Analysis of differentially expressed genes. We constructed a transcriptome sequencing library for the four stages of flower buds under two treatments, and 183.27 Gb clean reads were obtained. Clean reads of each sample were above 6.19 Gb, and the Q30 base percentage was above 93.28% (Supplementary Table S2). Sample PCA analysis shows that the samples collected in this study have a high identity (Supplementary Figure S2). The differentially expressed genes were identified by DESeq2 software, and the differentially expressed genes were found to have different expression trends in red-blue combination light treatment and control (Fig. 3A-C). Compared with T1, 2314 common DEGs and 611, 1367, and 3072 specific expression genes were found in grape buds identified in T2, T3, and T4. There were 2262, 3328, and 3342 upregulated genes and 1896, 3290, and 4310 down-regulated genes among T1 vs. T2, T1 vs. T3, and T1 vs. T4, respectively (Fig. 3D, G). Compared with S1, there were 1889 common DEGs and 591,1550,1613 unique expression genes in S2, S3, and S4. S1 vs. S2, S1 vs. S3, and S1 vs. S4, there were 1691, 2546, and 2116 up-regulated genes and 1736, 3131, and 3161 down-regulated genes (Fig. 3E, H). Analysis between T1 vs. S1, T2 vs. S2, T3 vs. S3, and T4 vs. S4 showed that 1214, 1292, 1744, and 1912 genes were up-regulated, and 819, 1263, 1983, and 674 genes were down-regulated at four different maturation stages (Fig. 3F, I). In the third stage, the up- and down-regulated genes increased significantly, indicating that these genes played a crucial regulatory role in the process of grape flower bud differentiation.
Annotation of DEGs. We performed GO functional annotation and KEGG pathway analysis of DEGs. Through GO functional annotation, 6129 genes were annotated as 50 functional branches of cell components, biological processes, and molecular functions. For all up- and down-regulated DEGs. The first ten annotated GO terms are binding (GO: 0005488), cell part (GO: 0044464), catalytic activity (GO: 0003824), cell process (GO: 0009987), metabolic process (GO: 0008152), membrane part (GO: 0044425), membrane (GO: 0016020), organelle (GO: 0043226), biological regulation (GO: 0065007) and response to stimulus (GO: 0050896). Among all the up-regulated DEGs, 1995, 1902, 1853, 1673, 1668, 1394, 1173, 1102, 773, and 441 annotated these 10 GO terms. In the down-regulated DEGs, the numbers were 1783, 1684, 1521, 1445, 1412, 1374, 1204, 953, 747 and 418, respectively (Supplementary Figure S3, Supplementary Table S3).
KEGG pathway analysis showed that the plant hormone signal transduction path-way (map04075) played an essential role in grape flower bud differentiation. The plant hormone signal transduction pathway was enriched in the four stages under red-blue combination light treatment and control in the down-regulated DEGs. In addition, other enrichment pathways include flavonoid biosynthesis (map00941), flavonoid and flavonol biosynthesis (map00944), circadian rhythm-plant (map04712), and glycine, serine, and threonine metabolism (map00260). The plant hormone signal transduction pathway was also significantly enriched in the four stages under red-blue combination light treatment and control in the up-regulated genes. In addition, the most enriched pathways are plant-pathogen interaction (map04626), and other enrichment pathways include MAPK signaling pathway-plant (map04016), glycerophospholipid metabolism (map00564), phenylpropanoid biosynthesis (map00940), and monoterpenoid biosynthesis (map00902) (Fig. 4, Supplementary Table S4). The results showed that grape flower bud differentiation was a very complex biological process. In addition to plant hormone signal transduction, MAPK signal transduction, phenylpropanoid biosynthesis, flavonoid biosynthesis, and circadian rhythm-plant are also involved in this process.
DEGs related to plant hormone signal transduction. Previous studies have shown that grape flower bud differentiation is closely related to plant hormone signaling. In this study, we found that DEGs in plant hormone signal transduction are mainly concentrated in auxin, cytokinin, gibberellin, and abscisic acid pathways (Fig. 5), 36 EDGs involved in these pathways include auxin pathway TIR1 (VIT_14s0030g0124), AUX/IAA (VIT_05s0020g01070, VIT_09s0002g05160, VIT_05s0020g04690, VIT_05s0049g01970), ARF (VIT_11s0016g00640), GH3 (VIT_07s0104g00800, VIT_03s0091g0031) and SAUR (VIT_19s0085g00010, VIT_09s0002g00670, VIT_15s0048g00530, VIT_01s0146g00180, VIT_03s0038g01150); cy-tokinin pathway CRE1 (VIT_12s0057g00690), B-ARR (VIT_17s0000g10100), A-ARR (VIT_08s0007g05390, VIT_17s0000g07580, VIT_01s0026g00940, VIT_13s0067g03510, VIT_18s0001g02540); gibberellin pathway GID2 (VIT_07s0129g01000), TF (VIT_07s0005g02510, VIT_14s0060g00260); abscisic acid pathway PYR/PYL (VIT_15s0046g01050), PP2C (VIT_16s0022g02210, VIT_06s0004g05460, VIT_16s0050g02680), SnRK2 (VIT_07s0197g00080, VIT_12s0035g00310) and ABF (VIT_18s0072g00470, VIT_03s0063g00310, VIT_18s0001g10450, VIT_04s0069g01150, VIT_12s0034g00110). Further analysis showed that the expression of TIR1, AUX/IAA, and ARF genes was relatively high in the early stage of flower bud differentiation, while the expression was down-regulated in the later stage of differentiation. The expression of GH3 was gradually increased in flower bud differentiation. In addition, the expression trend of the SAUR gene was utterly different between the red-blue combination light and control. The expression of the A-ARR gene was high at the early stage of flower bud differentiation and then decreased, while the expression trend was opposite under the red-blue combina-tion light. The expression trend of the TF gene in the control and red-blue combination light treatments was utterly different. The former was highly expressed at the early stage of flower bud differentiation and was low at the late stage, while the latter remained at a low throughout the flower bud differentiation stage. In the control, the expression of the PP2C gene increased with the process of flower bud differentiation, while in the red-blue combination light treatment, the expression decreased with the process of flower bud dif-ferentiation. The expression trends of the ABF gene in the two groups were different. The control's expression of GBF4 (VIT_18s0072g00470) and AI5L2 (VIT_04s0069g01150) re-mained at a low level, while the expression in the red-blue combination light treatment increased with the process of flower bud differentiation, reaching the highest in the third stage. AI5L7 (VIT_03s0063g00310) and AI5L5 (VIT_18s0001g10450) showed an upward trend in the control and gradually decreased under the red-blue combination light. The results showed that these signaling genes played an important role in grape flower bud differentiation.
qRT-PCR validates gene expression profiles. To validate our sequencing results, DEGs involved in plant hormone signal transduction, flavonoid biosynthesis and MAPK signal transduction pathways were randomly selected for qPCR analysis, including ARR4 (VIT_01s0026g00940), PIF3 (VIT_14s0060g00260), GBF4 (VIT_18s0072g00470), HST (VIT_09s0018g01190), C75A1 (VIT_06s0009g02840), CAMT (VIT_03s0063g00140), CML46 (VIT_14s0108g01000), YODA (VIT_02s0025g03850), M2K5 (VIT_09s0018g01820). qRT-PCR results showed that the expression patterns of these genes were consistent with those in RNA-seq data, with the correlation coefficient R = 0.82. These results showed that the gene expression pattern revealed by RNA-seq data was reliable and could be used for further analysis (Fig. 6).
Screening through WGCNA for the transcription factors regulating grape flower bud differentiation. In this study, a total of 24 samples from four stages of grape flower bud differentiation under red-blue combination light treatment and control were used for weighted WGCNA analysis (Supplementary Fig. 4S). Through dynamic genes changes in different developmental stages and correlation analysis between samples, possible transcription factors regulating flower bud differentiation were discussed. Firstly, the expression patterns of 25844 DEGs extracted from transcriptome sequencing were analyzed by WGCNA, and they were divided into 19 modules (Fig. 7A, Supplementary Table S5) according to the similarity of expression patterns. These modules can be divided into two main branches (one for two modules, the other for 17 modules) (Fig. 7B). The correlation between the expression patterns of each module and different stages of grape flower bud differentiation was analyzed. The results showed that the modules ‘red’, ‘green’ and ‘magenta’ were highly correlated with the third stage of the fastest flower bud differentiation under red-blue combined light treatment, indicating that these modules were closely related to the flower bud differentiation of grapes (Fig. 7C). A total of 2373 genes were found in these modules, and the gene association networks of 2373 genes were constructed. According to the degree of connection, the first 50 genes are considered Hub genes. Among these 50 Hub genes, seven transcription factors were found, including ERF family EF110 (VIT_18s0072g00260), ABR1 (VIT_07s0031g01980), bHLH family BH025 (VIT_00s0824g00020), BH025 (VIT_00s0927g00010), WRKY family WRK48 (VIT_05s0077g00730), and CAMTA3 (VIT_07s0141g00250). NF-X1family NFXL2 (VIT_13s0067g00920) and bZIP family GBF4 (VIT_18s0072g00470) (Supplementary Figure S5, Supplementary Table S6). As highly connected Hub genes, these transcription factors may regulate flower bud differentiation of grape.
The bZIP family genes are related to the ABA signal. In order to further analyze the transcription factors regulated by the GBF4 gene in the gibberellin pathway, the genes related to GBF4 in the ‘green’ module with edge weight (≥ 0.4) are selected for analysis (Fig. 8, Supplementary Table S7). It was found that 42 genes were regulated by GBF4 (VIT_18s0072g00470), including hub transcription factor WRKY family WRK48 (VIT_05s0077g00730), ERF family EF110 (VIT_18s0072g00260), ABR1 (VIT_07s0031g01980), CAMTA family CAMTA3 (VIT_07s0141g00250), HSF family HSFA3 (VIT_08s0007g0390) and other non-Hub transcription factor genes have a certain regulatory relationship with the GBF4 gene.