Genome-wide transcriptome profiling provides insights into floral bud development of summer-flowering Camellia azalea

Abstract

The transition from vegetative to reproductive growth in woody perennials involves pathways controlling flowering timing, bud dormancy and outgrowth in responses to seasonal cues. However little is known about the mechanism governing the adaptation of signaling pathways to environmental conditions in trees. Camellia azalea is a rare species in this genus flowering during summer, which provides a unique resource for floral timing breeding. Here we reported a comprehensive transcriptomics study to capture the global gene profiles during floral bud development in C. azalea. We examined the genome-wide gene expression between three developmental stages including floral bud initiation, floral organ differentiation and bud outgrowth, and identified nine co-expression clusters with distinctive patterns. Further, we identified the differential expressed genes (DEGs) during development and characterized the functional properties of DEGs by Gene Ontology analysis. We showed that transition from floral bud initiation to floral organ differentiation required changes of genes in flowering timing regulation, while transition to floral bud outgrowth was regulated by various pathways such as cold and light signaling, phytohormone pathways and plant metabolisms. Further analyses of dormancy associated MADS-box genes revealed that SVP- and AGL24- like genes displayed distinct expression patterns suggesting divergent roles during floral bud development.

Figure 1. Morphological analysis of floral bud development in C. azalea.

(a–b), apical buds at stage 1 (less than 4 mm) with no detectable changes to vegetative bud; (c–d), apical buds at stage 2 (5–7 mm) in which reproductive development was initiated. Arrows indicated fasciation of apical meristem suggesting the differentiation of floral organs. (e–f), apical bud at stage 3 in which floral buds began organ differentiation and outgrowth. White bar 50 μm; Black bar 200 μm.

Figure 2. Statistics of de novo assembly of transcriptome.

(a), Distribution of length of all contigs. (b), Distribution of length of all Unigenes. (c), The statistics of de novo assembly of transcriptome.

Figure 3. Global analysis of transcriptome datasets of biological replicates and samples.

(a), A dengrogram graph showing global relationships of samples. (b), A bar plot describing number of expressed transcripts after filtering. Samples from 1 to 9 stands for S1-rep1, S1-rep2, S1-rep3, S2-rep1, S2-rep2, S2-rep3, S3-rep1, S3-rep2, S3-rep3.

Figure 4. K-Means clustering of gene expression profiles.

(a), the centroids of nine clusters with different expression patterns. Numbers on the top are the number of genes for each cluster; and red numbers are cluster labels. (b), a heat map plot of nine clusters display the relative expression levels of centroids. Numbers on the right are corresponding to cluster labels in (a).

Figure 5. Analysis of DEGs between floral developmental stages.

(a), A venn diagram of number of unique and common DEGs between different stages. (b), A hierarchical clustering graph of DEGs found between S1–S3 and S2–S3 but not S1–S2. Upper panel showing the DEGs with highest expression levels at stage 3; lower panel showing two clusters with lowest expression levels at stage 3.

Figure 6. GO enrichment of DEGs between floral developmental stages.

(a), the scatterplot of enriched GO terms between stage 1 and stage 2; (b), the scatterplot of enriched GO terms between stage 1 and stage 3; (c), the scatterplot of enriched GO terms between stage 2 and stage 3.

Figure 7. Expression patterns of key flowering regulators.

Gene symbols were derived from TAIR and expression levels of putative orthologs were plotted. The mean values of RPKM of 15 orthologs of cry1, phyB, phyA, VIN3, SVP, AGL24, FVE, FT/TFL1, CO, FD, MAF/FLC, SOC1, AP1, SEP4, and DEF were plotted with standard deviations.

Figure 8. Phylogenetic and expression analyses of MADS-box genes.

(a), A phylogenetic tree containing 19 MADS-box genes from C. azalea and 58 MADS-box genes from Arabidopsis as well as two MADS-box genes from C. japonica. Cyan triangles indicate genes from C. azalea. The red clade indicates the dormancy associated MADS-box genes. Red circles and color squares are corresponding to floral regulators in Arabidopsis: AT5G10140-FLC/MAF, AT2G45660-SOC1, AT4G22950-AGL19, AT4G24540-AGL24, AT2G22540-SVP. (b), Expression patterns of MADS-box genes from C. azalea.