Comprehensive transcriptome analysis reveals distinct regulatory programs during vernalization and floral bud development of orchardgrass (Dactylis glomerata L.)

Abstract

Background: Vernalization and the transition from vegetative to reproductive growth involve multiple pathways, vital for controlling floral organ formation and flowering time. However, little transcription information is available about the mechanisms behind environmental adaption and growth regulation. Here, we used high-throughput sequencing to analyze the comprehensive transcriptome of Dactylis glomerata L. during six different growth periods.

Results: During vernalization, 4689 differentially expressed genes (DEGs) significantly increased in abundance, while 3841 decreased. Furthermore, 12,967 DEGs were identified during booting stage and flowering stage, including 7750 up-regulated and 5219 down-regulated DEGs. Pathway analysis indicated that transcripts related to circadian rhythm, photoperiod, photosynthesis, flavonoid biosynthesis, starch, and sucrose metabolism changed significantly at different stages. Coexpression and weighted correlation network analysis (WGCNA) analysis linked different stages to transcriptional changes and provided evidence of inner relation modules associated with signal transduction, stress responses, cell division, and hormonal transport.

Conclusions: We found enrichment in transcription factors (TFs) related to WRKY, NAC, AP2/EREBP, AUX/IAA, MADS-BOX, ABI3/VP1, bHLH, and the CCAAT family during vernalization and floral bud development. TFs expression patterns revealed intricate temporal variations, suggesting relatively separate regulatory programs of TF modules. Further study will unlock insights into the ability of the circadian rhythm and photoperiod to regulate vernalization and flowering time in perennial grass.

Keywords: Dactylis glomerata L.; Flowering regulation; High-throughput sequencing; Orchardgrass; Transcriptome; Vernalization.

Fig. 1

The morphological photographs of young inflorescence and stem. a Photographs of flower primordium after vernalization. b, c and d, the young inflorescence after flower primordium initiation for (b) 5 days (c) 10 days and (d) 3 weeks. e and g, Photographs of (g) stem and (e) transection of stem before young inflorescence formation. f and h, Photographs of (h) internodes and (f) transection of internodes after young inflorescence formation

Fig. 2

Transcriptional relationship between samples. a Number of up- and down-regulated genes in five pairwise sampling stages, including stage V_DON vs stage BV_DON (V vs BV), stage AV_DON vs stage V_DON (AV vs V), stage VG_DON vs stage AV_DON (VG vs AV), stage BH_DON vs stage VG_DON (BH vs VG), and stage H_DON vs stage BH_DON (H vs BH). b Principal component analysis based on all expressed genes, showing six distinct groups of samples: BV_DON_rep1, BV_DON_rep2, BV_DON_rep3 (light green circle); V_DON_rep1, V_DON_rep2, V_DON_rep3 (blue regular triangle); AV_DON_rep1, AV_DON_rep2, AV_DON_rep3 (brown cross); VG_DON_rep1, VG_DON_rep2, VG_DON_rep3 (purple cross); BH_DON_rep1, BH_DON_rep2, BH_DON_rep3 (khaki rhombus), H_DON_rep1, H_DON_rep2, H_DON_rep3 (green inverted triangle). c Cluster dendrogram, showing the global relationship between biological replicates. The y-axis shows the degree of variance. d Number of expressed unigenes and respective expression levels in each sample type, based on the FPKM of biological replicates. Sample labels are as follows: BV, before vernalization; V, vernalization; AV, after vernalization; VG, vegetative growth; BH, before heading; H, heading. DON refers to the orchardgrass cultivated varity DONATA (Registered No.398)

Fig. 3

Clusters expression associated with stages. The x-axis shows the sampling stages, y-axis shows the relative expression.Sample labels are as follows: BV, before vernalization; V, vernalization; AV, after vernalization; VG, vegetative growth; BH, before heading; H, heading

Fig. 4

WGCNA of genes at six stages. a Hierarchical cluster tree shows coexpression modules, identified via WGCNA. Each leaf in the tree represents one gene. Major tree branches constitute 14 modules labeled by different colors. b This heatmap shows the gene relative expression of different modules in six stages, the y-axis represents the relative expression of modules and the x-axis shows the different stages. c Visualization of the eigengene network represents the relationships among the modules and the clinical trait weight. The hierarchical clustering dendrogram of the eigengenes shows the relationships among the modules. Heatmap shows the correlation of different modules, and the deeper red color represents the higher correlation. Sample labels are as follows: BV, before vernalization; V, vernalization; AV, after vernalization; VG, vegetative growth; BH, before heading; H, heading

Fig. 5

Transcription factor profiling according to RNA-seq data. The heat map shows the expression of transcription factor families that are overrepresented among coexpression clusters. The y-axis represents transcription factor families: WRKY transcription factor families (a); NAC transcription factor families (b); AP2/EREBP transcription factor families (c); Alfin-like transcription factor families (d); AUX/IAA transcription factor families (e); MADS-BOX transcription factor families (f); bHLH transcription factor families (g); ABI3/VP1 transcription factor families (h); CCAAT transcription factor families (i), and the x-axis shows the different stages. Sample labels are as follows: BV, before vernalization; V, vernalization; AV, after vernalization; VG, vegetative growth; BH, before heading; H, heading

Fig. 6

Putative schematic network of bolting and flowering regulation in orchardgrass. Arrows indicate positive regulation. The heat map shows the relative expression of candidate genes in different stages, the y-axis represents identified candidate genes, and the x-axis shows the different stages. These identified candidate genes are involved in various flowering pathways according to previous reports. Sample labels are as follows: BV, before vernalization; V, vernalization; AV, after vernalization; VG, vegetative growth; BH, before heading; H, heading

Fig. 7

qRT-PCR validation. The abscissa represents the different sampling time, ordinate represents the expression. The bar with oblique stripes represents the relative expression base on qRT-PCR results and the bar with faillette base on RNA-seq results. The bottom title represents the different genes. Sample labels are as follows: BV, before vernalization; V, vernalization; AV, after vernalization; VG, vegetative growth; BH, before heading; H, heading