Integration of small RNAs and transcriptome sequencing uncovers a complex regulatory network during vernalization and heading stages of orchardgrass (Dactylis glomerata L.)

Abstract

Background: Flowering is a critical reproductive process in higher plants. Timing of optimal flowering depends upon the coordination among seasonal environmental cues. For cool season grasses, such as Dactylis glomerata, vernalization induced by low temperature provides competence to initiate flowering after prolonged cold. We combined analyses of the transcriptome and microRNAs (miRNAs) to generate a comprehensive resource for regulatory miRNAs and their target circuits during vernalization and heading stages.

Results: A total of 3,846 differentially expressed genes (DEGs) and 69 differentially expressed miRNAs were identified across five flowering stages. The expression of miR395, miR530, miR167, miR396, miR528, novel_42, novel_72, novel_107, and novel_123 demonstrated significant variations during vernalization. These miRNA targeted genes were involved in phytohormones, transmembrane transport, and plant morphogenesis in response to vernalization. The expression patterns of DEGs related to plant hormones, stress responses, energy metabolism, and signal transduction changed significantly in the transition from vegetative to reproductive phases.

Conclusions: Five hub genes, c136110_g1 (BRI1), c131375_g1 (BZR1), c133350_g1 (VRN1), c139830_g1 (VIN3), and c125792_g2 (FT), might play central roles in vernalization response. Our comprehensive analyses have provided a useful platform for investigating consecutive transcriptional and post-transcriptional regulation of critical phases in D. glomerata and provided insights into the genetic engineering of flowering-control in cereal crops.

Keywords: Dactylis glomerata; Flowering; Orchardgrass; Transcriptome; Vernalization-response; miRNA.

Fig. 1

Summary of differentially expressed miRNAs and genes. a Differential expressed miRNAs in five comparisons. b Differential expressed genes in five comparisons. The abscissa represents the different stages, ordinate represents the gene number

Fig. 2

A combined view of expressions levels between differentially expressed miRNAs and their target genes in Dactylis glomerata L. at five developmental stages. a The differential expression of miRNAs and (b) their targets. c The expression profile of miR156, miR172, SPL family and AP2 family genes. d The expression profile of miR396 and GRF family genes. e The expression profile of miR160, miR167 and ARF family genes. f The expression profile of miR398, HSFs and HSPs family genes. The horizontal axis showed the different developmental stages and the vertical axis showed the miRNAs and genes. The separate heatmap labeled by “Tem” indicated the change of temperature

Fig. 3

Verification by qRT-PCR of DEGs and differential expressed miRNAs. GAPDH and U6 were used as reference genes. The expression levels of each mRNA and miRNA were normalized by comparison with their expression with BV stage. The abscissa represents the different stages, ordinate represents the relative expression. The bar with oblique stripes represents the relative expression base on qRT-PCR results and the bar with faillette base on sequencing results. Figure a-h indicated the expression of eight DEGs and fig. i-p indicated 8 differential expressed miRNAs, the bottom title represents the gene and miRNA name, respectively

Fig. 4

Relationships of changes in gene expression based on transcriptome sequencing. AV-VE-up, up-regulated genes at after vernalization stage compared with vernalization stage. AV-VE-down, down-regulated genes at after vernalization stage compared with vernalization stage. VE-BV-up, up-regulated genes at vernalization stage compared with before vernalization stage. VE-BV-down, down-regulated genes at vernalization stage compared with before vernalization stage

Fig. 5

The floral primordium and young inflorescence of Dactylis glomerata after vernalization stimulation. a and (b) The floral primordium. c The young inflorescence. Arrows in (a) and (b) point to the floral primordium; arrows in (c) point to the young inflorescence

Fig. 6

The coexpression subnetwork of BRI1, BZR1, VRN1, VIN3 and FT. The rectangular frame indicated the different functional categories. The dotted arrow showed the potential miRNA-target regulation

Fig. 7

Analysis of gene expression in plant hormones pathways from growth stages BV to HT. a The putative network between GA, ABA and BR pathway. Line with arrows indicate positive regulation and line with blunt end indicated negative regulation. Dashed indicated the potential regulation. b The heatmap showed the gene expression involved in the network