Identification and characterization of regulatory pathways involved in early flowering in the new leaves of alfalfa (Medicago sativa L.) by transcriptome analysis

Abstract

Background: Alfalfa (Medicago sativa L.) is a perennial legume extensively planted throughout the world as a high nutritive value livestock forage. Flowering time is an important agronomic trait that contributes to the production of alfalfa hay and seeds. However, the underlying molecular mechanisms of flowering time regulation in alfalfa are not well understood.

Results: In this study, an early-flowering alfalfa genotype 80 and a late-flowering alfalfa genotype 195 were characterized for the flowering phenotype. Our analysis revealed that the lower jasmonate (JA) content in new leaves and the downregulation of JA biosynthetic genes (i.e. lipoxygenase, the 12-oxophytodienoate reductase-like protein, and salicylic acid carboxyl methyltransferase) may play essential roles in the early-flowering phenotype of genotype 80. Further research indicated that genes encode pathogenesis-related proteins [e.g. leucine rich repeat (LRR) family proteins, receptor-like proteins, and toll-interleukin-like receptor (TIR)-nucleotide-binding site (NBS)-LRR class proteins] and members of the signaling receptor kinase family [LRR proteins, kinases domain of unknown function 26 (DUF26) and wheat leucine-rich repeat receptor-like kinase10 (LRK10)-like kinases] are related to early flowering in alfalfa. Additionally, those involved in secondary metabolism (2-oxoglutarate/Fe (II)-dependent dioxygenases and UDP-glycosyltransferase) and the proteasome degradation pathway [really interesting new gene (RING)/U-box superfamily proteins and F-box family proteins] are also related to early flowering in alfalfa.

Conclusions: Integrated phenotypical, physiological, and transcriptomic analyses demonstrate that hormone biosynthesis and signaling pathways, pathogenesis-related genes, signaling receptor kinase family genes, secondary metabolism genes, and proteasome degradation pathway genes are responsible for the early flowering phenotype in alfalfa. This will provide new insights into future studies of flowering time in alfalfa and inform genetic improvement strategies for optimizing this important trait.

Keywords: Alfalfa; Flowering time; Hormone; New leaves; Transcriptome.

Fig. 1

Phenotypic characterization of genotypes 195 and 80 of alfalfa. a, The phenotype of genotype 195 and 80 plants grown in a controlled climate chamber at the time of sampling (25 days after plants). b and c, Tissues harvested for transcriptome analysis. ML, mature leaves; NL, new leaves including the apical meristem; FB, flower buds

Fig. 2

Contents of endogenous IAA (a), ABA (b), SA (c), and JA (d) in genotype 195 and 80 at the time of sampling. Different letters indicate significant differences among ML (mature leaves), NL (new leaves including the apical meristem), and FB (flower buds) based on the LSD test (P < 0.05). Data are shown as the mean ± standard deviation (SD) of three replicates

Fig. 3

The bar chart (a) and Venn diagram (b) of differentially expressed genes (DEGs) in genotypes 195 and 80

Fig. 4

MapMan display of the functional categories of DEGs between NL and ML in genotypes 195 (a) and 80 (b) upon the flowering of genotype 80. Squares represent DEGs; red and blue indicate up- and downregulated genes, respectively

Fig. 5

Transcript profiles of hormone-related genes in genotypes 195 and 80. The color scale indicates log2-transformed fold changes in gene expression levels in NL compared with ML, and in FB compared with NL or ML in genotypes 195 and 80. Red, blue, and gray denote upregulation, downregulation, and no change in expression, respectively

Fig. 6

Transcript profiles of pathogenesis-related genes in genotypes 80 and 195. The color scale indicates log2-transformed fold changes in expression levels of NL compared with ML, and FB compared with NL or ML in genotypes 195 and 80. Red, blue, and gray denote upregulation, downregulation, and no change in expression, respectively

Fig. 7

MapMan display of the coordinated changes in the expression levels of genes involved in the receptor kinase signaling pathway in genotypes 195 and 80 upon the flowering of genotype 80. Shown are DEGs between NL and ML in genotypes 195 (a) and 80 (b). Squares represent DEGs; red and blue indicate up- and downregulated genes, respectively

Fig. 8

MapMan display of coordinated changes in the expression levels of genes involved in the ubiquitin-dependent degradation pathway in genotypes 195 and 80 upon the flowering of genotype 80. Shown are DEGs between NL and ML in genotypes 195 (a) and 80 (b). Squares represent DEGs; red and blue indicate up- and downregulated genes, respectively

Fig. 9

The transcript profiles of genes involved in the ubiquitin-dependent degradation pathway in genotypes 80 and 195. The color scale indicates log2-transformed fold changes in gene expression levels in NL compared with ML and in FB compared with NL or ML in genotypes 195 and 80. Red, blue, and gray denote upregulation, downregulation, and no change in expression, respectively

Fig. 10

Comparison between the results of the qRT-PCR and RNA-seq analyses of selected DEGs. The color scale indicates log2-transformed fold changes in gene expression levels. Red, blue, and gray denote upregulation, downregulation, and no change in expression, respectively

Fig. 11

Diagram of a proposed regulatory network for early flowering in alfalfa