Genome-wide identification of growth-regulating factor transcription factor family related to leaf and stem development in alfalfa

Abstract

Growth-regulating factors (GRFs) play crucial roles in plant growth and stress response. To date, there have been no reports of the analysis and identification of the GRF transcription factor family in alfalfa. In this study, we identified 27 GRF family members from alfalfa (Medicago sativa L.) "Xinjiang Daye", and analyzed their physicochemical properties. Based on phylogenetic analysis, these MsGRFs were divided into five subgroups, each with a similar gene structure and conserved motifs. MsGRFs genes are distributed on 23 chromosomes, and all contain QLQ and WRC conserved domains. The results of the collinearity analysis showed that all MsGRFs are involved in gene duplication, including multiple whole-genome duplication or segmental duplication and a set of tandem duplication, indicating that large-scale duplication is important for the expansion of the GRF family in alfalfa. Several hormone-related and stress-related cis-acting elements have been found in the promoter regions of MsGRFs. Some MsGRFs were highly expressed in young leaves and stems, and their expression decreased during development. In addition, the leaf size of different varieties was found to vary, and MsGRF1 to 4, MsGRF18 to 20, and MsGRF22 to 23 were differentially expressed in large and small leaf alfalfa varieties, suggesting that they are critical in the regulation of leaf size. The results of this study can benefit further exploration of the regulatory functions of MsGRFs in growth and development, and can identify candidate genes that control leaf size development.

Keywords: alfalfa; expression profile; gene family; growth-regulating factor; leaf size.

FIGURE 1

Phylogenetic analysis of growth-regulating factors (GRFs) from Medicago sativa L. (Ms), Glycine max (Gm) and Arabidopsis thaliana (At). MEGA 7.0 software was employed to construct a neighbor-joining phylogenetic tree with 1,000 bootstrap replications. Subgroups are highlighted with different colors.

FIGURE 2

Analysis on phylogenetic relationships, motifs, and gene structure of growth-regulating factor genes from Medicago sativa. (A) Phylogenetic tree of 27 MsGRFs in alfalfa. (B) Conserved motif arrangements of MsGRFs. The motifs are indicated in different colored boxes with different numbers. Motifs 1 and 2 represent WRC and QLQ domains, respectively. (C) Exon-intron organizations of MsGRFs. Blue boxes indicate exons; black lines indicate introns.

FIGURE 3

The distribution of MsGRFs on alfalfa chromosomes. The green bars represent each chromosome, and the black lines label the position of each MsGRF gene.

FIGURE 4

Synteny analysis of MsGRFs genes in alfalfa. Red lines indicate the replicated MsGRFs gene pairs in alfalfa.

FIGURE 5

The cis-acting element contained in the 2 kb promoter sequence of the MsGRF gene. Different cis-elements are indicated by different colored rectangles and placed in the matching position on the promoter.

FIGURE 6

Expression profiling of MsGRFs. (A) Expression profiles of MsGRFs at different developmental stages of stem internodes. Each stem internode starting from the apex is used as a developmental stage, labeled as Stem-1 to 8. (B) Expression profiles of MsGRFs at different developmental stages of leaves clustered into A to F six clades. L1 to L4 indicates the leaf development according to leaf position, and the first leaf that has not fully unfolded is regarded as the first stage of leaf development (L1). (C,D) qRT-PCR quantification of gene expression levels of selected MsGRF genes from (A,B) displayed in a column chart. The different letters (a, b, c, etc.) indicate the significant difference at P < 0.05 by Student’s t-test analysis.

FIGURE 7

Leaf morphology and lower epidermal cells observed under microscope. (A) Morphological observation of large (X, “Xinjiang Daye”) and small (N, “Nei 1 × Nei 2”) leaves. Scale bar, 1 cm. (B) Epidermal cells of large (X, “Xinjiang Daye”) and small (N, “Nei 1 × Nei 2”) leaves under microscope. Scale bar, 50 μm. (C) The average area of a single epidermal cell in the leaf of “Xinjiang Daye” and “Nei 1 × Nei 2”. (D) Estimates of the average total cells number in a single leaf of “Xinjiang Daye” and “Nei 1 × Nei 2”. The letters (a, b) indicate the significant difference at P < 0.05 by Student’s t-test. The L4 stage leaves were used for the observation.

FIGURE 8

Quantification of the expression levels of selected MsGRFs in the leaves of X, “Xinjiang Daye” and N, “Nei 1 × Nei 2” using qRT-PCR. Vertical bars indicate standard deviation. The asterisk (*) indicates the significant difference at P < 0.05 by Student’s t-test.