Genome-Wide Identification and Analysis of the WRKY Transcription Factor Family Associated with Leaf Senescence in Alfalfa

Abstract

Leaves are the most significant parts of forage crops such as alfalfa. Senescence is the terminal stage of leaf development and is controlled by an integrated myriad of endogenous signals and environmental stimuli. WRKY transcription factors (TFs) play essential roles in regulating leaf senescence; however, only a few studies on the analysis and identification of the WRKY TF family in Medicago Sativa have been reported. In this study, we identified 198 WRKY family members from the alfalfa (M. sativa L.) cultivar 'XinjiangDaye' using phylogenetic analysis and categorized them into three subfamilies, Groups I, II, and III, based on their structural characteristics. Group II members were further divided into five subclasses. In addition, several hormone- and stress-related cis-acting elements were identified in the promoter regions of MsWRKYs. Furthermore, 14 aging-related MsWRKYs genes from a previous transcriptome in our laboratory were selected for RT-qPCR validation of their expression patterns, and subsequently cloned for overexpression examination. Finally, MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141 were confirmed to cause leaf yellowing in Nicotiana benthaminana using a transient expression system. Our findings lay a groundwork for further studies on the mechanism of M. sativa leaf aging and for the creation of new germplasm resources.

Keywords: Medicago sativa; WRKY; expression profile; leaf senescence.

Figure 1

A neighbor-joining phylogenetic tree was constructed using MEGA11.0 software with 1000 boot-strap replications, by comparing WRKY TFs from M. sativa L. (Ms) and Arabidopsis thaliana (At). Various highlighted colors correspond to the different subgroups.

Figure 2

Analysis of phylogenetic relationships, motifs, and gene structure of WRKY TFs from M. sativa. (a) Phylogenetic tree of 198 MsWRKYs in M. sativa. The colors highlighted the different subgroups are same as that in Figure 1. (b) Conserved motif arrangements of MsWRKYs. The motifs are highlighted in various colored boxes. Motif 1 represents the WRKY domain. (c) Exon-intron organizations of MsWRKYs. Green boxes indicate exons; black lines indicate introns.

Figure 3

The 2 kb promoter sequences of the MsWRKY gene contain various cis-acting elements. Different colored rectangles indicate different cis-elements, positioned according to their locations within the promoters.

Figure 4

A heatmap displaying the RNA−Seq data for 198 MsWRKYs, with expression levels normalized by row using the Z−Scores algorithm. The color scale on the right of the heatmap shows relative expression, with the color gradient from blue to red indicating increased expression levels. X0 (top not fully unfolded leaf), X1 (top fully unfolded first leaf), X2 (top fully unfolded second leaf), X3 (top fully unfolded third leaf), X4 (bottom leaf with senescent symptom); D0, D1, D2, D4, D6 (leaves treated in the dark for 0, 1, 2, 4 and 6 days); S1, S2, S4, S6 (leaves treated with salt for 1, 2, 4 and 6 days). Fourteen genes selected for RT-qPCR were labeled with an asterisk.

Figure 5

RT-qPCR results of 14 MsWRKYs in the process of leaf senescence under (a) natural condition (X0, X1, X2, X3, X4 represent different stages of leaf development), (b) dark stress (D0, D1, D2, D4, D6 represent 0, 1, 2, 4, and 6 days of dark treatment), (c) salt stress (S1, S2, S4, S6 represent 1, 2, 4, and 6 days of the 150 mM NaCl treatment). The error bars indicate the standard deviation of three biological replicates. Relative expression was calculated using the 2^–ΔCT method. The data for gene expression are presented as the mean ± SD and were analyzed to detect significant differences by ANOVA using GraphPad Prism 8 (NS: not significant; * p < 0.05; ** p < 0.01) against D0 or X0.

Figure 6

Functional validation of selected MsWRKYs was conducted using an Agrobacterium-mediated transient expression assay. Symptoms of leaf senescence in representative Nicotiana benthamiana leaves appeared after infiltration with various constructs encoding MsWRKY5, MsWRKY66, MsWRKY92, MsWRKY141, MsSGR and an empty vector with YFP. Positive control: SGR. Negative control: empty vector with YFP. Bar = 1 cm.