Genome-wide identification and functional analysis of the GRAS gene family in Medicago lupulina L

Abstract

Background: Transcription factors encoded by GRAS genes play pivotal roles in a wide range of biological processes, including signal transduction, plant growth and development, and responses to various abiotic and biotic stresses. Nevertheless, to date, the GRAS gene family has not been previously identified or analyzed in Medicago lupulina L.

Results: In this study, bioinformatic approaches were used to analyze genomic data of M. lupulina obtained from our laboratory, resulting in the identification of 52 GRAS genes (designated as MlGRASs). We predicted and analyzed the physicochemical properties, subcellular localization, protein structures, phylogenetic relationships, gene structures, and sequence characteristics of these genes. Phylogenetic analysis showed that 52 MlGRASs were categorized into eight different subfamilies: LILIUM LONGIFLORUM SCR-LIKE (LISCL), SHORT ROOT (SHR), PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1), SCARECROW-LIKE 3 (SCL3), DELLA, HAIRY MERISTEM (HAM), LATERAL SUPPRESSOR (LS) and SCARECROW (SCR). Notably, we identified 10 M. lupulina GRAS genes belonging to the PAT1 subfamily. Experimental results from RT-qPCR analysis demonstrated that MlPAT1s genes were expressed in leaves, stems and roots of M. lupulina. Moreover, in the graft union, the expression levels of most MlPAT1s were higher than those in the wild type M. lupulina. To further predict the potential functions of MlPAT1s genes, we performed molecular cloning and transcriptional activation assays on MlGRAS3/31/35/62/65. The transcriptional activation assay revealed that 35 S:GFP-MlGRAS31/35/62/65 positively regulated the expression of MlDOF3.4, a gene implicated in cell proliferation and regeneration.

Conclusions: This study provided a theoretical framework for further investigation into the roles of PAT1 proteins in M. lupulina.

Keywords: GRAS gene; Medicago lupulina L; PAT1.

Fig. 1

The phylogenetic tree of GRAS protein in Arabidopsis thaliana, Medicago truncatula and Medicago lupulina. AtGRAS, MtGRAS and MlGRAS protein sequences were aligned by MEGA v.11.0 with the ClustalW method, and the tree was performed with the Neighbor-Joining (NJ) method. Different colors represent different subfamilies

Fig. 2

Phylogenetic tree and distribution of conserved motifs of the 52 MlGRAS proteins. 52 MlGRAS protein sequences were aligned by MEGA v.11.0 with the ClustalW method, and the tree was performed with the Neighbor-Joining (NJ) method. The conserved motifs within the MlGRAS proteins were identified using the MEME suite. The 20 unique MEME-motifs are presented in boxes of various colors. The scale at the bottom can be utilized to approximate the length of the protein

Fig. 3

The phylogeny tree of AtPAT1, MtPAT1 and MlPAT1. AtPAT1, MtPAT1 and MlPAT1 protein sequences were aligned by MEGA v11.0 with the ClustalW method, and the tree was performed with the Neighbor-Joining (NJ) method. The phylogenetic tree was divided into seven groups based on the clustering relationships among the AtPAT1, MtPAT1 and MlPAT1 proteins, which were labeled as Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, and Group 7. Different colors represent different groups

Fig. 4

Alignment of the 10 MlGRAS protein homeodomain. The alignment was conducted utilizing the ClustalW alignment within the MEGA11 software, and the results were visualized employing GeneDoc. The highly conserved region of the GRAS proteins can be categorized into five distinct motifs: LHR I, VHIID, LHR II, PFYRE, RVER, and SAW, as depicted above. * indicates the separation of every twenty amino acid residues

Fig. 5

Protein motif and conserved domain of PAT1 in M. lupulina. (A) Conserved motifs in M. lupulina PAT1 proteins identified by MEME. Each motif is indicated by a specific color. 52 MlGRAS protein sequences were aligned by MEGA v11.0 with the ClustalW method, and the tree was performed with the Neighbor-Joining (NJ) method. The conserved motifs within the MlGRAS proteins were identified using the MEME suite. The 10 unique MEME-motifs are presented in boxes of various colors. The scale at the bottom can be utilized to approximate the length of the protein. (B) Prediction of three-dimensional conformation of the conserved domain of M. lupulina PAT1 proteins through SWISS-MODEL

Fig. 6

The expression levels of PAT1 subfamily members in leaf, stem and root tissues of M. lupulina using quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis. Error bars represent the standard deviations from three biological replicates. The reference gene used in qRT-qPCR is MlACTIN

Fig. 7

The expression levels of PAT1 subfamily members in WT and Graft Union of M. lupulina using quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis. Error bars represent the standard deviations from three biological replicates. The reference gene used in RT-qPCR is MlACTIN

Fig. 8

The cis-acting element analysis of the putative promoter of 10 MlPAT1 genes. The distribution of cis-acting elements within the 2000 bp upstream regions of MlPAT1 genes is depicted. The 16 unique cis-acting elements are presented in boxes of various colors

Fig. 9

Localization of MlPAT1s in tobacco cells. The MlGRAS3/31/35/62/653/31/35/62/65 proteins fused with green fluorescent protein (GFP) were transiently co-expressed in tobacco leaf cells to observe subcellular localization through laser confocal microscopy. Co-localization of GFP and DAPI fluorescence signals was assessed in Merged pictures. DAPI: DAPI fluorescence field, blue represents DAPI fluorescence signal; GFP: GFP fluorescence field, green represents GFP fluorescence signal; A Bright Field; Merged: compound field; Scale bar = 20 μm

Fig. 10

The dual-luciferase assay verified the relationship between MlGRAS3/31/35/62/65 and Pro-MlFOD3.4. (A) LUC fluorescence imaging. The promoter of MlDOF3.4 gene was fused upstream of the luciferase gene. The images of infected tobacco leaves were captured by Tanon-5200 Chemiluminescent Imaging System. (B) Measurement of relative luciferase activities in tobacco leaves. The 35 S:GFP empty vector was used as the control. (A) and (B) showed that 35 S:GFP-MlGRAS31/35/62/65 activate MlDOF3.4. The mean ± standard deviation (SD) from three replicates is shown. Asterisks indicate significant differences (*P < 0.05 by t-test)