Genome-Wide Identification of the GRAS Family Genes in Melilotus albus and Expression Analysis under Various Tissues and Abiotic Stresses

Abstract

The GRAS gene family is a plant-specific family of transcription factors, which play an important role in many metabolic pathways, such as plant growth and development and stress response. However, there is no report on the comprehensive study of the GRAS gene family of Melilotus albus. Here, we identified 55 MaGRAS genes, which were classified into 8 subfamilies by phylogenetic analysis, and unevenly distributed on 8 chromosomes. The structural analysis indicated that 87% of MaGRAS genes have no intron, which is highly conservative in different species. MaGRAS proteins of the same subfamily have similar protein motifs, which are the source of functional differences of different genomes. Transcriptome and qRT-PCR data were combined to determine the expression of 12 MaGRAS genes in 6 tissues, including flower, seed, leaf, stem, root and nodule, which indicated the possible roles in plant growth and development. Five and seven MaGRAS genes were upregulated under ABA, drought, and salt stress treatments in the roots and shoots, respectively, indicating that they play vital roles in the response to ABA and abiotic stresses in M. albus. Furthermore, in yeast heterologous expression, MaGRAS12, MaGRAS34 and MaGRAS33 can enhance the drought or salt tolerance of yeast cells. Taken together, these results provide basic information for understanding the underlying molecular mechanisms of GRAS proteins and valuable information for further studies on the growth, development and stress responses of GRAS proteins in M. albus.

Keywords: GRAS transcription factor; Melilotus albus; bioinformatics; gene expression; stress response.

Figure 1

Phylogenetic tree constructed using GRAS proteins from M. albus (red circle), M. truncatula (green square) and A. thaliana (blue triangle). The phylogenetic tree was constructed using MEGA 7.0 and using the neighbor–joining method.

Figure 2

Chromosomal distribution and position of 55 MaGRAS genes identified in the M. albus genome. Eight chromosomes are indicated in orange columns, and black lines indicate the position of each MaGRAS genes. Red lines represent the tandemly duplicated genes.

Figure 3

Distribution and synteny analysis of MaGRAS genes. In the figure, the 8 M. albus chromosomes are shown in green–colored partial circles, gene IDs are indicated at the top of each bar. Background gray lines indicate all M. albus genome synteny blocks, black lines indicate the duplicated MaGRAS genes, red lines represent the tandemly duplicated MaGRAS genes.

Figure 4

Exon–intron structure and distribution of conserved motifs of MaGRAS genes in M. albus: (A), MaGRAS proteins are categorized into 8 subfamilies, which are classified and labeled with different colors, including LISCL, SHR, PAT1, LAS, HAM, DELLA, SCR, and SCL3; (B), exon–intron structures of MaGRAS genes. Intron indicated by black line and CDS exon indicated by green boxes; (C), schematic diagram of the conserved motifs in the MaGRAS proteins. Each motif is represented by a number in the colored box. The black lines represent the non–conserved sequences. A total of 5 conserved domains and corresponding motifs in the GRAS proteins sequences are shown at the top. A scale of gene and protein length is shown at the bottom.

Figure 5

The cis–acting elements of promoter sequences (−2000 bp) of 55 MaGRAS genes are analyzed by PlantCARE in M. albus.

Figure 6

Expression analysis of the MaGRAS genes in different tissues: (A) a Heat map of all MaGRAS genes in different tissues based on transcriptome datasets, the expression values (FPKM) were normalized; (B) expression analysis of the MaGRAS genes in different tissues using qRT–PCR, the values shown are the means ± standard deviation of three replicates.

Figure 7

Expression of 55 MaGRAS genes in response to drought, salt and ABA treatments. Data were retrieved from transcriptome datasets, and the clustering was performed using TBtools. The heat map shows the relative transcript level of MaGRAS genes under drought, salt and ABA stresses. The expression values (FPKM) were normalized.

Figure 8

Gene expression analysis of six MaGRAS genes in shoots and roots under drought, salt and ABA treatments using qRT–PCR. CK represents control. Red lines indicated the expression values (FPKM) from RNA–seq data and the displayed values show the means ± standard deviation of three replicates.

Figure 9

Drought and salt stresses tolerance analysis of MaGRAS12, MaGRAS33 and MaGRAS34 in a yeast expression system, using yeast with empty pYES2 vector as control.