Genome-Wide Analysis of CCA1-Like Proteins in Soybean and Functional Characterization of GmMYB138a

Abstract

Plant CIRCADIAN CLOCK ASSOCIATED1 (CCA1)-like proteins are a class of single-repeat MYELOBLASTOSIS ONCOGENE (MYB) transcription factors generally featured by a highly conserved motif SHAQK(Y/F)F, which play important roles in multiple biological processes. Soybean is an important grain legume for seed protein and edible vegetable oil. However, essential understandings regarding CCA1-like proteins are very limited in soybean. In this study, 54 CCA1-like proteins were identified by data mining of soybean genome. Phylogenetic analysis indicated that soybean CCA1-like subfamily showed evolutionary conservation and diversification. These CCA1-like genes displayed tissue-specific expression patterns, and analysis of genomic organization and evolution revealed 23 duplicated gene pairs. Among them, GmMYB138a was chosen for further investigation. Our protein-protein interaction studies revealed that GmMYB138a, but not its alternatively spliced isoform, interacts with a 14-3-3 protein (GmSGF14l). Although GmMYB138a was predominately localized in nucleus, the resulting complex of GmMYB138a and GmSGF14l was almost evenly distributed in nucleus and cytoplasm, supporting that 14-3-3s interact with their clients to alter their subcellular localization. Additionally, qPCR analysis suggested that GmMYB138a and GmSGF14l synergistically or antagonistically respond to drought, cold and salt stresses. Our findings will contribute to future research in regard to functions of soybean CCA1-like subfamily, especially regulatory mechanisms of GmMYB138a in response to abiotic stresses.

Keywords: 14-3-3; CCA1-like proteins; MYB; protein interaction; soybean.

Figure 1

Phylogenetic analysis of CCA1-like subfamily between soybean and Arabidopsis. Phylogenetic trees were generated using: protein matrix (A); and CDS (Coding sequence) matrix (B), and groups are indicated by roman letters (I-VII/VI). The percent identity between duplicated gene pair is listed, and GmMYB138a (Glyma.02g026300) is marked with a star.

Figure 2

Chromosomal localization and duplication of CCA1-like genes in soybean. Each colored box refers to a chromosome, and chromosome numbers are shown beside each chromosome. The approximate localization of each soybean CCA1-like gene is indicated by a short black line. Tandem duplication is marked with star. The orange lines indicate the linkage group with segmentally duplicated gene pairs, and segmental duplication regions were estimated using the Plant Genome Duplication Database.

Figure 3

Expression patterns of CCA1-like genes in soybean. The transcript profiling data of CCA1-like genes in different soybean tissues were retrieved from Phytozome database (Available online: http://www.phytozome.net) for heat map generation. The colored scale bar beside the heat map indicates gene expression level. SAM, shoot apical meristem. GmMYB138a (Glyma.02g026300) is marked with a star.

Figure 4

Interaction of GmMYB138a with GmSGF14l. (A) Schematic diagram for two spliced transcripts of Glyma.02g026300 (GmMYB138a and GmMYB138b) at protein and gene levels. Black lines represent the introns, while black boxes refer to the exons of two spliced transcripts in the gene structures. In the protein structures, amino acids are indicated by black line. MYB domain is marked with rectangle, while sexangle refer to ZnF_C2HC domain; (B) The investigation of interaction between GmSGF14l and GmMYB138a/b using yeast two-hybrid assay. Yeast cells were co-transformed with combination of DNA-binding domain (BK, Bait) and activation domain (AD, Prey) fused constructs as indicated. A series of 5 µL of diluted yeast suspension culture co-transformed with bait and prey constructs was spotted onto synthetic defined (SD) selection plates. Growth on SD/-Leu/-Trp indicates the presence of both the vectors, while growth on SD/-Leu/-Trp/-His/-Ade shows interaction between prey and bait. The different combinations with GmMYB138a or GmMYB138b/BK or AD are negative controls; (C) The BiFC assay showing interaction between GmYB138a and GmSGF14l. Tobacco leaves were co-transformed with GmYB138a and GmSGF14l proteins fused to the N- or C-terminal half of yellow fluorescent protein followed by confocal microscopy. Scale bar is shown in 40 μm.

Figure 5

Multiple sequence alignment of deduced amino acid sequence of GmMYB138a with its homologs in different species. Amino acid sequences were aligned by ClustalW, and imported in Genedoc for shading. Identical amino acid residues are shown in black. If the conserved percent was up to 80% and 60%, the amino acid residues were shaded with gray and light gray colors, respectively. The MYB domains of CCA1-like proteins are indicated by lines. The consensus sequence SHAQK(Y/F)F is marked with stars.

Figure 6

Expression analysis of GmMYB138a in different soybean tissues and developmental seeds. Total RNA extracted from soybean root, stem, leaf, nodule, flower, embryo (10, 20, 30, 40, 50 and 60 days after pollination) were used for qPCR analysis. Three biological replicates and three technical replicates for each biological replicate were carried out. The standard error of the mean is represented by an error bar. The data were normalized against SUBI-3.

Figure 7

Dimer formation of GmMYB138a in planta: (A) BiFC analysis showing homodimer formation of GmMYB138a in vivo; and (B) prediction of GmMYB138a heterodimer using STRING. MYB114, Glyma.03g261800; MYB118, Glyma.10g048500. Scale bar is shown in 40 μm.

Figure 8

Expression analysis of GmMYB138a and GmSGF14l in response to abiotic stresses. Ten-day-old soybean seedlings were exposed to stress treatment as indicated below. Gene expression analysis was conducted by qRT-PCR using gene specific primers. (A) Gene expression pattern of GmMYB138a and GmSGF14l in seedlings exposed to cold stress for 0, 6, 12, 24, 48 and 72 h; (B) Gene expression pattern of GmMYB138a and GmSGF14l in seedlings exposed to drought stress for 0, 2, 4, 6, 8 and 10 days; (C) Gene expression pattern of GmMYB138a and GmSGF14l in seedlings exposed to salinity stress for 0, 6, 12, 24, 48 and 72 h. Error bars indicate SE (Standard error) of two biological and three technical replicates. Values were normalized against SUBI-3. Significant differences are denoted by asterisks (p < 0.01).