Genome-Wide Identification and Analysis of R2R3-MYB Genes Response to Saline-Alkali Stress in Quinoa

Abstract

Soil saline-alkalization inhibits plant growth and development and seriously affects crop yields. Over their long-term evolution, plants have formed complex stress response systems to maintain species continuity. R2R3-MYB transcription factors are one of the largest transcription factor families in plants, widely involved in plant growth and development, metabolism, and stress response. Quinoa (Chenopodium quinoa Willd.), as a crop with high nutritional value, is tolerant to various biotic and abiotic stress. In this study, we identified 65 R2R3-MYB genes in quinoa, which are divided into 26 subfamilies. In addition, we analyzed the evolutionary relationships, protein physicochemical properties, conserved domains and motifs, gene structure, and cis-regulatory elements of CqR2R3-MYB family members. To investigate the roles of CqR2R3-MYB transcription factors in abiotic stress response, we performed transcriptome analysis to figure out the expression file of CqR2R3-MYB genes under saline-alkali stress. The results indicate that the expression of the six CqMYB2R genes was altered significantly in quinoa leaves that had undergone saline-alkali stress. Subcellular localization and transcriptional activation activity analysis revealed that CqMYB2R09, CqMYB2R16, CqMYB2R25, and CqMYB2R62, whose Arabidopsis homologues are involved in salt stress response, are localized in the nucleus and exhibit transcriptional activation activity. Our study provides basic information and effective clues for further functional investigation of CqR2R3-MYB transcription factors in quinoa.

Keywords: CqMYB2Rs; metabolism; stress response; transcription factors; transcriptome analysis.

Figure 1

Conservative domain analysis of CqR2R3-MYB proteins. The green box represents MYB_DNA binding domain.

Figure 2

Phylogenetic analysis of R2R3-MYB proteins. A maximum-likelihood phylogenetic tree containing 65 CqR2R3-MYB proteins in quinoa and 126 in Arabidopsis thaliana. At, Arabidopsis thaliana; Cq, Chenopodium quinoa. The subgroups were distinguished by different colors. S1, subgroup 1.

Figure 3

Analysis of conservative motifs and gene structure of CqR2R3-MYB family members. (A) Conservative motif analysis of CqR2R3-MYB proteins. Different colors represent different motifs. (B) Gene structure of CqR2R3-MYBs. UTR, untranslated region.

Figure 4

Analysis of cis-acting elements in CqR2R3-MYB promoters. The categorized groups are indicated by color bars. The score which indicates the occurrence frequency of each cis-acting element in each promoter is displayed by number inside the circle, the depth of the circle’s color is proportional to the score, and the corresponding relationship between numbers and colors is shown by a color scale in lower panel.

Figure 5

The expression pattern of CqR2R3-MYB genes. (A) The expression levels of all CqR2R3-MYB genes in quinoa leaves that had undergone saline–alkali stress. CK1, CK2, and CK3 are the three replicates of the CK group. Treat1, Treat2, and Treat3 are the three replicates of the carbonate-treatment group. (B) The log₂ fold change of the six CqR2R3-MYB genes with the greatest changes in expression levels. (C) RT-qPCR validation of the CqR2R3-MYB genes in (B). UBQ9 (AUR62020068) was used as an endogenous control. Three independent experiments per sample, three replicates per experiment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student’s t-test.

Figure 6

GO enrichment analysis of CqR2R3-MYB genes in quinoa leaves that had undergone saline–alkali stress. (A) Go enrichment analysis of TOP50 of CqR2R2-MYB genes. (B) The enriched GO terms in Biology Process of the differentially expressed CqR2R2-MYBs. Rectangles indicate the most significant terms. Rectangle and oval colors represent the relative significances, dark red indicates the most significant, p < 0.0001.

Figure 7

Determination of subcellular localization and autoactivation activity of CqR2R3-MYBs. (A) Subcellular localization of GFP-CqMYBs was detected by transient expression in tobacco leaves. The plasmids harboring GFP-CqR2R3-MYBs were transformed into GV3101 and infiltrated to tobacco leaves. The subcellular localization of GFP-CqR2R3-MYB fusion proteins were observed after DAPI staining. (B) Analysis of autoactivation activity. The pGBKT7 plasmids harboring GFP-CqR2R3-MYBs were transformed with pGADT7 vector into strain AH109, respectively, and grown on SD/-Leu/-Trp medium. The autoactivation activity was examined on SD/-Leu/-Trp/-His/-Ade medium. pGBKT7 empty, a negative control; AtbHLH112, a positive control.