Identification, expression analysis of quinoa betalain biosynthesis genes and their role in seed germination and cold stress

Abstract

Betalains provide Chenopodium quinoa bright color, and the key enzyme genes for betalain biosynthesis include CYP76AD, DODA, and GTs. In this study, 59 CqCYP76AD, CqDODA and CqGTs genes in quinoa were identified and characterized by gene structural characteristics, phylogenetic relationships and gene expression patterns. The CqCYP76AD genes were divided into ɑ, β and γ types, CqDODA into ɑ and β types, and CqGTs into CqcDOPA5GT, CqB5GT and CqB6GT types according to phylogenetic relationships. The analysis of co-linearity identified eight pairs of duplicated genes which were subjected to purifying selection during evolution. CqCYP76AD and CqDODA, as well as CqcDOPA5GT and CqB5GT may have been evolutionarily linked in genetic inheritance, based on gene location and gene structure study. The tissue expression specificity of CqCYP76AD, CqDODA, and CqGTs genes in response to seed germination and cold stress was studied by RNA-Seq data. The genes CqCYP76AD, CqDODA, and CqGTs were involved in betalain biosynthesis and cold stress. CqCYP76AD, CqDODA, CqcDOPA5GT and CqB5GT gene sequences were consistent in the eight quinoa samples and showed significant variations in expression. In contrast, the inconsistency between changes in gene expression and betalain accumulation indicates that other factors may influence betalain biosynthesis in quinoa. This study offers the theoretical basis for the roles of the CqCYP76AD, CqDODA, and CqGTs genes in betalain biosynthesis and cold stress in quinoa, as well as a guide for the full utilization of betalains in quinoa plants.

Keywords: Chenopodium quinoa; betalain biosynthesis; expression analysis; gene cloning; gene identification.

Figure 1.

Betalain biosynthesis pathway (adapted from Alfonso Timoneda, et al 2019). The red colours indicate the type of enzyme required in betalain biosynthesis.

Figure 2.

Phylogenetic analysis of CqCYP76AD (a), CqDODA (b) and CqGts (c) with the other plant species. (a) the blue line indicates α evolutionary branch, the green line indicates β evolutionary branch and the red line indicates γ evolutionary branch. (b) the purple line indicates α evolutionary branch, the others belong to β evolutionary branch. (c) the red line indicates CqcDOPA5GT evolutionary branch, the blue line indicates CqB5GT evolutionary branch and the green line indicates CqB6GT evolutionary branch. Blue label background indicates genes from C. quinoa.

Figure 3.

Distribution and duplication of segments of CqCYP76AD, CqDODA and CqGts genes in C. quinoa.

Figure 4.

Conserved motifs, conserved structural domains and gene structures of CqCYP76AD, CqDODA and CqGts genes based on phylogenetic relationships in C. quinoa. (a) CqCYP76AD gene characterization. (b) CqDODA gene characterization. (c) CqGts gene characterization.

Figure 5.

Analysis of the cis-acting elements of the CqCYP76AD, CqDODA and CqGts genes.

Figure 6.

Heat map of quinoa betalain synthesis gene expression. (a, b, c) expression of CqCYP76AD gene members in different tissues, seed germination and cold stress. (d, e, f) expression of CqDODA gene members in different tissues, seed germination and cold stress. (g, h, i) expression of CqGts gene members in different tissues, seed germination and cold stress.

Figure 7.

Secondary and tertiary structure of the quinoa betalain synthesis genes. (a) Secondary and tertiary structure of the CqCYP76AD gene in C.Quinoa. (b) Secondary and tertiary structure of the CqDODA gene in C.Quinoa. (c) Secondary and tertiary structure of the CqcDOPA5GT gene in C.Quinoa. (d) Secondary and tertiary structure of the CqB5GT gene in C.Quinoa.

Figure 8.

Analysis of betacyanin contents in stems and leaves of different phenotypes of C. quinoa.

Figure 9.

Expression patterns of quinoa betalain synthesis genes in stems and leaves of different traits. (a) expression pattern of the CqCYP76AD gene in different coloured quinoa stems and leaves. (b) expression pattern of the CqDODA gene in different coloured quinoa stems and leaves. (c) expression pattern of the CqcDOPA5GT gene in different coloured quinoa stems and leaves. (d) expression pattern of the CqB5GT gene in different coloured quinoa stems and leaves. Different lowercase letters indicate significant differences at P < 0.05.