Identification and expression analysis of the GLK gene family in tea plant (Camellia sinensis) and a functional study of CsGLK54 under low-temperature stress

Abstract

The Golden2-like (GLK) transcription factor family is a significant group of transcription factors in plantae. The currently available studies have shown that GLK transcription factors have been studied mainly in chloroplast growth and development, with fewer studies in abiotic stress regulation. In this study, all tea plant GLK transcription factors were identified for the first time in tea plants, and genome-wide identification, phylogenetic analysis, and thematic characterization were performed to identify 66 GLK transcription factors in tea plants. These genes are categorized into seven groups, and an amino acid sequence comparison analysis is performed. This study revealed that the structure of GLK genes in tea plants is highly conserved and that these genes are distributed across 14 chromosomes. Collinearity analysis revealed 17 pairs of genes with fragment duplications and one pair of genes with tandem duplications, and the analysis of Ka/Ks ratios indicated that most of the genes underwent negative purifying selection. Analysis of promoter cis-elements revealed that the promoters of tea plant GLK genes contain a large number of cis-acting elements related to phytohormones and stress tolerance. In addition, a large number of genes contain LTR elements, suggesting that tea plant GLK genes are involved in low-temperature stress. qRT‒PCR analysis revealed that the expression of CsGLK17, CsGLK38, CsGLK54, CsGLK11 and CsGLK60 significantly increased and that the expression of CsGLK7 and CsGLK13 decreased in response to low-temperature induction. Taken together, the results of the transcription profile analysis suggested that CsGLK54 may play an important regulatory role under low-temperature stress. The subcellular localization of CsGLK54 was in the nucleus. Furthermore, CsGLK54 positively regulated the transcription levels of the NbPOD and NbSOD genes under low-temperature stress, which led to an increase in POD and SOD enzyme activities and a decrease in MDA content. These findings provide valuable insights into the regulatory mechanism of low-temperature stress in tea plants.

Keywords: Camellia sinensis; Functional validation; GLK family; Genome-wide analysis; Low-temperature stress.

Figure 1

Phylogenetic analysis of GLK genes in C. sinensis and Arabidopsis thaliana; the pentagrams represent Arabidopsis thaliana, and the triangles represent C. sinensis.

Figure 2

Phylogenetic evolutionary tree (A), conserved motif (B) and gene structure (C) analysis of the C. sinensis GLK gene family.

Figure 3

In the conserved regions of the C. sinensis GLK gene family, cyan represents ≥ 50%, pink represents ≥ 75%, and dark blue represents 100%.

Figure 4

Chromosome localization analysis of the C. sinensis GLK gene family.

Figure 5

Collinearity analysis of the C. sinensis GLK gene family; red indicates tandem replication events, and blue indicates fragment replication events.

Figure 6

Cis-element analysis of C. sinensis GLK gene promoters. The binding sites in the promoter region are represented by boxes of different colors (A), and the graph shows the number of binding sites (B).

Figure 7

FPKM expression profile of the C. sinensis GLK gene after low-temperature stress and room temperature recovery based on RNA-Seq data. The data were subjected to log2 processing. The color scale on the left side represents the relative expression values, CK: control, CA: 10 °C treatment, DA: room temperature recovery.

Figure 8

Relative expression levels of 8 selected GLK genes under cold stress. The bars represent the mean values of three replicates ± standard errors, and different letters indicate significant differences at different time points according to one-way ANOVA and least significant difference (LSD) tests (P < 0.05).

Figure 9

Subcellular localization of CsGLK54. Fluorescence signals from the nuclei of tobacco epidermal cells labeled with 4′,6-diamidino-2-phenylindole (DAPI).

Figure 10

Validation of the transient conversion function of CSGLK54 in this type of tobacco: (A) semiquantitative validation (Supplementary Fig. 10A); (B) relative electrical conductivity determination; (C) MDA content determination; (D–F) SOD, POD, and CAT enzyme content determination; (G) quantitative validation of NbSOD, NbPOD, and NbCAT. The data are presented as the means ± standard errors of three biological replicates. Student’s t test and significance tests were performed (*, P < 0.05; **, P < 0.01; ***, P < 0.001).