Genome-Wide Identification of Proline Transporter Gene Family in Non-Heading Chinese Cabbage and Functional Analysis of BchProT1 under Heat Stress

Abstract

Non-heading Chinese cabbage prefers cool temperatures, and heat stress has become a major factor for reduced yield. The proline transporter protein (ProT) is highly selective for proline transport, contributing to the heat tolerance of non-heading Chinese cabbage. However, there has been no systematic study on the identification and potential functions of the ProT gene family in response to heat stress in non-heading Chinese cabbage. We identified six BchProT genes containing 11-12 transmembrane helices characteristic of membrane proteins through whole-genome sequencing. These genes diverged into three evolutionary branches and exhibited similarity in motifs and intron/exon numbers. Segmental duplication is the primary driving force for the amplification of BchProT. Notably, many stress-related elements have been identified in the promoters of BchProT using cis-acting element analysis. The expression level of BchProT6 was the highest in petioles, and the expression level of BchProT1 was the highest under high-temperature stress. Subcellular localization indicated their function at cell membranes. Heterologous expression of BchProT1 in Arabidopsis plants increased proline transport synthesis under heat-stress conditions. This study provides valuable information for exploring the molecular mechanisms underlying heat tolerance mediated by members of the BchProT family.

Keywords: ProT; genome-wide analysis; heat stress; non-heading Chinese cabbage.

Figure 1

The gene–replication relationship between BchProTs. Outer ring representation gene density: red: higher expression; yellow: lower expression. The green fonts are 10 chromosomes A01–10, the blue lines represent collinear gene pairs, and the gray areas are collinear regions.

Figure 2

BchProTs collinearity analysis with Arabidopsis and Chinese cabbage. The gray lines in the background represent collinear blocks of the genome, and the red lines represent homologous ProT gene pairs.

Figure 3

A schematic diagram of gene structure and conserved motif of the BchProT gene family. (A) Motif distribution. Twelve conserved motifs in BchProT proteins are indicated by multiple colored boxes. Different colors boxes represent different conservative motifs. (B) The gene structure. Purple box represents UTR, green box represents CDS, and grey line represents intron.

Figure 4

BchProTs with a multispecies phylogenetic tree. The outer ring of different colors represents different groups of BchProTs gene family. Blue star represents Arabidopsis thaliana, red circle represents Brassica camptris, purple triangle represents Solanum lycopersicum, yellow square represents Triticum aestivum L., and green triangle represents Oryza sativa. The number on the branch of the evolutionary tree represents the Bootstrap value.

Figure 5

The analysis results of BchProT genes promoter sequence. (A) Functional classification of predicted cis-elements in BchProT promoter regions. % represents the percentage of the total number of components. (B) Quantity heatmap of stress responsiveness.

Figure 6

BchProT gene family expression. (A) Different tissue sites. (B) Heat stress (40 °C for 12 h). ** indicates extremely significant difference (p value < 0.01). All bars represent means ± SD (n = 3).

Figure 7

BchProT1 subcellular localization. Note: green fluorescence (GFP); visible light (BF); merge field (Merge); and scale bar = 100 μm.

Figure 8

Ectopic expression of BchProT1 in Arabidopsis. (A) Heat stress Arabidopsis phenotype. Red circles highlight the phenotypic changes of wild Arabidopsis under heat stress. (B) Heat stress Arabidopsis MDA and Proline content. WT represents wild-type plants; OE represents highly expressed transgenic lines; Heat represents (40 °C for 12 h). ** indicates extremely significant difference (p value < 0.01). All bars represent means ± SD (n = 3).