Genome-Wide Identification, Characterization, Evolutionary Analysis, and Expression Pattern of the GPAT Gene Family in Barley and Functional Analysis of HvGPAT18 under Abiotic Stress

Abstract

Glycerol-3-phosphoacyltransferase (GPAT) is an important rate-limiting enzyme in the biosynthesis of triacylglycerol (TAG), which is of great significance for plant growth, development, and response to abiotic stress. Although the characteristics of GPAT have been studied in many model plants, little is known about its expression profile and function in barley, especially under abiotic stress. In this study, 22 GPAT genes were identified in the barley genome and divided into three groups (I, II, III), with the latter Group III subdivided further into three subgroups based on the phylogenetic analysis. The analyses of conserved motifs, gene structures, and the three-dimensional structure of HvGPAT proteins also support this classification. Through evolutionary analysis, we determined that HvGPATs in Group I were the earliest to diverge during 268.65 MYA, and the differentiation of other HvGPATs emerged during 86.83-169.84 MYA. The tissue expression profile showed that 22 HvGPAT genes were almost not expressed in INF1 (inflorescence 1). Many functional elements related to stress responses and hormones in cis-element analysis, as well as qRT-PCR results, confirm that these HvGPAT genes were involved in abiotic stress responses. The expression level of HvGPAT18 was significantly increased under abiotic stress and its subcellular localization indicated its function in the endoplasmic reticulum. Various physiological traits under abiotic stress were evaluated using transgenic Arabidopsis to gain further insight into the role of HvGPAT18, and it was found that transgenic seedlings have stronger resistance under abiotic stress than to the wild-type (WT) plants. Overall, our results provide new insights into the evolution and function of the barley GPAT gene family and enable us to explore the molecular mechanism of functional diversity behind the evolutionary history of these genes.

Keywords: Hordeum vulgare L.; abiotic stress; classification; evolution; expression pattern; functional analysis; gene family; glycerol-3-phosphate acyltransferase (GPAT).

Figure 1

Chromosomal localization of the HvGPAT genes on the 7 barley chromosomes. Chromosome numbers were shown at the top of each vertical bar. On the left side of the chromosome is the physical location, and on the right side is the name of the HvGPAT genes (chr: chromosomes).

Figure 2

Phylogenetic and evolutionary analysis of HvGPAT proteins. (A) The phylogenetic tree was constructed in three taxa: barley (Hordeum vulgare), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa) using the N-J method with 1000 bootstrap replicates. (B) Evolutionary map of GPAT genes in Oryza sativa (OsGPATs), Brassica napus (BdGPATs), Zea may (ZmGPATs), Arabidopsis thaliana (AtGPATs), and Hordeum vulgare (HvGPATs).

Figure 3

The protein sequence alignment, conserved motif, and gene structure of the HvGPAT genes. (A) Alignment of HvGPAT proteins and identification of conserved amino acid motifs. The multiple sequence alignment of the 22 GPAT proteins was performed in the CLUSTALX program. The four conserved acyltransferase amino acid motifs (Blocks I–IV) are boxed, and residues critical for enzyme catalysis and substrate binding are highlighted by a black triangle. Red, green and yellow in the sequence indicate that the sequence alignment is from high to low. * indicates consistent sequence. (B) Conserved motif and exon–intron gene structure of HvGPAT genes. The conserved motifs were identified by MEME. Motifs were indicated by different colored boxes with the motif number, while non-conserved sequences were represented by grey lines. Length of motifs was exhibited proportionally. Gene structure analysis was performed using the exon–intron length. The length of exons and introns of each HvGPAT gene was displayed proportionally. 0 1 2: intron phase.

Figure 4

Expression profiles of the HvGPAT genes in different tissues. The RNA-seq data of 16 tissues in different developmental stages of barley seedlings were obtained from the BARLEX database at the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). X-axis: mRNA levels in 16 different tissues and life stages of barley; EMB: 4-day embryos; ROO1: roots from seedlings (10 cm shoot stage); LEA: shoots from seedlings (10 cm shoot stage); INF1: young developing inflorescences (5 mm); INF2: developing inflorescences (1–1.5 cm); NOD: developing tillers, 3rd internode (42 DAP); CAR5: developing grain (5 DAP); CAR15: developing grain (15 DAP); ETI: etiolated seedling, dark cond; LEM: inflorescences, lemma (42 DAP); LOD: inflorescences, lodicule (42 DAP); PAL: dissected inflorescences, palea (42 DAP); EPI: epidermal strips (28 DAP); RAC: inflorescences, rachis (35 DAP); ROO2: roots (28 DAP); and SEN: senescing leaves (56 DAP) were used to analyze tissues expression pattern. The expression level was shown in color as a scale: red represents high expression level and green represents low expression level. A and B stands for two different branches.

Figure 5

Prediction of cis-acting elements of the HvGPAT promoters. Many cis-acting elements were detected in the promoter region of each HvGPAT gene and different colors and shapes represent different cis-acting elements.

Figure 6

Expression profiles of 11 HvGPAT genes under different abiotic stresses. Data were normalized to the actin gene, and vertical bars indicate the standard deviation. The relative expression levels of the HvGPAT genes under different abiotic stresses were measured by qRT–PCR. The mean (±SE) expression values were calculated from three independent biological replicates and three technical replicates (* p < 0.01).

Figure 7

Effect of abiotic stress on phenotype and physiology of WT and HvGPAT18 transgenic Arabidopsis thaliana lines. (A) Seedlings at 20 days after transfer to MS, MS with 4 °C, MS + 200 mM mannitol plates, or MS + 130mM NaCl, (B) germination rates, (C) Root length, (D) PRO (Proline) content, (E) CAT (hydrogen peroxidase) activity. Different lowercase letters indicate a significant difference at the 0.05 level; the same lowercase letters indicate a significant difference at the 0.05 level.