Cloning and molecular characterization of Triticum aestivum ornithine amino transferase (TaOAT) encoding genes

Abstract

Background: Ornithine aminotransferase (OAT, EC:2.6.1.13), alternatively known as ornithine delta aminotransferase (δOAT), is a pyridoxal phosphate (PLP)-dependent enzyme involved in the conversion of ornithine into glutamyl-5-semi-aldehyde (GSA) and vice versa. Up till now, there has been no study on OAT in wheat despite the success of its isolation from rice, maize, and sorghum. This study focuses on identification and molecular characterization of OAT in wheat.

Results: In total, three homeologous OAT genes in wheat genome were found on chromosome group 5, named as TaOAT-5AL, TaOAT-5BL, and TaOAT-5DL. Sequence alignment between gDNA and its corresponding cDNA obtained a total of ten exons and nine introns. A phylogenetic tree was constructed and results indicated that OATs shared highly conserved domains between monocots and eudicots, which was further illustrated by using WebLogo to generate a sequence logo. Further subcellular localization analysis indicated that they functioned in mitochondria. Protein-protein interactions supported their role in proline biosynthesis through interactions with genes, such as delta 1-pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), involved in the proline metabolic pathway. Promoter analysis exposed the presence of several stress responsive elements, implying their involvement in stress regulation. Expression profiling illustrated that TaOAT was highly induced in the wheat plants exposed to drought or salt stress condition. Upregulated expression of TaOATs was observed in stamens and at the heading stage. A potential role of TaOAT genes during floret development was also revealed. Furthermore, the transgenic plants overexpressing TaOAT showed enhanced tolerance to drought stress by increasing proline accumulation. In addition, salt tolerance of the transgenic plants was also enhanced.

Conclusion: TaOATs genes were involved in proline synthesis and nitrogen remobilization because they interacted with genes related to proline biosynthesis enzymes and arginine catabolism. In addition, TaOAT genes had a role in abiotic stress tolerance and a potential role in floret development. The results of this study may propose future research in the improvement of wheat resistance to abiotic stresses.

Keywords: Drought tolerance; Floret development; Ornithine aminotransferase; Salt tolerance; Wheat.

Fig. 1

Alignment of the cDNAs sequences of TaOAT genes amplified from the hexaploid wheat cultivar Fielder. In total there are four alleles named TaOAT-5AL-1, TaOAT-5AL-2, TaOAT-5BL, and TaOAT-5DL with sizes of 1497 bp, 1287 bp, 1407 bp, and 1422 bp, respectively. The TaOAT-5AL-1 contained in-frame stop codon indicated by red box that caused the incomplete translation of protein. The TaOAT-5AL-2, TaOAT-5BL, and TaOAT-5DL showed complete translation of proteins. Intron-exon junction is divided by red lines. Dotted lines indicate the deletion

Fig. 2

Structure of TaOAT genes in common wheat. The transcripts viz TaOAT-5AL-2, TaOAT-5BL, and TaOAT-5DL show ten exons and nine introns while transcript viz TaOAT5AL-1 shows the disrupted gene structure due to the retention of the 9th intron that fused with the 9th exon causing the larger 9th exon as compared to the other transcripts. Gray, yellow and blue colors respectively represent introns, exon and UTRs. The red arrow and line show the start (ATG) and stop codon (TAA), respectively

Fig. 3

Determination of chromosomal location of TaOAT genes in hexaploid wheat using specific primers and nullitetrasomic lines. The absence of the band 864 bp in size in lane 1 shows that the primer is specific to TaOAT-5AL due to the absence of chromosome 5A in N5A/T5B. Similarly, the absence of the bands 882 and 840 bp in size in lane 2 and 3 shows the specificity of the primers to TaOAT-5BL and TaOAT-5DL as chromosomes 5B and 5D are absent in N5B/T5A and N5D/T5A lines, respectively. M: DL2000 DNA ladder (TianGen Biotech. Beijing Co., Ltd.); 1–4: Chinese Spring chromosome group 5 nullitetrasomic lines where N represents the null chromosome, and T represents the tetra chromosome in which 2 chromosomes replaced or shuffled the respective absent two chromosomes (1: N5A/T5B, 2: N5B/T5A, 3: N5D/T5A, and 4: Chinese Spring)

Fig. 4

Subcellular localization of TaOAT protein. Green fluorescence indicates the GFP signals from TaOAT C terminal fused GFP vector, red indicates mitochondria stained with mitotracker dye, and yellow shows the merged signals of GFP and mitotracker. The size for scale bar is 5 μm

Fig. 5

Protein-protein interaction of TaOAT with other wheat proteins. a The interacting protein partners predited by STRING database. The TaOAT-5BL (Traes_5BL_1D3F1BCC2.1) amino acid sequence was used to search for its interacting genes in wheat. Nodes represent the single protein coding locus and edges represent the meaningful interaction that shared the same functions. Color nodes represent the first shell of interactors while white nodes represent the second shell of interactors. Empty nodes represent the protein of unknown 3D structure while filled nodes represent the known or predicted 3D structure. b Confirmation of the predicted interaction results by the yeast two hybrid. The figure on the left hand side represents the yeast growth on two absent (Leu/Trp) medium. The figure on the right hand side represents the yeast growth on three absent (Leu/Trp/His) medium. The specific interaction was determined by the growth of transformants on three absent (Leu/Trp/His) medium. 10⁰ represent the original culture sample. 10^− 1–10^− 3 showed 10 fold serial dilution of the sample

Fig. 6

Expression profiles of wheat OAT genes in different tissues and developmental stages. a Expression pattern of TaOAT in different tissues. b Expression pattern of TaOAT at different developmental stages. c The seven spikelet-developmental stages according to Kirby and Appleyard (1987). The stages, from left to right, are: terminal spikelet stage (TS), white anther stage (WA), green anther stage (GA), yellow anther stag (YA), tipping stage (TP), heading stage (HD), and anthesis stage (AN). d Relative expression at the seven floret-developmental stages. Three biological replicates were averaged and the statistical analysis was performed using IBM-SPSS statistic 20. Small letters in bold represent significant differences between groups at α < 0.05 using Duncan’s multiple range tests. Bars indicate the standard error of the mean

Fig. 7

Expression pattern analyses by qRT-PCR of TaOAT genes upon PEG and NaCl treatment. a Transcript levels of TaOAT genes in wheat seedlings exposed to 50% PEG-4000 for different time periods. b Transcript levels of TaOAT genes in wheat seedling exposed to 200 mM NaCl stress for different time periods. Three biological replicates were averaged and statistical analysis was performed using IBM-SPSS statistic 20. Small letters in bold represent significant differences between groups at α < 0.05 using Duncan’s multiple range tests. Bars indicate the standard error of the mean

Fig. 8

Vector construction and drought tolrenace test of TaOAT-5BL overexpressing transgenic wheat lines. a Schematic structure of expression vector used for Agrobacterium-mediated transformation. b Semi-quantitative PCR profiles for TaOAT-5BL in transgenic lines and their wild type Fielder. c Phenotype of TaOAT-5BL transgenic lines and wild type Fielder before and after drought stress. d Free proline content measured in the transgenic lines and wild type Fielder before and after drought stress. FC represents wild type Fielder and OE-F7, OE-F8, and OE-F9 are TaOAT-5BL overexpressing transgenic lines derived from Fielder. Data was analyzed using IBM-SPSS statistic 20. Small letters in bold represent significant differences between groups at α < 0.05 using Duncan’s multiple range tests. Bars indicate the standard error of the mean

Fig. 9

Salt tolerance test of TaOAT-5BL overexpressing transgenic lines and their wild type Fielder in 150 mM NaCI containing medium. a Salt tolerance phenotype of TaOAT-5BL transgenic lines and wild type Fielder cultured for 30 days. b Survival rate of TaOAT-5BL transgenic lines and wild type Fielder cultured 30 days. c Relative expression analysis of TaOAT-5BL in transgenic lines and wild type Fielder cultured for 30 days. d Root length of the surviving plants cultured for 30 days. e Shoot length of the surviving plants cultured for 30 days. FC represents wild type Fielder and OE-F7, OE-F8, and OE-F9 are TaOAT-5BL overexpressing transgenic lines derived from Fielder. The data was average of three replications and analyzed using IBM-SPSS statistic 20. Small letters in bold represent significant differences between groups at α < 0.05 using Duncan’s multiple range tests. Bars indicate the standard error of the mean