Isolation and structure analysis of TaOAT genes in hexaploid wheat
Sequence retrieval from the International Wheat Genome Sequencing Consortium (IWGSC) database using AtOAT accession At5g46180 as query resulted in a total of three scaffolds that matched our query, namely, TGACv1_scaffold_374190, TGACv1_scaffold_404925, and TGACv1_scaffold_435304, which were located on the long arm of chromosome group 5 with e-values of 1e-30, 7e-27 and 4e-24, respectively. Predictions of the open reading frame (ORF) of the three candidate genes’ gDNA/cDNA lengths were 4568/1419 bp, 4276/1488 bp, and 4446/1422 bp, respectively. No variation was found between the sequence of the common wheat cultivar Fielder and the reference sequence of Chinese Spring. However, two transcript variants of TaOAT-5AL were revealed. These were named TaOAT-5AL-1 and TaOAT-5AL-2 and characterized by 1497 bp and 1287 bp in cDNA length, respectively. Compared to TaOAT-5AL-2, TaOAT-5AL-1 contained an additional 120-bp insertion encompassing an in-frame stop codon, which resulted in a premature protein (Fig. 1). The additional insertion was genotypically confirmed by sequencing results from six cultivars used in this study. TaOAT-5AL-2 was identical to the reference sequence based on the sequencing results. Interestingly, there were six splice variants in the T. dicoccoides database and the TaOAT-5AL-2 transcripts showed high similarity to two of these variants, TRIDC5AG054810.2 and TRIDC5AG054810.3 (Additional file 1: Figure S1). After sequence alignment, we found that TaOAT-5AL-2 had higher identity to TRIDC5AG054810.2. We suspect that during the evolution of hexaploid wheat, it retained two alternative-spliced variants for TaOAT-5AL. On the other hand, only one transcript has been found for TaOAT-5BL and one for TaOAT-5DL. In addition, TaOAT-5BL had 1407 bp instead of 1488 bp due to an 81-bp deletion, which is the same as the putative transcript in the ensemble Plants database with an accession ID of TraesCS5B02G376900.1. There was no difference in bp and length for TaOAT-5DL transcript. We have submitted these sequences to the National Center for Biotechnology Information (NCBI) database and their accession numbers are MK942062, MK942063, MK680533 and MK748213 for TaOAT-5AL-1, TaOAT-5AL-2, TaOAT-5BL, and TaOAT-5DL, respectively.
The exon-intron boundaries were determined by comparing the gDNA sequence in each genome with the full-length cDNA sequences of TaOAT-5AL-1, TaOAT-5AL-2, TaOAT-5BL, and TaOAT-5DL. In total, the TaOAT gene contained ten exons and nine introns. The TaOAT-5AL gene had two types of transcripts which were likely a consequence of alternative splicing. TaOAT-5AL-1 consisted of nine exons due to the retention of the 9th intron, resulting in the formation of a premature protein (Fig. 2). Sequence analysis showed a nucleotide transition (T → C) occurred in the gDNA of TaOAT-5AL (the transition is indicated by the red box in Additional file 1: Figure S2), which breaks the classic boundary of intron splicing (5′-GT(N)nAG-3′; where N represents any nucleotide and n represents a random number). The transition likely caused the retention of the 9th intron in TaOAT-5AL-1 corresponding to that in TaOAT-5BL and TaOAT-5DL (Fig. 2). However, TaOAT-5AL-2 has the same structure as TaOAT-5BL and TaOAT-5DL (Fig. 1). The three genes have 87.68 and 87.18% identity at the gDNA and cDNA levels, respectively.
Chromosomal and subcellular localization of TaOAT genes and their encoding proteins in wheat
To confirm the chromosomal location of TaOAT genes in wheat, primers specific to each TaOAT gene were designed and the localization was performed by using three Chinese Spring nullitetrasomic lines related to chromosome group 5 as templates for the PCR assay in which Chinese Spring (CS) was used as a control. No band was obtained in lane 1 for N5A/T5B when using the specific primers of TaOAT-5AL, in lane 2 for N5B/T5A when using the specific primers of TaOAT-5BL, and in lane 3 for N5D/T5A when using the specific primers of TaOAT-5DL (Fig. 3). The absence of these bands suggests a deletion of the gene due to the corresponding chromosome removal. Therefore, the three genes, TaOAT-5AL, TaOAT-5BL, and TaOAT-5DL, were experimentally assigned to chromosome 5A, 5B, and 5D, respectively, in hexaploid wheat.
Subcellular localization of plant OAT proteins of 65 species was predicted using TargetP. Most of the plant OATs (83%) were targeted to the mitochondria (Additional file 2: Table S1). Because OATs are thought to be highly conserved enzymes and previous reports have shown that AtOAT and OsOAT are targeted to mitochondria [8, 9], we speculated that TaOAT proteins also functions in mitochondria, which was supported by the high probability of 0.9707 obtained using Mitoprot in this study. Moreover, the transient expression of TaOAT-fused GFP signals was only observed in the mitochondria of wheat protoplasts; the merging of mitochondrion-specific dye with GFP signals indicated mitochondrion-targeting of TaOAT proteins (Fig. 4).
Phylogenetic and promoter analysis of wheat OAT genes
The phylogenetic analysis showed that there were two distinguished groups, one among the monocots (indicated by red) and the other among the eudicots (indicated by blue) (Additional file 1: Figure S3). The OATs from cereals like maize, sorghum, and rice formed a distinguished but small cluster and shared high similarity. The homologous genes in wheat showed a close similarity with its respective ancestral group, for example TaOAT-5AL showed high similarity with Triticum urartu and TaOAT-5DL showed high similarity with Aegilops tauschii, implying that gene structure and function were shared by common wheat and its wild relatives. Overall, these results suggest that OAT from common wheat and its wild relatives remained highly conserved during evolution. In contrast, the larger group of dicot OAT genes formed five sub-groups, indicating that dicot OAT genes have evolved more diverse functions. The conservation of the targeted genes among monocot and dicot species was also illustrated in a sequence logo created using WebLogo (Additional file 1: Figure S4).
In this study, 1000 base pairs upstream of the start condon of TaOATs were selected for the prediction of cis-elements using an online tool. The results showed that many of the predicted elements were stress-responsive, including abscisic acid responsive element (ABRE), MYB (Myeloblastosis) cis-elements, ROS-related motifs (G-box and W-box), ethylene-responsive element (ERE), heat shock element (HSE), APETALA2-like (AP-2-like) element and low temperature responsive (LTR) element (Additional file 1: Figure S5). The G-box (CACGTG) element is involved in responses to light, abscisic acid, methyl-jasmonate and anaerobiosis. The G-box also has a role in ethylene induction as well as in seed-specific expression. Additionally, the G-box also functions as an ABRE (ABA-responsive element) [14, 15]. Both ABRE and G-box elements provide the binding sites for bZIP transcription factors (TFs) that regulate stress responses. Both ABRE and G-box have been shown to be present in the three wheat OAT genes of this study. The W box is present in TaAOT-5AL and TaOAT-5DL which interacts with TFs belonging to the WRKY family. The ABRE present in all TaOAT genes, DREs (dehydration responsive elements) in TaOAT-5AL and TaOAT-5DL, and LTR in TaOAT-5DL provide the binding sites for NAC genes, implying TaOATs role in both salinity and drought stress response. In addition, the AP-2-like domain has been found in TaOAT-5DL, which supports its role in floret development. These findings suggest that TaOATs have potential roles in plant responses to drought, salinity and pathogen stresses.
Protein-protein interactions realted to wheat OAT
According to the STRING database, TaOAT interacts with Traes_1BL_31105367B.1 (delta 1-pyrroline-5-carboxylate synthetase), Traes_2BS_E836C5A07.1 (an uncharacterized protein in the arginase family), Traes_3B_1E5C683B5.1 (pyrroline-5-carboxylate reductase that belongs to the pyrroline-5-carboxylate reductase family), Traes_3DL_EB6A17449.1 (pyrroline-5-carboxylate reductase), Traes_4BL_E4445BC35.1 (a regulatory subunit of cyclin-dependent kinases), Traes_5BL_6E095245A.1 (arginine decarboxylase belonging to the Orn/Lys/Arg decarboxylase class-II family, SpeA subfamily), and many uncharacterized proteins (Fig. 5a, Additional file 3). Interestingly, Traes_2BS_E836C5A07.1 is wheat arginase (TaARG) gene which have been functionally characterized in our previous publication [16]. In this study, TaOAT-5BL protein showed the interaction with 2BS_E836C5A07.1 (TaARG) by STRING database and this interaction was also experimentally tested by yeast two hybrid assay (Fig. 5b) which demonstrated the positive interaction of both genes. These results also supported the predicted interaction. The interaction of TaOAT with P5CS and P5CR supports the role of TaOAT in proline biosynthesis. Additionally, the interaction of TaOAT-5BL with the proteins in the arginase family (TaARG-2BS) implies the involvement of TaOAT in arginine metabolism (Fig. 5b; Additional file 1: Figure S6).
Expression profile of the wheat OAT gene in different tissues and developmental stages
To investigate the expression patterns of TaOATs, quantitative reverse transcription PCR (qRT-PCR) was performed in different tissues of wheat line Fielder. High TaOAT transcript levels were observed in stamens; moderate expression occurred in the leaf, seed, stem, and glume; and very low expression occurred in the root, pistil and palea (Fig. 6a). The expression pattern of TaOAT is similar to those of OsOAT [8]. The relative expression of transcripts in leaves gradually upregulated until it peaked at the heading stage and then decreased at the grain filling stage (Fig. 6b). Results of OAT expression at spike-developmental stages were strong at tipping, heading, and anthesis stages with the highest expression at the heading stage (Fig. 6c, d). The high expression observed in the stamen and low expression observed at the anthesis stage suggest that TaOATs are likely to be involved in anther dehiscence.
Expression patterns of TaOAT genes induced by exogenous PEG and NaCl
To refine the influence of abiotic stresses (drought and salt stresses) on expression of TaOATs, three drought-tolerant cultivars and three drought-susceptible cultivars were utilized and their expression levels of TaOATs were compared. The expression level at 0 h was set as the reference for comparisons in the data analysis (Fig. 7). The expression trends of TaOATs under PEG and NaCl stresses were similar despite their different responses in different wheat cultivars. For example, upregulated expression of TaOATs was more obvious in drought tolerant wheat cultivars than that in drought susceptible ones. The general expression trend of TaOATs first increased then decreased over time in both stress treatments of PEG and NaCl exposure. Expression peaked once at 10 h and another time at 20 h due to the 50% PEG-4000 treatment and then decreased at 40 h (Fig. 7a). Similarly, peaks of expression were observed at 20 h and 40 h due to the exposure to 200 mM NaCl stress (Fig. 7b). These results clearly suggested that TaOATs play a significant role in drought and salt stress.
Generation of stable transgenic lines and drought tolerance test
Totally, 35 independent transgenic wheat plants were obtained by Agrobacterium-mediated transformation, among which 30 were found to be positive with transgenes TaOAT-5BL by PCR detection and bar by a QuickStix Kit (Additional file 1: Figure S7). Six stable independent transgenic lines were obtained in T2 generation and 3 of them named as OE-F7, OE-F8, and OE-F9 were used to perform functional analysis. Semi-quantitative PCR analysis demonstrated that the transgenic lines showed significantly higher expression of TaOAT-5BL than wild type Fielder (Fig. 8b).
The transgenic lines were subjected to water withholding at three-leaf-stage to test the contribution of TaOAT-5BL on drought tolerance. Seventeen days after water stress, wild type Fielder was severly effected in growth by drought as compared to its corresponding transgenic lines (Fig. 8c). As OAT is predicted to be involved in proline biosynthesis, free proline content was measured at normal and stress conditions. The results showed that there is no difference in proline content between transgenic plants and the wild type under normal condition; under drought stress condition, the transgenic plants accumulated more proline than the wild type plants. These results suggested the involvevment of TaOAT-5BL in proline biosynthesis under drought stress condition.
Transgenic plants showed enhanced tolerance to salt stress in vitro condition
On 150 mM salt containing medium, the mature embryos of transgenic plants germinated with a rate of 71–85% while the mature embryos of the wild type germinated only with a rate of 27% (Additional file 1: Figure S8). Thirty days after inoculation on the salt medium, survival rate was 35–40% for the transgenic plants and 12% for the wild type plants (Fig. 9a, b). Additionally, the trangenic plants showed faster growth, longer and denser roots than the wild type plants (Fig. 9c, d). Relative expression analysis demonstrated that TaOAT-5BL was greatly up-regulated in the transgenic plants as compare to the wild type plants in response to salt stress condition (Fig. 9e). These results clearly depicted that TaOAT-5BL ehnanced salt tolerance of transgenic plants due to its high expression.