Isolation and evolution of TaPRX-2A
In order to obtain further insights into class III peroxidase (PRX) evolution, we performed identification, classification and gene structures of class III PRXs. We searched 12 plants (S. moellendorffii,Z. mays, B. distachyon, T. aestivum, Ae. tauschii, T. dicoccoides, V. vinifera, T. urartu, O. sativa, A. thaliana, P. patens and C. reinhardtii) by HMMER 3.1 and Pfam 32.0 in batch mode with the PRX domain ( Additional file 1: Table S1, and Additional file 2: Table S2). We excluded the atypical PRXs of these 12 plants with less than 50% alignment of PRX domain in the following analysis (Additional file 3: Table S3). The classification of these PRXs was based on two methods, HMM scan and NJ phylogenetic tree (Additional file 2: Table S2 and Additional file 4: Figure S1). The exon-intron structures within the PRX domain were also diagrammed in 12 investigated plants ( Additional file 5: Figure S2).
Among them, we cloned one member of the TaPRXs in the wheat cultivar “Sumai 3”, and named it TaPRX-2A. The predicted TaPRX-2A ORF is 1026 bp, and the deduced TaPRX-2A protein comprises 342 amino acid residues. We studied this clone in NCBI by BLAST and found that it was a T. aestivum PRX gene (GenBank: AJ878510.1) in cultivar “Cheyenne”. We studied this clone in our identified T. aestivum class III PRX gene family by our local BLAST, and found one sequence (TraesCS2A02G573900.1.cds1) from subfamily VI PRXs with 100% sequence similarity. In order to investigate the evolution of this clone, we conducted a phylogenetic tree and gene structures of subfamily VI PRXs in 12 plants (Fig. 1a). As shown, this clone (TraesCS2A02G573900.1.cds1) in T. aestivum contained only one exon, while four other wheat and Ae. Tauschii homologous PRXs (Tdi_TRIDC2AG080470.2, Ata_AET2Gv21275100.1, Tae_TraesCS2B02G613900.1.cds1, Tdi_TRIDC2BG088710.2) in this clan of the NJ tree also contained only one exon, hinting that this one exon structure of these five PRXs might have formed before the Triticum-Aegilops split in evolution (Fig. 1b). Based on the analysis of exon-intron gene structure diagrams (Additional file 5: Figure S2), we inferred an evolutionary model of this T. aestivum PRX (TraesCS2A02G573900.1.cds1) about exon fusion (Fig. 2a). As shown, P. patens PRX (Pp3c19_20780V3.3) contained the conserved exon-intron structure within the “001” exon phases in the PRX domain. The “001” exon-intron structures could also be found in most subfamilies V-XVIII. Two S. moellendorffii PRXs (Smo_EFJ32905 and Smo_EFJ15769) retained this “001” exon-intron structure. However, the last two exons might have experienced the exon fusion event before the monocot-eudicot split. Indeed, subfamily VI PRX of the two investigated eudicots (A. thaliana and V. vinifera) contained only one member (Ath_AT1G71695.1 and Vvi_VIT_18s0072g00160.t01), and their exon-intron structures changed into “00”. Subfamily VI PRX of monocots also contained this “00” structure. For instance, rice PRX (Osa_Os04t0688200 − 01) contained this “00” structure. These three exons might have fused into one exon before the Triticum-Aegilops split in evolution to become the ancestor of TraesCS2A02G573900.1.cds1. Interestingly, the alignment and exon phases of these PRXs supported our evolutionary model about exon fusion events (Fig. 2b).
Expression patterns of TaPRX-2A in various tissues and with hormone treatments
To detect the expression patterns of TaPRX-2A we perfomed the qRT-PCR in different tissues and with various hormone treatments. The results showed that this clone exhibited expressions in roots, stems and leaves, and significantly high expression levels in root tissue comparing with leaf and stem tissues (Fig. 3a). In order to determine the stress effects on the expression of this TaPRX-2A clone, the qRT-PCR with various hormone treatments were performed (Fig. 3). The results showed that the expressions of TaPRX-2A were induced by drought (PEG6000), NaCl and H2O2 treatments, and the expression levels reached the perk in 6 h (hour) after treatments (Fig. 3b,c and d). We also detected the expression patterns of TaPRX-2A in four phytohormones (salicylic acid (SA), methyljasmonic acid (MeJA), indole-3-acetic acid (IAA) and abscisic acid (ABA) treatments (Fig. 3e-h). As shown in Fig. 3e, this TaPRX-2A clone exhibited approximately 2.5-fold upregulation at 1 h after SA treatment (Fig. 3e). Similarly, the expression levels of TaPRX-2A reached the peak at 3 h and 6 h after JA and ABA treatment, respectively (Fig. 3f, h). However, the expression levels of TaPRX-2A remained almost the same at 0–6 h after IAA treatment, but exhibited about 1.5-fold upregulation at 12 h (Fig. 3g). These results showed that this TaPRX-2A clone was involved in various abiotic stress responses and plays important role in plant stress responses.
Subcellular localization of the TaPRX-2A protein
To characterize the function of TaPRX-2A, the ORF of TaPRX-2A was fused to pBIN35S-eGFP vector with the CaMV 35S promoter (Additional file 6: Figure S3). The pBIN35S:eGFP and recombinant construct pBIN35S:TaPRX-2A:eGFP vector were transformed into tobacco leaf cells by Agrobacterium infection. We observed the epidermal cells of injected N. benthamiana leaves by confocal microscope and found that TaPRX-2A gene was localized mainly in the nuclei. In addition, the pBIN35S-TaPRX-2A-eGFP and pBIN35S-eGFP vector were transformed into onion epidermal cells. Consistent with the location results of tobacco epidermal cells, the TaPRX-2A gene was also located on in the nuclei. In summary, we showed that TaPRX-2A is a nuclear protein.
TaPRX-2A enhanced the salt tolerance in transgenic wheat
To further confirm the function of TaPRX-2A in salinity stress responses, TaPRX-2A-overexpressing wheats were constructed. Firstly, we tested the salinity tolerance between TaPRX-2A-overexpressing wheat lines (TaOE1, TaOE2 and TaOE3) and wild-type (WT) wheats. The result showed that there were no obvious phenotypical differences between the WT and TaPRX-2A-overexpressing wheat lines under normal conditions. However, the TaPRX-2A-overexpressing wheat lines showed better growth under salinity conditions comparing with the WT. Moreover, the leaves of WT wheats began to turn yellow and wilt comparing with the TaPRX-2A-overexpressing transgenic lines under salinity stress (Fig. 4a). We found that the survival rate of WT plants was only 40% after salt treatment, whereas the survival rates of TaPRX-2A-overexpressing transgenic lines TaOE1, TaOE2 and TaOE3 were 63.6%, 57.6% and 63%, respectively, (Fig. 4b). Moreover, we also compared the shoot lengths, relative water content (RWC) and root lengths between WT plants and TaPRX-2A-overexpressing transgenic lines under salt treatment (Fig. 4c, d and e). The results showed that TaPRX-2A-overexpressing transgenic lines contained longer shoot length and more RWCs than WT plants. However, the root lengths between them were almost the same. Take together, these results indicated that TaPRX-2A dramatically enhanced the salt tolerance in wheat.
To explore further mechanism of the TaPRX-2A involving in the salt stress response, some imperative physiological-biochemical indices were measured between transgenic wheats and WT wheats (Fig. 5a-d). Under salt treatment, we found that TaPRX-2A-overexpressing transgenic lines contained significantly lower MDA contents than WT, but more soluble sugar, proline and soluble protein contents. Moreover, the proline contents of transgenic lines are approximately 2-fold than WT (Fig. 5c). These results suggested that TaPRX-2A-overexpressing transgenic lines increased the osmotic and oxidative stress tolerance, resulting in improved the tolerances to salt.
TaPRX-2A regulates ROS scavenging and the expression of stress-related genes in transgenic wheat
Tolerance to oxidative stress was associated with tolerance to abiotic stresses [30, 40, 41]. The function of TaPRX-2A was confirmed in reducing ROS levels in transgenic lines under salinity stress [4]. As major indicators of the ROS level, O2− and H2O2 contents were assayed in this study. Subsequently, we detected the accumulation of O2− and endogenous H2O2 between transgenic plants and WT lines with NBT (Nitroblue Tetrazolium) and 3-diaminobenzidine (DAB) staining, respectively. Under salt treatment, O2− (stained by blue with NBT) and the H2O2 (stained by brown with DAB) levels were significantly lower in TaPRX-2A-overexpressing transgenic lines than WT (Fig. 6a−d). In addition, the activities of SOD, POD, and CAT antioxidant enzymes were also measured between TaPRX-2A-overexpressing transgenic lines and WT plants. The results indicated that the transgenic plants contained significantly higher activities of these three antioxidant enzymes than the WT plants (Fig. 6e−g).
To determine whether the expressions of stress-responsive genes were involved in the function of TaPRX-2A under salt stresses, some stress-related genes (including dehydration-responsive proteins such as RD22; thaumatin-like proteins such as TLP4; ABA-inducing gene ABAI; germin-like proteins such as GLP4; glutathione S-transferases such as GST22; and the genes encoding ROS-scavenging enzymes (FeSOD, CuSOD, CAT, APX) were selected to detect their expressions by using qRT-PCR (Fig. 7). The results showed that almost all these stress-related genes in TaPRX-2A-overexpressing lines contained more expression levels than WT under salinity stresses. However, the expression levels of the CuSOD gene showed almost the same between WT and transgenic lines under salinity stresses. In addition, we found that the expressions of some stress-related genes, such as RD22, ABAI, CAT and APX, were lower in the WT plants than in TaPRX-overexpressing transgenic lines under normal conditions. In conclusion, these results indicated that TaPRX-2A may improve the salt tolerances by enhancing the stress-responsive genes transcription levels.