Isolation and evolution of TaPRX-2A
In order to obtain further insights into evolutionary conservation or divergence among class III peroxidases (PRXs), we performed identification, classification and gene structures of class III PRXs. PRXs of 12 plants (T. aestivum, Triticum dicoccoides, Triticum urartu, Aegilops tauschii, Brachypodium distachyon, O. sativa, Z. mays, A. thaliana, Vitis vinifera, Selaginella moellendorffii, Physcomitrella patens and Chlamydomonas reinhardtii) were identified by HMMER 3.1 and Pfam 32.0 in batch mode with the PRX domain (peroxidase.hmm, PF00141.23) (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, HMMER3.1 scan and neighbour-joining (NJ) phylogenetic reconstruction (Additional file 2: Table S2 and Additional file 4: Figure S1). The exon-intron structures within the PRXs domain were also diagrammed in 12 investigated plants ( Additional file 5: Figure S2).
Among them, we cloned one member (named TaPRX-2A) of the PRXs in the wheat cultivar “Sumai 3”. The predicted TaPRX-2A ORF is 1026 bp, and the deduced TaPRX-2A protein comprises 342 amino acid residues. BLAST results at NCBI showed that a PRX gene (GenBank: AJ878510.1) in T. aestivum cultivar “Cheyenne” contained the minimum E value. Our local BLAST against identified PRXs of 12 plants showed that one T. aestivum PRX (TraesCS2A02G573900.1.cds1) from subfamily VI contained 100% sequence similarity with TaPRX-2A. In order to investigate the evolution of this clone, we reconstructed a small NJ phylogenetic tree only containing subfamily VI PRXs from 12 plants, and compared their structural features (Fig. 1a-b). As shown, the exon-intron structure of this T. aestivum clone (TraesCS2A02G573900.1.cds1) was one-exon structure, while the other four wheat and Ae. tauschii homologous PRXs (Tdi_TRIDC2AG080470.2, Ata_AET2Gv21275100.1, Tae_TraesCS2B02G613900.1.cds1, Tdi_TRIDC2BG088710.2) in this clade were also one-exon structure, suggesting that this one-exon structure might have originated in these PRXs before the Triticum-Aegilops split (Fig. 1b).
Based on the phylogenetic and exon-intron structure analysis (Additional file 5: Figure S2), we proposed an evolutionary model to infer the origin of TaPRX-2A (TraesCS2A02G573900.1.cds1), which was involved in the processes of exon fusion (Fig. 2a). Moreover, we concluded that two rounds of exon fusion events occurred during the Angiosperm and Gramineae emergence. The first exon fusion event (4 exons changed into 3 exons) occurred during the Angiosperm emergence. An ancestral sequence resembling P. patens PRX (Pp3c19_20780V3.3) contained a conserved exon-intron structure within four exons and the “001” exon phases near PRX domain. This four-exon structures within “001” exon phases retained in the ancestral sequences resembling two S. moellendorffii PRXs (Smo_EFJ32905 and Smo_EFJ15769). However, the exon-intron structures of PRXs in A. thaliana (Ath_AT1G71695.1), V. vinifera (Vvi_VIT_18s0072g00160.t01) and O. sativa (Osa_Os04t0688200−01) changed into the three-exons within “00” exon phases, suggesting that a exon fusion event might have happened in the last two exons of four-exon structures within “001” exon phases before the monocot-eudicot split. The second exon fusion event (3 exons changed into 2 exons or 1 exon) occurred during the Gramineae emergence. As shown in Fig. 2a, the first two exons in three-exon structure within “00” exon phases could have fused, and changed into the two-exon structure within “0” exon phase (B. distachyon, KQJ85452). Similarly, the last two exons could have also fused (T. aestivum, TraesCS2A02G574400.1; T. urartu, TRIUR3_03591-P1). Even all of the three exons could have fused, and became into a single exon structrure (Ae. Tauschii, Ata_AET2Gv21275100.1; T. dicoccoides, Tdi_TRIDC2AG080470.2, Tdi_TRIDC2BG088710.2; T. aestivum, TraesCS2A02G573900.1.cds1, Tae_TraesCS2B02G613900.1.cds1). The alignments of these PRXs within the breakpoints of exon fusion events supported our evolutionary model (Fig. 2b).
In order to confirm these PRX sequences for TaPRX-2A evolutionary model, we checked them for cDNA-level evidences in RNA-seq data from seven plants, including P. patens, A. thaliana, V. vinifera, B. distachyon, Ae. tauschii, T. dicoccoides and T. aestivum (Additional file 6: Table S4). We did not detected them in S. moellendorffii and T. urartu because their GFF3 annotation files were just in scaffolds, not in chromosomes. The results showed that most of the PRX sequences (except VIT_18s0072g00160.t01 and TraesCS2A02G574400.1) from seven plants were detected in RNA-seq data (FPKM and coverage values in “information” column of Additional file 6: Table S4), hinting that TaPRX-2A evolution model of exon fusion may happen during the plant evolution.
We also detected positive selection of PRX domain sequences in TaPRX-2A and other 12 homologous PRXs by using PAML 4.9 (Table 1). According to the LRT (likelihood ratio test) of site-specific models, model M2a (selection) was more significantly higher than M1a (neutral) (df=2, 2ΔlnL=68.4, P<0.005), indicating that some amino acid sites underwent positive selection during evolution. The M7-M8 comparison (df=2, 2ΔlnL=7.47, P<0.025) also supported the assertion of positive selection. These positively selected sites were found by using Naive Empirical Bayes (NEB) and Bayes Empirical Bayes (BEB) analysis (Additional file 7: Figure S3a,b). Two (95 E and 185 K, refer to sequence: Smo_EFJ32905) and six positively selected sites (95 E, 110 S, 117 Q, 135 E, 185 K and 212 R) were found in M2a and M8 model, respectively. Ancestral sequences in evolutionary nodes were also inferred by PAML 4.9 and MEGAX (Additional file 7: Figure S3).
Expression patterns of TaPRX-2A in various tissues and stress treatments
To detect the expression patterns of TaPRX-2A in response to stress-related signaling, we performed qRT-PCR in different tissues (leaf, stem, and root) and with different stress treatments (PEG6000, NaCl, H2O2, SA, MeJA, IAA, and ABA). The results showed that TaPRX-2A was differentially expressed in roots, stems and leaves, with significantly higher expression levels in root tissue compared with that of leaf and stem tissues (Fig. 3a). Then, we checked the expression patterns of TaPRX-2A by using qRT-PCR in treatments of PEG6000, NaCl and H2O2. The results showed that the expressions of TaPRX-2A were induced by PEG6000, NaCl and H2O2 treatments, and the expression levels reached a perk at 6 h (hour) after treatments (Fig. 3b,c and d). We also examined the expression patterns in treatments of four phytohormones. As shown in Fig. 3e, TaPRX-2A exhibited approximately 2.5-fold upregulation at 1 h after SA treatment (Fig. 3e). Similarly, the expression levels of TaPRX-2A reached a peak at 6 h after JA and ABA treatments (Fig. 3f, h). However, the expression levels of TaPRX-2A remained relatively unchanged throughout 0-6 h after IAA treatment, but exhibited an approximate 1.5-fold up-regulation at 12 h (Fig. 3g). These results showed that this TaPRX-2A was involved in various abiotic stress responses.
Subcellular localization of the TaPRX-2A protein
To characterize the function of TaPRX-2A, the ORF of TaPRX-2A was fused to a pBIN35S-eGFP vector under the control of a CaMV 35S promoter (Additional file 8: Fig. S4a). The pBIN35S:eGFP empty vector control and the pBIN35S:TaPRX-2A:eGFP recombinant vector construct were transformed into tobacco leaf cells by Agrobacterium infiltration. We observed the epidermal cells of injected N. benthamiana leaves by confocal microscopy, and found that TaPRX-2A:eGFP was mainly localized in nuclei (Additional file 8: Fig. S4b1-d2). In addition, the pBIN35S-TaPRX-2A-eGFP and pBIN35S-eGFP vector were transformed into onion epidermal cells. Consistent with the localization results observed in tobacco epidermal cells, the TaPRX-2A:eGFP was also mainly localized in nuclei of the onion epidermal cells (Additional file 8: Fig. S4b3-d4). Moreover, the prediction of web server cNLS showed that five NLSs (nuclear localization signals) sequences existed in TaPRX-2A (Additional file 9: Figure S5).
TaPRX-2A enhanced the salt tolerance in transgenic wheat
To further confirm the function of TaPRX-2A in salt stress responses, we transformed wheat cultivar “KN199” with a TaPRX-2A-overexpression and constructed three independent transgenic lines (TaOE1, TaOE2 and TaOE3). The expression profile of TaPRX-2A was analyzed in TaPRX-2A transgenic lines through qRT-PCR. The results showed that transgenic lines exhibited higher expression level than wild type (WT) plants (Additional file 10: Fig. S6a). Then, we measured the peroxidase activity in three independent transgenic lines and WT. The peroxidase activity was higher in transgenic lines than WT (Additional file 10: Fig. S6b). Taken together, we concluded that TaPRX-2A overexpression caused high peroxidase activity in transgenic lines.
Then, we measured the phenotypic differences between transgenic lines (three independent lines, TaOE1, TaOE2 and TaOE3) and WT in salt stress conditions. Under non-stress condition, no visibly phenotypic difference was observed between TaOE1-3 and WT. Under salt condition, the TaOE lines showed stronger growth comparing with the WT. In addition, the WT leaves turned yellow and wilted under salt stress, while the TaOE leaves still remained green (Fig. 4a). We also found that the survival rate of WT plants was only 40% after salt treatment, whereas the survival rates among TaOE1, TaOE2, and TaOE3 plants were 63.6%, 57.6%, and 63%, respectively (Fig. 4b). We then compared the shoot lengths, relative water content (RWC), and root lengths between WT and TaOE plants under salt treatment (Fig. 4c, d and e). The results showed that transgenic lines exhibited longer shoot length and higher RWC than WT plants. However, no significant difference in root lengths was observed between WT and transgenic lines. Taken together, these results indicated that TaPRX-2A overexpression dramatically enhanced the salt tolerance in wheat.
To further explore mechanisms underlying TaPRX-2A-mediated response to salt stress response, we measured physiological-biochemical indices between TaOE and WT plants under non-stress and salt stress conditions (Fig. 5a-d). Under salt treatment, TaOE lines contained significantly lower malondialdehyde (MDA) content than WT, but higher soluble sugar, proline, and soluble protein contents. Moreover, the proline contents of transgenic lines were approximately 2-fold greater than that of WT (Fig. 5c). These results suggested that overexpression of TaPRX-2A increased the contents of metabolites which were necessary for osmotic and oxidative stress tolerance in wheat cultivars “KN199”, thus resulting in improving tolerance to salt.
TaPRX-2A regulates ROS scavenging and the expression of stress-related genes in transgenic wheat
Previous studies showed that the tolerance to oxidative stress was associated with plant physiological response to abiotic stresses [35,55,56]. We therefore examined the function of TaPRX-2A in reducing ROS levels in transgenic lines under salt stress. As major indicators of the ROS level, we assayed the accumulation of O2- and H2O2 for comparison between TaOE and WT lines using Nitroblue Tetrazolium (NBT) and 3-diaminobenzidine (DAB) staining. Under salt treatment, we found that levels of O2- (stained blue with NBT) and H2O2 (stained brown by DAB) were significantly lower in TaOE transgenic lines than in WT (Fig. 6a-d). In addition, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) antioxidant enzymes were also measured between TaOE and WT plants. The results indicated that the transgenic plants contained higher activities of SOD, CAT and POD antioxidant enzymes than the WT plants (Fig. 6e-g).
To determine whether stress-responsive genes were associated with enhancing the salt tolerance by TaPRX-2A, We checked the expression patterns of various stress-related genes in TaOE plants by using qRT-PCR (Fig. 7). These stress-related genes (encoding dehydration-responsive protein, RD22; thaumatin-like protein, TLP4; ABA-inducing, ABAI; germin-like protein, GLP4; glutathione S-transferase, GST22; and the genes encoding ROS-scavenging enzymes FeSOD, CuSOD, and CAT) were reported to be response to various abotic stresses. The results showed that majority of these stress-related genes had higher expression in TaOE lines than in WT under salt stress. However, expression of CuSOD was not significantly different between WT and transgenic lines under salinity stress. In addition, we found lower expression of some stress-related genes in WT plants under non-stress conditions, including RD22, ABAI, and CAT. Taken together, these results indicated that TaPRX-2A overexpression may improve salt tolerance in wheat by enhancing the transcription levels of stress-responsive genes.