Isolation and evolution of TaPRX-2A
To obtain further insights into the evolutionary conservation or divergence of genes among class III PRXs, we identified, classified, and described the 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 that showed <50% alignment with the PRX domain (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 examined in the 12 plants ( Additional file 5: Figure S2).
Among them, we cloned one member (named TaPRX-2A) of the PRXs obtained from the wheat cultivar “Sumai 3.” The predicted TaPRX-2A open reading frame (ORF) is 1026 bp, and the deduced TaPRX-2A protein comprises 342 amino acid residues. BLAST (basic local alignment search tool) results from the National Center for Biotechnology Information (NCBI) showed that a PRX gene (GenBank: AJ878510.1) in the T. aestivum cultivar “Cheyenne” contained the minimum E value. Our local BLAST against the identified PRXs of the 12 plants showed that the T. aestivum PRX TraesCS2A02G573900.1.cds1 from subfamily VI contained 100% sequence similarity with TaPRX-2A. To investigate the evolution of this clone, we reconstructed a small NJ phylogenetic tree only containing subfamily VI PRXs from these 12 plants and compared their structural features (Fig. 1a, b). As shown, the exon–intron structure of the T. aestivum clone (TraesCS2A02G573900.1.cds1) was a one-exon structure, whereas the other four wheat and Ae. tauschii homologous PRXs (Tdi_TRIDC2AG080470.2, Ata_AET2Gv21275100.1, Tae_TraesCS2B02G613900.1.cds1, and Tdi_TRIDC2BG088710.2) in this clade also had a one-exon structure, suggesting that this one-exon structure 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 determine the origin of TaPRX-2A (TraesCS2A02G573900.1.cds1), which was involved in the processes of exon fusion (Fig. 2a). This model suggests that two rounds of exon fusion events occurred during Angiosperm and Gramineae emergence. The first exon fusion event (four exons became three) occurred during 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 the PRX domain. This four-exon structure within the “001” exon phase was 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-exon structure within the “00” exon phases, suggesting the occurrence of an exon fusion event in the last two exons of the four-exon structures within the “001” exon phases before the monocot–eudicot evolutionary split. The second exon fusion event (three exons changed into two or one exon) occurred during Gramineae emergence. As shown in Fig. 2a, the first two exons in the three-exon structure within the “00” exon phases may have fused and changed into the two-exon structure within the “0” exon phase (B. distachyon, KQJ85452). Similarly, the last two exons could have fused (T. aestivum, TraesCS2A02G574400.1; T. urartu, TRIUR3_03591-P1) or all the three exons could have fused, thus merging into a single-exon structure (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 proposed evolutionary model (Fig. 2b).
To confirm these PRX sequences for the TaPRX-2A evolutionary model, we determined the cDNA-level evidences in RNA-sequencing (RNA-seq) data from seven plants (P. patens, A. thaliana, V. vinifera, B. distachyon, Ae. tauschii, T. dicoccoides, and T. aestivum) (Additional file 6: Table S4). We did not determine these evidences in S. moellendorffii and T. urartu because their GFF3 annotation files were just in scaffolds and not in chromosomes. The results showed that most PRX sequences (except VIT_18s0072g00160.t01 and TraesCS2A02G574400.1) from from seven plants were detected in RNA-seq data (FPKM and coverage values in the “information” column of Additional file 6: Table S4), suggesting that the occurrence of exon fusion events during plant evolution.
We also detected positive selection of PRX domain sequences in TaPRX-2A and 12 other homologous PRXs using PAML 4.9 (Table 1). According to the likelihood ratio test of site-specific models, the M2a (selection) model was significantly higher than M1a (neutral) (degrees of freedom (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 hypothesis of positive selection. These positively selected sites were identified using Naive Empirical Bayes and Bayes Empirical Bayes analyses (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 the M2a and M8 models, 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 the roots, stem, and leaves, with significantly higher expression levels in root tissues than in leaf and stem tissues (Fig. 3a). Then, we checked the expression patterns of TaPRX-2A by qRT–PCR in treatments of PEG 6000, NaCl, and H2O2. The results showed that the expressions of TaPRX-2A were induced by PEG 6000, NaCl, and H2O2 treatments and the expression levels reached a peak at 6 h after treatments (Fig. 3b, c, and d). We also examined the expression patterns 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 upregulation at 12 h (Fig. 3g). These results suggested the involvement of TaPRX-2A 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 the cell 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 the nuclei of onion epidermal cells (Additional file 8: Fig. S4b3-d4). Moreover, the prediction of web server cNLS showed that five nuclear localization signal (NLS) sequences were present in TaPRX-2A (Additional file 9: Figure S5).
TaPRX-2A enhanced salt tolerance in transgenic wheat
To further confirm the function of TaPRX-2A in responses against salt stress in plants, we transformed the wheat cultivar “KN199” with 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 the transgenic lines exhibited a higher expression level than wild-type (WT) plants (Additional file 10: Fig. S6a). We subsequently measured the PRX activity in three independent transgenic lines and WT and found that the activity was higher in transgenic lines than in WT (Additional file 10: Fig. S6b). Taken together, we concluded that TaPRX-2A overexpression caused high PRX activity in transgenic lines.
Then, we measured the phenotypic differences between transgenic lines (three independent lines (TaOE1, TaOE2, and TaOE3) and WT under salt stress conditions. Under non-stress conditions, no visibly phenotypic difference was observed between TaOE1-3 and WT. Under salt stress conditions, transgenic lines showed stronger growth compared with WT. In addition, the WT leaves turned yellow and wilted under salt stress, whereas 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 drastically enhanced salt tolerance in wheat.
To further explore mechanisms underlying TaPRX-2A-mediated response to salt stress, 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; they also contained higher soluble sugar, proline, and soluble protein contents. Moreover, the proline contents of transgenic lines were approximately 2-fold greater than those of the WT plants (Fig. 5c). These results suggested that overexpression of TaPRX-2A increased the contents of metabolites that 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 have demonstrated that the tolerance to oxidative stress is associated with the physiological response of the plant to abiotic stresses [35,55,56]. Therefore, we 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 the levels of O2− (stained blue with NBT) and H2O2 (stained brown by DAB) were significantly lower in transgenic lines than in WT plants (Fig. 6a-d). In addition, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) antioxidant enzymes were higher in the transgenic plants than in the WT plants (Fig. 6e-g).
To determine whether stress-responsive genes were associated with enhancing salt tolerance through TaPRX-2A, we determined the expression patterns of various stress-related genes in TaOE plants using qRT–PCR (Fig. 7). These stress-related genes (encoding dehydration-responsive protein, RD22; thaumatin-like protein, TLP4; ABA-inducing protein, ABAI; germin-like protein, GLP4; glutathione S-transferase, GST22; and the genes encoding the ROS-scavenging enzymes FeSOD, CuSOD, and CAT) were reported to be involved in the response to various abiotic stresses. Our results showed that the majority of these stress-related genes were more highly expressed in the TaOE lines than in the WT plants under salt stress, except CuSOD expression, which was not significantly different between the WT and transgenic lines. We also found that the expression of some stress-related genes, including RD22, ABAI, and CAT, was lower in the WT plants under non-stress conditions than in the transgenic plants. Taken together, these results suggested that TaPRX-2A overexpression may improve salt tolerance in wheat by enhancing the transcription levels of stress-responsive genes.