For the analysis of the tissue-specific expression of ScAPX6, six healthy plants of 10 months old of ROC22 were used, and then +1 leaf, root, bud, skin and pith were collected. The samples were fixed in liquid nitrogen and stored at −80°C until the extraction of total RNA.

For the abiotic treatment, uniform tissue cultured plantlets of ROC22 at the five or six leaf stage were transferred to water for one week and then treated by the following six stress conditions with root dipping of 5 mmol·L⁻¹ SA, 100 μmol·L⁻¹ MeJA, 100 μmol·L⁻¹ ABA, 10 μmol·L⁻¹ H₂O₂, 25.0% PEG 8000, 250 mmol·L⁻¹ NaCl, and 100 mmol·L⁻¹ copper chloride (CuCl₂), at 28°C with 16 h light and 8 h darkness (Su et al., 2014a). The whole plantlets under SA, MeJA, H₂O₂, PEG, and NaCl stresses were harvested at 0, 6, 12, and 24 h, respectively. Another set of plantlets under Cu stress was harvested at 0, 12, 24, and 48 h, respectively. Three plantlets per time point were gathered and immediately fixed in liquid nitrogen, and stored at −80°C until the extraction of total RNA.

Total RNA of the treated samples was extracted by Trizol® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The first-strand cDNA synthesis was performed using Prime-Script^TM RT Reagent Kit (TaKaRa, Dalian, China) following manufacturer's instructions and tested by 1% agarose gel electrophoresis.

The sequence of a putative APX6 unigene (ScAPX6) from our previous transcriptome data of sugarcane in response to SrMV infection was used to design the cloning primer APX6-1F/1R (Table 1). The first-strand cDNA of ROC22 was used as amplification template. The reverse transcription–polymerase chain reaction (RT-PCR) procedure was 94°C for 4 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 35 cycles; and 72°C for 10 min. RT-PCR products were gel-purified and cloned into pMD19-T vector (TaKaRa, Dalian, China), and then transformed into E. coli DH5α competent cells and sequenced (Sangon, Shanghai, China).

The open reading frame (ORF) of ScAPX6 with Gateway entry adapters attB1 and attB2 was amplified from the plasmid of pMD19-T-ScAPX6 by the primers (APX6-3F/3R) (Table 1). The touchdown PCR procedure was 94°C for 4 min; 94°C for 30 s, 70°C for 30 s and then each loop drop 0.5°C, 72°C for 1 min and 30 s, 35 cycles; and 72°C for 10 min. The PCR amplification products were gel-purified and transformed into the Gateway^@ donor vector of pDONR221 (Invitrogen, USA) following the manufacturer's instructions of Gateway® BP Clonase™ II Enzyme Mix (Invitrogen, USA). The mixture of BP reaction was transformed into DH5α competent cells and sequenced (Sangon, Shanghai, China). The positive plasmid pDONR221-ScAPX6 was achieved and then used for the constructions of prokaryotic expression vector and eukaryotic expression vector.

The ORF was translated and analyzed by ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Conserved domain of ScAPX6 was predicted by the SMART program (http://smart.embl-heidelberg.de/) and NCBI Conserved Domains Database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The ExPASy tool (http://us.expasy.org/tools) was used to predict the basic physical and chemical properties of ScAPX6. The cleavage sites of the signal peptides were predicted by SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/). Prediction of transmembrane helices in ScAPX6 protein was performed by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Psort software was used to predict the subcellular localization of ScAPX6. GOR IV software (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_gor4.html) was used to analyze the secondary structure of ScAPX6. The protein 3D model was predicted by SWISSMODEL software (http://swissmodel.expasy.org/). The homologous sequences of ScAPX6 were obtained using Blastp in NCBI. DNAMAN software was used for the multiple sequence alignment. The phylogenetic tree of ScAPX6 was constructed with amino acid sequences from other species by the neighbor-joining (NJ) method (1,000 bootstrap replicates) using the MEGA 6.06 (Saitou and Nei, 1987).

The ORF of ScAPX6 was amplified by the primer APX6–4F/4R, and then was inserted into the BsaI and Eco31I restriction sites of the pBWA(V)HS-ccdb-GLosgfp vector. Then the recombinant vector pBWA(V)HS-ScAPX6-GLosgfp and the chloroplast marker vector were co-transformed in rice protoplasts with PEG solution (40% W/V PEG 4000, 0.2 mol·L⁻¹ mannitol and 0.1 mol·L⁻¹ calcium chloride). The mixture was cultured in dark for 30 min, and then the protoplasts was gathered and cultured in dark for 16–24 h. The method of transformation of rice protoplasts was followed by Datta and Datta (1999). The subcellular localization of the fusion protein was observed by a confocal laser scanning microscope Leica TCS SP5 (Germany).

The plasmid of pDONR221-ScAPX6 was digested with AvaI and then gel-purified for LR reaction with prokaryotic expressive vector of pEZYHb according to the manufacturer's instructions of LR Clonase™ II Enzyme Mix (Invitrogen, USA). The recombinant plasmid of pEZYHb-ScAPX6 was transformed into the competent cells E. coli BL21 (DE3) and then induced by 1.0 mmol·L⁻¹ isopropyl β–D-thiogalactoside (IPTG) at 28°C for 0, 2, 4, and 8 h (Guo et al., 2008). LB medium with E. coli BL21 (blank) and BL21+pEZYHb (control) were induced by 1.0 mmol·L⁻¹ IPTG for 0 and 8 h, respectively. The collected bacterial protein was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Spot assay was conducted to study the responses of E. coli BL21 cells expressing the ScAPX6 gene under abiotic stress, such as NaCl, Cu and PEG. When OD₆₀₀ of E. coli BL21 cells in LB medium (containing 80 μg·mL⁻¹ ampicillin) reached to 0.6, IPTG with a concentration of 1.0 mmol·L⁻¹ was added, and then the cells were grown at 37°C for 12 h. The cultures were diluted to OD₆₀₀ = 0.6, and then diluted to two levels of 10⁻³ and 10⁻⁴ (Guo et al., 2012). Ten microliters from each level was spotted on LB plates containing NaCl (250, 500, and 750 mmol·L⁻¹), CuCl₂ (250, 500, and 750 μmol·L⁻¹) and PEG (15, 30, and 45%), respectively (Su et al., 2013). All plates were cultured in 37°C overnight and photographed.

SYBR Green Master (ROX) (Roche, China) and a 7500 qRT-PCR system (Applied Biosystems, South San Francisco, CA, USA) were applied to analyze gene expression levels of ScAPX6 in different tissues and in response to various stresses. The sequence-specific primer of ScAPX6 (APX6-2F/2R) (Table 1) was designed by Premier 5.0 software. The primer combination of clathrin adaptor complex (CAC) and cullin (CUL) (Table 1) was regarded as the internal control (Guo et al., 2014). The 20 μL reaction system containing 10 μL SYBR Green Master Mix, 0.8 μL each of 10 μmol·L⁻¹ upstream and downstream primers, 2 μL cDNA templates (20 × diluted cDNA) and 6.4 μL double distilled water. Each qRT-PCR was conducted in triplicate. The qRT-PCR procedure was 50°C for 2 min; 95°C for 10 min; 35 cycles of 95°C for 15 s, and 60°C for 1 min. The 2^−ΔΔCt method (Livak and Schmittgen, 2001) was employed to analyze the qRT-PCR data.

To study the role of ScAPX6 in response to pathogen infection and its hypersensitive reaction in plant, an overexpressed vector pEarleyGate 203-ScAPX6 was constructed by Gateway cloning technique according to the manufacturer's instructions of LR Clonase™ II Enzyme Mix (Invitrogen, USA). N. benthamiana leaves was inoculated with the vector of pEarleyGate 203-ScAPX6 by an Agrobacterium-mediated transient expression method conducted by Su et al. (2014b). Two important tobacco pathogens, Pseudomonas solanacearum and Fusarium solani var. coeruleum, were cultured in potato dextrose water (PDW) liquid medium at 28°C. When the two pathogens cells were cultured to an OD₆₀₀ of 0.8, they were separately infected into the treated leaves that were agroinfiltrated with pEarleyGate 203-ScAPX6 for 24 h. Then the 3,3′-diaminobenzidine (DAB) staining, trypan blue staining and transcript analysis of the eight tobacco immunity-associated marker genes (Table 1), were conducted by the treated N. benthamiana leaves according to Su et al. (2016). RT-PCR was used to detect whether ScAPX6 has been overexpressed in N. benthamiana, with the RNA of treated leaves and ScAPX6 specific primer (APX6-3F/3R, Table 1), the NtEF1-α was treated as control. RT-PCR procedure was 94°C for 4 min; 94°C for 30 s, 72°C for 30 s, 72°C for 2 min, 35 cycles; and 72°C for 10 min. All treatment materials were cultured at 24°C (16 h light/8 h darkness) and then photographed at 1 and 7 day (d) separately. Each test was repeated three times.

DAB and trypan blue staining were used for histochemical analysis of Agrobacterium-infiltrated leaves. The leaves was soaked in DAB-HCl solution (1.0 mg·mL⁻¹, pH 5.8), and then cultured in the dark for 12 h. The leaves were destained with 95% ethanol at 100°C for 10 min (Su et al., 2014a), and then was imaged for H₂O₂ detection with a stereoscopic microscope (Nikon, Tokyo, Japan) and a light microscope (Leica, Wetzlar, Germany). Three biological replicates were prepared. The leaves were also deal with trypan blue mixture, containing 10 mL lactic acid, 10 g phenol, 10 mL glycerol, 30 mL absolute ethanol, 10 mg trypan blue, and 10 mL distilled water, and then was boiled for 5 min. After staining, the leaves were soaked in a chloral hydrate solution (2.5 g·mL⁻¹) for decoloring (Dang et al., 2013). The blue color of the leaves for the cell death was also imaged with a stereoscopic microscope (Nikon, Tokyo, Japan) and a light microscope (Leica, Wetzlar, Germany).

In the present study, a full-length cDNA of APX6 unigene, which was named as ScAPX6 (GenBank Acc. No. KT907352), was isolated from ROC22. The cDNA sequence length of ScAPX6 was 1,086 bp (Figure S1) with a complete ORF (1,002 bp, from position 27 to position 1,025), encoding 333 amino acid residues. ScAPX6 had a molecular mass of 36.21 kDa and an isoelectric point (pI) of 6.91. CDD search of NCBI showed that ScAPX6 belonged to a member of the plant-peroxidase-like superfamily (Figure 1). The instability index of ScAPX6 protein was 48.10, suggesting that ScAPX6 might be an unstable acid hydrophilic protein (Walker, 2005). Secondary structure prediction of ScAPX6 predicted that the percentages of alpha-helix, random coil, and extended strand were 44.74, 42.94, and 12.31%, respectively.

Furthermore, SWISSMODEL program showed that the main spatial structures of ScAPX6 were alpha-helix and random coil (Figure 2). Comparing ScAPX6 with O. sativa Japonica Group APX6 (EAZ43377.1), S. italic APX6 (XP_004973913.1), and S. bicolor APX6 (XP_002445876.1), we found that the spatial structure of these four APX6 was basically in line with each other, suggesting that ScAPX6 owned high conservation of spatial structure with different plant species. Psort software predicted that ScAPX6 might be located in the chloroplast thylakoid membrane, plasma membrane, chloroplast stroma, and chloroplast thylakoid space with the probabilities of 71.9, 65.0, 56.1, and 56.1%, respectively.

According to the classification method by Teixeira et al. (2004), the phylogenetic tree was separated into three groups, including cytosolic isoforms, Apx-R isoforms and chloroplastic isforms (Figure 3). ScAPX6 was clustered into group chloroplastic isforms. Two APX proteins reported in Saccharum hybrid cultivar, which were ScAPX (AIG52216.1) and TAPX (AGD80596.1), were also clustered into the same clade as ScAPX6.

The recombinant vector pBWA(V)HS-ScAPX6-GLosgfp was constructed to understand the subcellular location of ScAPX6. The results showed that ScAPX6 and the chloroplast marker were located in the same place, so it was confirmed that ScAPX6 was located in the chloroplast, which is in accordance with the results of prediction (Figure 4).

The ScAPX6 gene was combined with the expression vector pEZYHb and then was transformed into E. coli BL21 cell. The SDS-PAGE analysis (Figure 5) showed that ScAPX6 was expressed as a recombinant protein in the BL21 cells. In Figure 5, after induced by 1.0 mmol·L⁻¹ IPTG at 28°C for 2, 4, and 8 h, an obvious accumulation protein (including the 6× His-tag) at approximate 55 kDa was observed.

It has been reported that the APX activity was up-regulated by abiotic stress, such as heavy metal, high salinity, drought, high temperature, and wounding (Shi et al., 2001). In this study, spot assay was performed to study the function of ScAPX6 in response to abiotic stress in vivo. The control (BL21+pEZY-Hb) and the gene-expressed cells (BL21+pEZY-Hb-ScAPX6) grew in LB plates containing NaCl, CuCl₂, and PEG were performed (Figure 6). It had been recorded that the recombinant ScAPX6 cells showed a more rapid growth than the control in LB plates with PEG and CuCl₂ supplement, but not with NaCl, suggesting that the overexpression of ScAPX6 in E. coli could enhance its tolerance to PEG and Cu stress.

qRT-PCR analysis showed that ScAPX6 was constitutively expressed in all five kinds of sugarcane tissues, including root, bud, skin, leaf, and pith, but with different expression levels (Figure 7). ScAPX6 showed the highest expression levels in the pith and leaf, and then on the skin, while the transcript in root was at the lowest level.

qRT-PCR analysis revealed that the ScAPX6 gene exhibited different expression characteristics in response to ABA, MeJA, SA, H₂O₂, PEG, NaCl, and Cu stimuli (Figures 8A,B). As shown in Figure 8A, the transcripts of ScAPX6 were remarkably up-regulated under the stresses of ABA and MeJA, and with the highest inducible expression levels at 6 h, which were 6.0- and 70.0-times higher than that of control, respectively. However, ScAPX6 was down-regulated during the SA treatment and rapidly decreased at 6 h. Under the stress of PEG, the expression of ScAPX6 showed no change at 6 h, and then decreased at 12 h. ScAPX6 was down-regulated after the treatment of H₂O₂ and NaCl, but was up-regulated by the CuCl₂. These results demonstrated that ScAPX6 might positively respond to ABA, MeJA, and Cu stresses but negatively respond to SA, H₂O₂, PEG, and NaCl stresses.

After transient overexpression of ScAPX6 in N. benthamiana leaves for 1 day (d), the transcripts of ScAPX6 were detected by qRT-PCR (Figure 9A). As shown in Figures 9A,B darker DAB staining color and more intense trypan blue staining cells were observed in ScAPX6 leaves than that in the control (35S::00) after infiltration for 2 and 6 d, respectively. The eight immunity-associated marker genes in N. benthamiana were induced by transient overexpression of ScAPX6 (Figure 9C). The hypersensitive response (HR) marker genes, NtHSR201 and NtHSR203, showed no change in transcript, while NtHSR515 was up-regulated. The expression level of SA-responsive gene NtPR2 remained unchanged, while NtPR-1a/c and NtPR3 and two ethylene synthesis dependent genes, NtEFE26 and NtAccdeaminase, were all down-regulated. Compared with the control leaves, 35S::ScAPX6 exhibited darker color reflecting high levels of H₂O₂ accumulation and intense hypersensitivity response.

To further investigate the response of ScAPX6 to pathogen, two tobacco pathogens, P. solanacearum and F. solani var. coeruleum, were separately injected into N. benthamiana containing 35S::ScAPX6 or the control. After inoculation with P. solanacearum, no disease symptom was found between 35S::ScAPX6 and the control leaves for 1 d, while 35S::ScAPX6 exhibited darker color than the control at 1 d by DAB staining. With elongated treatment time, although the DAB staining showed no difference between 35S::ScAPX6 and the control leaves, the leaves in the control showed slight yellow phenomenon and necrotic spot, while the 35S::ScAPX6 only exhibited the faint wilting symptom after inoculation at 7 d (Figure 9D). After challenging with P. solanacearum for 1 and 7 d (Figure 9E), the expression levels of NtHSR201, NtHSR203, and NtPR2, were unchanged or down-regulated in the control and 35S::ScAPX6 leaves. The transcripts of NtPR-1a/c and NtPR3 were significantly down-regulated at 1 d and increased at 7 d in the control leaves, but were down-regulated or remained unchanged in the 35S::ScAPX6 leaves. The expression levels of NtHSR515 and NtEFE26 were unchanged at 1 and 7 d and NtAccdeaminase was up-regulated at 7 d in the control leaves, while these three genes in the 35S::ScAPX6 leaves were all unchanged at 1 d and reached the peak values at 7 d after inoculation.

Likewise, for DAB staining, the 35S::ScAPX6 leaves showed darker color than the control after inoculation with F. solani var. coeruleum for 1 d and exhibited no difference at 7 d. No apparent disease symptom differences between 35S::ScAPX6 and the control were found at 1 d after inoculation (Figure 9F). Some symptoms, such as wilting, decay phenomenon and necrotic spot, were observed in the control leaves at 7 d, but not in the 35S::ScAPX6 leaves. After challenging with F. solani var. coeruleum for 1 and 7 d (Figure 9G), the expression levels of NtHSR201 and NtPR2 were unchanged or down-regulated in the control and 35S::ScAPX6 leaves. The transcripts of NtAccdeaminase, NtHSR203, and NtHSR515, were significantly up-regulated at 1 or 7 d in the control leaves, while stayed stable or showed a small rise in the 35S::ScAPX6 leaves. The transcripts of NtPR-1a/c, NtPR3, and NtEFE26, were down-regulated or remained unchanged in the control leaves, but were all significantly up-regulated in the 35S::ScAPX6 leaves.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.