A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane

Abstract

The L-ascorbate peroxidase 6 gene (APX6) is one of the most important genes for scavenging H₂O₂ and plays a vital role in plant resistance to environmental stresses. In this study, a novel ScAPX6 gene (GenBank Accession No. KT907352) was obtained from a sugarcane variety (ROC22). Bioinformatics analysis showed that ScAPX6 has a cDNA length of 1,086 bp and encoded 333 amino acid residues. Subcellular localization confirmed that ScAPX6 was located in the chloroplast. Enhanced growth of Escherichia coli BL21 cells that expressed ScAPX6 showed high tolerance under copper (Cu) stress. Real-time quantitative PCR analysis revealed that ScAPX6 was constitutively expressed wherein with the highest expression levels in sugarcane pith and leaf and the lowest in the root. ScAPX6 was down-regulated by salicylic acid (SA), hydrogen peroxide (H₂O₂), polyethylene glycol (PEG) and sodium chloride (NaCl) stimuli. Interestingly, it was significantly up-regulated under the stresses of abscisic acid (ABA) and methyl jasmonate (MeJA) wherein with the highest inducible expression levels at 6 h at 6.0- and 70.0-times higher, respectively than that of control. Overexpression of ScAPX6 in Nicotiana benthamiana leaves enhanced the resistance to the infection of tobacco pathogens Pseudomonas solanacearum and Fusarium solani var. coeruleum. These results implied that ScAPX6 might positively respond to ABA, MeJA, and Cu, but might negatively respond to the stresses of SA, H₂O₂, PEG, and NaCl. Keeping in view the current investigation, ScAPX6 could be associated with the hypersensitive response (HR) or immunity of sugarcane, which will provide a baseline for the function identification of sugarcane ScAPX6.

Keywords: L-ascorbate peroxidase 6 gene; biotic and abiotic stresses; subcellular localization; sugarcane; transient overexpression.

Figure 1

Nucleotide acid sequence and deduced amino acid sequence of sugarcane ScAPX6 gene obtained by RT-PCR. The start codon and termination codon were underlined in black. The primer used in RT-PCR was underlined in red line. The peroxidase like superfamily domain contains 222 amino acids (from 103 to 324) was highlighted in red. The amino acids highlighted in yellow represented the heme binding site. ^*, Stop codon.

Figure 2

Predicted 3D structure of ScAPX6. The plant-peroxidase-like domain was in red. Saccharum spp. hybrids (AMQ80947.1), Sorghum bicolor (XP_002445876.1), Setaria italica (XP_004973913.1), Oryza sativa Japonica Group (EAZ43377.1).

Figure 3

Phylogenetic analysis of deduced amino acid sequence from ScAPX6 and other ascorbate peroxidases proteins. The GenBank accession number of proteins were according to Teixeira et al. (2004) and downloaded from NCBI. The neighbor-joining method with 1,000 bootstrap replications was used.

Figure 4

Subcellular localization analysis of ScAPX6 in rice protoplasts. (a,d) green fluorescence; (b,f) visible light; (e) red fluorescence from chloroplast marker; (c,g) merged light.

Figure 5

Prokaryotic expression of pEZY-Hb-ScAPX6 fusion protein in Escherichia coli BL21 (DE3). M, protein marker; 1, BL21 cell induction for 8 h; 2, BL21 cell without induction; 3, pEZY-Hb without induction; 4, pEZY-Hb induction for 8 h; 5–8, pEZY-Hb-ScAPX6 induction for 8, 4, 2, and 0 h, respectively.

Figure 6

Spot assays of BL21+pEZY-Hb-ScAPX6 (b) and BL21+pEZY-Hb (control) (a) on LB plates with NaCl, PEG and CuCl₂. Isopropyl β-D-thiogalactoside (IPTG) was added to the cultures of BL21+pEZY-Hb-ScAPX6 and BL21+pEZY-Hb to induce the expression of recombinant protein. The cultures were adjusted to OD₆₀₀ = 0.6. Ten microliters from 10⁻³ (left side of the red line on the plate) to 10⁻⁴ (right side of the red line on the plate) dilutions were spotted onto LB plates without any supplement (CK) (A) or with NaCl (250, 500, and 750 mmol·L⁻¹) (B), PEG (15, 30, and 45%) (C) and CuCl₂ (250, 500, and 750 μmol·L⁻¹) (D), respectively. NaCl, sodium chloride; PEG, polyethylene glycol; CuCl₂, copper chloride.

Figure 7

Tissue-specific expression analysis of ScAPX6 in sugarcane. The error bars represented the standard error of each treating group (n = 3). Data were normalized to the CAC and CUL expression level. All data points were means ± SE (n = 3). Different lowercase letters indicate a significant difference, as determined by the Duncan's new multiple range test (p < 0.05).

Figure 8

Gene expression patterns of ScAPX6 in sugarcane under various plant hormones (A) and abiotic stresses (B). Data were normalized to the CAC and CUL expression level. All data points were means ± SE (n = 3). Different lowercase letters indicate a significant difference, as determined by the Duncan's new multiple range test (p < 0.05). ABA, abscisic acid; SA, salicylic acid; MeJA, methyl jasmonate; PEG, polyethylene glycol; H₂O₂, hydrogen peroxide; NaCl, sodium chloride; CuCl₂, copper chloride.

Figure 9

Transient overexpression of ScAPX6 in Nicotiana benthamiana leaves. (A) RT-PCR analysis of ScAPX6 in the N. benthamiana leaves after 1 d infiltration by Agrobacterium strain GV3101 carrying pEarleyGate 203-ScAPX6 and the empty vector (35S::00). (B) DAB (3,3′-diaminobenzidinesolution) staining and trypan blue staining of N. benthamiana leaves at 48 h and 6 d after Agrobacterium strain infiltration, respectively, (1) represented a stereoscopic microscope and (2) represented a light microscope. (C) The transcripts of eight immunity-associated marker genes in the N. benthamiana leaves at 24 h after infiltration. (D,F) Disease symptoms and DAB staining results of N. benthamiana leaves by P. solanacearum and F. solani var. coeruleum infection after infiltration with 35S::00 (control) or 35S::ScAPX6-containing Agrobacterium strain. Disease symptoms of infected leaves were observed at 1 and 7 d post-inoculation. (E,G) The transcripts of immunity-associated marker genes in the N. benthamiana leaves after inoculation with P. solanacearum or F. solani var. coeruleum for 1 and 7 d. NtEF1-α was used for normalization of the transcript levels. All data points were expressed as the mean ± SE (n = 3). Different lowercase letters indicate a significant difference, as determined by the Duncan's new multiple range test (p < 0.05). NtHSR201, NtHSR203, and NtHSR515, hypersensitive response marker genes; NtPR2, NtPR-1a/c, and NtPR3, a salicylic acid pathway-related gene; NtEFE26 and NtAccdeaminase, the ethylene synthesis-dependent genes. Control, the Agrobacterium strain carrying 35S::00.