The Role of Sugarcane Catalase Gene ScCAT2 in the Defense Response to Pathogen Challenge and Adversity Stress

Abstract

Catalases, which consist of multiple structural isoforms, catalyze the decomposition of hydrogen peroxide in cells to prevent membrane lipid peroxidation. In this study, a group II catalase gene ScCAT2 (GenBank Accession No. KF528830) was isolated from sugarcane genotype Yacheng05-179. ScCAT2 encoded a predicted protein of 493 amino acid residues, including a catalase active site signature (FARERIPERVVHARGAS) and a heme-ligand signature (RVFAYADTQ). Subcellular localization experiments showed that the ScCAT2 protein was distributed in the cytoplasm, plasma membrane, and nucleus of Nicotiana benthamiana epidermal cells. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the ScCAT2 gene was ubiquitously expressed in sugarcane tissues, with expression levels from high to low in stem skin, stem pith, roots, buds, and leaves. ScCAT2 mRNA expression was upregulated after treatment with abscisic acid (ABA), sodium chloride (NaCl), polyethylene glycol (PEG), and 4 °C low temperature, but downregulated by salicylic acid (SA), methyl jasmonate (MeJA), and copper chloride (CuCl₂). Moreover, tolerance of Escherichia coli Rosetta cells carrying pET-32a-ScCAT2 was enhanced by NaCl stress, but not by CuCl₂ stress. Sporisorium scitamineum infection of 10 different sugarcane genotypes showed that except for YZ03-258, FN40, and FN39, ScCAT2 transcript abundance in four smut-resistant cultivars (Yacheng05-179, YZ01-1413, YT96-86, and LC05-136) significantly increased at the early stage (1 day post-inoculation), and was decreased or did not change in the two smut-medium-susceptibility cultivars (ROC22 and GT02-467), and one smut-susceptible cultivar (YZ03-103) from 0 to 3 dpi. Meanwhile, the N. benthamiana leaves that transiently overexpressed ScCAT2 exhibited less severe disease symptoms, more intense 3,3'-diaminobenzidine (DAB) staining, and higher expression levels of tobacco immune-related marker genes than the control after inoculation with tobacco pathogen Ralstonia solanacearum or Fusarium solani var. coeruleum. These results indicate that ScCAT2 plays a positive role in immune responses during plant⁻pathogen interactions, as well as in salt, drought, and cold stresses.

Keywords: Sporisorium scitamineum; catalase; defense response; expression profiles; sugarcane.

Figure 1

Nucleotide acid sequence and the deduced amino acid sequence of ScCAT2. Red letters represented the specific amplification primer pair for ScCAT2. Catalase active site (FARERIPERVVHARGAS) and heme-ligand (RVFAYADTQ) signatures of ScCAT2 were underlined. * represented stop codon.

Figure 2

Protein sequence and homology of ScCAT2 with catalases from sugarcane and other plant spices. So, Saccharum officinarum; Sb, Sorghum bicolor; Si, Setaria italic; Al, Aeluropus litoralis; Os, Oryza sativa; Hv, Hordeum vulgare; and Ta, Triticum aestivum. The black foreground and white background represented non-similar residues. The blue foreground and cyan background indicated conservative residues. The black foreground and green background were blocks of similar residues. The red foreground and yellow background showed identical residues. The dark green foreground and white background depicted the weakly similar residues.

Figure 3

Phylogenetic reconstruction of ScCAT2 with catalases from sugarcane and other plant species. The maximum-likelihood method with 1000 bootstrap replication was used. Zm, Zea mays; Os, Oryza sativa; Hv, Hordeum vulgare; Ta, Triticum aestivum; Sb, Sorghum bicolor; Si, Setaria italic; Al, Aeluropus littoralis; So, Saccharum officinarum; Ss, Saccharum spontaneum; and Ea, Erianthus arundinaceus. ScCAT2 was marked with a red asterisk.

Figure 4

Subcellular localization of ScCAT2 and empty vector in Nicotiana benthamiana leaves after 2 days of infiltration. Images of epidermal cells captured using visible light, green fluorescence, blue fluorescence, and merged light. Red arrows 1, 2, and 3, indicated the plasma membrane, cytoplasm, and nucleus, respectively. Bar = 20 μm. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus.

Figure 5

Tissue-specific expression analysis of ScCAT2 in 10-month-old sugarcane Yacheng05-179 plants. Data were normalized to the expression level of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. All data points were expressed as means ± standard error (n = 3). Different lowercase letters indicated a significant difference (p < 0.05) compared to the control, as determined with Duncan’s test.

Figure 6

qRT-PCR analysis of the ScCAT2 expression in 10 different sugarcane genotypes after inoculation with Sporisorium scitamineum. The data were normalized to the expression level of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. All data points (normalized to the control) were expressed as means ± standard error (n = 3). Different lowercase letters indicated a significant difference (p < 0.05) compared to the control, as determined with Duncan’s test. Yacheng05-179, YZ01-1413, YT96-86, LC05-136, and YZ03-258 were smut-resistant cultivars (R). ROC22, GT02-467, and FN39 were smut-medium-susceptibility cultivars (MS). YZ03-103 and FN40 were smut-susceptible cultivars (S). dpi, days post-inoculation.

Figure 7

Relative expression of ScCAT2 after the application of exogenous plant hormone and abiotic stress. ScCAT2 transcript abundance in Yacheng05-179 tissue cultured plantlets was assessed in the presence of 100 μM ABA, 5 mM SA, 25 μM MeJA, 250 mM NaCl, 25% PEG, 100 μM CuCl₂, and 4 °C low temperature. The data were normalized to the expression level of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. All data points were expressed as the means ± standard error (n = 3). Different lowercase letters indicated a significant difference (p < 0.05) compared to the control, as determined with Duncan’s test. ABA, abscisic acid; SA, salicylic acid; MeJA, methyl jasmonate; NaCl, sodium chloride; PEG, polyethylene glycol; CuCl₂, copper chloride; and low temperature, 4 °C cold stress.

Figure 8

Spot assay of Rosetta + pET-32a (control) (a) and Rosetta + pET-32a-ScCAT2 (b) on LB plates with different concentrations of NaCl and CuCl₂ components. The cultures of Rosetta + pET-32a-ScCAT2 and Rosetta + pET-32a were supplemented with 1.0 mM isopropyl β-d-thiogalactoside to induce the expression of recombinant protein overnight. Then, the cultures were adjusted to an OD₆₀₀ = 0.6. Ten microliters from the 10⁻³ (left side of red line on plate) to 10⁻⁴ (right side of red line on plate) dilutions were spotted onto the LB basal plates (A) or with NaCl (250, 500, and 750 mM) (B), and CuCl₂ (250, 500, and 750 μM) (C). NaCl, sodium chloride; and CuCl₂, copper chloride.

Figure 9

Transient overexpression of ScCAT2 in Nicotiana benthamiana leaves. (A) RT-PCR analysis of ScCAT2 in the N. benthamiana leaves 2 days after infiltration with Agrobacterium strain GV3101 that carried the vector 35S::00 or 35S::ScCAT2. (B) DAB staining of N. benthamiana leaves 2 days after infiltration with 35S::ScCAT2-containing Agrobacterium strain to assess H₂O₂ production. (C) Relative expression level of nine tobacco immune-related marker genes in 35S::ScCAT2-transiently expressing leaves at 2 days after infiltration. The tobacco immune-related marker genes included the hypersensitive response marker genes NtHSR201, NtHSR203, and NtHSR515; the salicylic acid-related genes NtPR-1 and NtPR-1a/c; the jasmonic acid pathway-associated genes NtPR2 and NtPR3; and the ethylene synthesis-dependent genes NtEFE26 and NtAccdeaminase, and using NtEF1-α for data normalization. All data points were presented as the means ± standard error (n = 3). Different lowercase letters indicated a significant difference, as determined with Duncan’s test (p < 0.05). The empty vector 35S::00 and recombinant vector 35S::ScCAT2 were indicated by a and b, respectively. (1) and (2) represented images captured using a SONY camera and microscope, respectively. Bar = 1 mm.

Figure 10

The effect of transient overexpression of ScCAT2 in Nicotiana benthamiana leaves after inoculation with Ralstonia solanacearum. (A) The disease symptoms and DAB staining of N. benthamiana leaves at 1 day and 6 days post-inoculation with R. solanacearum. (B) Analysis of nine tobacco immune-related marker genes in the N. benthamiana leaves 1 day and 6 days after inoculation with R. solanacearum. The tobacco immune-related marker genes, which included the hypersensitive response marker genes NtHSR201, NtHSR203, and NtHSR515; the salicylic acid-related genes NtPR-1 and NtPR-1a/c; the jasmonic acid pathway-associated genes NtPR2 and NtPR3; and the ethylene synthesis-dependent genes NtEFE26 and NtAccdeaminase, and using NtEF1-α for data normalization. All data points were presented as means ± standard error (n = 3). Different lowercase letters indicated a significant difference, as determined with Duncan’s test (p < 0.05). The empty vector 35S::00 and recombinant vector 35S::ScCAT2 were indicated by a and b, respectively. (1) and (2) represented images captured using a SONY camera and microscope, respectively. Bar = 1 mm.

Figure 11

The effect of transient overexpression of ScCAT2 in Nicotiana benthamiana leaves after inoculation with Fusarium solani var. coeruleum. (A) The disease symptoms and DAB staining of N. benthamiana leaves 1 day and 6 days after inoculation with F. solani var. coeruleum. (B) Analysis of the nine tobacco immune-related marker genes in the N. benthamiana leaves after inoculation with F. solani var. coeruleum for 1 day and 6 days, respectively. The tobacco immune-related marker genes included the hypersensitive response marker genes NtHSR201, NtHSR203, and NtHSR515; the salicylic acid-related genes NtPR-1 and NtPR-1a/c; the jasmonic acid pathway-associated genes NtPR2 and NtPR3; and the ethylene synthesis-dependent genes NtEFE26 and NtAccdeaminase, and using NtEF1-α for data normalization. All data points were means ± standard error (n = 3). Different lowercase letters indicated a significant difference, as determined with Duncan’s test (p < 0.05). The empty vector 35S::00 and recombinant vector 35S::ScCAT2 were indicated by a and b, respectively. (1) and (2) represented images captured using a SONY camera and microscope, respectively. Bar = 1 mm.