Isolation of a Novel Peroxisomal Catalase Gene from Sugarcane, Which Is Responsive to Biotic and Abiotic Stresses

Catalase is an iron porphyrin enzyme, which serves as an efficient scavenger of reactive oxygen species (ROS) to avoid oxidative damage. In sugarcane, the enzymatic activity of catalase in a variety (Yacheng05–179) resistant to the smut pathogen Sporisorium scitamineum was always higher than that of the susceptible variety (Liucheng03–182), suggesting that catalase activity may have a positive correlation with smut resistance in sugarcane. To understand the function of catalase at the molecular level, a cDNA sequence of ScCAT1 (GenBank Accession No. KF664183), was isolated from sugarcane infected by S. scitamineum. ScCAT1 was predicted to encode 492 amino acid residues, and its deduced amino acid sequence shared a high degree of homology with other plant catalases. Enhanced growth of ScCAT1 in recombinant Escherichia coli Rosetta cells under the stresses of CuCl2, CdCl2 and NaCl indicated its high tolerance. Q-PCR results showed that ScCAT1 was expressed at relatively high levels in the bud, whereas expression was moderate in stem epidermis and stem pith. Different kinds of stresses, including S. scitamineum challenge, plant hormones (SA, MeJA and ABA) treatments, oxidative (H2O2) stress, heavy metal (CuCl2) and hyper-osmotic (PEG and NaCl) stresses, triggered a significant induction of ScCAT1. The ScCAT1 protein appeared to localize in plasma membrane and cytoplasm. Furthermore, histochemical assays using DAB and trypan blue staining, as well as conductivity measurement, indicated that ScCAT1 may confer the sugarcane immunity. In conclusion, the positive response of ScCAT1 to biotic and abiotic stresses suggests that ScCAT1 is involved in protection of sugarcane against reactive oxidant-related environmental stimuli.

过氧化氢酶（Catalase）是一类铁卟啉酶，可作为活性氧（reactive oxygen species, ROS）的高效清除剂，以规避氧化损伤。在甘蔗中，抗黑穗病病原菌Sporisorium scitamineum的品种（Yacheng05–179）的过氧化氢酶活性始终高于感病品种（Liucheng03–182），这表明甘蔗的过氧化氢酶活性可能与黑穗病抗性呈正相关。为在分子水平解析过氧化氢酶的功能，研究人员从受S. scitamineum侵染的甘蔗中分离得到ScCAT1的cDNA序列（GenBank登录号：KF664183）。预测显示，ScCAT1编码492个氨基酸残基，其推导的氨基酸序列与其他植物过氧化氢酶具有高度同源性。在CuCl₂、CdCl₂和NaCl胁迫条件下，表达ScCAT1的重组大肠杆菌Rosetta（Escherichia coli Rosetta）细胞的生长能力显著提升，表明该蛋白具有较高的胁迫耐受性。实时定量PCR（Q-PCR）结果显示，ScCAT1在芽中表达量相对较高，而在茎表皮和茎髓中表达量处于中等水平。多种胁迫处理，包括S. scitamineum侵染、植物激素（水杨酸（salicylic acid, SA）、茉莉酸甲酯（methyl jasmonate, MeJA）和脱落酸（abscisic acid, ABA））处理、氧化（H₂O₂）胁迫、重金属（CuCl₂）以及高渗（PEG和NaCl）胁迫，均能显著诱导ScCAT1的表达。亚细胞定位分析表明，ScCAT1蛋白定位于细胞膜和细胞质中。此外，通过二氨基联苯胺（3,3'-Diaminobenzidine, DAB）和台盼蓝（trypan blue）染色的组织化学检测，结合电导率测定，结果提示ScCAT1可能赋予甘蔗免疫抗性。综上，ScCAT1对生物和非生物胁迫的正向响应表明，该基因参与保护甘蔗免受活性氧相关的环境刺激侵害。