Responses of sweet potato peroxidases to sodium nitroprusside-mediated nitric oxide

To ascertain the response of sweetpotato peroxidases (PODs) to nitric oxide (NO), we treated the leaves of sweet potato with the NO generator sodium nitroprusside (SNP) and the NO scavenger carboxyl-PTIO (cPTIO). Exogenous application of more than 5 mM SNP caused damage to sweetpotato leaves at 24 h after treatment. The accumulation of NO in leaves was positively correlated with the SNP dose. The specific activity of PODs in sweet potato leaves was markedly increased by treatment with greater than 1 mM SNP for 24 h, whereas POD activity and accumulated NO content decreased to low levels by treatment with cPTIO. Expression analysis of POD genes in response to treatment with SNP and cPTIO revealed that major stress-inducible acidic genes, such as swpa1, swpa2, swpa3, and swpa4, were specifically regulated. These results indicate that increased NO levels in sweet potato leaves are closely linked to an improved defense capability mediated by stress-inducible PODs.     

Differential expression of 10 sweetpotato peroxidase genes in response to bacterial pathogen, Pectobacterium chrysanthemi.

To understand the function of each peroxidase (POD, EC 1.11.1.7) in terms of biotic stress, changes in POD specific activity and expression of 10 POD genes were investigated in four cultivars of sweetpotato (Ipomoea batatas) after infection with Pectobacterium chrysanthemi. POD specific activity (units mg(-1) protein) increased from 16 h after inoculation (HAI) in three varieties. POD activities of two cultivars, Shinwhangmi and White Star, reached a maximum level at 24 HAI by about three times compared to mock treatment (MT), and then decreased, whereas those of Zami and Yulmi continuously increased until 36 HAI. Native gel analysis revealed that one POD isoenzyme with a high electrophoretic mobility significantly increased in response to pathogen infection in all cultivars. Additionally, 10 POD genes displayed differential expression patterns upon bacterial infection by northern analysis. Several POD genes such as swpa2, swpa3, swpa4, swpa5, swpb1 were induced upon bacterial infection, but other genes were not. Particularly, swpa4 gene was markedly expressed in response to bacterial infection in four different cultivars, suggesting that this gene has a stress-inducible promoter. These results indicate that some specific POD isoenzymes are involved in defense in relation to pathogenesis of P. chrysanthemi in sweetpotato plants.     

Differential expression of 10 sweetpotato peroxidases in response to sulfur dioxide, ozone, and ultraviolet radiation

Secretory class III plant peroxidase (POD, EC 1.11.1.7) is believed to function in diverse physiological processes, including responses to various environmental stresses. To understand the function of each POD in terms of air pollutants and UV radiation, changes in POD activity and expression of 10 POD genes isolated from cell cultures of sweetpotato (Ipomoea batatas) were investigated in the leaves of sweetpotato after treatment with sulfur dioxide (SO(2) 500ppb, 8h/day for 5 days), ozone (O(3) 200ppb, 8h/day for 6 days), and ultraviolet radiation (UV-B 0.6mWm(-2) for 24h, UV-C 0.16mWm(-2) for 24h). All treatments significantly reduced the PSII photosynthetic efficiency (F(v)/F(m)). POD-specific activities (units/mg protein) were increased in leaves treated with SO(2) and O(3) by 5.2- and 7.1-fold, respectively, compared to control leaves. UV-B and UV-C also increased POD activities by 3.0- and 2.4-fold, respectively. As determined by RT-PCR analysis, 10 POD genes showed differential expression patterns upon treatment with air pollutants and UV radiation. Among the POD genes, swpa1, swpa2, and swpa4 were strongly induced following each of the treatments. Interestingly, basic POD genes (swpb1, swpb2, and swpb3) were highly expressed following SO(2) treatment only, whereas neutral swpn1 was highly induced following O(3) treatment only. These results indicated that some specific POD isoenzymes might be specifically involved in the defense mechanism against oxidative stress induced by air pollutants and UV radiation in sweetpotato plants.     

Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco

Plant peroxidases (POD) reduce hydrogen peroxide (H₂O₂) in the presence of an electron donor. Extracellular POD can also induce H₂O₂ production and may perform a significant function in responses to environmental stresses via the regulation of H₂O₂ in plants. We previously described the isolation of 10 POD cDNA clones from cell cultures of sweetpotato (Ipomoea batatas). Among them, the expression of the swpa4 gene was profoundly induced by a variety of abiotic stresses and pathogenic infections (Park et al. in Mol Gen Genome 269:542–552 2003; Jang et al. in Plant Physiol Biochem 42:451–455 2004). In the present study, transgenic tobacco (Nicotiana tabacum) plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated in order to assess the function of swpa4 in planta. The transgenic plants exhibited an approximately 50-fold higher POD specific activity than was observed in control plants. Both transient expression analysis with the swpa4-GFP fusion protein and POD activity assays in the apoplastic washing fluid revealed that the swpa4 protein is secreted into the apoplastic space. In addition, a significantly enhanced tolerance to a variety of abiotic and biotic stresses occurred in the transgenic plants. These plants harbored increased lignin and phenolic content, and H₂O₂ was also generated under normal conditions. Furthermore, they showed an increased expression level of a variety of apoplastic acidic pathogenesis-related (PR) genes following enhanced H₂O₂ production. These results suggest that the expression of swpa4 in the apoplastic space may function as a positive defense signal in the H₂O₂-regulated stress response signaling pathway.     

Molecular biology and application of plant peroxidase genes

Peroxidases are a family of isozymes found in all plants; they are heme-containing monomeric glycoproteins that utilize either H(2)O(2) or O(2) to oxidize a wide variety of molecules. These important enzymes are used in enzyme immunoassays, diagnostic assays and industrial enzymatic reactions. Peroxidase genes and their promoters can be used for molecular breeding of useful plants. Transgenic techniques have also been used to investigate the physiological and molecular functions of peroxidase genes in plants. Here, we review transgenic studies of peroxidase genes, including the functional analyses of the enzymes and their promoters. Regarding application of peroxidase genes, it has been reported that overexpression of the tomato TPX2 gene or the sweet potato swpa1 gene conferred increased salt-tolerance or oxidative-stress tolerance, respectively. The growth stimulation effect in transgenic tobacco and hybrid aspen upon overexpression of horseradish peroxidase gene is also discussed.     

Changes in activities of antioxidant enzymes and their gene expression during leaf development of sweetpotato

To understand the functions of antioxidant enzymes during leaf development in sweetpotato, we investigated the activities of several antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT). Significant increases were observed in the activities of SOD, POX and APX during the late stage of leaf development, whereas CAT activity increased during the early developmental stage. By RT-PCR analysis, various POX and APX genes showed differential expression patterns during leaf development. Four POX genes swpa3, swpa4, swpa6, swpb4 and one APX gene swAPX1 exhibited high levels of gene expression during the senescence stage of leaf development, but two POX genes, swpa1 and swpa7 were preferentially expressed at both the mature green and the late senescence stages of leaf development. These results indicate that hydrogen peroxide (H₂O₂)-related antioxidant enzymes are differentially regulated in the process of leaf development of sweetpotato.     

Heterologous expression and secretion of sweet potato peroxidase isoenzyme A1 in recombinant Saccharomyces cerevisiae

A vector system has been developed to express isoenzyme A1 of sweet potato peroxidase (POD) and was introduced into Saccharomyces cerevisiae. The system contains the signal sequence of Aspergillus oryzae -amylase to facilitate the extracellular secretion of peroxidase under the control of constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. In a batch culture using YNBDCA medium (yeast nitrogen base without amino acids 6.7 g l^–1, Casamino acids 5 g l^–1 and glucose 20 g l^–1), the recombinant strain expressed the swpa1 gene giving a secretion yield of POD activity of ca. 90% of total expressed peroxidase. Supplementation with PMSF (0.05 mM) and Casamino acids (5 g/50 ml) increased extracellular POD activity to nearly 10 kU ml^–1, equivalent to 1.5 kU g^–1 cell dry wt. This is 9 fold higher than that obtained in medium without PMSF. From SDS-PAGE and native-PAGE analyses POD has an M _r of 53 kDa.     

Effect of copper on peroxidase activity and lignin content in Raphanus sativus

Copper (Cu²⁺) significantly inhibits the growth of radish (Raphanus sativus) seedlings at the concentration of 1 μM. As far as the relationship between the growth of radish roots and peroxidase (POD) activity is concerned, the reduction of radish roots is correlated with the induction of cationic and anionic PODs. The data show that the increase of cationic PODs (pI 8.6 and pI 9.3) and anionic PODs (pI 5.1 and pI 3.5) activities was correlated with the rise in lignin content in Cu-treated tissues. In our investigation, among the radish root PODs, the cationic pI 8.6 POD isozyme displayed a high affinity (K_m of 57.9 μM) for syringaldazine and the similar value of catalytic efficiency jointly with the anionic pI 5.1 POD, 0.14 and 0.12 μM^–1 s^–1, respectively. The results suggest that the increase of cationic POD (pI 8.6) induced by Cu treatment can be a good candidate for lignification in radish roots.