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.     

Nitric oxide (NO) counteracts cadmium induced cytotoxic processes mediated by reactive oxygen species (ROS) in Brassica juncea: cross-talk between ROS, NO and antioxidant responses

Research on NO in plants has achieved huge attention in recent years mainly due to its function in plant growth and development under biotic and abiotic stresses. In the present study, we investigated Cd induced NO generation and its relationship to ROS and antioxidant regulation in Brassica juncea. Cd accumulated rapidly in roots and caused oxidative stress as indicated by increased level of lipid peroxidation and H₂O₂ thus, inhibiting the overall plant growth. It significantly decreased the root length, leaf water content and photosynthetic pigments. A rapid induction in intracellular NO was observed at initial exposures and low concentrations of Cd. A 2.74-fold increase in intracellular NO was recorded in roots treated with 25 μM Cd than control. NO effects on Malondialdehyde (MDA) content and on antioxidant system was investigated by using sodium nitroprusside (SNP), a NO donor and a scavenger, [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] (cPTIO). Roots pretreated with 5 mM SNP for 6 h when exposed to 25 μM Cd for 24 h reduced the level of proline, non-protein thiols, SOD, APX and CAT in comparison to only Cd treatments. However, this effect was almost blocked by 100 μM cPTIO pretreatment to roots for 1 h. This ameliorating effect of NO was specific because cPTIO completely reversed the effect in the presence of Cd. Thus, the present study report that NO strongly counteracts Cd induced ROS mediated cytotoxicity in B. juncea by controlling antioxidant metabolism as the related studies are not well reported in this species.     

Effects of salinity and nitric oxide donor sodium nitroprusside (SNP) on development and salt secretion of salt glands of Limonium bicolor

NO, as a signaling molecule, is involved in abiotic stresses. Limonium bicolor seedlings were treated with 200 mM NaCl combined with 0.05 mM SNP for 20 days to study the effects of NO on development and salt-secretion rates of salt glands. It was shown that the total number of salt glands on adaxial surfaces under condition of 200 mM NaCl containing 0.05 mM SNP treatment increased significantly compared with that under 200 mM NaCl treatment. Na⁺ secretion rate per leaf under 200 mM NaCl containing 0.05 mM SNP was significantly higher than that under 200 mM NaCl without SNP. However, there was no significant difference in salt-secretion rate of individual salt glands between 200 mM NaCl containing 0.05 mM SNP treatment and 200 mM NaCl treatment. Although there was no significant difference in salt-secretion rate of individual glands, Na⁺ concentration in the leaves treated with 200 mM NaCl solution containing SNP was significantly lower than that treated with 200 mM NaCl solution. Treatment with 200 mM NaCl solution containing SNP caused a remarkable increase in Na⁺ concentration in salt glands. Obviously, the efficiency of the secretion process per gland was enhanced by adding SNP to NaCl. The results showed NO may enhance the salt secretion by inducing more dermatogen cells to develop into salt glands and by enhancing the efficiency of the secretion process per gland.     

Nitric oxide regulation of leaf phosphoenolpyruvate carboxylase-kinase activity: implication in sorghum responses to salinity

Nitric oxide (NO) is a signaling molecule that mediates many plant responses to biotic and abiotic stresses, including salt stress. Interestingly, salinity increases NO production selectively in mesophyll cells of sorghum leaves, where photosynthetic C₄ phosphoenolpyruvate carboxylase (C₄ PEPCase) is located. PEPCase is regulated by a phosphoenolpyruvate carboxylase-kinase (PEPCase-k), which levels are greatly enhanced by salinity in sorghum. This work investigated whether NO is involved in this effect. NO donors (SNP, SNAP), the inhibitor of NO synthesis NNA, and the NO scavenger cPTIO were used for long- and short-term treatments. Long-term treatments had multifaceted consequences on both PPCK gene expression and PEPCase-k activity, and they also decreased photosynthetic gas-exchange parameters and plant growth. Nonetheless, it could be observed that SNP increased PEPCase-k activity, resembling salinity effect. Short-term treatments with NO donors, which did not change photosynthetic gas-exchange parameters and PPCK gene expression, increased PEPCase-k activity both in illuminated leaves and in leaves kept at dark. At least in part, these effects were independent on protein synthesis. PEPCase-k activity was not decreased by short-term treatment with cycloheximide in NaCl-treated plants; on the contrary, it was decreased by cPTIO. In summary, NO donors mimicked salt effect on PEPCase-k activity, and scavenging of NO abolished it. Collectively, these results indicate that NO is involved in the complex control of PEPCase-k activity, and it may mediate some of the plant responses to salinity.     

Effects of nitric oxide on the germination of cucumber seeds and antioxidant enzymes under salinity stress

Effects of exogenous nitric oxide (NO) on the germination and antioxidant enzyme during cucumber seed germination were investigated under salt stress. Seeds of cucumber (Cucumis sativus L. cv. Jinyou 1) were treated with distilled water or NaCl in the presence or absence of NO donor sodium nitroprusside (SNP) during germination. Excess 50 mM NaCl reduced significantly the seed germination rate in a short term and speed of germination. When salt concentration increased, germination of cucumber seed was reduced and the time needed to complete germination lengthened. Addition of exogenous SNP in salt solution attenuated the salt stress effects in a dose-dependent manner, as indicated by accelerating the seed germination, as well as weight increase of budding seeds, and 50 μM SNP was optimal concentration. At 150 mM NaCl, the 50 μM exogenous SNP significantly increased the activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and protein content, while decreased the contents of malondialdehyde (MDA). There were no obvious effects of exogenous NO on peroxidase (POD, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.6) activities under salt stress. Exogenous NO also increased the SOD and CAT isozyme expression under salt stress, which was in accordance with the improved antioxidant activities in the germinating seeds. The NO-induced salt stress resistance was associated with activated enzymes, and enhanced protein content, thus decreasing MDA content. It is concluded that exogenous NO treatment on cucumber seeds may be a good option to improve seed germination under saline conditions.     

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.     

Stomatal closure in sweet potato leaves induced by sulfur dioxide involves H2S and NO signaling pathways

Sulfur dioxide (SO₂) is a well-known and widespread air pollutant but it also acts as signaling molecule in various processes in animals. However, there is limited information on the role of SO₂ in plants except of its toxicity. Here we studied the role of SO₂ on stomatal movements in sweet potato (Ipomoea batatas) leaves. SO₂, generated by Na₂SO₃/NaHSO₃ solutions, was applied on epidermal strips. We found that the SO₂ donor induced stomatal closure in a dose-dependent manner. Rapid increases in endogenous hydrogen sulfide and nitric oxide content levels were observed in leaves after the treatment with the SO₂ donor. The SO₂-induced stomatal closure was reversed by the H₂S scavenger hypotaurine and the NO-specific scavenger cPTIO. Our results indicate that the SO₂-induced stomatal closure was likely mediated by the H₂S and NO signaling pathways.     

Presoaking with hemin improves salinity tolerance during wheat seed germination

The aim of this study was to investigate whether presoaking with hemin, an inducer of heme oxygenase-1 (HO-1), could alleviate salinity damage during wheat seed germination in comparison with the pretreatment of a well-known nitric oxide (NO) donor sodium nitroprusside (SNP). The results showed that, compared with the samples upon 150 mM NaCl salt stress alone, both 10 ??M hemin and 200 ??M SNP pretreatments could (1) significantly attenuate the inhibition of seed germination and thereafter seedling growth; (2) induce HO expression; (3) enhance amylase activity, thus accelerating the formation of reducing sugar and total soluble sugar; and (4) increase the potassium (K) to sodium (Na) ratio, particularly in the shoot parts. Hemin and SNP could also increase antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), and ascorbate peroxidase (APX), thus resulting in the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) content. Moreover, semi-quantitative RT-PCR and isozymatic analysis illustrated that hemin and SNP pretreatment were able to up-regulate the expression of Mn-SOD (especially) and Cu/Zn-SOD gene, and activate SOD isozymatic activities. Since the addition of the NO scavenger methylene blue (MB) differentially reversed the above effects, the protective roles of hemin might be related to the induction of endogenous NO signal. Meanwhile, hemin-driven NO production was confirmed. Together, these results indicated that hemin exerted an advantageous effect on enhancing salinity tolerance during wheat seed germination, which might interact with NO.     

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.     

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.     

Level nitric oxide (NO) and growth of roots of etiolated pea seedlings

Data regarding the interrelation of nitric oxide (NO) content in roots of 3-day-old etiolated pea seedlings and their growth under different concentrations of N-containing compounds were obtained. The concentration of exogenous compounds (sodium nitroprusside SNP, KNO₃, NaNO₂, L-arginine) rendering an inhibiting effect on the growth of roots were established, and the NO content in roots was determined at these concentration. It was shown that the inhibition of growth and highest NO content in the roots was determined with SNP (4 mM) and NaNO₂ (2 mM) during 24 h exposition of seedlings. This dependence was not established in combinations with KNO₃ (20 mM) and L-arginine (4 mM). We established that a NO scavenger, hemoglobin (4 μM), fully or partially removed the toxic effect of SNP, nitrate, and nitrite on growth. The effect of NO on the growth and the participation of N-containing compounds in generation of NO in roots of pea seedlings is discussed.