Yun-Yang Chao

Effect of magnesium deficiency on antioxidant status and cadmium toxicity in rice seedlings

Cadmium (Cd) is one of the most toxic heavy metals and inhibits physiological processes of plants. Magnesium (Mg) is known as one of the essential nutrients for plants. Mg deficiency in plants affects metabolic processes. Plants grown in the field may encounter several abiotic stresses, rather than a single stress. Thus, the relationship between Mg nutrition and Cd toxicity is of ecological importance. In this study, effects of Mg deficiency on antioxidant systems and Cd toxicity in rice seedlings were investigated. Mg deficiency significantly decreased Mg concentrations in shoot and roots of rice seedlings. However, fresh weight and dry weight of rice seedlings were not affected by Mg deficiency. The contents of ascorbate and glutathione (GSH), the ratio of GSH/oxidized glutathione, and the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in Mg-deficient leaves were higher than respective control leaves. Cd toxicity was judged by the decrease in biomass production, decrease in chlorophyll, and induction of oxidative stress. Based on these criteria, we demonstrated that Mg deficiency protected rice seedlings from Cd stress. Moreover, chlorophyll destruction by paraquat was higher in detached leaves from Mg-sufficient than Mg-deficient seedlings. Cd concentration was higher in Mg-deficient shoot and roots than their respective control shoot and roots, suggesting that the protective effect of Mg deficiency against Cd toxicity is not due to reduction of Cd uptake. Moreover, we observed that Cd-decreased Fe and Zn contents in Mg-deficient seedlings were more pronounced than that in Mg-sufficient seedlings. Of particular interest is the finding that the increase in OsIRT1, OsZIP1, and OsZIP3 transcripts caused by Cd in Mg-deficient roots was greater than that in control roots.

The decline in ascorbic acid content is associated with cadmium toxicity of rice seedlings.

Cadmium (Cd) toxicity of rice (Oryza sativa L. cv. Taichung Native 1) seedlings was evaluated by the decrease in chlorophyll content and the increase in malondialdehyde (MDA) in the second leaves of rice seedlings. CdCl2 (5 microM) treatment was accompanied by a decrease in the contents of ascorbic acid (AsA) and AsA + dehydroascorbate (DHA) and in the ratios of AsA/DHA in leaves. However, CdCl2 treatment resulted in an increase in DHA content in leaves. Moreover, the decrease in AsA content was prior to the occurrence of chlorosis and associated with the increase in MDA content in the leaves of seedlings treated with Cd. Pretreatment with 0.5 mM AsA or L-galactono-1,4-lactone (GalL), the biosynthetic precursor of AsA, for 6 h resulted in an increase in the contents of AsA and reduced glutathione (GSH), the ratios of AsA/DHA and GSH/oxidized glutathione, and the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in the leaves of rice seedlings. Quantitative RT-PCR was applied to quantify the mRNA levels for OsAPX and OsGR genes from rice leaves to examine the effect of AsA or GalL pretreatment on the expression of OsAPX and OsGR genes in rice leaves. The expression of OsAPX2, OsAPX3, OsAPX4, OsAPX5, OsAPX6, OsAPX7, and OsGR1 was increased by AsA or GalL pretreatment. Rice seedlings pretreated with AsA or GalL were observed to reduce the subsequent Cd-induced toxicity. Our results suggest that AsA content may play a role in regulating Cd toxicity of rice seedlings.

Salicylic acid-mediated hydrogen peroxide accumulation and protection against Cd toxicity in rice leaves

The role of H2O2 in salicylic acid (SA)-induced protection of rice leaves against subsequent Cd toxicity was investigated. SA pretreatment resulted in an increase in the contents of endogenous SA, as judged by the expression of OsWRKY45 (a SA responsive gene), and H2O2 in rice leaves. Diphenyleneiodonium (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented SA-increased H2O2 production, suggesting that NADPH oxidase is a H2O2-generating enzyme in SA-pretreated rice leaves. DPI and IMD also inhibited SA-increased activities of superoxide dismutase (SOD), ascorbate peroixdase (APX), and glutathione reductase (GR) activities, but had no effect on SA-increased catalase (CAT) activity. Moreover, SA-induced protection against subsequent Cd toxicity could also be prevented by DPI and IMD. The inhibitory effect of DPI and IMD on SA-induced protection against subsequent Cd toxicity could be reversed by exogenous H2O2. All these results suggested that SA-induced protection against subsequent Cd toxicity is mediated through H2O2. This conclusion is supported further by the observations that exogenous H2O2 application resulted in an increase in SOD, APX, and GR activities, but not CAT activity and a protection against subsequent Cd toxicity of rice leaves.

Involvement of glutathione in heat shock– and hydrogen peroxide–induced cadmium tolerance of rice (Oryza sativa L.) seedlings

The role of reduced glutathione (GSH) in heat shock (HS)- and H2O2-induced protection of rice (Oryza sativa L., cv. Taichung 1) seedlings from Cd stress was investigated. HS- and H2O2-pretreatment resulted in an increase in GSH content in leaves of rice seedlings. Addition of exogenous GSH under non-HS conditions, which resulted in an increase in GSH in leaves, enhanced subsequent Cd tolerance of rice seedlings. Pretreatment with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, which effectively inhibited GSH content induced by HS and H2O2, reduced subsequent Cd tolerance. Furthermore, the effect of BSO on HS- and H2O2-induced GSH accumulation and toxicity by subsequent Cd stress can be reversed by the addition of GSH. The time-course analyses of HS in rice seedlings demonstrated that the accumulation of H2O2 preceded the increase in GSH. Based on the data obtained in this study, it could be concluded that the early accumulation of H2O2 during HS signals the increase in GSH content, which in turn protects rice seedlings from oxidative damage caused by Cd.

Involvement of hydrogen peroxide in heat shock- and cadmium-induced expression of ascorbate peroxidase and glutathione reductase in leaves of rice seedlings

Hydrogen peroxide (H2O2) is considered a signal molecule inducing cellular stress. Both heat shock (HS) and Cd can increase H2O2 content. We investigated the involvement of H2O2 in HS- and Cd-mediated changes in the expression of ascorbate peroxidase (APX) and glutathione reductase (GR) in leaves of rice seedlings. HS treatment increased the content of H2O2 before it increased activities of APX and GR in rice leaves. Moreover, HS-induced H2O2 production and APX and GR activities could be counteracted by the NADPH oxidase inhibitors dipehenylene iodonium (DPI) and imidazole (IMD). HS-induced OsAPX2 gene expression was associated with HS-induced APX activity but was not regulated by H2O2. Cd-increased H2O2 content and APX and GR activities were lower with than without HS. Cd did not increase the expression of OsAPX and OsGR without HS treatment. Cd increased H2O2 content by Cd before it increased APX and GR activities without HS. Treatment with DPI and IMD effectively inhibited Cd-induced H2O2 production and APX and GR activities. Moreover, the effects of DPI and IMD could be rescued with H2O2 treatment. H2O2 may be involved in the regulation of HS- and Cd-increased APX and GR activities in leaves of rice seedlings.

Heme oxygenase is involved in nitric oxide- and auxin-induced lateral root formation in rice.

Lateral root (LR) development performs the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of nitric oxide (NO), auxin, and hemin (Hm) on LR formation in rice. Treatment with Hm [a highly effective heme oxygenase (HO) inducer], sodium nitroprusside (SNP, an NO donor), or indole-3-butyric acid (IBA, a naturally occurring auxin) induced LR formation and HO activity. LR formation and HO activity induced by SNP and IBA but not Hm was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. As well, Hm, SNP, and IBA could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) and hemoglobin (the carbon monoxide/NO scavenger) reduced LR number and HO activity induced by Hm, SNP, and IBA. Our data suggest that HO is required for Hm-, auxin-, and NO-induced LR formation in rice.

Na+ but not Cl− or osmotic stress is involved in NaCl-induced expression of Glutathione reductase in roots of rice seedlings

Glutathione reductase (GR, EC 1,6.4.2) is an important reactive oxygen species-scavenging enzyme. The present study examined the relative importance of Na(+), Cl(-), and the osmotic component in NaCl-induced expression of Oryza sativa glutathione reductase (OsGR) genes in rice roots. Semi-quantitative RT-PCR was used to quantify the mRNA levels for one cytosolic (OsGR2) and two chloroplastic (OsGR1 and OsGR3) isoforms of GR identified in the rice genome. The expression of OsGR2 and OsGR3 but not OsGR1 was increased in rice roots treated with NaCl. Treatment with 150 mM NaCl and 150 mM NaNO(3) affected OsGR2 and OsGR3 induction similarly, which suggests that Na(+) but not Cl(-) is required for the NaCl-induced expression of OsGR2 and OsGR3. We also show that Na(+) but not Cl(-) is required for NaCl-enhanced GR activity and hydrogen peroxide (H(2)O(2)) production in rice roots. In addition to its component of ion toxicity, salt concentration in soil results in an osmotic effect. Here, we show that OsGR2 and OsGR3 expression, GR activity, and H(2)O(2) content were not affected at a concentration of mannitol iso-osmotic with 150 mM NaCl. NaCl-induced OsGR2 and OsGR3 in rice roots could be associated with Na(+) but not an osmotic component.

Heme oxygenase is involved in H2O2-induced lateral root formation in apocynin-treated rice

Key messageApocynin is a natural organic compound structurally related to vanillin. We demonstrated that hydrogen peroxide and heme oxygenase participated in apocynin-induced lateral root formation in rice.AbstractApocynin, also known as acetovanillone, is a natural organic compound structurally related to vanillin. Information concerning the effect of apocynin on plants is limited. In this study, we examined the effect of apocynin on lateral root (LR) formation in rice. Treatment with apocynin induced LR formation and increased H2O2 production, but had no effect on nitric oxide production. Diphenyleneiodonium chloride, an inhibitor of H2O2 generating NADPH oxidase, was effective in reducing apocynin-induced H2O2 production and LR formation. Apocynin treatment also increased superoxide dismutase activity and decreased catalase activity. H2O2 application was able to increase the number of LRs. Moreover, H2O2 production caused by H2O2 and apocynin was localized in the root area corresponding to the LR emergence. Treatment with H2O2 and apocynin also increased heme oxygenase (HO) activity and induced OsHO1 mRNA expression. Lateral root formation and HO activity induced by H2O2 and apocynin were reduced by Zn protoporphyrin IX (the specific inhibitor of HO). Our data suggest that both H2O2 and HO are required for apocynin-induced LR formation in rice.

Methyl jasmonate-induced lateral root formation in rice: the role of heme oxygenase and calcium.

Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of methyl jasmonate (MJ) on LR formation in rice. Treatment with MJ induced LR formation and heme oxygenase (HO) activity. As well, MJ could induce OsHO1 mRNA expression. Zinc protoporphyrin IX (the specific inhibitor of HO) and hemoglobin [the carbon monoxide/nitric oxide (NO) scavenger] reduced LR formation, HO activity and OsHO1 expression. LR formation and HO activity induced by MJ was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-oxide. The effects of Ca(2+) chelators, Ca(2+)-channel inhibitors, and calmodulin (CaM) antagonists on LR formation induced by MJ were also examined. All these inhibitors were effective in reducing the action of MJ. However, Ca(2+) chelators and Ca(2+) channel inhibitors induced HO activity when combining with MJ further. It is concluded that Ca(2+) may regulate MJ action mainly through CaM-dependent mechanism.

Calcium deficiency increases Cd toxicity and Ca is required for heat-shock induced Cd tolerance in rice seedlings

While growing in the field, plants may encounter several different forms of abiotic stress simultaneously, rather than a single stress. In this study, we investigated the effects of calcium (Ca) deficiency on cadmium (Cd) toxicity in rice seedlings. Calcium deficiency alone decreased the length, fresh and dry weight, and the Ca concentration in shoots and roots. Also, the content of glutathione (GSH), the ratio of GSH/oxidized glutathione, and the activity of catalase were lower in Ca-deficient leaves compared to control leaves. Exogenous Cd caused a decrease in the contents of chlorophyll and protein, and induced oxidative stress. Based on these stress indicators, we found that Ca deficiency enhanced Cd toxicity in rice seedlings. Under exogenous Cd application, internal Cd concentrations were higher in Ca-deficient shoots and roots than in the respective controls. Moreover, we observed that Ca deficiency decreased heat-shock (HS) induced expression of HS protein genes Oshsp17.3, Oshsp17.7, and Oshsp18.0 in leaves thereby weakening the protection system and increasing Cd stress. In conclusion, Ca deficiency enhances Cd toxicity, and Ca may be required for HS response in rice seedlings.