A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.).

In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins.

Heme oxygenase is involved in H(2)O (2)-induced lateral root formation in apocynin-treated rice.

KEY MESSAGE : Apocynin 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. Apocynin, 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 H(2)O(2) production, but had no effect on nitric oxide production. Diphenyleneiodonium chloride, an inhibitor of H(2)O(2) generating NADPH oxidase, was effective in reducing apocynin-induced H(2)O(2) production and LR formation. Apocynin treatment also increased superoxide dismutase activity and decreased catalase activity. H(2)O(2) application was able to increase the number of LRs. Moreover, H(2)O(2) production caused by H(2)O(2) and apocynin was localized in the root area corresponding to the LR emergence. Treatment with H(2)O(2) and apocynin also increased heme oxygenase (HO) activity and induced OsHO1 mRNA expression. Lateral root formation and HO activity induced by H(2)O(2) and apocynin were reduced by Zn protoporphyrin IX (the specific inhibitor of HO). Our data suggest that both H(2)O(2) and HO are required for apocynin-induced LR formation in rice.

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.

Foliar Application of Rhodopseudomonas palustris Enhances the Rice Crop Growth and Yield under Field Conditions.

Anthropogenic activities causing climate change and other environmental effects are lowering crop yield by deteriorating the growing environment for crops. Rice, a globally important cereal crop, is under production threat due to climate change and land degradation. This research aims to sustainably improve rice growth and yield by using Rhodopseudomonas palustris, a plant growth-promoting bacteria that has recently gained much attention in crop production. The experiment was set up in two fields, one as a control and the other as a PNSB-treated field. The foliar application of treatment was made fortnightly until the end of the vegetative stage. Data on the growth, yield, and antioxidant enzymes were collected weekly. The results of this experiment indicate no significant differences in the plant height, root volume, average grain per panicle, biological yield, grain fertility, and antioxidant enzyme activity between the PNSB-treated and untreated plants. However, a significant increase in the tiller number, leaf chlorophyll content and lodging resistance were noted with PNSB treatment. Likewise, PNSB-treatment significantly increased root length (25%), root dry weight (57%), productive tillers per plants (26%), average grains per plant (38%), grain yield (33%), 1000 grain weight (1.6%), and harvest index (41%). Hence, from this research, it can be concluded that foliar application of PNSB on rice crops under field conditions improves crop growth and yield, although it does not affect antioxidant enzyme activity.

Different Drought-Tolerant Mechanisms in Quinoa (Chenopodium quinoa Willd.) and Djulis (Chenopodium formosanum Koidz.) Based on Physiological Analysis.

The purpose of this experiment is to study the effects of treatment with 90% (28.5% volumetric water content (VWC)), 75% (24% VWC), 50% (16% VWC), and 25% (8% VWC) of water requirements on the growth of two djulis (Chenopodium formosana Koidz) varieties (red: RP and yellow: OR) and one quinoa (Chenopodium quinoa Willd) varieties (PI). The results showed that drought stress (8% VWC) significantly reduced plant growth and relative water content, and increased H2O2 and MDA content in C. formosana and C. quinoa. The most significant increase in these parameters was detected in the OR variety. The antioxidant enzymes, such as SOD, APX, and GR activities of PI variety under drought treatment (8% VWC), are significantly increased, while GR activity of C. formosana also increased significantly. Additionally, C. formosana and PI variety remained at a stable AsA/DHA ratio, but the GSH/GSSG ratio decreased during drought treatment. Moreover, drought stress increased total soluble sugars and proline content in the PI variety. However, C. formosana proline content was extremely significantly enhanced, and only the OR variety increased the total soluble sugar content at the same time during the vegetative growth period. In summary, C. formosana and C. quinoa have different drought tolerance mechanisms to adapt to being cultivated and produced under severe drought conditions.

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

BACKGROUND: Cadmium (Cd) is one of the most toxic heavy metals and inhibits physiological processes of plants. Potassium (K) is an essential macronutrient in plants. K deficiency and Cd stress represent two different abiotic stress conditions that occur in the field simultaneously. In this study, effects of K deficiency on antioxidant status and Cd toxicity in rice seedlings were investigated. RESULTS: K deficiency significantly decreased K concentration in shoots and roots. However, fresh weight and dry weight of rice seedlings were not affected by K deficiency. The activities of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase) in K-deficient leaves were higher than respective control leaves. However, K deficiency had no effect on the content of antioxidants (ascorbate and glutathione). Cd toxicity was judged by the decrease in biomass production, chlorosis, and induction of oxidative stress. Based on these criteria, we demonstrated that K deficiency protected rice seedling from Cd stress. Moreover, chlorophyll concentration was higher in K-deficient shoots and roots than their respective control shoots and roots. CONCLUSIONS: Our results indicated that K deficiency protects rice seedlings from Cd toxicity. This protective effect of K deficiency is mainly due to enhanced antioxidant enzyme activities but not inhibition of Cd uptake.

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.

Cobalt chloride-induced lateral root formation in rice: the role of heme oxygenase.

Lateral roots (LRs) perform the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. Recent findings suggest that heme oxygenase (HO) plays an important role in LR development. In this study, we examined the effect of cobalt chloride (CoCl2) on LR formation and HO expression in rice. Treatment with CoCl2 induced LR formation and HO activity. We further observed that CoCl2 could induce the expression of OsHO1 but not OsHO2. CoCl2-increased HO activity occurred before LR formation. Zinc protoporphyrin IX (ZnPPIX, the specific inhibitor of HO) and hemoglobin (the carbon monoxide/nitric oxide scavenger) reduced LR formation, HO activity, and OsHO1 expression. Application of biliverdin, a product of HO-catalyzed reaction, to CoCl2-treated rice seedlings reversed the ZnPPIX-inhibited LR formation and ZnPPIX-decreased HO activity. CoCl2 had no effect on H2O2 content and nitric oxide production. Moreover, application of ascorbate, a H2O2 scavenger, failed to affect CoCl2-promoted LR formation and HO activity. It is concluded that HO is required for CoCl2-promoted LR formation in rice.

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.

Biliverdin-promoted lateral root formation is mediated through heme oxygenase in rice.

In this study, we examined the effect of biliverdin (BV), a product of heme oxygenase (HO) catalyzed reaction, on lateral root (LR) formation in rice. Treatment with BV induced LR formation and HO activity. As well, BV, could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) reduced LR number, HO activity and OsHO1 mRNA level induced by BV. Our data suggest that HO is required for BV-induced LR formation in rice.

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.

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.

Exposure of rice seedlings to heat shock protects against subsequent Cd-induced decrease in glutamine synthetase activity and increase in specific protease activity in leaves.

In the present study, we investigated the effect of heat shock (HS) on the subsequent Cd-induced decrease in the activity of glutamine synthetase (GS) and increase in the specific activity of protease in rice leaves. HS exposure of rice seedlings for 3h in the dark was effective in reducing subsequent Cd-induced decrease in the activity of glutamine synthetase and increase in the specific activity of protease. The effect of HS can be mimicked by pretreatment of rice seedlings with exogenous H(2)O(2) or reduced glutathione (GSH) under non-HS conditions. We also found that HS protected against subsequent Cd-induced decrease in the activity of GS and increase in the specific activity of protease can be counteracted by imidazole, a NADPH oxidase inhibitor. Pretreatment with buthione sulfoximine (a GSH synthesis inhibitor) under HS conditions enhanced subsequent Cd effects on the activity of GS and the specific activity of protease. Moreover, the effect of BSO can be reversed by the addition of GSH. The mechanisms of the protective effect of HS effect against subsequent Cd effects are discussed.

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.

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.