Mari Iwaya-Inoue

Reactive Oxygen Species Are Involved in Gibberellin/Abscisic Acid Signaling in Barley Aleurone Cells

Reactive oxygen species (ROS) act as signal molecules for a variety of processes in plants. However, many questions about the roles of ROS in plants remain to be clarified. Here, we report the role of ROS in gibberellin (GA) and abscisic acid (ABA) signaling in barley (Hordeum vulgare) aleurone cells. The production of hydrogen peroxide (H2O2), a type of ROS, was induced by GA in aleurone cells but suppressed by ABA. Furthermore, exogenous H2O2 appeared to promote the induction of α-amylases by GA. In contrast, antioxidants suppressed the induction of α-amylases. Therefore, H2O2 seems to function in GA and ABA signaling, and in regulation of α-amylase production, in aleurone cells. To identify the target of H2O2 in GA and ABA signaling, we analyzed the interrelationships between H2O2 and DELLA proteins Slender1 (SLN1), GA-regulated Myb transcription factor (GAmyb), and ABA-responsive protein kinase (PKABA) and their roles in GA and ABA signaling in aleurone cells. In the presence of GA, exogenous H2O2 had little effect on the degradation of SLN1, the primary transcriptional repressor mediating GA signaling, but it promoted the production of the mRNA encoding GAMyb, which acts downstream of SLN1 and involves induction of α-amylase mRNA. Additionally, H2O2 suppressed the production of PKABA mRNA, which is induced by ABA:PKABA represses the production of GAMyb mRNA. From these observations, we concluded that H2O2 released the repression of GAMyb mRNA by PKABA and consequently promoted the production of α-amylase mRNA, thus suggesting that the H2O2 generated by GA in aleurone cells is a signal molecule that antagonizes ABA signaling.

Hydrogen peroxide spraying alleviates drought stress in soybean plants

To ascertain the effect of exogenously applied hydrogen peroxide (H2O2) on drought stress, we examined whether the spraying of soybean leaves with H2O2 would alleviate the symptoms of drought stress. Pre-treatment by spraying leaves with H2O2 delayed foliar wilting caused by drought stress compared to leaves sprayed with distilled water (DW). Additionally, the relative water content of drought-stressed leaves pre-treated with H2O2 was higher than that of leaves pre-treated with DW. Therefore, we analyzed the effect of H2O2 spraying on photosynthetic parameters and on the biosynthesis of oligosaccharides related to water retention in leaves during drought stress. Under conditions of drought stress, the net photosynthetic rate and stomatal conductance of leaves pre-treated with H2O2 were higher than those of leaves pre-treated with DW. In contrast to DW spraying, H2O2 spraying immediately caused an increase in the mRNA levels of d-myo-inositol 3-phosphate synthase 2 (GmMIPS2) and galactinol synthase (GolS), which encode key enzymes for the biosynthesis of oligosaccharides known to help plants tolerate drought stress. In addition, the levels of myo-inositol and galactinol were higher in H2O2-treated leaves than in DW-treated leaves. These results indicated that H2O2 spraying enabled the soybean plant to avoid drought stress through the maintenance of leaf water content, and that this water retention was caused by the promotion of oligosaccharide biosynthesis rather than by rapid stomatal closure.

Regulation of soybean seed germination through ethylene production in response to reactive oxygen species

BACKGROUND AND AIMS Despite their toxicity, reactive oxygen species (ROS) play important roles in plant cell signalling pathways, such as mediating responses to stress or infection and in programmed cell death, at lower levels. Although studies have indicated that hydrogen peroxide (H(2)O(2)) promotes seed germination of several plants such as Arabidopsis, barley, wheat, rice and sunflower, the role of H(2)O(2) in soybean seed germination is not well known. The aim of this study therefore was to investigate the relationships between ROS, plant hormones and soybean seed germination. METHODS An examination was made of soybean seed germination, the expression of genes related to ethylene biosynthesis, endogenous ethylene contents, and the number and area of cells in the root tip, using N-acetylcysteine, an antioxidant, to counteract the effect of ROS. KEY RESULTS H(2)O(2) promoted germination, which N-acetylcysteine suppressed, suggesting that ROS are involved in the regulation of soybean germination. H(2)O(2) was produced in the embryonic axis after imbibition. N-Acetylcysteine suppressed the expression of genes related to ethylene biosynthesis and the production of endogenous ethylene. Interestingly, ethephon, which is converted to ethylene, and H(2)O(2) reversed the suppression of seed germination by N-acetylcysteine. Furthermore, morphological analysis revealed that N-acetylcysteine suppressed cell elongation at the root tip, and this suppression was also reversed by ethephon or H(2)O(2) treatments, as was the case in germination. CONCLUSIONS In soybean seeds, ROS produced in the embryonic axis after imbibition induce the production of endogenous ethylene, which promotes cell elongation in the root tip. This appears to be how ROS regulate soybean seed germination.

Increased Anion Channel Activity Is an Unavoidable Event in Ozone-Induced Programmed Cell Death

Background Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. Principal Findings By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O3 treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O3-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O3, Ca2+ influx and NADPH-oxidase generated reactive oxygen species (ROS) in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O3; namely, H2O2 generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. Significance Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O3-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s) for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation.

NADPH oxidases act as key enzyme on germination and seedling growth in barley (Hordeum vulgare L.)

Reactive oxygen species (ROS) play an important role in seed germination. Although hydrogen peroxide (H2O2), a type of ROS, enhances the germination rate of various plant seeds, little is known about the mechanism. NADPH oxidases catalyze the production of superoxide anion (O2-) that is one of the ROS and the enzymes regulate plant development. We, therefore, investigated the role of NADPH oxidases in seed germination and seedling growth in barley (Hordeum vulgare L.). The production of O2- was observed both in embryo and aleurone layers in barley seeds treated with distilled water (DW). However, it was suppressed in seeds treated with diphenylene iodonium (DPI) chloride, NADPH oxidase inhibitor. Moreover, DPI markedly delayed germination and remarkably suppressed α-amylase activity in barley seeds, indicating the importance of NADPH oxidases in germination of barley seeds. The gene expression and the enzyme activity of NADPH oxidases gradually increased after imbibition, and the enzyme activities were closely correlated with seedling growth after imbibition. Besides, DPI markedly suppressed the seedling growth. These results indicated that NADPH oxidases perform a crucial function in germination and seedling growth in barley. These facts clearly reveal that O2- produced by NADPH oxidases after imbibition regulates seed germination and seedling growth in barley.

Silicon enhances growth independent of silica deposition in a low-silica rice mutant, lsi1

To examine whether silica bodies are essential for silicon-enhanced growth of rice seedlings, we investigated the response of rice, Oryza sativa L., to silicon treatment. Silicic acid treatment markedly enhanced the SPAD (soil plant analytical development) values of leaf blades and the growth and development of leaves and lateral roots in cvs. Hinohikari and Oochikara, and a low-silicon mutant, lsi1. Combination of ethanol–benzene displacement and staining with crystal violet lactone enabled more detailed histochemical analysis to visualize silica bodies in the epidermis under bright-field microscopy. Supply of silicon induced the development of motor cells and silica bodies in epidermal cells in Hinohikari and Oochikara but not or marginal in lsi1. X-ray analytical microscopy detected silicon specifically in the leaf sheath, the outermost part of the stem, and the leaf blade midrib, suggesting that silicon is distributed to tissues involved in maintaining rigidity of the plant to prevent lodging, rather than being passively deposited in growing tissues. Silicon supplied at high dose accumulated in all rice seedlings and enhanced growth and SPAD values with or without silica body formation. Silicon accumulated in the cell wall may play an important physiological role different from that played by the silica deposited in the motor cell and silica bodies.

Hydrogen peroxide scavenging regulates germination ability during wheat (Triticum aestivum L.) seed maturation

Hydrogen peroxide (H2O2) promotes seed germination of cereal plants and ascorbic acid which acts as antioxidant suppresses the germination of wheat seeds, but the role of H2O2 scavenging on germination during seed maturation has not been demonstrated. We investigated relationship of germination, ascorbate, H2O2 scavenging enzymes and sensitivity to ascorbic acid (AsA) maturing seeds of two typical wheat (Triticum aestivum L.) cultivars, cvs. Shirogane-Komugi and Norin61. Shirogane-Komugi had marked high germination ability than Norin61 during seed maturation. Although the H2O2 content had no difference in the two cultivars, sensitivity to AsA of Norin61 seeds was higher than that of Shirogane-Komugi seeds during seed maturation. The sensitivity to AsA closely correlated with germination characteristic in the two cultivars. Especially, at 28 days after pollination (DAP), sensitivity to AsA in Norin61 seeds was remarkably high. At that stage, no significant differences were observed in endogenous AsA level, ascorbate peroxidase (APX, EC 1.11.1.11) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) activities in the two cultivars. However, catalase (CAT, EC 1.11.1.6) activity and CAT mRNA in Norin61 were remarkably higher than in Shirogane-Komugi. Sensitivity to AsA at 35 and 42 DAPs kept high levels in Norin61, and endogenous AsA and CAT activity in the seeds were significantly higher than in Shirogane-Komugi. These results revealed a direct correlation between germination and antioxidant sensitivity during the developmental stages of wheat seeds.

Influence of low/high temperature on water status in developing and maturing rice grains.

Abstract Temperature limits productivity and kernel quality of rice grains. Water status of rice (Oryza sativa L. cv. Hinohikari) grains grown at 20, 25 and 30ºC was evaluated by NMR relaxation times (T1 and T2) in relation to kernel quality during the period of grain development and maturation. During milky and dough stages, 7 to 15 days after flowering (DAF), T1 values of long and short fractions in rice grains grown at 20ºC slightly increased but those grown at 30ºC decreased markedly. In rice grains grown at 25 and 30ºC, T2 values of long and short fractions were about 100 and 10 ms, respectively, until 22 DAF but rapidly decreased to about 1 ms and 20 µs, respectively, at 29 DAF (yellow-ripe stage). This means that these grains had free water until 22 DAF , but only loosely bound water and bound water thereafter. On the other hand, in the grains grown at 20ºC, T2 values of long and short fractions were about 100 and 10 ms, respectively, until 29 DAF, indicating that free water was maintained for seven days longer than at higher temperatures. The grains grown at 30ºC had white-back kernels in 85% of them and no perfect kernels, while the grains grown at 25 and 20ºC had perfect kernels in 68 and 27% of them, respectively. In contrast, the grains grown at 20ºC had notched-belly kernels in 28% of them though the grains grown at 25 and 30ºC had only a few and no notched-belly kernels, respectively. The present study revealed that the changes in T1 of rice grains closely related with the quantity of water until mid-mature stage, while T2 was more sensitive diagnostic indicator for accumulation of dry matter and quality of kernels, which were influenced by low/high temperature stresses.

Changes in NMR Relaxation of Rice Grains, Kernel Quality and Physicochemical Properties in Response to a High Temperature after Flowering in Heat-Tolerant and Heat-Sensitive Rice Cultivars

Abstract Rice productivity is related to the ability of plants to adapt to heat stress. The heat-tolerant cultivars ‘Nikomaru’ and ‘Chikushi 64’ and heat-sensitive cultivar ‘Hinohikari’ were grown at 30ºC and 25ºC for 49 days after flowering. At 30ºC, only a few white immature kernels were produced in ‘Nikomaru’ and ‘Chikushi 64’, but about 22% of grains had immature kernels in ‘Hinohikari’. The high temperature(30ºC) caused no significant changes in grain dry weight, water content, and the NMR T1 value during the early ripening stage in ‘Nikomaru’ and ‘Chikushi 64’. It also did not affect grain development, especially with respect to the nucellar epidermis, in ‘Nikomaru’ and ‘Chikushi 64’, but caused clear cessation of development of the nucellar epidermis at 14 days after flowering in ‘Hinohikari’. In addition, high temperature decreased the amylose content and increased hardnessvs. adhesion ratio of cooked rice in both ‘Nikomaru’ and ‘Chikushi 64’ resulting a softer, less sticky texture, but not in ‘Hinohikari’. The maximum viscosity and breakdown values were increased, and final viscosity decreased at 30ºC in all three cultivars. These results suggested that starch in the endosperm of grains changed from a fluid state to a doughy state more slowly in ‘Nikomaru’ and ‘Chikushi 64’ than in ‘Hinohikari’, in which the water content and NMR relaxation time decreased, and transported assimilates accumulated slowly during grain development.

A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination.

Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells.