Heat shock protein 70 is associated with duration of cell proliferation in early pod development of soybean.

Pod is an important organ for seed production in soybean. Pod size varies among soybean cultivars, but the mechanism is largely unknown. Here we reveal one of the factors for pod size regulation. We investigate pod size differences between two cultivars. The longer pod of 'Tachinagaha' is due to more cell number than in the short pod of 'Iyodaizu'. POD SIZE OF SOYBEAN 8 (GmPSS8), a member of the heat shock protein 70 (HSP70) family, is identified as a candidate gene for determining pod length in a major QTL for pod length. Expression of GmPSS8 in pods is higher in 'Tachinagaha' than 'Iyodaizu' and is highest in early pod development. The difference in expression is the result of an in/del polymorphism　which includes an enhancer motif. Treatment with an HSP70 inhibitor reduces pod length and cell number in the pod. Additionally, shorter pods in Arabidopsis hsp70-1/-4 double mutant are rescued by overexpression of GmPSS8. Our results identify GmPSS8 as a target gene for pod length, which regulates cell number during early pod development through regulation of transcription in soybean. Our findings provide the mechanisms of pod development and suggest possible strategies enhancing yield potential in soybean.

Mechanism of delayed seed germination caused by high temperature during grain filling in rice (Oryza sativa L.).

High temperature during grain filling considerably reduces yield and quality in rice (Oryza sativa L.); however, how high temperature affects seed germination of the next generation is not yet well understood. Here, we report that seeds from plants exposed to high temperature during the grain filling stage germinated significantly later than seeds from unstressed plants. This delay remained even after dormancy release treatments, suggesting that it was not due to primary seed dormancy determined during grain filling. In imbibed embryos of heat-stressed seeds, expression of abscisic acid (ABA) biosynthesis genes (OsNCEDs) was higher than in those of control seeds, whereas that of ABA catabolism genes (OsABA8'OHs) was lower. In the aleurone layer, despite no change in GA signaling as evidenced by no effect of heat stress on OsGAMYB gene expression, the transcripts of α-amylase genes OsAmy1C, OsAmy3B, and OsAmy3E were significantly down-regulated in heat-stressed seeds in comparison with controls. Changes in promoter methylation levels were consistent with transcriptional changes of ABA catabolism-related and α-amylase genes. These data suggest that high temperature during grain filling results in DNA methylation of ABA catabolism-related and α-amylase gene promoters, delaying germination of heat-stressed seeds.

Effect of Seedling Nitrogen Condition on Subsequent Vegetative Growth Stages and Its Relationship to the Expression of Nitrogen Transporter Genes in Rice.

Nitrogen (N) deficiency is one of the most common problems in soils, limiting crop growth and production. However, the effects of N limitation in seedlings on vegetative growth remain poorly understood. Here, we show that N limitation in rice seedlings restricted vegetative growth but not yield. Aboveground parts were affected mainly during the period of tillering, but belowground parts were sensitive throughout vegetative growth, especially during panicle development. At the tillering stage, N-limited plants had a significantly lower N content in shoots, but not in roots. On the other hand, N content in roots during the panicle development stage was significantly lower in N-limited plants. This distinct response was driven by significant changes in expression of N transporter genes during growth. Under N limitation, N translocation from roots to shoots was greatly sped up by systemic expression of N transporter genes to obtain balanced growth. N limitation during the seedling stage did not reduce any yield components. We conclude that the N condition during the seedling stage affects physiological responses such as N translocation through the expression of N transporter genes.

Mechanisms of Maturation and Germination in Crop Seeds Exposed to Environmental Stresses with a Focus on Nutrients, Water Status, and Reactive Oxygen Species.

Environmental stresses can reduce crop yield and quality considerably. Plants protect cell metabolism in response to abiotic stresses at all stages of their life cycle, including seed production. As the production of vigorous seeds is important to both yield and crop growth, we analyzed causes of yield loss and reduced grain quality in staple crops exposed to environmental stresses such as drought and temperature extremes, with a focus on the remobilization of nutrients and water status during seed filling. Because water is one of the factors that limit seed development, seeds must have mechanisms that allow them to withstand water loss during seed maturation. In addition, analysis of the effects of reactive oxygen species (ROS) on transcription regulation and signaling should help to elucidate the regulation of seed dormancy and germination. In this review, we focus on nutrient remobilization, water mobility, plant hormones (gibberellins, abscisic acid, and ethylene), and ROS in sink and source organs and describe how rice, wheat, barley, soybean, and cowpea plants control seed maturation and germination under environmental stresses.

Reactive oxygen species induced by heat stress during grain filling of rice (Oryza sativa L.) are involved in occurrence of grain chalkiness.

Heat stress during grain filling increases rice grain chalkiness due to increased activity of α-amylase, which hydrolyzes starch. In rice and barley seeds, reactive oxygen species (ROS) produced after imbibition induce α-amylase activity via regulation of gibberellin (GA) and abscisic acid (ABA) levels during seed germination. Here, we examined whether ROS is involved in induction of grain chalkiness by α-amylase in developing rice grains under heat stress. To elucidate the role of ROS in grain chalkiness, we grew post-anthesis rice plants (Oryza sativa L. cv. Koshihikari) under control (25°C) or heat stress (30°C) conditions with or without antioxidant (dithiothreitol) treatment. The developing grains were analyzed for expression of NADPH oxidases, GA biosynthesis genes (OsGA3ox1, OsGA20ox1), ABA catabolism genes (OsABA8'OH1, OsABA8'OH2) and an α-amylase gene (OsAmy3E), endogenous H2O2 content and the grain quality. In grains exposed to heat stress, the expression of NADPH oxidase genes (especially, OsRbohB, OsRbohD, OsRbohF and OsRbohI) and the ROS content increased. Heat stress also increased the expression of OsGA3ox1, OsGA20ox1, OsABA8'OH1, OsABA8'OH2 and OsAmy3E. On the other hand, dithiothreitol treatment reduced the effects of heat stress on the expression of these genes and significantly reduced grain chalkiness induced by heat stress. These results suggest that, similar to cereal seed germination mechanism, ROS produced under heat stress is involved in α-amylase induction in maturating rice grains through GA/ABA metabolism, and consequently caused grain chalkiness.

Effects of light quality on pod elongation in soybean (Glycine max (L.) Merr.) and cowpea (Vigna unguiculata (L.) Walp.).

Soybean pods are located at the nodes, where they are in the shadow, whereas cowpea pods are located outside of the leaves and are exposed to sunlight. To compare the effects of light quality on pod growth in soybean and cowpea, we measured the length of pods treated with white, blue, red or far-red light. In both species, pods elongated faster during the dark period than during the light period in all light treatments except red light treatment in cowpea. Red light significantly suppressed pod elongation in soybean during the dark and light periods. On the other hand, the elongation of cowpea pods treated with red light markedly promoted during the light period. These results suggested that the difference in the pod set sites between soybean and cowpea might account for the difference in their red light responses for pod growth.

The Interrelationship between Abscisic Acid and Reactive Oxygen Species Plays a Key Role in Barley Seed Dormancy and Germination.

Seed dormancy is one of the adaptive responses in the plant life cycle and an important agronomic trait. Reactive oxygen species (ROS) release seed dormancy and promote seed germination in several cereal crops; however, the key regulatory mechanism of ROS-mediated seed dormancy and germination remains controversial. Here, we focused on the relationship between hydrogen peroxide (a ROS) and abscisic acid (ABA) in dormant and non-dormant barley seeds. The hydrogen peroxide (H2O2) level produced in barley seed embryos after imbibition was higher in non-dormant seeds than in dormant seeds. H2O2 regulated the ABA content in the embryos through ABA-8'-hydroxylase, an ABA catabolic enzyme. Moreover, compared with non-dormant seeds, in dormant seeds the activity of NADPH oxidase, which produces ROS, was lower, whereas the activity of catalase, which is a H2O2 scavenging enzyme, was higher, as was the expression of HvCAT2. Furthermore, precocious germination of isolated immature embryos was suppressed by the transient introduction of HvCAT2 driven by the maize (Zea mays) ubiquitin promoter. HvCAT2 expression was regulated through an ABA-responsive transcription factor (HvABI5) induced by ABA. These results suggest that the changing of balance between ABA and ROS is active in barley seed embryos after imbibition and regulates barley seed dormancy and germination.

Role of reactive oxygen species produced by NADPH oxidase in gibberellin biosynthesis during barley seed germination.

NADPH oxidase catalyzes the production of the superoxide anion (O2(-)), a reactive oxygen species (ROS), and regulates the germination of barley (Hordeum vulgare L.). Diphenyleneiodonium (DPI) chloride, an NADPH oxidase inhibitor, delayed barley germination, and exogenous H2O2 (an ROS) partially rescued it. Six enzymes, ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20-oxidase (GA20ox) and GA3-oxidase (GA3ox), catalyze the transformation of trans-geranylgeranyl diphosphate to active gibberellin, which promotes germination. Exogenous H2O2 promoted the expressions of HvKAO1 and HvGA3ox1 in barley embryos. These results suggest that ROS produced by NADPH oxidase are involved in gibberellin biosynthesis through the regulation of HvKAO1 and HvGA3ox1.

Nitrogen redistribution and its relationship with the expression of GmATG8c during seed filling in soybean.

It is well known that some nitrogen in the vegetative organs is redistributed to the seeds during seed filling in soybean (Glycine max [L.] Merrill). This redistribution is considered to affect the seed yield of soybean. However, it is still not clear when the nitrogen moves from the vegetative part to the seeds, and the relationship between nitrogen redistribution and leaf senescence has not been clarified. The soybean variety Fukuyutaka was grown in the experimental field of Saga University, Japan from 22 July to 31 October, 2014. After the first flower stage (R1), the plant samples were collected weekly and were separated into leaf, petiole, stem, podshell and seed. The nitrogen concentrations in each plant part were determined. Fresh leaf samples were provided for the determination of soluble protein and autophagy gene GmATG8c expression. The nitrogen that accumulated in the vegetative parts reached its highest level at 60days after sowing (DAS), then began to decrease at 73DAS (R6). This decrease is considered to be the consequence of nitrogen redistribution from the vegetative parts to the seeds. The movement of nitrogen from the vegetative parts to the seeds was estimated to occur at around 73DAS (R6). At this stage, leaf SPAD values, leaf nitrogen, and soluble protein concentrations began to decrease simultaneously, suggesting the onset of leaf senescence. Furthermore, the expression of the autophagy gene GmATG8c in the leaves increased dramatically from 73 to 85DAS, which is the duration of nitrogen redistribution. The results suggest that the nitrogen redistribution from the vegetative parts to the seeds could be one of the initiating factors of leaf senescence, and the autophagy gene GmATG8c was associated with this process.

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.

Programmed cell death in barley aleurone cells is not directly stimulated by reactive oxygen species produced in response to gibberellin.

The cereal aleurone layer is a secretory tissue that produces enzymes to hydrolyze the starchy endosperm during germination. We recently demonstrated that reactive oxygen species (ROS), produced in response to gibberellins (GA), promoted GAMyb expression, which induces α-amylase expression in barley aleurone cells. On the other hand, ROS levels increase during programmed cell death (PCD) in barley aleurone cells, and GAMyb is involved in PCD of these cells. In this study, we investigated whether the ROS produced in response to GA regulate PCD directly by using mutants of Slender1 (SLN1), a DELLA protein that negatively regulates GA signaling. The wild-type, the sln1c mutant (which exhibits gibberellin-type signaling even in the absence of GA), and the Sln1d mutant (which is gibberellin-insensitive with respect to α-amylase production) all produced ROS in response to GA, suggesting that ROS production in aleurone cells in response to GA is independent of GA signaling through this DELLA protein. Exogenous GA promoted PCD in the wild-type. PCD in sln1c was induced even without exogenous GA (and so without induction of ROS), whereas PCD in Sln1d was not induced in the presence of exogenous GA, even though the ROS content increased significantly in response to GA. These results suggest that PCD in barley aleurone cells is not directly stimulated by ROS produced in response to GA but is regulated by GA signaling through DELLA protein.

Assimilate translocation and expression of sucrose transporter, OsSUT1, contribute to high-performance ripening under heat stress in the heat-tolerant rice cultivar Genkitsukushi.

High temperature reduces the grain quality of rice, a situation likely to become more frequent because of global warming. We studied the effects of high-temperature stress on grain quality of heat-tolerant cultivar 'Genkitsukushi' and heat-sensitive cultivar 'Tsukushiroman'. When day/night temperatures were 31/26 °C from heading until maturity, the grain quality of 'Genkitsukushi' was rated at the first inspection grade (high quality), whereas 'Tsukushiroman' showed a remarkable increase in the percentage of white immature kernels (low quality). Nonstructural carbohydrate content in the stem of 'Genkitsukushi' the early maturation was significantly higher than in 'Tsukushiroman' and greatly decreased under high temperature. From 14 to 21 days after heading, the expression of the sucrose transporter gene, OsSUT1, was higher in the stem of 'Genkitsukushi' grown under high temperature than in 'Tsukushiroman'. In addition, the expression of OsSUT1 in the grains of 'Genkitsukushi' was significantly higher than in 'Tsukushiroman' during the ripening period. These results indicate that sugar transport functions more effectively in 'Genkitsukushi' than in 'Tsukushiroman', and that the effectiveness of sugar transport contributes to maintaining high grain quality in 'Genkitsukushi' under high-temperature conditions.

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.

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.

The ethylene signal mediates induction of GmATG8i in soybean plants under starvation stress.

In higher plants, autophagy-related genes (ATGs) appear to play important roles in development, senescence, and starvation responses. Hormone signals underlying starvation-induced gene expression are involved in the expression of ATGs. An effect of starvation stress on the expression of ATGs and ethylene-related genes in young seedlings of soybean (Glycine max [L.] Merr. cv. Fukuyutaka) was analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression levels of GmATG8i and GmATG4 increase in a starvation medium, but at a null or marginal level in a sucrose/nitrate-rich medium. The expression of GmACC synthase and GmERF are also upregulated in the starvation medium. In addition, immunoblot revealed that ethylene insensitive 3 (Ein3), an ethylene-induced transcription factor are accumulated in seedlings subjected to severe starvation stress. These results indicate that starvation stress stimulates the expression of GmATG8i and ethylene signal-related genes. Since the ethylene signal is involved in senescence and various environmental stresses, it is possible that starvation stress-induced autophagy is partly mediated by the ethylene 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.

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 O(3) treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O(3)-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O(3), Ca(2+) 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 O(3); namely, H(2)O(2) 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 O(3)-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.

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

Hydrogen peroxide (H(2)O(2)) promotes seed germination of cereal plants and ascorbic acid which acts as antioxidant suppresses the germination of wheat seeds, but the role of H(2)O(2) scavenging on germination during seed maturation has not been demonstrated. We investigated relationship of germination, ascorbate, H(2)O(2) 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 H(2)O(2) content had no difference in the two culti-vars, 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.