Climate change due to global warming is now affecting agricultural production worldwide. In rice, one of the most important crops, water limitation due to irregular rainfall in rainfed lowlands during crop growth limits yield. Dry direct-sowing has been proposed as a water-efficient approach to cope with water stress during rice growth, but poor seedling establishment due to drought during germination and emergence is a problem. Here, we germinated indica rice cultivars Rc348 (drought tolerant) and Rc10 (drought sensitive) under osmotic stress induced by PEG to elucidate mechanisms of germination under drought. Rc348 had higher germination rate and germination index under severe osmotic stress of −1.5 MPa, above those of Rc10. Rc348 showed up-regulated GA biosynthesis, down-regulated ABA catabolism, and up-regulated α-amylase gene expression in imbibed seeds under PEG treatment compared to that of Rc10. During germination, reactive oxygen species (ROS) play important roles in antagonism between gibberellic acid (GA) and abscisic acid (ABA). Embryo of Rc348 treated with PEG had significantly greater expression of NADPH oxidase genes and higher endogenous ROS levels, together with significantly increased endogenous GA₁, GA₄ and ABA contents compared to that of Rc10. In aleurone layers treated with exogenous GA, expression of α-amylase genes was higher in Rc348 than in Rc10, and expression of NADPH oxidase genes was enhanced with significantly higher ROS content in Rc348, suggesting higher sensitivity of GA to ROS production and starch degradation in aleurone cells of Rc348. These results suggest that the osmotic stress tolerance of Rc348 is due to enhancement of ROS production, GA biosynthesis, and GA sensitivity, resulting in a higher germination rate under osmotic stress.

Abstract The extensive and intensive use of herbicides has resulted in the spread of herbicide-resistant weeds in many crop production systems; therefore, it is imperative to devise new organic weed control methods. Recently, the application of spent coffee grounds (SCG) in agricultural fields has been found to inhibit plant growth and germination and is thus considered a potentially effective weed control measure. This study aimed to evaluate the effects of different amounts and methods of SCG application on weed growth through field experiments. The field experiments were conducted in an upland field converted from a paddy in western Japan. The results show that the plow-in application of over 10 kg m⁻² of SCG and mulching application of 20 kg m⁻² decreased the weed dry weight compared with the control. In addition, the growth of weed species of families other than Gramineae, such as wingleaf primrose-willow and horseweed, was not significantly affected by SCG application. Weed species of families other than Gramineae are dominant in some upland fields. Hence, the inhibitory effect of SCG on weeds may be lower in original upland fields than in the upland field converted from paddy field that was investigated in the present study. Overall, this study demonstrated that the plow-in application of 10 kg m⁻² of SCG every 4 mo was effective for weed control in an upland field converted from a paddy field. Because SCG worked against grass weeds under the specific conditions in this study, it would be valuable to explore other potential applications of this novel means of weed control.

Serial sectioning transmission electron microscopy (ssTEM) is a classical method of 3D reconstruction using serial sections obtained with an ultramicrotome. However, producing a long ribbon with homogeneity is difficult. Here, ultramicrotome movement was suspended after producing a ribbon of 15-30 serial sections (cutting intervals, 100 nm), and then, the ribbon was mounted on an individual one-slot grid. However, as this ssTEM method may include influencing factors such as incorrect intervals of section thickness and distortion of sections, which is produced by cutting sections using a diamond knife and beam interaction under TEM observation, qualitative and quantitative data on rice mesophyll cells and chloroplasts were compared with those obtained from a focused ion beam scanning electron microscopy (FIB-SEM) (cutting intervals, 50 nm). No structural distortion in 3D models was observed. In addition, no significant differences in the volume and surface area were observed between the two methods. The surface to volume ratio was significantly affected by the increase in section thickness, but not the difference of methodologies. Our method was useful for observing large volumes of plant cells and organelles, leading to the identification of various sizes and types of chloroplasts. The formation of a chloroplast pocket, which is a structure surrounding other intracellular compartments, was confirmed in rice leaves grown under moderate growth conditions using the ssTEM method. As only four out of 90 chloroplasts formed pocket structures, the formation was considered to be rare under the applied moderate growth conditions.

We have investigated the effectiveness of a new soybean fertilization technique, named crack fertilization, which involves the application of nodule bacteria on biochar to soybean roots through cracks formed between planting rows during midterm tillage. In the present study, the factors and timing of crack fertilization were investigated in an upland field converted from a paddy. The variables investigated were: 1) crack formation without the application of any agricultural materials, and 2) the application of biochar or 3) nodule bacteria on biochar into cracks. The treatment periods were: 1) before sowing, 2) during midterm tillage, and 3) during both periods. The combination of crack fertilization and reduced tillage was also tested. The method of crack fertilization that increased yield was the combination of crack formation and the application of biochar, and the most effective period for the treatment was before sowing in the reduced tillage field. Seed yields in conventional and reduced tillage fields were comparable in the upland field converted from the paddy. These results suggest that the application of biochar into cracks after scratching the soil surface to remove weeds before sowing is a practical method of increasing soybean yield in upland fields converted from paddies.

Excess salinity inhibits the metabolism of various systems and induces structural changes, especially in chloroplasts. Although the chloroplast body seems to swell under salinity stress as observed by conventional transmission electron microscopy, previous studies are limited to 2-D data and lack quantitative comparisons because specimens need to be sliced into ultrathin sections. This study shows three-dimensionally the structural changes in a whole mesophyll cell responding to salinity stress by serial sectioning with a focused ion beam scanning electron microscope (FIB-SEM) and compares the differences in chloroplast structures based on reconstructed models possessing accurate numerical voxel values. Leaf blades of rice plants treated with 100 mm NaCl or without (control) for 4 d were fixed chemically and embedded in resin. The specimen blocks were sectioned and observed using the FIB-SEM, and then the sliced image stacks were reconstructed into 3-D models by image processing software. On the transverse sections of rice mesophyll cells, the chloroplasts in the control leaves appeared to be elongated meniscus lens shaped, while those in the salt-treated leaves appear to be expanded oval shaped. The 3-D models based on serial sectioning images showed that the chloroplasts in the control cells spread like sheets fitted to the shape of the cell wall and in close contact with the adjacent chloroplasts. In contrast, those in the salt-stressed cells curled up into a ball and fitted to cell protuberances without being in close contact with adjacent chloroplasts. Although the shapes of chloroplasts were clearly different between the two treatments, their volumes did not differ. The 3-D reconstructed models of whole rice mesophyll cells indicated that chloroplasts under salt stress conditions were not swollen but became spherical without increasing their volume. This is in contrast to findings of previous studies based on 2-D images.

*These authors contributed equally to this work.

We investigated the invagination structure of a chloroplast that surrounds organelles such as mitochondria and peroxisomes within a thin layer of chloroplast stroma, which is called a chloroplast pocket. In this study, chloroplast pockets were observed in rice plants subjected to salinity stress but not under moderate growth condition. They included cytosol, transparent structure, lipid bodies, mitochondria, and peroxisomes. We constructed the three-dimensional architecture of chloroplast pockets by using serial images obtained by transmission electron microscopy and focused ion beam-scanning electron microscopy. Three types of chloroplast pockets were observed by transmission electron microscopy: Organelles were completely enclosed in a chloroplast pocket (enclosed type), a chloroplast pocket with a small gap in the middle part (gap type), and a chloroplast pocket with one side open (open type). Of the 70 pockets observed by serial imaging, 35 were enclosed type, and 21 and 14 were gap and open types, respectively. Mitochondria and peroxisomes were often in contact with the chloroplast pockets. Focused ion beam-scanning electron microscopy revealed chloroplasts with a sheet structure partially surrounding peroxisomes. This fact suggests that chloroplasts might construct large sheet structures that would be related to the formation of chloroplast pockets.

Aims: Rice is often cultivated in drought-prone regions causing growth inhibition. Therefore, we investigated whether close mixed planting of rice with pearl millet would mitigate these effects. Methods: We used pot and lysimeter to evaluate whether close mixed planting with pearl millet is more effective than single-stand planting in suppressing the growth inhibition of rice (NERICA4) under drought. Results: Close mixed planting only slightly alleviated the growth inhibition of rice under drought in the pot experiment and the first year lysimeter experiment, but mitigated this in the second year lysimeter experiment due to the lower levels of competition. Deuterated water applied from the bottom of the pot and lysimeter was present at a higher concentration in the xylem sap of rice under close mixed planting than under single-stand planting, and this tendency was enhanced by drought. Partitioning of the available water between the two crops was also observed under close mixed planting, with the rice and pearl millet depending on water from the soil surface and deep soil, respectively. Conclusions: Close mixed planting with pearl millet effectively mitigates the growth inhibition of rice under drought due to the increased access to deep water and the partitioning of available water between the two crops.

The authors have proposed the close mixed planting technique using mixed seedlings of two different crop species that results in close tangling of their root systems. Especially, the combination of drought-adaptive upland crops (e.g. pearl millet or sorghum) and flood-adaptive lowland crop of rice would be beneficial to overcome the drought and flood conditions and to reduce the risks of crop failure. In our previous studies, we found that upland crop yield losses by flood stress was mitigated by mix-cropped rice, owing to the oxygen gas released from the rice roots into the aqueous rhizosphere. In the present study, we conducted two experiments to assess whether mixed cropping a drought-resistant cereal, pearl millet, would improve the performance of co-growing drought-susceptible crop, rice under drought conditions. In the field experiment, some grains were obtained from the rice plants mix-cropped with pearl millet under drought condition. However, no rice matured in the single cropping system. In the model experiment using deuterium analysis, it was confirmed that water absorbed by pearl millet roots from deep soil layer was utilized by rice, suggesting that mix-cropped rice could withstand drought stress and complete grain filling using water released into the upper soil layer by hydraulic lift.

Dry direct-seeded rice (DSR) cultivation is widely spreading in tropical Asia, but drought and nutrient deficiency stresses often cause crop failure in rainfed lowlands. The objective of this study was to dissect the physio-morphological characteristics associated with crop establishment and early vigour of DSR under drought and P deficiency conditions in the Philippines. It was found that new drought-resistant cultivars bred for DSR (Rc348 and Rc192) had faster germination and sprout growth than popular irrigated rice cultivars (Rc222 and Rc10) under soil water deficit due to rapid moisture acquisition by the germinating seeds from drying soils. There was a significant correlation between seed moisture content and the reduction in seed dry weight, and between reduction in seed dry weight and shoot elongation under both control and drought stress treatments at the germination stage. At the seedling stage, the root growth of Rc348 under drought tended to be more vigorous with its higher root-to-shoot ratio compared to Rc222 and Rc10. The seedling vigour of Rc348 under P deficiency was also greater than that of Rc222 due to its greater root growth and P uptake. The yields of Rc348 and Rc192 grown under rainfed condition at the target drought-prone site where a dry spell of 13 days occurred during crop establishment were higher (4.0–4.1 t ha−1) than the yield of Rc10 (3.0 t ha−1). These results suggest that quick germination and seedling vigour with quick root anchorage and great nutrient uptake capacity, even with limitations of soil moisture and nutrients, would be important traits for DSR in rainfed lowlands.

Raffinose (sucrosylgalactoside oligosaccharide) is a water soluble carbohydrate and accumulates in response to abiotic stresses in plants. Plant raffinose synthases are poorly characterized, and the genes involved in raffinose biosynthesis are unknown in sugar beet. Here, we report the isolation of two genes encoding raffinose synthase (BvRS1 and BvRS2) as well as a gene encoding galactinol synthase (BvGolS1) from sugar beet. BvRS1 and BvRS2 show high homologies to Arabidopsis raffinose synthase AtRS5. BvRS1 and BvGolS1 were expressed in Escherichia coli. Crude extracts showed the activities of raffinose synthase and galactinol synthase. The Km values of BvRS1 for galactinol and sucrose and the Km values of BvGolS1 for UDP-galactose and myo-inositol were determined. The expression levels of BvRS1 were significantly higher than that of BvRS2. The mRNA for BvRS1 was rapidly induced by cold stress whereas the mRNA for BvRS2 was slowly induced by cold and salt stresses. These data suggest that BvRS1 and BvRS2 encode raffinose synthase genes responsible to cold and salt stress, respectively.

Mixed cropping is a cultivation method widely practiced in tropical regions. The newly developed close mixed planting technique mitigates the flood stress of drought-adapted upland cereal species by co-growing rice (Oryza sativa) plants under field flood conditions. We tested the hypothesis that O-2 was transferred from rice to upland crops using the model system of hydroponic culture. To confirm the hypothesis, the phenomena of O-2 absorption and release by plants were evaluated in a water culture condition without soil. Experiments were conducted in a climate chamber to estimate the amount of O-2 released from the roots of rice and pearl millet (Pennisetum glaucum) under both O-2 rich (20.0 +/- .0% conc. in phase I) and O-2-free dark (.8 +/- .0% conc. in phase II) conditions. The total O-2 change (between the two phases) in a single planting of rice and pearl millet was significantly higher than that of the mixed planting of rice and pearl millet, which indicated that O-2 was transferred from rice to pearl millet under a water culture condition. The result indicated that approximately 7 +/-mu M O-2 g fresh root weight(-1) h(-1) was transferred between the two plant species. O 2 transfer was confirmed between the two plant species in a mix cultured in water, implying its contribution to the phenomenon that improved the physiological status of drought-adapted upland crops under field flood conditions.

Background and Aims Ultrathin sections of rice leaf blades observed two-dimensionally using a transmission electron microscope (TEM) show that the chlorenchyma is composed of lobed mesophyll cells, with intricate cell boundaries, and lined with chloroplasts. The lobed cell shape and chloroplast positioning are believed to enhance the area available for the gas exchange surface for photosynthesis in rice leaves. However, a cell image revealing the three-dimensional (3-D) ultrastructure of rice mesophyll cells has not been visualized. In this study, a whole rice mesophyll cell was observed using a focused ion beam scanning electron microscope (FIB-SEM), which provides many serial sections automatically, rapidly and correctly, thereby enabling 3-D cell structure reconstruction. Methods Rice leaf blades were fixed chemically using the method for conventional TEM observation, embedded in resin and subsequently set in the FIB-SEM chamber. Specimen blocks were sectioned transversely using the FIB, and block-face images were captured using the SEM. The sectioning and imaging were repeated overnight for 200500 slices (each 50nm thick). The resultant large-volume image stacks (x = 25 mu m, y = 25 mu m, z = 10-25 mu m) contained one or two whole mesophyll cells. The 3-D models of whole mesophyll cells were reconstructed using image processing software. Key Results The reconstructed cell models were discoid shaped with several lobes around the cell periphery. The cell shape increased the surface area, and the ratio of surface area to volume was twice that of a cylinder having the same volume. The chloroplasts occupied half the cell volume and spread as sheets along the cell lobes, covering most of the inner cell surface, with adjacent chloroplasts in close contact with each other. Conclusions Cellular and sub-cellular ultrastructures of a whole mesophyll cell in a rice leaf blade are demonstrated three-dimensionally using a FIB-SEM. The 3-D models and numerical information support the hypothesis that rice mesophyll cells enhance their CO2 absorption with increased cell surface and sheet-shaped chloroplasts.

We investigated the alleviative effects of mixed cropping using ice plant, which is one of the saltaccumulating halophytes, on the damage and growth inhibition of cowpea, which is not tolerant to high salinity. Three cropping patterns (mono cropping of cowpea and ice plant and their combination) were tested. The plants were treated with 0, 100, 200 and 300 mM NaCl for 14 days (consecutive NaCl). The plants were also treated with NaCl for 3 days, followed by 2 weeks (shortterm recovery) and 1 month (long-term recovery) recovery. Salinity levels for short-term recovery were similar to those of the consecutive experiment, while the concentration of long-term recovery was 250 mM. The alleviative effects of mixed cropping in the consecutive NaCl experiment were observed at 200 and 300 mM NaCl. Mixed cropping significantly reduced the Na content in the cowpea leaves at 200 and 300 mM NaCl compared with mono cropping. In addition, the Na content in the soil of mix-cropped cowpea at 200 and 300 mM NaCl was statistically lower than that of the mono cropping. Mixed cropping was effective to recover from high concentration of NaCl in the experiments of short-and long-term recovery. These results indicate that mixed cropping with a halophyte could be effective in mitigating the damage and growth inhibition of a glycophyte not only under salinity but also under recovery periods.

Flash floods, erratically striking semi-arid regions, often cause field flooding and soil anoxia, resulting in crop losses on food staples, typically pearl millet (Pennisetum glaucum L.) and sorghum (Sorghum bicolor (L) Moench). Recent glasshouse studies have indicated that rice (Oryza spp.) can enhance flood stress tolerance of co-growing dryland cereals by modifying their rhizosphere microenvironments via the oxygen released from its roots into the aqueous rhizosphere. We tested whether this phenomenon would be expressed under field flood conditions. The effects of mix-planting of pearl millet and sorghum with rice on their survival, growth and grain yields were evaluated under controlled field flooding in semi-arid Namibia during 2014/2015-2015/2016. Single-stand and mixed plant treatments were subjected to 11-22 day flood stress at the vegetative growth stage. Mixed planting increased plant survival rates in both pearl millet and sorghum. Grain yields of pearl millet and sorghum were reduced by flooding, in both the single-stand and mixed plant treatments, relative to the non-flooded upland yields, but the reduction was lower in the mixed plant treatments. In contrast, flooding increased rice yields. Both pearl millet rice and sorghum rice mixtures demonstrated higher land equivalent ratios, indicating a mixed planting advantage under flood conditions. These results indicate that mix-planting pearl millet and sorghum with rice could alleviate flood stress on dryland cereals. The results also suggest that with this cropping technique, rice could compensate for the dryland cereal yield losses due to field flooding. (C) 2016 The Authors. Published by Elsevier B.V.

We investigated the soybean productivity of double cropping and relay intercropping in farmer's fields for three years using two black soybean cultivars of Kurozukin and Tanbaguro, which are used for the first and second crop, respectively. Kurozukin is the early maturing cultivar for vegetable soybean harvest and Tanbaguro is the late-maturing cultivar for the harvest of vegetable soybean and seeds. The yield of the first crop (Kurozukin) was similar to the mono cropping regardless of cropping patterns. However, the yield of the second crop (Tanbaguro) was affected by cropping patterns. The yield of Tanbaguro in double cropping was prone to decrease by late sowing. The late sowing was induced by the late sowing and late harvesting of Kurozukin because of the low temperature in April and the large amount of precipitation in rainy season, respectively. In relay intercropping that Tanbaguro was sown between the rows of Kurozukin at about one month before the harvest of Kurozukin, the yield of Tanbaguro was similar to the mono cropping and the competition with Kurozukin was not observed. Thus, the land equivalent ratio value of double cropping was lower than that of relay intercropping. These results suggest that relay intercropping is more useful cultivation system than double cropping to increase the annual soybean production.

Waterlogging is the constraint for soybean growth and yield, because soybean is often cultivated in upland fields converted from paddy in Japan. However, efficient cultivation techniques for alleviating the adverse effects have not been developed. We have proposed the new soybean cultivation technique named crack fertilization which enables yield increase due to enhancing new root growth and N acquisition by increasing nodulation. Waterlogging induces N deficiency due to the suppression of nutrient uptake by the inhibition of root growth and nodule activity. Thus, it is hypothesized that crack fertilization would be effective to alleviate the inhibition of soybean growth and yield. The soybean cultivar of Sachiyutaka was planted in 1/5000 a Wagner pots and root boxes. Two separate waterlogging treatments were imposed to soybean plants at different growth stages, V1 and R4, and crack fertilization was done at V3. After these treatments, soybean plants were sampled at R5 in 2012 and 2013 experiments, respectively. Waterlogging at V1 and R4 inhibited the growth and yield of soybean and nodule growth, and the decreases in physiological parameters of soybean such as photosynthesis, chlorophyll content, and xylem sap exudation rate were observed. The adverse effects of waterlogging at V1 were alleviated by crack fertilization at V3, whereas crack fertilization could not alleviate the adverse effects of waterlogging at R4. Thus, crack fertilization after waterlogging at early vegetative stage would be the cultivation technique that enables to alleviate the adverse effects.

Recently, the occurrences of extreme flooding and drought, often in the same areas, have increased due to climate change. Wetland plant species are known to oxygenate their rhizospheres by releasing oxygen (O-2) from their roots. We tested the hypothesis that wetland species could help upland species under flood conditions; that is, O-2 released from the wetland crop roots would ameliorate rhizosphere O-2-deficient stress and hence facilitate upland crop root function. Flooding tolerance of upland-adapted staple crops-pearl millet (Pennisetum glaucum) and sorghum (Sorghum bicolor) mix-cropped with rice (Oryza spp.) was investigated in glasshouse and laboratory. We found a phenomenon that strengthens the flood tolerance of upland crops when two species-one wetland and one drought tolerant-were grown using the mixed cropping technique that results in close tangling of their root systems. This technique improved the photosynthetic and transpiration rates of upland crops subjected to flood stress (O-2-deficient nutrient culture). Shoot relative growth rates during the flooding period (24 days) tended to be higher under mixed cropping compared with single cropping. Radial oxygen loss from the wetland crop roots might be contributed to the phenomenon observed. Mixed cropping of wet and dryland crops is a new concept that has the potential to overcome flood stress under variable environmental conditions. (C) 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

This paper examined the route of water supply into soybean nodules through the new visualization technique of time of flight secondary ion mass spectrometry (Tof-SIMS) cryo system, and obtained circumstantial evidence for the water inflow route. The maximum resolution of the Tof-SIMS imaging used by this study was 1.8 mu m (defined as the three pixel step length), which allowed us to detect water movement at the cellular level. Deuterium-labeled water was supplied to soybean plants for 4 h and the presence of deuterium in soybean nodules was analyzed by the Tof-SIMS cryo system. Deuterium ions were found only in the endodermis tissue surrounding the central cylinder in soybean nodules. Neither xylem vessels nor phloem complex itself did not indicate any deuterium accumulation. Deuterium-ion counts in the endodermis tissue were not changed by girdling treatment, which restricted water movement through the phloem complex. The results strongly indicated that nodule tissues did not receive water directly from the phloem complex, but received water through root cortex apoplastic pathway from the root axis. (C) 2015 Elsevier GmbH. All rights reserved.

Crack fertilization is a soybean cultivation technique for nodulation control in which midterm subsoiling is used to supply fertilizing materials to deep soil just before the flowering stage. This study examined the effects of fertilizing materials and the continuous application of nodulation control, on soybean yield enhancement in two field experiments. The survival of nodule bacteria in deep soil was also evaluated by a bioassay of nodule bacteria in a root box. When the nodule bacteria on biochar were continuously applied without any other chemical fertilizers for three successive years, seed weight was significantly heavier being up to 1.34 times that of the control. The application of nodulation control in the previous year but not in the experimental year did not have residual effects on seed weight. The enhancement of seed weight in a farm field converted from a paddy was much lower. This may be partly attributed to the midterm tillage practice, which destroys the crack structure after the nodulation control, together with soil water status and cultivar differences. Nodule growth and nitrogen fixation activities significantly increased in the soybean plants grown on the soil collected from the subsoil to which nodule bacteria on biochar had been applied the previous year. This suggests that nodule bacteria in the subsoil survived in the biochar habitat for at least a year after application. These results indicate that nodulation control by the crack fertilization technique leads to yield enhancement when nodule bacteria on biochar are continuously applied.

The use of coffee grounds in crop fields were evaluated in terms of crop growth enhancement, soil improvement, and weed control during four successive cropping seasons for two years. Six summer and three winter green manure crops were grown from June 2009 to May 2011. In the first cropping season, the growth of all green manure crops was significantly inhibited by the application of 10 kg m(-2) of coffee grounds. However, the inhibitory effects spontaneously diminished after the second cropping season (about 12 months later), and the growth of guinea grass, sorghum and sunflower was about 2-fold higher than that of the control. The application of horse manure at 10 kg m(-2) effectively alleviated the inhibitory effects, even though the high concentration of coffee grounds was included. Although top dressing application of coffee grounds at 16 kg m(-2) permitted weed control, the impact maintained enough only for half a year. Coffee grounds application effectively increased both carbon and nitrogen contents of soil and reduced CN ratio. The soil amendment effects were significantly higher in terms of nitrogen enrichment and CN ratio improvement as compared with the horse manure application. These results indicated that coffee grounds are useful to enhance long term crop growth, short duration weed control, and soil improvement in agricultural fields by considering the inhibitory effects on the plant growth for half year after the application. Agricultural use of coffee grounds was also discussed in term of fallow periods in crop rotation.

An appropriate combination of rice cultivar and cropping system that maximizes water use efficiency (WUE) may improve yield of rainfed lowland rice. In the paddy field, a large amount of water is consumed by evaporation during the early growth period, and it can be reduced by canopy coverage especially in semi-arid regions. Therefore, we evaluated the role of canopy coverage in WUE of rice in the early growth period in semi-arid region. A pot experiment was conducted in Namibia to investigate the genotypic and species difference in WUE, and another pot and a field experiment were conducted to investigate the effects of planting density on WUE. Although no significant difference was observed among species, the mean WUE was in the decreasing order of Oryza sativa, and Oryza glaberrima followed by the interspecific progenies including NERICA. In contrast, there was a significant difference in WUE at the genotypic level. Highly tillering genotypes such as WAB1159-2-12-11-5-1 and WITA 2 showed a high WUE. Furthermore, WUE was significantly correlated with the number of tillers (R-2 = 0.453), and higher planting density resulted in a higher WUE. In contrast, stomatal conductance had no significant correlation with WUE (R-2 = 0.081). Thus, the physical conditions affected by number of tillers and planting density had greater impacts on WUE than physiological characteristics such as stomatal conductance. The suppression of surface water evaporation by coverage was significant, probably contributing to WUE improvement. To increase WUE in semi-arid regions, we recommend the increase of canopy coverage and higher planting density.

Chloroplast protrusions (CPs) are often observed under environmental stresses, but their role has not been elucidated. The formation of CPs was observed in the leaf of rice plants treated with 75 mm NaCl for 14 d. Some CPs were almost separated from the main chloroplast body. In some CPs, inner membrane structures and crystalline inclusions were included. Similar structures surrounded by double membranes were observed in the cytoplasm and vacuole. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was detected in CPs and the similar structures in the cytoplasm and vacuole. These results suggest that CP is one of the pathways of Rubisco exclusion from chloroplasts into the cytoplasm under salinity, and the exclusions could be transported to vacuole for their degradation.

The localization of salt-induced H2O2 accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl3 staining at ultrastructure level. When the 3-week-old rice plants were affected by 100 mM NaCl for 14 days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H2O2 accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl3 were mainly observed especially around the swelling of thylakoids. H2O2 accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H2O2 accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H2O2 under salinity is responsible for the changes in chloroplast ultrastructure. H2O2 accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H2O2 and the production is light dependent under salinity.

We analyzed the response of transcripts corresponding to ascorbate peroxidase (APX) and catalase (CAT) under salinity in the basal region of the rice leaf, which is tolerant to salinity compared with the apical region. In the NaCl treated plants, the transcript levels of CATB, CATC, APX1, APX4, APX6 and APX7 increased. The transcript level of APX2 was comparable to that of the control, but the transcript level of APX8 was slightly decreased by salinity. The activity of dehydroascorbate reductase decreased by salinity. These results suggest that the increase in CAT activity observed in our previous study is due to the enhancement of transcript levels of CATB and CATC, and the increase in the transcript level of APX1, APX4, APX6 and APX7 may contribute to maintain APX activity under salinity. The enhancement of the enzyme activities involved in regeneration of ascorbate under salinity is needed to increase APX activity and salinity tolerance in rice plants.

We investigated the mechanisms of increased sensitivity to Na+ in the apical and basal regions of the rice leaf under salinity. Three-week-old plants were created with 200 mM NaCl in hydroponic culture for 3 d. Segments 6 cm in length were obtained from the apical and basal regions of the fully expanded uppermost leaves (6th leaf blades) as old and young tissues, respectively. In the plants exposed to 200 mM NaCl, Nitro blue tetrazolium (NBT) reducing activity, and H2O2 and Malondialdehyde (MDA) contents significantly increased, accompanied by the swelling of thylakoids and destruction of thylakoid membranes in the apical regions. However, no indication of oxidative damages was observed in the basal region, even though the Na+ content in the basal region was comparable to that in the apical region. In the apical region, the capacity to scavenge H2O2 was lower than that in the basal region due to decrease in the constitutive levels of ascorbate peroxidase and guaiacol peroxidase. In addition, the activities of antioxidant enzymes except superoxide dismutase and guaiacol peroxidase decreased drastically after 48 hr of exposure to NaCl. By contrast., the activities of catalase and glutathione reductase in the basal region increased compared with those in the control, and other antioxidant enzymes did not decrease under salinity during the experimental period. These results suggest that: the capacity to scavenge reactive oxygen species decreased with age, and thus the apical region of the leaf blade suffered severer damage by Na+ than the basal region.

It has been reported that glycinebetaine (betaine) is synthesized in response to abiotic stresses via a two-step oxidation of choline in which choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are involved. Here we show that significant amounts of betaine, > 20 mu mol/gFW, accumulated in young leaves of Beta vulgaris even under normal growth conditions, whereas levels in old leaves, cotyledons, hypocotyls, and roots were low. Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. By contrast, the levels of BADH were high in all tissues. Exogenously supplied choline was converted into betaine in old leaves, but levels were significantly tower in young leaves under the same conditions. When d(11)-betaine was applied exogenously to old leaves, it was translocated preferentially into young leaves and roots. In response to salt stress, betaine levels increased in all tissues, but most significantly increased in young leaves. The levels of CMO increased in various tissues, but were low in young leaves. A betaine transporter gene was isolated. Its expression was more strongly induced in old leaves than in young leaves. Based on these data, we discussed the rote of CMO and betaine transporter under stress and non-stress conditions. (C) 2009 Elsevier GmbH. All rights reserved.

The seedlings of Oryza saliva L. cv. Nipponbare grown by hydroponic culture for 3 wks were treated with 75, 100, 150 and 200 mM NaCl for 14, 14, 6 and 3 days, respectively, and examined for chloroplast ultrastructure in the region where chlorophyll fluorescence had been recorded. NaCl treatment decreased the ratio of variable to maximum chlorophyll fluorescence yield (F(v)/F(m)) and caused swelling of thylakoids. The swelling of thylakoids was quantified by the percentage of the length of swollen thylakoids to the total length of thylakoids. This value was increased with increasing NaCl concentration. Although F(v)/F(m) decreased at all concentrations of NaCl, the minimal fluorescence yield F, was not increased by the treatment with 75 or 100 mM NaCl. The percentage of the length of swelling was low at 75 and 100 mM NaCl. On the other hand, F(0) increased and the swelling of thylakoids was prominent with 150 and 200 mM NaCl treatment. These results suggest that the decrease in F(v)/F(m) due to the increase in F(0) under salt stress correlates with the ultrastructural damage. The decrease in F(v)/F(m) due to the increase in F(0) is expected to be useful as an indicator to evaluate the damage in chloroplasts, especially in thylakoid membranes, under salinity.

We investigated the effects of pretreatment with a low concentration of methyl viologen (MV) on the salinity-induced chloroplast degeneration in rice seedlings. The seedlings grown in hydroponic culture containing nutrient solution for 3 wks were treated with 100 nM MV mixed in the hydroponic culture for 3 days, and then with 200 mM NaCl without MV for 3 days. In the plants without MV pretreatment, the chlorophyll content drastically decreased during the NaCl treatment accompanied by swelling of thylakoids and destruction of thylakoid membranes. These damages were alleviated by the pretreatment with MV. The activities of CuZn-SOD and Fe-SOD, which localize in chloroplasts, increased under salt stress in both plants with and without MV pretreatment. In the plants under salt stress without MV pretreatment, ascorbate peroxidase (APX) activity did not differ from that of control. However, in MV-pretreated plants, APX activity under salt stress was about 1.2- to 1.3-fold higher than that of the control. Catalase (CAT) activity in NaCl treated plants was decreased to 52% of the control and the reduction in CAT activity was suppressed by MV pretreatment. These results suggest that MV reduced the damages by salt stress in chloroplasts by increasing APX activity and preventing the decrease in CAT activity.

We investigated the kinds of active oxygen species leading to the destruction of chloroplast ultrastructure in salt-stressed rice plants. After the seedlings were grown for 3 wks, leaf segments (5 mm square) were cut from the middle portion of the 5th leaves. Leaf segments were incubated in 200 mM NaCl under dark or light conditions for 24 hr. The chlorophyll content in the leaf segments drastically decreased in light between 12- and 24 hr in 200 mM NaCl, but, no reduction was observed in the dark. In electron microscopic studies, 200 mM NaCl caused swelling of thylakoids and destruction of thylakoid membranes in light. On the other hand, no ultrastructural changes were observed under dark condition. In one experiment, leaf segments were incubated in 200 mM NaCl for 24 hr in light after preincubation with antioxidants for 12 hr in light. Pretreatment with ascorbate and benzoate, which scavenge H 2O2 and •OH, respectively, effectively suppressed the reduction of chlorophyll content and the destruction of chloroplasts by NaCl in light. However, Tiron and DABCO, which scavenge O2- and 1O2, respectively, could not suppress the effects of salt stress in light. Fe-SOD activity was increased about eight time by salt stress (200 mM NaCl), but, catalase activity was reduced to 69% of the control and ascorbate peroxidase activity was not affected by NaCl. These results suggested that salt-induced injury in chloroplasts is dependent on light, and that H 2O2 and •OH are responsible for the deleterious effects of salt stress on chlorophyll content and chloroplast ultrastructure.

We investigated the effects of drought stress on the ultrastructure of chloroplasts in rice plants, After the seedlings were grown in a glasshouse for 1 month, they were treated for drought stress using two methods. One drought treatment was imposed by reducing the water supply to the plants for 1 month. The other was imposed by withholding water for 2 weeks to examine the withering process of leaves by drought stress. The ultrastructural changes of chloroplasts in bundle sheath cells were more prominent than those in mesophyll cells under both drought stress treatments. Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) content in bundle sheath chloroplasts reduced more dramatically than in mesophyll chloroplasts by drought stress. Although a slight swelling of thylakoids was sometimes observed in bundle sheath chloroplasts in moderate stress for 1 month, the thylakoids were less affected by drought stress than chloroplast envelope. These results suggest that chloroplasts in bundle sheath cells were more sensitive to drought stress than those in mesophyll cells and the thylakoids were less damaged by drought stress compared with chloroplast envelope.

Ionic and osmotic effects of salinity on the ultrastructure of chloroplasts in salt-treated rice seedlings were investigated. After rice seedlings were grown in hydroponic culture for three weeks, they were treated with NaCl and polyethylene glycol (PEG) 4000 both at a water potential of -1.0 MPa for 3 days. The most notable difference in ultrastructural change between NaCl and PEG treatment was observed in the damage in chloroplast membranes. NaCl induced swelling of thylakoids and caused only a slight destruction of the chloroplast envelope. PEG caused severe destruction of the chloroplast envelope compared with NaCl, however thylakoids did not swell. Our observations suggested that in salt-treated rice plants, the ionic effects induced swelling of thylakoids and the osmotic effects caused the destruction of chloroplast envelope.

イネの塩感受性品種の日本晴と耐塩性品種であるPokkaliを用い、3週齢の個体に100 mMのNaClストレスを4日間処理した植物体を用いて電子顕微鏡固定を実施した。それぞれの品種において、対照区と塩処理区のサンプルを用い、150 nm間隔で1500枚程度の連続切片をカバーガラス6～7枚に分けて回収した。この連続切片を用いて、維管束鞘細胞を含む維管束周辺の連続画像を卓上SEM (TM4000, Hitachi)で撮影した。 日本晴とPokkaliの対照区の観察を行ったところ、維管束鞘細胞の中には多数の葉緑体が確認でき、維管束とは逆側の細胞壁に沿って配置されていた。さらに、維管束鞘葉緑体が配置されている部分には葉肉細胞が隣接しておらず、維管束鞘葉緑体の大部分は細胞間隙に面するように配置されていた。そのため、二酸化炭素を積極的に吸収できる配置になっていることが予想された。ミトコンドリアは、維管束鞘葉緑体と膜接触するように配置されるとともに、維管束側の細胞壁にも配置されており、維管束側のアポプラストに物質を搬出するためのエネルギー源になっていることが予想された。 維管束鞘細胞の内側には、一層の柔細胞が確認できた。内部にはチラコイド膜を有する色素体やミトコンドリアが多数確認できた。この柔細胞において、道管の横に隣接する細胞だけが、他の柔細胞よりも細胞質が密でミトコンドリアが多く存在していた。さらに、道管には二次壁が発達しているが、この密な柔細胞との間だけ、部分的に二次壁の肥厚が見られず、それに対応して柔細胞側の細胞壁も薄くなって壁孔を形成していた。道管と隣接する維管束鞘細胞との間には、これらの構造は見られなかったことから、この密になっている柔細胞が、水や無機塩類の通過に対して重要な役割を担っている可能性が示唆された。

気候変動による水環境の極端化は、すでに半乾燥地では顕在化しており、日本のような温帯圏でも、近い将来、頻発することが予想できる。本研究では、代表者らが開発途上の独自の栽培概念、接触混植と亀裂施肥という2 つの考え方を応用することにより、洪水と干ばつの両者に対応可能な栽培技術を創出することを目指し、以下の試験を現地の共同研究者らとともに開始した。 ① 接触混植試験：ナミビア大学オゴンゴ校内に設置されており、水環境が制御可能なコンクリート水田において、イネ、カウピー、ソルガムを用いた接触混植試験を開始した。本試験は湛水ストレス下で、これらの作物の接触混植における土地等価比率（混作の収量性）が最も高い組み合わせを明らかにすることを目的としている。 ② カウピー品種選抜試験：イネやソルガムとの接触混植に適したカウピー品種を選抜することを目的として、ナミビア大学オゴンゴ校内でカウピー4品種を用いたポット試験を開始した。接触混植においてイネやソルガムとの競合ができるだけ小さいカウピー品種を選抜する予定である。 ③ 亀裂施肥試験：ナミビア大学オゴンゴ校内に設置されている実験畑において、カウピーを用いた亀裂施肥試験を開始した。本試験を開始するにあたって、現地のトラクターを用いて新型の亀裂施肥機を作成し、走行試験を行った。本試験はナミビア国の砂質土壌において、亀裂施肥による湛水ストレス緩和効果を定量化することを目的としている。 ④ 隣国のボツワナはナミビアと同様な自然・社会環境を持つため、南部アフリカにおける拠点形成を目的としてボツワナ大学とも共同研究を開始した。まずナミビア国に導入したイネ品種群をナミビア大学からボツワナ大学に植物検疫手続きを経て送付した。次に、それらのイネの種子増殖を開始した。潜在的な稲作圃場を農水省研究部、ボツワナ大学研究者とともに視察し、今後の共同研究を打ち合わせた。

水ストレス環境に適応した耐性イネ品種およびイネ栽培技術の開発のためには、干ばつ・洪水条件下の発芽・出芽に関する分子生理学・解剖学・遺伝学的機構の解明が重要である。フィールドでの乾燥条件におけるイネ出芽の品種間差異の確認を進めるとともに、室内実験（閉鎖系実験）の条件整理の検討を行った。また、フィールド実験では、イネの深播き耐性（乾燥土壌条件下の土中出芽性）に関する品種間差異の確認とこれに関与する生理形態形質の探索も進めた。一方、閉鎖系実験では、種子発芽に関するin vitro試験を展開し、ストレス下の発芽における遺伝子発現と細胞内部形態変化の詳細な動態を追跡するためのストレス処理方法を統一した。細胞内部形態変化の観察については、アリューロン細胞の三次元像の構築に向けて、電顕固定を行い、ウルトラミクロトームを用いて連続切片の作製を行った。アリューロン細胞の内側からデンプン粒が蓄積しており、その細胞に差し掛かると連続切片の作製が困難になる問題に直面している。今後、固定や樹脂包埋の手法を改善するなど、連続切片取得に向けた工夫を行う予定である。オルガネラの三次元像構築や接触面の定量法は、立体像構築のソフトウェアであるImageProを用いた方法を確立できた。そのため、アリューロン細胞の画像を取得次第、画像解析を実施する予定である。また、ストレス下の出芽に優れる品種・劣る品種を交配し、新規の遺伝解析集団作成も進めている。ストレス耐性品種×感受性品種のF2集団から戻し交配集団を準備していく予定である。

イネ葉肉細胞内の葉緑体、ミトコンドリア、ペルオキシソームにおける相互の膜接触を基にオルガネラ配置を観察した。観察した対照区のミトコンドリアにおいて、93%のミトコンドリアはペルオキシソームと膜接触をして集団を形成し、その集団は2つ以上の葉緑体の間に位置していた (Cluster)。しかし、6.6%のミトコンドリアはペルオキシソームと膜接触をしていなかった。ペルオキシソームと膜接触をしないミトコンドリアのうち、3.6%は2つの葉緑体の間に配置されていた (Bridge)。一方、3.0%のミトコンドリアは1つの葉緑体とだけ接触していた (Alone)。対照環境において、全てのペルオキシソームはミトコンドリアと膜接触をして集団を形成し、Clusterに配置されていた。 PEGストレス下において、ミトコンドリアやペルオキシソームの数は有意に増加していたが、配置は対照区と同様に分類できた。しかし、その割合は大きく異なり、ミトコンドリアにおいてClusterの位置が64%、Bridgeが19%、Aloneが17%であった。さらに、他のオルガネラと膜接触をしないミトコンドリア (Solo)も4.9%存在した。葉緑体とミトコンドリアの膜接触面積は増加したが、BridgeとAloneの位置に配置しているミトコンドリアが、その増加に大きく貢献していた。ペルオキシソームもPEGストレス下において配置変化が観察された。Clusterが91%になり、BridgeとAloneの位置にいるペルオキシソームが、それぞれ5.2と0.8%に増加していた。また、Soloに位置するペルオキシソームが3.4%に増加していた。葉緑体とペルオキシソームの膜接触面積が有意に増加していたが、Clusterに位置するペルオキシソームがその増加に大きく貢献していた。

ミトコンドリアやペルオキシソームが細長いストロマを含む構造で葉緑体に取り囲まれる構造は、葉緑体ポケットとよばれている。葉緑体が作る細長い突起状の構造であるstromuleが関与すると予想される。そのため、3次元立体像を構築することで、stromuleとポケット構造の関係を調べた。TEMとFIB-SEMを用いて葉緑体ポケット構造を含む葉緑体の連続画像を取得し、3次元再構築法によって観察した。その結果、葉緑体が大きなシート構造を作り、それがポケット構造を作っていることが明らかとなった。また、ポケット構造で取り囲まれているオルガネラとして、ミトコンドリアとペルオキシソームが一番多く観察された。

本研究では、イネの耐塩性向上を目指して、イネの耐塩性に重要であると考えられる組織耐性の作物生理学的意義および機構を調べた。その結果、幼苗期および分げつ期のいずれにおいても組織耐性を有するイネ品種は塩障害および成育阻害が軽微であり、塩感受性品種と比較して耐塩性を示した。一方塩吸収蓄積程度は塩感受性品種と同程度であり、組織耐性と塩吸収抑制能は独立していることが明らかになった。またその機構は過酸化脂質の増加を抑制するなど酸化ストレス耐性に関わることが示唆された。本研究で用いた日本のイネ品種は塩害下で組織耐性を示さなかったことから、組織耐性は日本イネ品種の耐塩性向上にも役に立つことが期待された。

本研究では、外部から根粒菌を投与し、ダイズに人為的に根粒着生を促すことで増収を可能にする根粒着生制御技術 (亀裂施肥)の有用性を明らかにすることを目的とした。さらに、北海道と本州という異なる気象条件で亀裂施肥を実施し、本技術の普遍性を検討した。寒冷地である北海道では、本技術の十分な増収効果は得られなかった。しかし、本州では、畑地と水田転換畑において増収効果が確認された。畑地では中耕時に条間に亀裂に沿って炭のみを投与する処理、水田転換畑では、播種前の条直下根粒菌を付与した炭を投与する処理が、最も増収効果が高かった。

本研究では、湿害後の過剰活性酸素生成機構をオルガネラレベルで特定することを目的とした。播種後1週間のダイズに1週間の湿害を与え、湿害解除翌日にO2-の発生部位を観察したところ、主根の中央部に染色が観察された。その部分をCeCl3染色により電験レベルで過酸化水素局在を観察したところ、皮層細胞の細胞膜に染色が観察された。生育初期に湿害を与えたダイズの生育を播種後98日目に観察したところ、湿害を受けたダイズは対照区と比較して葉の黄化が早まった。また、生育も著しく低下していた。これらの結果から、ダイズが生育初期に受けた湿害は、湿害直後だけではなく、生育後半の生育にも影響を及ぼすことが明らかとなった。

砂漠国ナミビアには、隣国からの洪水により雨期の間だけ形成される広大な季節湿地がある。本研究では、これまで作物生産に活用されてこなかった水資源に注目し、新たに稲作を導入することにより、水資源開発と環境保全との両立を検討した。その結果、半乾燥地におけるトウジンビエ在来農法と稲作とが融和しうることと、低位収量レベルであれば水環境を改変しない稲作導入が可能であることを示唆した。

CeCl3法およびDAB法を改良し電子顕微鏡レベルで細胞内の過酸化水素の分布を観察する方法を開発した。CeCl3法では、固定前の葉組織に5mM CeCl3をpH7. 2で1時間処理した後、固定し包埋試料を作製する方法が有効であった。DAB法では2時間Karnovski液で固定した後10mMのDABをpH8. 0で1時間処理し、オスミウム固定を行って包埋試料を作製する方法が有効であった。塩ストレス処理を行ったイネ葉緑体において、グラナチラコイドの周囲などに過酸化水素が検出された。

植物が塩ストレスを受けると地上部の塩含量の増加に伴い障害発現が起こるが,塩含量と障害は一致しないことが示唆されている.組織の齢が若いと塩含量が高い場合においても障害程度が低いことから,障害程度は組織の齢に依存していることが考えられる.しかし,若い組織における塩ストレス障害抑制機構は調べられていない.本研究では,イネ(Oryza sativa L.)の葉の基部・先端部の齢の違いを利用し,障害発現と塩の蓄積量の関係を明らかにすることを目的とした.また,抗酸化酵素活性,mRNAの発現を調べることで,部位における塩ストレス耐性の違いの原因が抗酸化酵素にあるかどうかを調べることを目的とした.塩ストレス下における抗酸化酵素活性の変化を調べたところ,先端部では塩含量の増加に伴いスーパーオキシドラジカル,過酸化水素の増加が観察された.また,障害を観察したところ,脂質過酸化物の増加,葉緑体の形態変化が観察された.また,障害発現と同時にスーパーオキシドディスムターゼとグアヤコールペルオキシダーゼ以外の全ての抗酸化酵素活性の低下が観察された.一方,基部では塩含量が先端部と同程度に増加しても障害は観察されなかった.抗酸化酵素活性を測定したところ,塩処理開始直後からカタラーゼとグルタチオンペルオキシダーゼの活性増加が観察され,カタラーゼにおいてはmRNAの発現も増加していた.このことから,抗酸化酵素の中でもカタラーゼとグルタチオンペルオキシダーゼがイネの塩ストレス障害抑制に重要な働きをしていることが示唆された.これらの結果から,若い組織では塩含量が増加しても抗酸化酵素活性を上昇させ,活性酸素を効率的に消去する耐塩性機構が備わっており,その機構は加齢に伴って失われていくことが示唆された.塩ストレス下における抗酸化酵素活性の増加抑制には加齢が関与していることから,今後,加齢に伴って抗酸化酵素活性を抑制する因子の探索を試みることでさらなる耐塩性強化が可能になることが示唆された。