Hiroshi Miyake

In maize, the structure of bundle sheath cell (BSC) chloroplasts is less subject to salinity stress than that of mesophyll cell (MC) chloroplasts. To elucidate the difference in sensitivity to salinity, antioxidant capacities and localization of reactive oxygen species were investigated in both chloroplasts. Transmission electron microscopic observation showed that O₂⁻ localization was found in both chloroplasts under salinity, but the accumulation was much greater in MC chloroplasts. H₂O₂ localization was observed only in MC chloroplasts of salt‐treated plants. In isolated chloroplasts, the activities of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) were increased by salinity. While the enhancement of SOD activity was similar in both chloroplasts, the increase of APX and DHAR activities were more pronounced in BSC chloroplasts than in MC chloroplasts. Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and glutathione reductase (GR, EC 1.6.4.2) were undetectable in BSC chloroplasts, while they increased in MC chloroplasts under salinity. Although ascorbate content increased by salinity only in BSC chloroplasts, glutathione content increased significantly in both chloroplasts, and was higher in MC chloroplasts than in BSC chloroplasts. The content of thiobarbituric acid‐reactive substances, which is an indicator of lipid peroxidation, was significantly increased by salinity in both chloroplasts. These results suggested O₂⁻‐scavenging capacity was comparable between both chloroplasts, whereas H₂O₂‐scavenging capacity was lower in MC chloroplasts than in BSC chloroplasts. Moreover, the increased lipid peroxidation under salinity was associated with the structural alteration in MC chloroplasts, while it had less impact on the structure of BSC chloroplasts.

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.

Distribution of Na⁺ along the root axis under salinity stress was analyzed in two rice (Oryza sativa L.) cultivars with different salt resistance (salt-sensitive IR 24 and salt-resistant Pokkali). Rice plants were grown hydroponically and NaCl was applied with nutrient solution at concentrations of 0, 25 and 50 mM for 7 d after germination. The distribution of Na⁺ in roots under salinity was analyzed by the cryo time-of-flight secondary ion mass spectrometry (cryo TOF-SIMS). The Na⁺ content in the root was higher in salt-sensitive IR 24 than in salt-resistant Pokkali under NaCl stress. The content was highest at the root tip and was decreased basipetally along the root axis. The difference in Na⁺ content between the cultivars was apparent in all regions from the root tip.

The effect of NaCl stress on the structure of leaf chloroplasts was investigated in several NAD-Malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) type C4 plant species. Seedlings of the monocot species, except Zoysia japonica, grown in 300 mL pots were subjected to salt stress by adding 50 mL of 3% NaCl solution per day to the soil for 5 d after the fourth leaf blades were fully developed. Z. japonica and the dicot species, Amaranthus tricolor, were also treated with 3% NaCl in a similar manner from 5 wk after germination. Salt stress negatively affected the growth, chlorophyll content and chloroplast structure in all the species. At the ultrastructure level, swelling of thylakoids and disruption of envelopes were more or less observed in mesophyll cell (MC) chloroplasts after salt treatment. The structure of bundle sheath cell (BSC) chloroplasts, on the other hand, was hardly damaged under salt condition although stromal and starch areas were considerably decreased. Furthermore, s...

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 andCATC, and the increase in the transcript levelof APX1, APX4, APX6 and APX7 may contribute to maintain APX activity under salinity. Theenhancement of the enzyme activities involved in regeneration of ascorbate under salinity is needed to increase APX activity and salinity tolerance in rice plants.

Effects of NaCl on the growth, ion content, root cap structure and Casparian band development were examined in four rice (Oryza sativa L.) cultivars with different salt resistance (salt-sensitive indica-type IR 24 and japonica-type Nipponbare and salt-resistant indica-type Nona Bokra and Pokkali). Experiments were conducted to find the differences in salinity resistance during early seedling and developed seedling stages among the cultivars. For salinity treatment, sodium chloride (NaCl) was added to nutrient solution at concentrations of 0, 25 and 50 mM for 7 days from germination to the 7th day (early seedling stage) or from the 7th day to 14th day (developed seedling stage). Growth inhibition by salinity was more prominent in the early seedling stage than in the developed seedling stage. Based on the growth, the order of the sensitivity was IR24 > Nipponbare > Nona Bokra > Pokkali. The growth of NaCl-treated rice cultivars relative to control was significantly and negatively correlated with the...

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 treated 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 a...