Koji Yamane

Differential effect of NaCl and polyethylene glycol on the ultrastructure of chloroplasts in rice seedlings

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.

Pretreatment with Antioxidants Decreases the Effects of Salt Stress on Chloroplast Ultrastructure in Rice Leaf Segments (Oryza sativa L.)

Abstract 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 H2O2 and ˁOH, 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 H2O2 and ˁOH are responsible for the deleterious effects of salt stress on chlorophyll content and chloroplast ultrastructure.

Correlation between Chloroplast Ultrastructure and Chlorophyll Fluorescence Characteristics in the Leaves of Rice (Oryza sativa L.) Grown under Salinity

Abstract The seedlings of Oryza sativa 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 (Fv/Fm) 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 Fv/Fm decreased at all concentrations of NaCl, the minimal fluorescence yield F0 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, F0 increased and the swelling of thylakoids was prominent with 150 and 200 mM NaCl treatment. These results suggest that the decrease in Fv/Fm due to the increase in F0 under salt stress correlates with the ultrastructural damage. The decrease in Fv/Fm due to the increase in F0 is expected to be useful as an indicator to evaluate the damage in chloroplasts, especially in thylakoid membranes, under salinity.

Salt-induced chloroplast protrusion is the process of exclusion of ribulose-1,5-bisphosphate carboxylase/oxygenase from chloroplasts into cytoplasm in leaves of rice.

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.

Antioxidant Capacity and Damages Caused by Salinity Stress in Apical and Basal Regions of Rice Leaf

Abstract 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 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.

Preferential accumulation of betaine uncoupled to choline monooxygenase in young leaves of sugar beet--importance of long-distance translocation of betaine under normal and salt-stressed conditions.

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 micromol/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 lower 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 role of CMO and betaine transporter under stress and non-stress conditions.

Bundle sheath chloroplasts of rice are more sensitive to drought stress than mesophyll chloroplasts.

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.

Pretreatment with a Low Concentration of Methyl Viologen Decreases the Effects of Salt Stress on Chloroplast Ultrastructure in Rice Leaves (Oryza sativa L.)

Abstract 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.

Transcription Profiles of Genes Encoding Catalase and Ascorbate Peroxidase in the Rice Leaf Tissues under Salinity

Abstract 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.

Mixed cropping has the potential to enhance flood tolerance of drought-adapted grain crops

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 (O2) from their roots. We tested the hypothesis that wetland species could help upland species under flood conditions; that is, O2 released from the wetland crop roots would ameliorate rhizosphere O2-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 (O2-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.