Michio Kawasaki

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.

Light Dependency of Salinity-Induced Chloroplast Degradation

Abstract The contents of Na, K, CI, chlorophyll and the foliar ultrastructure of rice seedlings grown in NaCl solution at various concentrations were investigated under light and dark conditions. The seedlings were first grown in water for 7 d under a light condition and then in NaCl solutions at various concentrations for 24 h under a light or dark condition. The Na and CI contents in the 3rd leaves increased as the concentration of NaCl in the culture solution increased, and were significantly higher under a light condition than under a dark condition. The K content was scarcely influenced by the NaCl concentration under both conditions. The chlorophyll content in the 3rd leaves of the seedlings decreased as the NaCl concentrations of the culture solution increased under a light condition but not under a dark condition. In the 3rd leaves of the seedlings grown in the NaCl solution under a light condition, the thylakoids of chloroplasts in mesophyll cells were swollen and showed a wavy configuration. Under a dark condition, however, the thylakoids appeared intact under saline conditions although the leaves accumulated a large amount of Na and CI than in a light condition. The present study suggests that the damages in the chloroplasts, such as a decrease in the chlorophyll content and the degradation of thylakoids, were caused by a light-dependent reaction and not directly by accumulation of excess salt.

Differential Sensitivity of Chloroplasts in Mesophyll and Bundle Sheath Cells in Maize, an NADP-Malic Enzyme-Type C4 Plant, to Salinity Stress

Abstract The changes in chloroplast ultrastructure and the contents of chlorophyll, Na and K in response to salinity stress were investigated in leaves of maize, an NADP-malic enzyme-type C4 plant species possessing dimorphic chloroplasts. The seedlings were treated with 0, 1, 2 or 3% NaCl for three or five days under a light or dark condition. In both light and dark conditions, the dry weight of salt-treated plants decreased as NaCl concentration increased. Chlorophyll and K contents of the second leaf blade decreased as NaCl concentration increased under the light condition but not under the dark condition. Na content of the second leaf blade was significantly higher at high NaCl concentrations under both light and dark conditions. However, Na content was much lower under the dark condition than light condition. Higher concentrations (2 and 3%) of NaCl significantly increased the size of plastoglobules, decreased the number and size of starch granules and altered the chloroplast ultrastructure. Under the light condition, mesophyll cell (MC) chloroplasts appeared more sensitive to the damaging effect of salinity than the bundle sheath cell (BSC) chloroplasts. MC chloroplasts became more globular in shape and showed swollen and disorganized thylakoids and reduced thickness of grana by salinity. BSC chloroplasts were less affected by salinity than MC chloroplasts. Although chloroplast size and number and size of starch granules were reduced, there was no structural distortion in the thylakoids of BSC chloroplasts. However, the thickness of grana was increased by salinity. Under the dark condition, the chloroplast structure was less affected by salinity. Though the envelope of BSC chloroplasts was occasionally damaged, the thylakoids in both MC and BSC chloroplasts were preserved under salinity stress. The present study suggests that the chloroplast damage caused by salinity is light-dependent and MC chloroplasts are more sensitive to salinity than BSC chloroplasts.

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.

Relationship between Salinity-Induced Damages and Aging in Rice Leaf Tissues

Abstract Segments of rice leaves at different nodal positions were incubated in NaCl solutions for various periods, and the chlorophyll content, Na content, CI content, Na/K ratio and the ultrastructure of excised leaf tissues were examined. The chlorophyll content of the leaf tissues decreased with increasing NaCl concentration and incubation period. Na and CI contents of the leaf tissues also increased with increasing NaCl concentration, but the decrease in chlorophyll content by salt stress was greater in old than in young tissues even when both tissues contained comparable amounts of Na and CI. The presence of benzylaminopurine (BAP) alleviated the salt stress-induced decrease in chlorophyll content, but did not significantly affect the element contents. Ultrastructural damages were apparent in the chloroplasts of the leaf tissues subjected to salt stress. In 0.1% NaCl-treated old leaf tissues, the thylakoids were swollen, the envelope was partly destroyed causing leakage of the chloroplast contents. However, these damages were alleviated by the addition of BAP to the NaCl solution. In young leaf tissues, the thylakoids were swollen by incubation in 1.0% NaCl solution, but no structural distortion was observed in a 0.1% NaCl solution even without BAP added. The present study suggests that the leaf tissues were damaged by an increasing salt content and became more sensitive to salt stress with advancing leaf age. BAP seemed to alleviate the damages by salt stress through retardation of leaf aging.

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.

Salinity Stress Induces Granal Development in Bundle Sheath Chloroplasts of Maize, an NADP-Malic Enzyme-Type C4 Plant

Abstract Zea mays is an NADP-malic enzyme (ME)-type C4 plant. The C4 plants of this type are attractive species for ultrastructural and physiological studies because they possess reduced grana in bundle sheath cell (BSC) chloroplasts. The present study evaluated the effect of salinity on granal development in BSC chloroplasts of maize. The plants were grown in soil media and after the second leaf was fully developed they were irrigated with four different concentrations (0, 1, 2 and 3%) of NaCl for 5 d. Ultrastructure, quantitative properties of chloroplasts and chlorophyll fluorescence parameters were evaluated. Granal stacking in BSC chloroplasts was induced by treatment with 2 or 3% NaCl. In contrast, granal stacking in mesophyll cell (MC) chloroplasts was reduced and disorganized by the NaCl treatment due to swelling of thylakoid. In control plants, only 2% of grana in BSC chloroplasts contained more than three thylakiods. In the plants treated with 3% NaCl, however, 66% of grana contained more than three thylakoids in BSC chloroplasts. The maximum number of thylakoids in grana of BSC chloroplasts in the control and 3% NaCl-treated plants, was 4 and 16 respectively. The granal index in BSC chloroplasts of 3% NaCl-treated plants was more than three times higher than that in the control plants. Chlorophyll fluorescence parameter analysis showed that the maximal quantum yield (Fv/Fm), the effective quantum yield of PSII (ΦPSII) and PSII-driven electron transport rate (ETR) decreased with the increase of salinity stress. These results suggest that the suppression mechanism of granal development in BSC chloroplasts of maize is influenced by salinity.

Relationship between the Distribution of Na and the Damages Caused by Salinity in the Leaves of Rice Seedlings Grown under a Saline Condition

Abstract The distributions of Na and chlorophyll in the leaves of rice (Oryza sativa L.) seedlings grown under a saline condition were examined in relation to the anatomical changes caused by salinity. In salt-treated plants, the Na content was higher in older leaves and basal part of the leaf. In the 4th leaf of salt-treated plants, Na content was the highest in the middle part of the leaf sheath and decreased toward the tip of the leaf blade. The chlorophyll content in the 4th leaf was decreased by salt treatment at the tip and middle parts of the leaf blade, whereas it was unaffected in the leaf sheath and at the base of the leaf blade. Electron microscopic studies revealed that salt-treatment caused plasmolysis, vesiculation of cellular membranes and degradation of cytoplasm at the tip of the leaf blade, but scarcely caused such alterations at the base of the leaf blade. The present study suggests that the damage by salinity correlates more strongly with the age of the tissue than the Na content of the tissue.

Strictness of the Centrifugal Location of Bundle Sheath Chloroplasts in Different NADP-ME Type C4 Grasses

Abstract C4 plants have many attractive traits for crops, but their structural and functional relationships are complicated. C4 plants are different in bundle sheath cell (BSC) chloroplast location (centrifugal or centripetal) among species. The effects of light intensity on the centrifugal location of BSC chloroplasts were investigated in four grass species of NADP malic enzyme (NADP-ME) type (Zea mays, Echinochloa utilis, Sorghum bicolorand Eriachne aristidea)by light and electron microscopy. Furthermore, the degree of granal development was examined to investigate the relation between BSC chloroplast location and dependence of BSC chloroplasts on the reducing power of mesophyll cells. We investigated BSC chloroplast location grown under high intensity light (HL) (600 μ mol m-2s-1), low intensity light (LL) (2.5 μ mol m-2s-1) and dark conditions and counted the number of granal thylakoids per granum. Although BSC chloroplasts of maize maintained the centrifugal position under all light conditions, the centrifugal location of other species was disturbed under LL and in the dark. Granal development in BSC chloroplasts in the plants grown under HL was suppressed, although the suppression in Z maysand S. bicolorwas more prominent than in other two species. These findings indicate that there is a difference in the strictness of centrifugal location of BSC chloroplasts among NADP-ME type C4 grass species and the strictness had no relation to the degree of granal development in BSC chloroplasts.