Shiro Mitsuya

Effects of sodium chloride on foliar ultrastructure of sweet potato (Ipomoea batatas Lam.) plantlets grown under light and dark conditions in vitro

Summary The effects of sodium chloride on foliar ultrastructure of sweet potato plantlets grown under light and dark conditions in vitro were examined. Ultrastructural changes induced by salt stress under light conditions occurred in the following order: (1) vacuolation, development and partial swelling of ER; (2) decrease in mitochondrial cristae and swelling of mitochondria, with an increase of the vesicles released from the Golgi stacks; (3) vesiculation and fragmentation of tonoplast; and (4) degradation of cytoplasm by the mixture of cytoplasmic and vacuolar matrices. In the mesophyll, thylakoid membranes of chloroplasts were swollen and most were lost following severe salt stress. Under dark condition, however, inner membranes of etioplasts maintained their ultrastructural integrity. The present study suggests that degradation of cytoplasm and cell organelles, except thylakoid membranes of chloroplast, occurred as a result of light-independent salt stress, and degradation of thylakoid membranes of chloroplast in the mesophyll occurred as a result of salt-induced oxidative stress.

Enzymatic characterization of peroxisomal and cytosolic betaine aldehyde dehydrogenases in barley

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is an important enzyme that catalyzes the last step in the synthesis of glycine betaine, a compatible solute accumulated by many plants under various abiotic stresses. In barley (Hordeum vulgare L.), we reported previously the existence of two BADH genes (BBD1 and BBD2) and their corresponding proteins, peroxisomal BADH (BBD1) and cytosolic BADH (BBD2). To investigate their enzymatic properties, we expressed them in Escherichia coli and purified both proteins. Enzymatic analysis indicated that the affinity of BBD2 for betaine aldehyde was reasonable as other plant BADHs, but BBD1 showed extremely low affinity for betaine aldehyde with apparent K(m) of 18.9 microM and 19.9 mM, respectively. In addition, V(max)/K(m) with betaine aldehyde of BBD2 was about 2000-fold higher than that of BBD1, suggesting that BBD2 plays a main role in glycine betaine synthesis in barley plants. However, BBD1 catalyzed the oxidation of omega-aminoaldehydes such as 4-aminobutyraldehyde and 3-aminopropionaldehyde as efficiently as BBD2. We also found that both BBDs oxidized 4-N-trimethylaminobutyraldehyde and 3-N-trimethylaminopropionaldehyde.

Disruption of RCI2A leads to over-accumulation of Na + and increased salt sensitivity in Arabidopsis thaliana plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na+ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na+ ions in the cell and shows increased Na+ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na+ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na+ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na+ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO3, Na2SO4, KCl, KNO3, K2SO4 or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl2, MgCl2, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na+ and K+ ions in plants, and contributes to salt tolerance.

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.

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.

Salt Stress Causes Peroxisome Proliferation, but Inducing Peroxisome Proliferation Does Not Improve NaCl Tolerance in Arabidopsis thaliana

The PEX11 family of peroxisome membrane proteins have been shown to be involved in regulation of peroxisome size and number in plant, animals, and yeast cells. We and others have previously suggested that peroxisome proliferation as a result of abiotic stress may be important in plant stress responses, and recently it was reported that several rice PEX11 genes were up regulated in response to abiotic stress. We sought to test the hypothesis that promoting peroxisome proliferation in Arabidopsis thaliana by over expression of one PEX11 family member, PEX11e, would give increased resistance to salt stress. We could demonstrate up regulation of PEX11e by salt stress and increased peroxisome number by both PEX11e over expression and salt stress, however our experiments failed to find a correlation between PEX11e over expression and increased peroxisome metabolic activity or resistance to salt stress. This suggests that although peroxisome proliferation may be a consequence of salt stress, it does not affect the ability of Arabidopsis plants to tolerate saline conditions.

OsBADH1 is possibly involved in acetaldehyde oxidation in rice plant peroxisomes

Although rice (Oryza sativa L.) produces little glycine betaine (GB), it has two betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) gene homologs (OsBADH1 and OsBADH2). We found that OsBADH1 catalyzes the oxidation of acetaldehyde efficiently, while the activity of OsBADH2 is extremely low. The accumulation of OsBADH1 mRNA decreases following submergence treatment, but quickly recovers after re‐aeration. We confirmed that OsBADH1 localizes in peroxisomes. In this paper, a possible physiological function of OsBADH1 in the oxidation of acetaldehyde produced by catalase in rice plant peroxisomes is discussed.

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.

Characterization of a novel glycinebetaine/proline transporter gene expressed in the mestome sheath and lateral root cap cells in barley

The accumulation of glycinebetaine (GB) is one of the adaptive strategies to adverse salt stress conditions. Although it has been demonstrated that barley plants accumulate GB in response to salt stress and various studies focused on GB synthesis were performed, its transport mechanism is still unclear. In this study, we identified a novel gene, HvProT2, encoding Hordeum vulgare GB/proline transporter from barley plants. Heterologous expression in yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of HvProT2 was highest for GB, intermediate for proline and lowest for γ-aminobutyric acid. Transient expression of fusions of HvProT2 and green fluorescent protein in onion epidermal cells revealed that HvProT2 is localized at the plasma membrane. Relative quantification of mRNA level of HvProT2 using semi-quantitative reverse transcription-polymerase chain reaction analysis showed that HvProT2 is constitutively expressed in both leaves and roots, and the expression level was higher in old leaves than young leaves and roots. Moreover, we found that HvProT2 was expressed in the mestome sheath and lateral root cap cells. We discussed the possible involvement of HvProT2 for salt stress tolerance.