Masaaki Yoshiba

Difference in the distribution and speciation of cellular nickel between nickel‐tolerant and non‐tolerant Nicotiana tabacum L. cv. BY‐2 cells

To evaluate Ni dynamics at the subcellular level, the distribution and speciation of Ni were determined in wild-type (WT) and Ni-tolerant (NIT) tobacco BY-2 cell lines. When exposed to low but toxic levels of Ni, NIT cells were found to contain 2.5-fold more Ni (14% of whole-cell Ni values) in their cell walls than WT cells (6% of whole-cell Ni values). In addition to higher levels of Ni in the apoplast, a higher proportion (94%) of symplastic Ni was localized in the vacuoles of NIT cells than in the vacuoles of WT cells (81%). The concentration of cytosolic Ni in the NIT cells was significantly lower (18 nmol g(-1) FW) than that in the WT cells (85 nmol g(-1) FW). In silico simulation showed that 95% of vacuolar Ni was in the form of Ni-citrate complexes, and that free Ni(2+) was virtually absent in the NIT cells. On the other hand, the amount of free metal ions was markedly increased in WT cells because free citrate was depleted by chelation of Ni. A protoplast viability assay using BCECF-AM further demonstrated that the main mechanism that confers strong Ni tolerance was present in the symplast as opposed to the cell wall.

Iron deficiency causes zinc excess in Zea mays.

Abstract Iron deficiency stress causes a severe reduction in plant growth. Although Fe deficiency causes an imbalance in divalent heavy metal nutrients, the mechanisms underlying the growth reduction caused by this imbalance remain unclear. We investigated Zn uptake and accumulation in maize under Fe-deficient conditions. Under Fe-deficient conditions, Zn uptake was 15-fold higher and Zn accumulation was 16-fold higher than that under normal nutrient conditions. The Zn content of maize leaves under Fe-deficient conditions was >0.4 mg g−1 dry weight, which was higher than the content of plants grown in a nutrient solution containing 50 µM ZnCl2. Plant growth under conditions of both Fe and Zn deficiency was significantly higher than that under only Fe-deficient conditions. Moreover, Fe deficiency increased the thiol content of the plant. These results indicate that Fe deficiency causes excess uptake and accumulation of Zn, and that the stress resulting from the Zn overload accelerates growth reduction in maize.

Growth response of Suaeda salsa (L.) Pall to graded NaCl concentrations and the role of chlorine in growth stimulation

Abstract Growth response of a halophyte species, Suaeda salsa (L.) Pall, to graded NaCl concentrations was examined under water culture conditions. Growth increased with increasing NaCl concentration from 2 to 200 mol m−3, but decreased at NaCl concentrations above 200 mol m−3. Maximum growth was attained at 50 to 200 mol m−3. The role of Na and Cl in the growth stimulation by NaCl was examined by growing S. salsa in nutrient solutions with or without Na and Cl separately at 5 and 50 mol m−3. The growth stimulation induced by Cl was greater than that induced by Na, and Na did not significantly induce growth stimulation. The effect of Na or Cl on O2 evolution from leaves was examined under 5 and 50 mol m−3 concentrations using an oxygen electrode. Oxygen evolution from leaves in –Cl treatments was smaller than that in +Cl treatments both at 5 and 50 mol m−3. The O2 evolution in Na treatments with Cl was similar to that at NaCl. These results indicated that the mechanism of growth stimulation induced by Cl was mainly an increased photosystem II of photosynthesis in leaves. The contribution of Na on the growth stimulation of S. salsa by NaCl was smaller than Cl.

Characteristics of Na+ and K+ absorption in Suaeda salsa (L.) Pall.

Suaeda salsa can grow well in saline soils with high sodium (Na)-low potassium (K) concentrations. While high Na accumulation in plant tissues plays an important role in osmoregulation and maintenance of water absorption for normal growth of Suaeda salsa, characteristics of Na and K absorption in S. salsa grown in such saline soils have not been elucidated. We thus examined Na and K absorption characteristics of S. salsa grown in nutrient solutions of graded sodium chloride (NaCl) with 1 mmol L−1 K or of graded potassium chloride (KCl) with 1 mmol L−1 Na. The K accumulation capacity in S. salsa plant tissues was greater than the Na accumulation capacity. Although Na showed no competitive effects on K absorption, K application suppressed Na absorption drastically. Selectivity for K over Na in the absorption system became higher than that for Na over K concomitantly with increased Na or K concentration in the medium, indicating that Suaeda salsa is able to absorb K effectively with absorption of a large amount of Na. Sodium absorption was significantly decreased by tetra-ethylammonium (typical K channel inhibitor). Calcium (Ca) application increased not Na absorption but K absorption, resulting in enhancement of selectivity for K over Na. We speculate Na absorption pathways in S. salsa are partly mediated by AKT1 (Arabidopsis K transporter 1) type K channel and LCT (a low-affinity cation transporter) and NSCCs (non-selective cation channels) are not the major Na absorption pathway. These results may provide an explanation for the fact that S. salsa can grow well under saline soils with high Na-low K concentrations.

Variations in salt tolerance of reed canarygrass (Phalaris arundinacea L.) plants grown at sites with different degrees of cattle urine contamination

Abstract Variations in the salt tolerance of reed canarygrass (RCG) plants grown at sites with different degrees of cattle urine contamination were investigated. Cattle urine overflowed the urine reservoir and invaded the grassland. Soils and RCG plants were collected from sampling spots selected along the urine flowing line from 0 (contaminated site beside the urine reservoir) to 100 m. With increasing distance from the reservoir, the contents of K, Na, Ca, Mg and NO3-N in the soil and Na, K and NO3-N in the collected RCG plants decreased. The RCG plants were grown in a standard solution to obtain a large number of clones and seeds for the next generation. After the RCG clones and their next generation were subjected to water culture to the fifth leaf stage, NaCl was applied to the solution to investigate their salt tolerance. The salt tolerance, which was the highest in the RCG clones and their next generation for the plants collected from the site beside the reservoir, decreased with increasing distance from the reservoir. Under saline water culture conditions, Na and free-proline contents in shoots, K content in shoots and roots, water potential of leaves and plasmalemma ATPase activity of roots increased in the RCG clones and their next generation along with a higher salt tolerance. In conclusion, responses to saline conditions were similar between the RCG clones and the next generation that were cross-pollinated. It was considered that, even within a confined range of the grassland, salt-tolerant RCG plants that had adapted to the urine-contaminated soils were selected, and that high salt tolerance was genetically controlled. It appears likely that the maintenance of the water potential and K-absorbing function under NaCl stress contributed to the high salt tolerance.

Contribution of iron associated with high-molecular-weight substances to the maintenance of the SPAD value of young leaves of barley under iron-deficient conditions

Abstract In higher plants, it is well known that the retranslocation of iron from old leaves to young leaves is difficult; as a result, iron deficiency leads to interveinal chlorosis, particularly in the young leaves. However, in the case of barley, young chlorotic leaves can grow under conditions of long-term iron deficiency. Previously, we have reported that the distribution and retranslocation characteristics of iron in barley may be better adapted to iron deficiency than those in rice. Furthermore, barley maintained a relatively high chlorophyll index (SPAD value) even when its iron content was not higher than that of rice. In this study, we aimed to predict the chemical form of iron that contributes to the physiologically available iron in barley leaves. To examine the correlation between plant growth and the SPAD value with the amount of fractionated iron, we cultured plant materials in a culture solution containing various iron concentrations. We compared these correlations among barley, rice and sorghum and among three barley cultivars. To compensate for the amount of mugineic acid phytosiderophores (MAs) in the culture solution, we cultured different plant species in the same container. The results revealed that the amount of soluble iron associated with the high-molecular-weight substances (MW >10,000) correlated with the SPAD value of the young barley leaves and the R2 value (determination coefficient) of barley was higher than the values of rice and sorghum.

Comparison of Ca Effect on the Salt Tolerance of Suspension Cells and Intact Plants of Tobacco (Nicotiana tabacum L., cv. Bright Yellow-2)

Salt tolerance and the effect of Ca application on the salt tolerance were compared between suspension culture cells and intact plants of tobacco (Nicotiana tabacum L., cv. Bright Yellow-2). The suspension culture cells and intact plants of tobacco were grown in a culture medium without NaCl and with 100 mM NaCl containing 0.3 or 10 mM CaCl2. At 7 d after the treatment, the dry weight of the cells and intact plants grown in the medium containing 100 mM NaCl with 0.3 mM CaCl2 decreased and the decreasing rate of the weight was much higher in the intact plants. In the medium containing 100 mM NaCl with 10 mM CaCl2, the growth of the cells and intact plants was improved, and the improvement of the growth by Ca application was much more significant in the cultured cells. Sodium content in the intact plants markedly increased with the increase of the NaCl concentration, compared to that in the cells. Calcium application decreased the Na content both in the cells and intact plants and the decreasing rate was higher in the cells than in the intact plants. Thus, it was suggested that the growth inhibition and the increase of the Na content under 100 mM NaCl conditions were more pronounced in intact plants, and that the alleviatory effect of Ca on the growth inhibition was much more conspicuous in the cells. Although the contents of total Ca and pectic acid-binding Ca in the cells and plant roots decreased with the increase of the NaCl concentration, they increased by Ca application. The activity of plasmalemma ATPase of the cells and plant roots decreased in the medium containing 100 mM NaCl with 0.3 mM CaCl2, but increased by Ca application, regardless of the NaCl concentration. In summary, Ca application to the culture medium containing 100 mM NaCl alleviated the growth inhibition, and the difference in the growth inhibition induced by 100 mM NaCl between the suspension culture cells and intact plants was closely related to that in the Na content. It appears that the alleviatory effect of Ca on the growth inhibition might be caused mainly by the enhancement of the Na-excluding function. The high content of pectic acid-binding Ca on the cell wall also contributed moderately to the alleviatory effect of Ca through the decrease of apoplasmic Na translocation.

Comparison of the functions of the barley nicotianamine synthase gene HvNAS1 and rice nicotianamine synthase gene OsNAS1 promoters in response to iron deficiency in transgenic tobacco

Abstract Barley (Hordeum vulgare L.) nicotianamine synthase gene (HvNAS1) expression in barley is strongly induced by Fe deficiency in the roots and rice (Oryza sativa L.) nicotianamine synthase gene (OsNAS1) expression in rice is induced by Fe deficiency both in the roots and in the shoots. In dicots, NAS genes are not strongly induced by Fe deficiency, and they function to maintain Fe homeostasis. Rice OsNAS1promoter::GUS or barley HvNAS1promoter::GUS was introduced into tobacco (Nicotiana tabacum L.) and tissue specificities and systemic regulation of their expression were compared. A split-root experiment revealed that the HvNAS1 promoter exhibited functions similar to those of Fe-acquisition-related genes in tobacco roots, suggesting that this promoter responded to certain Fe-deficiency systemic signals and to the Fe concentration in the rhizosphere. The HvNAS1 promoter might harbor a type of universal system of gene expression for Fe acquisition. However, the OsNAS1 promoter did not respond to local application of Fe to the roots and induced GUS activities in mature leaves in response to Fe deficiency. This promoter might possess numerous types of cis-acting sequences that are involved in Fe metabolism.

Enhancement of nitrate reduction by chlorine application in Suaeda salsa (L.) Pall

Abstract We have previously reported that one of the mechanisms of growth stimulation of the halophyte Suaeda salsa (L.) Pall by NaCl is partly attributed to increased activity of photosystem II by Cl application. In the present study, the effect of Na and/or Cl application on the reduction and assimilation of nitrate in S. salsa was examined by growing plants in nutrient solutions with the following four treatments: –Na·–Cl, +Na·–Cl, –Na·+Cl and +Na·+Cl at 5 mol m−3. Growth was stimulated by the application of Cl, but not Na. The total amount of nitrogen absorbed did not differ among treatments. However, nitrate accumulation in the plant, particularly in the stem, was much higher in the –Na·–Cl and +Na·–Cl treatments than in the –Na·+Cl and +Na·+Cl treatments. In contrast, the accumulation of crude protein in all plant tissues and of amino acids in the leaves was higher in the –Na·+Cl and +Na·+Cl treatments than in the –Na·–Cl and +Na·–Cl treatments. Thus, nitrate reduction and assimilation were stimulated by Cl application, irrespective of Na application. Increased nitrate reduction was considered to be the main cause of the increased nitrate assimilation induced by Cl. Mechanisms of stimulation of nitrate reduction and assimilation are discussed.

Behavior of nitrogen in soil and uptake by the rice plant

Direct seeded rice is becoming important in developing countries as a result of increased labor costs for transplanting rice. The objective of this research was to study the N availability in a gray alluvial soil applied with 15N labeled (NH4)2SO4 (AmS) and/or rice straw (RS) by in vitro incubation and pot experiments with rice (Oryza sativa L.). Extractable inorganic N (NH4 +-N and NO3 --N) in incubated soils were determined under flooding and 60% water holding capacity (WHC) at 15 and 30°C for 16 weeks. The 60%WHC treatments significantly increased N mineralization, especially at 30°C. However, under flooding, applied 15N was immobilized. Plants total dry matter production was significantly the same for RS treatments. However, with AmS treatment, IR36 had significantly higher dry matter yield. Straw and fertilizer application improved uptake of soil N at harvest time. At panicle initiation stage however, uptake of soil N under the control was significantly higher than the straw treatment as a result of immobilization. Application of fertilizer N with the rice straw, reduced soil N immobilization caused by application of the straw alone.