Minobu Kasai

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves.

SummaryPlasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(Km(Ca2+)=0.4 μm) and ATP(Km(ATP)=3.9 μm), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl− or NO3−. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl− was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO43− which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl−-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl−-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl−-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.

Response of the Plant Root to Aluminum Stress : Analysis of the Inhibition of the Root Elongation and Changes in Membrane Function

Pea root elongation was strongly inhibited in the presence of a low concentration of Al (5 μM). In Al-treated root, the epidermis was markedly injured and characterized by an irregular layer of cells of the root surface. Approximately 30% of total absorbed Al accumulated in the root tip and Al therein was found to cause the inhibition of whole root elongation. Increasing concentrations of Ca2+ effectively ameliorated the inhibition of root elongation by Al and 1 mM of CaCl2 completely repressed the inhibition of root elongation by 50 μM Al. The ameliorating effect of Ca2+ was due to the reduction of Al uptake.H+-ATPase and H+-PPase activity as well as ATP and PPidependent H+ transport activity of vacuolar membrane vesicles prepared from barley roots increased to a similar extent by the treatment with 50 μM AlCl3. The rate of increase of the amount of H+-ATPase and H+-PPase was proportional to that of protein content measured by immunoblot analysis with antibodies against the catalytic subunit of the vacuolar H+-ATPase and H+-PPase of mung bean. The increase of both activities was discussed in relation to the physiological tolerance mechanism of barley root against Al stress.

Regulation of leaf photosynthetic rate correlating with leaf carbohydrate status and activation state of Rubisco under a variety of photosynthetic source/sink balances

There is evidence suggesting that in plants changes in the photosynthetic source/sink balance are an important factor that regulates leaf photosynthetic rate through affects on the leaf carbohydrate status. However, to resolve the regulatory mechanism of leaf photosynthetic rate associated with photosynthetic source/sink balance, information, particularly on mutual relationships of experimental data that are linked with a variety of photosynthetic source/sink balances, seems to be still limited. Thus, a variety of manipulations altering the plant source/sink ratio were carried out with soybean plants, and the mutual relationships of various characteristics such as leaf photosynthetic rate, carbohydrate content and the source/sink ratio were analyzed in manipulated and non-manipulated control plants. The manipulations were removal of one-half or all pods, removal of one-third or two-third leaves, and shading of one-third or one-half leaves with soybean plants grown for 8 weeks under 10 h light (24 degrees C) and 14 h darkness (17 degrees C). It was shown that there were significant negative correlations between source/sink ratio (dry weight ratio of attached leaves to other all organs) and leaf photosynthetic rate; source/sink ratio and activation ratio (percentage of initial activity to total activity) of Rubisco in leaf extract; leaf carbohydrate (sucrose or starch) content and photosynthetic rate; carbohydrate (sucrose or starch) content and activation ratio of Rubisco; amount of protein-bound ribulose-1,5-bisphosphate (RuBP) in leaf extract and leaf photosynthetic rate; and the amount of protein-bound RuBP and activation ratio of Rubisco. In addition, there were significant positive correlations between source/sink ratio and leaf carbohydrate (sucrose or starch) content; source/sink ratio and the amount of protein-bound RuBP; carbohydrate (sucrose or starch) content and amount of protein-bound RuBP and the activation ratio of Rubisco and leaf photosynthetic rate. The plant water content, leaf chlorophyll and Rubisco contents were not affected significantly by the manipulations. There is a previous report in Arabidopsis thaliana that the amount of protein-bound RuBP in leaf extract correlates negatively with the activation ratio of Rubisco in the leaf extract. Therefore, the results obtained from the manipulation experiments indicate that there is a regulatory mechanism for the leaf photosynthetic rate that correlates negatively with leaf carbohydrate (sucrose and starch) status and positively with the activation state of Rubisco under a variety of photosynthetic source/sink balances.

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H+-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H+-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K+ for 40 min under various conditions. The rate of efflux of K+ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H+-ATPase of the plasma membrane, increased the efflux of K+. Al repressed this efflux at low pH, probably through an effect on K+ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K+ irrespective of the presence of vanadate. Ca2+ also had a repressive effect on the efflux of K+ at low pH. The effect of Ca2+, greater in Scout, might be related to the regulation of the net influx of H+, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H+, which in turn is counteracted by the efflux of K+ and H+. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H+ and the efflux of K+ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.

Stimulation of H+ extrusion and plasma membrane H+-ATPase activity of barley roots by ammonium-treatment

Abstract H+ extrusion and the plasma membrane H+-ATPase activity were analyzed using barley roots exposed to ammonium. H+ extrusion from roots was enhanced 4 h after the addition of 5 mol m-3 ammonium to the medium. However, the stimulation of the plasma membrane H+-ATPase activity occurred at least 8 h after the addition of ammonium. Furthermore, this stimulation was found to be caused by the increase in the content of H+-ATPase proteins based on the results of immunoassay. These results indicate that H+ was actively extruded to the medium through the increase in the amount of plasma membrane H+-ATPase proteins, when barley roots were exposed to ammonium for more than 8 h. Thus, it is assumed that in the long term exposure to ammonium nitrogen, ammonium-induced stimulation of the H+-ATPase activity may be important to maintain intracellular pH in root cells under the acidic conditions associated with the uptake of ammonium.

Determination of sugar phosphates and nucleotides related to photosynthetic metabolism by high-performance anion-exchange liquid chromatography with fluorometric and ultraviolet detection

A high-performance anion-exchange liquid chromatography system was constructed to identify sugar phosphates and nucleotides involved in photosynthetic metabolism. First sugar phosphates and nucleotides were separated by a gradient elution with boric acid and sodium phosphate, then they were detected by a fluorescence detector (as fluorescent derivatives with arginine) and UV detector, respectively. Eight authentic sugar phosphates and 11 authentic nucleotides could be analyzed using the system. The applicability of the system to the determination of the corresponding sugar phosphates and nucleotides in extracts from only five soybean leaf discs (8.95 cm2) was shown.

Stimulation of plasma membrane h+-transport activity in barley roots by salt stress

Abstract Plasma membrane vesicles were prepared from barley roots (Hordeum vulgare cv. Kikaihadaka) using the phase partitioning method. H+ -transport activity could be measured without membrane inversion treatment, since the plasma membrane vesicles showed a latency value of approximately 50% for the activity. Following the exposure to NaCl, alterations of the plasma membrane H+ -ATPase and H+ -transport activity were investigated. After barley roots were treated with 200 mol m−3 NaCl for 1 d, both plasma membrane H+-ATPase activity and H+ -transport activity assayed in the presence of nitrate were reduced as compared with those of control roots. However, in the salinized roots, the H+ -transport activity measured with chloride was higher than that of control roots, though the H+-ATPase activity was lower. These results suggest that the permeability of the plasma membrane to chloride may increase by salt stress.

Photosynthesis and micro-environmental factors in a spring ephemeral,Erythronium japonicum, from native and open habitats

AbstractEnvironmental factors affecting photosynthetic activity of the typical vernal speciesErythronium japonicum Decne were examined on the floor of a deciduous broad-leavedQuercus mongolica forest (Q.m. stand) and on bare land left undisturbed for 4 years after forest clearing (bare stand). Daytime solar irradiation and air and leaf temperatures at the bare stand were significantly higher than those at theQ.m. stand. The relative air humidity was very low and did not differ much between the stands, although the leaf-air vapor pressure differences (VPD) at the bare stand were about twice as high as those at theQ.m. stand. The plants at both stands were supplied with sufficient soil water throughout their growing season by a large snowmelt. However, the aboveground parts of the plants at the bare stand were subjected to much more severe heat stress, caused by the strong radiations and high leaf temperatures, and water stress, caused by the highly transpiring conditions, than those at theQ.m. stand. When the radiation on leaves, leaf temperatures and VPD in the assimilation chamber were changed from those observed at theQ.m. stand to those at the bare stand, the photosynthetic rate and stomatal conductance fell significantly. However, the rate and conductance were immediately restored to the respective values near those measured under the conditions at theQ.m. stand when only VPD was dropped to the value similar to that observed at theQ.m. stand. These results indicate that the photosynthetic rate of the plants at the bare stand was lowered largely by a decrease in stomatal conductance. The internal CO2 partial pressure % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfKttLearuqr1ngBPrgarmWu51MyVXgatC% vAUfeBSjuyZL2yd9gzLbvyNv2CaeHbd9wDYLwzYbItLDharyavP1wz% ZbItLDhis9wBH5garqqtubsr4rNCHbGeaGqiVu0Je9sqqrpepC0xbb% L8F4rqqrFfpeea0xe9Lq-Jc9vqaqpepm0xbba9pwe9Q8fs0-yqaqpe% pae9pg0FirpepeKkFr0xfr-xfr-xb9adbaqaaeGaciGaaiaabeqaam% aaeaqbaaGcbaGaeiikaGIaemiuaa1aaSbaaSqaaGqaaiab-neadjab% -9eapnaaBaaameaacqWFYaGmaeqaaaWcbeaakiabcMcaPaaa!4134!

Effect of Sink-Limitation on Leaf Photosynthetic Rate and Related Characteristics in Soybean Plants

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