Shin-ichi Nakamura

Thioredoxin h is one of the major proteins in rice phloem sap

Sieve tubes play important roles in the transfer of nutrients as well as signals. Hundreds of proteins were found in pure phloem sap collected from rice (Oryza sativa L. cv. Kantou) plants through the cut ends of insect stylets. These proteins may be involved in nutrient transfer and signal transduction. To characterize the nature of these proteins, the partial amino-acid sequence of a 13kDa protein, named RPP13-1, that was abundant in the pure phloem sap was determined. A cDNA clone of 687 bp, containing an open reading frame of 122 amino acids, was isolated using corresponding oligonucleotides as a probe. The deduced amino-acid sequence was very similar to that of the ubiquitous thiol redox protein, thioredoxin. The consensus sequences of thioredoxins are highly conserved. No putative signal peptide was identified. Antiserum against wheat thioredoxin h cross-reacted with RPP13-1 in the phloem sap of rice plants. RPP131 produced in Escherichia coli was reactive to antiserum against wheat thioredoxin h. Both E. coli-produced RPP13-1 and the phloem sap proteins catalyzed the reduction of the disulfide bonds of insulin in the presence of dithiothreitol. These results indicate that an active thioredoxin is a major protein translocating in rice sieve tubes.

Tracing Cadmium from Culture to Spikelet: Noninvasive Imaging and Quantitative Characterization of Absorption, Transport, and Accumulation of Cadmium in an Intact Rice Plant

We characterized the absorption and short-term translocation of cadmium (Cd) in rice (Oryza sativa ‘Nipponbare’) quantitatively using serial images observed with a positron-emitting tracer imaging system. We fed a positron-emitting 107Cd (half-life of 6.5 h) tracer to the hydroponic culture solution and noninvasively obtained serial images of Cd distribution in intact rice plants at the vegetative stage and at the grain-filling stage every 4 min for 36 h. The rates of absorption of Cd by the root were proportional to Cd concentrations in the culture solution within the tested range of 0.05 to 100 nm. It was estimated that the radial transport from the culture to the xylem in the root tissue was completed in less than 10 min. Cd moved up through the shoot organs with velocities of a few centimeters per hour at both stages, which was obviously slower than the bulk flow in the xylem. Finally, Cd arrived at the panicles 7 h after feeding and accumulated there constantly, although no Cd was observed in the leaf blades within the initial 36 h. The nodes exhibited the most intensive Cd accumulation in the shoot at both stages, and Cd transport from the basal nodes to crown root tips was observed at the vegetative stage. We conclude that the nodes are the central organ where xylem-to-phloem transfer takes place and play a pivotal role in the half-day travel of Cd from the soil to the grains at the grain-filling stage.

Application of glutathione to roots selectively inhibits cadmium transport from roots to shoots in oilseed rape

Glutathione is a tripeptide involved in various aspects of plant metabolism. This study investigated the effects of the reduced form of glutathione (GSH) applied to specific organs (source leaves, sink leaves, and roots) on cadmium (Cd) distribution and behaviour in the roots of oilseed rape plants (Brassica napus) cultured hydroponically. The translocation ratio of Cd from roots to shoots was significantly lower in plants that had root treatment of GSH than in control plants. GSH applied to roots reduced the Cd concentration in the symplast sap of root cells and inhibited root-to-shoot Cd translocation via xylem vessels significantly. GSH applied to roots also activated Cd efflux from root cells to the hydroponic solution. Inhibition of root-to-shoot translocation of Cd was visualized, and the activation of Cd efflux from root cells was also shown by using a positron-emitting tracer imaging system (PETIS). This study investigated a similar inhibitory effect on root-to-shoot translocation of Cd by the oxidized form of glutathione, GSSG. Inhibition of Cd accumulation by GSH was abolished by a low-temperature treatment. Root cells of plants exposed to GSH in the root zone had less Cd available for xylem loading by actively excluding Cd from the roots. Consequently, root-to-shoot translocation of Cd was suppressed and Cd accumulation in the shoot decreased.

Effect of cadmium on the chemical composition of xylem exudate from oilseed rape plants (Brassica napus L.)

Abstract Phytoremediation is a good technique for removing cadmium (Cd) from farmland soils. To remove Cd from these soils effectively, it is necessary for Cd ions to be transported to the shoot organs for later harvest. However, the mechanism of Cd translocation to shoot organs via xylem vessels has not yet been elucidated. We selected oilseed rape plants (Brassica napus L.) and established a method to collect xylem exudates from these plants. After 3 days of Cd treatment (10 µmol L−1 and 30 µmol L−1) the Cd concentrations in the xylem exudates were approximately 6.5 µmol L−1 and 16 µmol L−1, respectively. The detection of Cd in the xylem exudate indicated that Cd was moving to shoot organs via xylem vessels. The effect of these Cd treatments on the amino acid, organic acid and protein composition of xylem exudates from oilseed rape plants was investigated. The level of amino acids and organic acids detected was enough to bind Cd transported via the xylem. Sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis revealed that proteins with molecular weights of 36 kDa and 45 kDa clearly increased in the exudates with Cd treatment. The possibility that these compounds are binding Cd in the xylem exudates was discussed.

Isolation of novel types of Arabidopsis mutants with altered reactions to cadmium: cadmium-gradient agar plates are an effective screen for the heavy metal-related mutants

We are interested in elucidating the molecular mechanisms underlying plant reactions to the toxic heavy metal cadmium (Cd). To this end, we devised a new screening strategy using agar plates with a gradient of Cd concentrations, termed Cd-gradient agar plates (CGAPs), to isolate Arabidopsis mutants that displayed altered reactions to the metal. Arabidopsis M2 seeds, derived from ethyl methanesulfonate (EMS) treated seeds, were germinated on the CGAPs such that the primary root of each seedling elongated against increasing concentrations of Cd on the surface of the plate. Under these conditions, the lengths of the primary roots reliably demonstrated the degree of Cd tolerance of individual seedlings. The use of CGAPs also allowed close observation of the root reaction of each seedling to Cd without causing lethal damage. The screen identified three mutant lines, MRC-32, MRC-22 and MRC-26, which showed distinctly different characteristics. MRC-32 plants exhibited enhanced tolerance to Cd and contained Cd at higher concentrations than wild-type (WT) plants treated with the heavy metal. The whole root system of MRC-22 plants showed a Cd-phobic response. MRC-26 plants accumulated less Cd in their aboveground tissues than WT plants, suggesting that they were defective in transporting the heavy metal from roots to aboveground tissues. We also determined the likely chromosomal location of each mutation.

Analysis of phloem exudate collected from fruit-bearing stems of coconut palm: Palm trees as a source of molecules circulating in sieve tubes

Abstract Sieve tubes have been attracting widespread research interest because of their possible role in mediating physiological signals within the whole plant. However, progress in research into the function of sieve tubes has been limited by the low volume of sap available. To overcome this problem, we attempted to collect phloem exudate from tropical coconut palm trees (Cocos nucifera L. cv. Namhom). As much as 3 to 15 mL of exudate per hour was collected from the cut surface of the plants fruit-bearing stem. Our analyses revealed that the characterized profiles of sugars (sucrose: 339 mM), amino acids (total concentration: 17.1 mM), cations (potassium: 48.3 mM), and proteins (total concentration: 0.1 /-lg /-lL-1) in the exudate were mostly consistent with those of phloem sap or phloem exudate collected from rice plants, castor bean plants, etc. This exudate was assumed to reflect the composition of the phloem sap from the source organs of coconut palm trees. The large volume of exudate collected contributed significantly to the analyses of the various compounds in the stream of sieve tubes.

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using 15N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes.

Detection of calmodulin and calmodulin-binding proteins in pure phloem sap of rice plants

Abstract Calcium-ligand blots using 45Ca2+ revealed that there were at least four calcium-binding proteins (17, 18.5, 48 and 52 kDa) in the phloem sap of rice plants. Furthermore, an anti-spinach calmodulin antibody cross-reacted strongly with an 18.5 kDa protein from the phloem sap. When electrophoresed on native gels, the anti-calmodulin cross-reacting protein migrated more slowly in the presence of calcium than in its absence. Based on these results, we concluded that the cross-reacting protein was a calmodulin. An 125I-CaM overlay assay revealed that two calmodulin-binding proteins (20 kDa and 40 kDa) were present in the rice phloem sap. Based on these data, it is possible that a calmodulin signal cascade is present in the sieve tubes of rice plants.

Application of glutathione and dithiothreitol to oil seed rape (Brassica napus L.) roots affects cadmium distribution in roots and inhibits Cd translocation to shoots

ABSTRACT Previously we have shown that site-specific application of glutathione (GSH) to roots inhibits cadmium (Cd) translocation from roots to shoots. In this study, we investigated the effects of chemical compounds containing sulfur, such as dithiothreitol (DTT), potassium sulfate (K2SO4) and a GSH synthesis inhibitor, buthionine sulfoximine (BSO), on the behavior of Cd in oilseed rape plants (Brassica napus L.). We found that DTT, just like GSH, also inhibited Cd translocation from roots to shoots significantly. However, the inhibitory effects of Cd behaviors differed between GSH and DTT. Cd efflux from root cells was activated by GSH and inactivated by DTT. Positron imaging experiments revealed that there are minor differences in the effects of GSH and DTT on Cd adsorption to the surface of roots. These results provide insights into the function of GSH involved in the inhibitory effects of Cd translocation from roots to shoots.

Effects of Glutathione Concentration in the Root Zone and Glutathione Treatment Period on Cadmium Partitioning in Oilseed Rape Plants

Glutathione is a sulfur-containing peptide involved in various aspects of plant metabolism. Glutathione is also known to have effects on heavy metal responses in plants. In our previous work, we have found glutathione, applied to roots site-specifically, inhibited cadmium (Cd) translocation from roots to shoots and Cd accumulation in shoots in oilseed rape plants. In addition, we succeeded in visualizing inhibition of root-to-shoot translocation of Cd by using a positron-emitting tracer imaging system (PETIS). In this work, the effects of glutathione concentration in the root zone (hydroponic solution) and the glutathione treatment period on Cd partitioning in oilseed rape plants were investigated. Our experimental results demonstrated that glutathione, exceeding a certain concentration in the root zone, is needed to trigger inhibition of Cd translocation, and that treatment time from the start of glutathione application had different effects on Cd partitioning in oilseed rape plants.