Noninvasive imaging of hollow structures and gas movement revealed the gas partial-pressure-gradient-driven long-distance gas movement in the aerenchyma along the leaf blade to submerged organs in rice.

Rice (Oryza sativa) plants have porous or hollow organs consisting of aerenchyma, which is presumed to function as a low-resistance diffusion pathway for air to travel from the foliage above the water to submerged organs. However, gas movement in rice plants has yet to be visualized in real time. In this study involving partially submerged rice plants, the leaves emerging from the water were fed nitrogen-13-labeled nitrogen ([13 N]N2 ) tracer gas, and the gas movement downward along the leaf blade, leaf sheath, and internode over time was monitored. The [13 N]N2 gas arrived at the bottom of the plant within 10 min, which was 20 min earlier than carbon-11 photoassimilates. The [13 N]N2 gas movement was presumably mediated by diffusion along the aerenchyma network from the leaf blade to the root via nodes functioning as junctions, which were detected by X-ray computed tomography. These findings imply the diffusion of gas along the aerenchyma, which does not consume energy, has enabled plants to adapt to aquatic environments. Additionally, there were no major differences in [13 N]N2 gas movement between paddy rice and deepwater rice plants, indicative of a common aeration mechanism in the two varieties, despite the difference in their response to flooding.

Shoot base responds to root-applied glutathione and functions as a critical region to inhibit cadmium translocation from the roots to shoots in oilseed rape (Brassica napus).

Glutathione (GSH) is a tripeptide involved in controlling heavy metal movement in plants. Our previous study showed that GSH, when site-specifically applied to plant roots, inhibits Cd translocation from the roots to shoots in hydroponically cultured oilseed rape (Brassica napus) plants. A factor that led to this inhibitory effect was the activation of Cd efflux from root cells. To further investigate the molecular mechanism triggered by root-applied GSH, Cd movement was non-invasively monitored using a positron-emitting tracer imaging system. The Cd absorption and efflux process in the roots were visualized successfully. The effects of GSH on Cd efflux from root cells were estimated by analyzing imaging data. Reanalysis of image data suggested that GSH applied to roots, at the shoot base, activated Cd return. Cutting the shoot base significantly inhibited Cd efflux from root cells. These experimental results demonstrate that the shoot base plays an important role in distributing Cd throughout the plant body. Furthermore, microarray analysis revealed that about 400 genes in the roots responded to root-applied GSH. Among these, there were genes for transporter proteins related to heavy metal movement in plants and proteins involved in the structure modification of cell walls.

Effects of enhancing endogenous and exogenous glutathione in roots on cadmium movement in Arabidopsis thaliana.

Glutathione (GSH) is a thiol-containing compound involved in many aspects of plant metabolism. In the present study, we investigated how enhancing endogenous and exogenous GSH affects cadmium (Cd) movement and distribution in Arabidopsis plants cultured hydroponically. Transgenic Arabidopsis plants with a strong ability to synthesize GSH in roots were generated　by transforming the gene encoding the bifunctional γ-glutamylcysteine synthetase-glutathione synthetase enzyme from Streptococcus thermophiles (StGCS-GS). Enhancing endogenous and exogenous GSH decreased the Cd translocation ratio in different ways. Only exogenous GSH significantly inhibited Cd translocation from roots to shoots in wild-type and transgenic Arabidopsis plants. Our study demonstrated that GSH mainly functions outside root cells to inhibit Cd translocation from roots to shoots.

On-line rapid purification of [13N]N2 gas for visualization of nitrogen fixation and translocation in nodulated soybean.

Accurate analysis of N fixation in leguminous crops requires determination of N utilization within an intact plant; however, most approaches require tissue disassembly. We developed a simple and rapid technique to generate high-purity and high-yield [13N]N2 gas and obtained real-time images of N fixation in an intact soybean plant. The purification efficiency was ∼81.6% after decay correction. Our method provides accurate signals of N fixation and allows free changes to the tracer gas composition to suit different experimental designs.

Elevated glutathione synthesis in leaves contributes to zinc transport from roots to shoots in Arabidopsis.

Glutathione (GSH) is a vital compound involved in several plant metabolic pathways. Our previous study indicated that foliar GSH application can increase zinc (Zn) levels in leafy vegetables. The objective of this study was to determine the mode of action of GSH as it relates to Zn transport from roots to shoots. Two types of transgenic Arabidopsis plants with genes for GSH synthesis, including StGCS-GS or AtGSH1 driven by the leaf-specific promoter of chlorophyll a/b-binding protein (pCab3) gene were generated. Both types of transgenic Arabidopsis plants showed significant increases in shoot GSH concentrations compared to the wild type (WT). Monitoring 65Zn movement by positron-emitting tracer imaging system (PETIS) analysis indicated that the 65Zn amount in the shoots of both types of transgenic Arabidopsis plants were higher than that in the WT. GSH concentration in phloem sap was increased significantly in WT with foliar applications of 10 mM GSH (WT-GSH), but not in transgenic Arabidopsis with elevated foliar GSH synthesis. Both types of transgenic Arabidopsis with elevated foliar GSH synthesis and WT-GSH exhibited increased shoot Zn concentrations and Zn translocation ratios. These results suggest that enhancement of endogenous foliar GSH synthesis and exogenous foliar GSH application affect root-to-shoot transport of Zn.

Kinetic Analysis of Zinc/Cadmium Reciprocal Competitions Suggests a Possible Zn-Insensitive Pathway for Root-to-Shoot Cadmium Translocation in Rice.

BACKGROUND: Among cereals, rice has a genetic propensity to accumulate high levels of cadmium (Cd) in grains. Xylem-mediated root-to-shoot translocation rather than root uptake has been suggested as the main physiological factor accounting for the genotypic variation observed in Cd accumulation in shoots and grains. Several evidence indicate OsHMA2 - a putative zinc (Zn) transporter - as the main candidate protein that could be involved in mediating Cd- and Zn-xylem loading in rice. However, the specific interactions between Zn and Cd in rice often appear anomalous if compared to those observed in other staple crops, suggesting that root-to-shoot Cd translocation process could be more complex than previously thought. In this study we performed a complete set of competition experiments with Zn and Cd in order to analyze their possible interactions and reciprocal effects at the root-to-shoot translocation level. RESULTS: The competition analysis revealed the lack of a full reciprocity when considering the effect of Cd on Zn accumulation, and vice versa, since the accumulation of Zn in the shoots was progressively inhibited by Cd increases, whereas that of Cd was only partially impaired by Zn. Such behaviors were probably dependent on Cd-xylem loading mechanisms, as suggested by: i) the analysis of Zn and Cd content in the xylem sap performed in relation to the concentration of the two metals in the mobile fractions of the roots; ii) the analysis of the systemic movement of (107)Cd in short term experiments performed using a positron-emitting tracer imaging system (PETIS). CONCLUSIONS: Our results suggest that at least two pathways may mediate root-to-shoot Cd translocation in rice. The former could involve OsHMA2 as Zn(2+)/Cd(2+) xylem loader, whereas the latter appears to involve a Zn-insensitive system that still needs to be identified.

A kinetic analysis of cadmium accumulation in a Cd hyper-accumulator fern, Athyrium yokoscense and tobacco plants.

Cadmium (Cd) accumulations in a Cd hyper-accumulator fern, Athyrium yokoscense (Ay), and tobacco, Nicotiana tabacum (Nt), were kinetically analysed using the positron-emitting tracer imaging system under two medium conditions (basal and no-nutrient). In Ay, maximumly 50% and 15% of the total Cd accumulated in the distal roots and the shoots under the basal condition, respectively. Interestingly, a portion of the Cd in the distal roots returned to the medium. In comparison with Ay, a little fewer Cd accumulations in the distal roots and clearly higher Cd migration to the shoots were observed in Nt under the basal condition (maximumly 40% and 70% of the total Cd, respectively). The no-nutrient condition down-regulated the Cd migration in both species, although the regulation was highly stricter in Ay than in Nt (almost no migration in Ay and around 20% migration in Nt). In addition, the present work enabled to estimate physical and physiological Cd accumulation capacities in the distal roots, and demonstrated condition-dependent changes especially in Ay. These results clearly suggested occurrences of species-/condition-specific regulations in each observed parts. It is probable that integration of these properties govern the specific Cd tolerance/accumulation in Ay and Nt.

Nitrate facilitates cadmium uptake, transport and accumulation in the hyperaccumulator Sedum plumbizincicola.

The aims of this study are to investigate whether and how the nitrogen form (nitrate (NO3 (-)) versus ammonium (NH4 (+))) influences cadmium (Cd) uptake and translocation and subsequent Cd phytoextraction by the hyperaccumulator species Sedum plumbizincicola. Plants were grown hydroponically with N supplied as either NO3 (-) or NH4 (+). Short-term (36 h) Cd uptake and translocation were determined innovatively and quantitatively using a positron-emitting (107)Cd tracer and positron-emitting tracer imaging system. The results show that the rates of Cd uptake by roots and transport to the shoots in the NO3 (-) treatment were more rapid than in the NH4 (+) treatment. After uptake for 36 h, 5.6 (0.056 µM) and 29.0 % (0.290 µM) of total Cd in the solution was non-absorbable in the NO3 (-) and NH4 (+) treatments, respectively. The local velocity of Cd transport was approximately 1.5-fold higher in roots (3.30 cm h(-1)) and 3.7-fold higher in shoots (10.10 cm h(-1)) of NO3 (-)- than NH4 (+)-fed plants. Autoradiographic analysis of (109)Cd reveals that NO3 (-) nutrition enhanced Cd transportation from the main stem to branches and young leaves. Moreover, NO3 (-) treatment increased Cd, Ca and K concentrations but inhibited Fe and P in the xylem sap. In a 21-day hydroponic culture, shoot biomass and Cd concentration were 1.51 and 2.63 times higher in NO3 (-)- than in NH4 (+)-fed plants. We conclude that compared with NH4 (+), NO3 (-) promoted the major steps in the transport route followed by Cd from solution to shoots in S. plumbizincicola, namely its uptake by roots, xylem loading, root-to-shoot translocation in the xylem and uploading to the leaves. S. plumbizincicola prefers NO3 (-) nutrition to NH4 (+) for Cd phytoextraction.

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.

Real-time imaging and analysis of differences in cadmium dynamics in rice cultivars (Oryza sativa) using positron-emitting 107Cd tracer.

BACKGROUND: Rice is a major source of dietary intake of cadmium (Cd) for populations that consume rice as a staple food. Understanding how Cd is transported into grains through the whole plant body is necessary for reducing rice Cd concentrations to the lowest levels possible, to reduce the associated health risks. In this study, we have visualized and quantitatively analysed the real-time Cd dynamics from roots to grains in typical rice cultivars that differed in grain Cd concentrations. We used positron-emitting 107Cd tracer and an innovative imaging technique, the positron-emitting tracer imaging system (PETIS). In particular, a new method for direct and real-time visualization of the Cd uptake by the roots in the culture was first realized in this work. RESULTS: Imaging and quantitative analyses revealed the different patterns in time-varying curves of Cd amounts in the roots of rice cultivars tested. Three low-Cd accumulating cultivars (japonica type) showed rapid saturation curves, whereas three high-Cd accumulating cultivars (indica type) were characterized by curves with a peak within 30 min after 107Cd supplementation, and a subsequent steep decrease resulting in maintenance of lower Cd concentrations in their roots. This difference in Cd dynamics may be attributable to OsHMA3 transporter protein, which was recently shown to be involved in Cd storage in root vacuoles and not functional in the high-Cd accumulating cultivars. Moreover, the PETIS analyses revealed that the high-Cd accumulating cultivars were characterized by rapid and abundant Cd transfer to the shoots from the roots, a faster transport velocity of Cd to the panicles, and Cd accumulation at high levels in their panicles, passing through the nodal portions of the stems where the highest Cd intensities were observed. CONCLUSIONS: This is the first successful visualization and quantification of the differences in whole-body Cd transport from the roots to the grains of intact plants within rice cultivars that differ in grain Cd concentrations, by using PETIS, a real-time imaging method.

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.