Nondestructive characterization of diseased Chinese chive leaves using X-ray intensity ratios with microbeam synchrotron radiation X-ray fluorescence spectrometry.

The Chinese chive (Allium tuberosum) is a core crop grown in Kochi Prefecture, Japan. However, withering symptoms occur during greenhouse growing, which have a negative impact on crop management Chinese chive leaves with physiological disorders (PD) or necrotic streak disease (ND) present with withering as typical blight symptoms. Excess or deficiency of elements may cause such withering in Chinese chive leaves with PD. Therefore, visualizing the elemental distribution in plant bodies may help clarify the cause of this withering. In this study, using synchrotron radiation X-ray fluorescence (SR-XRF) imaging, we examined the elemental distribution conditions in healthy Chinese chive leaves without withering, those that withered due to PD, and those that withered due to ND. Segmentation analysis of inductively coupled plasma-optical emission spectroscopy (ICP-OES) was performed on the SR-XRF imaged Chinese chive leaves and the data from the two analytical methods were compared. SR-XRF imaging provided more detailed data on elemental distribution compared with segmentation analysis using ICP-OES. Based on the SR-XRF imaging results, the X-ray intensity ratios for Ca/K, Fe/Mn, and Zn/Cu were calculated. These findings support that the Ca/K, Fe/Mn, and Zn/Cu X-ray intensity ratios can be used in the early detection of withered leaves and to predict the factors causing withering.

Duplication of a manganese/cadmium transporter gene reduces cadmium accumulation in rice grain.

Global contamination of soils with toxic cadmium (Cd) is a serious health threat. Here we found that a tandem duplication of a gene encoding a manganese/Cd transporter, OsNramp5, was responsible for low-Cd accumulation in Pokkali, an old rice cultivar. This duplication doubled the expression of OsNramp5 gene but did not alter its spatial expression pattern and cellular localization. Higher expression of OsNramp5 increased uptake of Cd and Mn into the root cells but decreased Cd release to the xylem. Introgression of this allele into Koshihikari, an elite rice cultivar, through backcrossing significantly reduced Cd accumulation in the grain when cultivated in soil heavily contaminated with Cd but did not affect both grain yield and eating quality. This study not only reveals the molecular mechanism underlying low-Cd accumulation but also provides a useful target for breeding rice cultivars with low-Cd accumulation.

Photosynthetic Parameters Show Specific Responses to Essential Mineral Deficiencies.

In response to decreases in the assimilation efficiency of CO2, plants oxidize the reaction center chlorophyll (P700) of photosystem I (PSI) to suppress reactive oxygen species (ROS) production. In hydro-cultured sunflower leaves experiencing essential mineral deficiencies, we analyzed the following parameters that characterize PSI and PSII: (1) the reduction-oxidation states of P700 [Y(I), Y(NA), and Y(ND)]; (2) the relative electron flux in PSII [Y(II)]; (3) the reduction state of the primary electron acceptor in PSII, QA (1 - qL); and (4) the non-photochemical quenching of chlorophyll fluorescence (NPQ). Deficiency treatments for the minerals N, P, Mn, Mg, S, and Zn decreased Y(II) with an increase in the oxidized P700 [Y(ND)], while deficiencies for the minerals K, Fe, Ca, B, and Mo decreased Y(II) without an increase in Y(ND). During the induction of photosynthesis, the above parameters showed specific responses to each mineral. That is, we could diagnose the mineral deficiency and identify which mineral affected the photosynthesis parameters.

A member of cation diffusion facilitator family, MTP11, is required for manganese tolerance and high fertility in rice.

MAIN CONCLUSION: Rice MTP11 is the trans-Golgi-localized transporter that is involved in Mn tolerance with MTP8.1, and it is required for normal fertility. Rice (Oryza sativa L.) is one of the most manganese (Mn)-tolerant species, and it is able to accumulate high levels of this metal in the leaves without showing toxic symptoms. The metal tolerance protein 8.1 (MTP8.1), a member of the Mn-cation diffusion facilitator (CDF) family, has been shown to play a central role in high Mn tolerance by sequestering Mn into vacuoles. Recently, rice MTP11 was identified as an Mn transporter that is localized to Golgi-associated compartments, but its exact role in Mn tolerance in planta has not yet been understood. Here, we investigated the role of MTP11 in rice Mn tolerance using knockout lines. Old leaves presented higher levels of constitutively expressed MTP11 than other tissues and MTP11 expression was also found in reproductive organs. Fused MTP11:green fluorescent protein was co-localized to trans-Golgi markers and differentiated from other Golgi-associated markers. Knockout of MTP11 in wild-type rice did not affect tolerance and accumulation of Mn and other heavy metals, but knockout in the mtp8.1 mutant showed exacerbated Mn sensitivity at the vegetative growth stage. Knockout of MTP11 alone resulted in decreased grain yield and fertility at the reproductive stage. Thus, MTP11 is a trans-Golgi localized transporter for Mn, which plays a role in Mn tolerance through intracellular Mn compartmentalization. It is also required for maintaining high fertility in rice.

Characterization of rice KT/HAK/KUP potassium transporters and K+ uptake by HAK1 from Oryza sativa.

Plant high-affinity K+ (HAK) transporters are divided into four major clusters. Cluster I transporters, in particular, are thought to have high-affinity for K+. Of the 27 HAK genes in rice, eight HAK transporters belong to cluster I. In this study, we investigated the temporal expression patterns during K+ deficiency and K+ transport activity of these eight HAK transporters. The expression of seven HAK genes except OsHAK20 was detected. Expression of OsHAK1, OsHAK5 and OsHAK21 was induced in response to K+ deficiency; however, that of other genes was not. Six of the eight HAK transporters-OsHAK1, OsHAK5, OsHAK19, OsHAK20, OsHAK21, and OsHAK27-complemented the K+-transporter-deficient yeast or bacterial strain. Further, the yeast cells expressing OsHAK1 were more sensitive to Na+ than those expressing OsHAK5. Mutant analysis showed that the high-affinity K+ uptake activity was almost undetectable in oshak1 mutants in a low-K+ medium (0.02 mM). In addition, the high-affinity K+ uptake activity of wild-type plants was inhibited by mild salt stress (20 mM NaCl); however, Na+ permeability of OsHAK1 was not detected in Escherichia coli cells. The high-affinity K+ uptake activity by leaf blades was detected in wild-type plants, while it was not detected in oshak1 mutants. Our results suggest that OsHAK1 and OsHAK5 are the two important components of cluster I corresponding to low-K+ conditions, and that the transport activity of OsHAK1, unlike that of OsHAK5, is sensitive to Na+. Further, OsHAK1 is suggested to involve in foliar K+ uptake.

Rice reduces Mn uptake in response to Mn stress.

Rice (Oryza sativa L) is one of the most Mn-tolerant crops that can grow in submerged paddy fields, where the Mn concentration in soil solution is very high due to reduction. Although a large part of Mn is transferred from the roots to the shoot in rice, the roots are constantly exposed to high Mn concentrations in submerged paddies. Thus, mechanisms for preventing Mn overaccumulation in the cytoplasm of root cells are necessary. Recently, we showed that two cation diffusion facilitators, MTP8.1 and MTP8.2, play a crucial role in Mn tolerance in rice roots by sequestering Mn in vacuoles. Moreover, we observed that disruption of MTP8.1 and MTP8.2 resulted in reduced Mn accumulation under excess Mn. In the present study, we examined the effects of disruption of MTP8.1 and MTP8.2 on Mn uptake and determined that this phenotype is caused by a rapid and significant reduction of Mn uptake in response to excess Mn. Previously, we showed that Mn export from root cells through MTP9 was promoted by high Mn. Together, these findings suggest that optimal Mn concentration in rice roots is maintained by reduced uptake, vacuolar sequestration, and extrusion by cation diffusion facilitators.

The Tonoplast-Localized Transporter MTP8.2 Contributes to Manganese Detoxification in the Shoots and Roots of Oryza sativa L.

Manganese (Mn) cation diffusion facilitators (Mn-CDFs) play important roles in the Mn homeostasis of plants. In rice, the tonoplast-localized Mn-CDF metal tolerance protein 8.1 (MTP8.1) is involved in Mn detoxification in the shoots. This study functionally characterized the Mn-CDF MTP8.2 and determined its contribution to Mn tolerance. MTP8.2 was found to share 68% identity with MTP8.1 and was expressed in both the shoots and roots, but its transcription level was lower than that of MTP8.1. Transient expression of the MTP8.2:green fluorescent protein (GFP) fusion protein and immunoblotting studies indicated that MTP8.2 was also localized to the tonoplast. MTP8.2 expression in yeast conferred tolerance to Mn but not to Fe, Zn, Co, Ni or Cd. MTP8.2 knockdown caused further growth reduction of shoots and roots in the mtp8.1 mutant, which already exhibits stunted growth under conditions of excess Mn. In the presence of high Mn, the MTP8.2 knockdown lines of the mtp8.1 mutant showed lower root Mn concentrations, as well as lower root:total Mn ratios, than those of wild-type rice and the mtp8.1 mutant. These findings indicate that MTP8.2 mediates Mn tolerance along with MTP8.1 through the sequestration of Mn into the shoot and root vacuoles.

A polarly localized transporter for efficient manganese uptake in rice.

Manganese is an essential metal for plant growth. A number of transporters involved in the uptake of manganese from soils, and its translocation to the shoot, have been identified in Arabidopsis and rice. However, the transporter responsible for the radial transport of manganese out of root exodermis and endodermis cells and into the root stele remains unknown. Here, we show that metal tolerance protein 9 (MTP9), a member of the cation diffusion facilitator family, is a critical player in this process in rice (Oryza sativa). We find that MTP9 is mainly expressed in roots, and that the resulting protein is localized to the plasma membrane of exo- and endodermis cells, at the proximal side of these cell layers (opposite the manganese uptake transporter Nramp5, which is found at the distal side). We demonstrate that MTP9 has manganese transport activity by expression in proteoliposomes and yeast, and show that knockout of MTP9 in rice reduces manganese uptake and its translocation to shoots. We conclude that at least in rice MTP9 is required for manganese translocation to the root stele, and thereby manganese uptake.

Mn tolerance in rice is mediated by MTP8.1, a member of the cation diffusion facilitator family.

Manganese (Mn) is an essential micronutrient for plants, but is toxic when present in excess. The rice plant (Oryza sativa L.) accumulates high concentrations of Mn in the aerial parts; however, the molecular basis for Mn tolerance is poorly understood. In the present study, genes encoding Mn tolerance were screened for by expressing cDNAs of genes from rice shoots in Saccharomyces cerevisiae. A gene encoding a cation diffusion facilitator (CDF) family member, OsMTP8.1, was isolated, and its expression was found to enhance Mn accumulation and tolerance in S. cerevisiae. In plants, OsMTP8.1 and its transcript were mainly detected in shoots. High or low supply of Mn moderately induced an increase or decrease in the accumulation of OsMTP8.1, respectively. OsMTP8.1 was detected in all cells of leaf blades through immunohistochemistry. OsMTP8.1 fused to green fluorescent protein was localized to the tonoplast. Disruption of OsMTP8.1 resulted in decreased chlorophyll levels, growth inhibition in the presence of high concentrations of Mn, and decreased accumulation of Mn in shoots and roots. However, there was no difference in the accumulation of other metals, including Zn, Cu, Fe, Mg, Ca, and K. These results suggest that OsMTP8.1 is an Mn-specific transporter that sequesters Mn into vacuoles in rice and is required for Mn tolerance in shoots.

Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of Thlaspi caerulescens.

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.

Physiological, genetic, and molecular characterization of a high-Cd-accumulating rice cultivar, Jarjan.

Cadmium (Cd) in rice is a major source of Cd intake for people on a staple rice diet. The mechanisms underlying Cd accumulation in rice plant are still poorly understood. Here, we characterized the physiology and genetics of Cd transport in a high-Cd-accumulating cultivar (Jarjan) of rice (Oryza sativa). Jarjan showed 5- to 34-fold higher Cd accumulation in the shoots and grains than the cultivar Nipponbare, when it was grown in either a non-Cd-contaminated or a Cd-contaminated soil. A short-term uptake experiment showed no significant difference in Cd uptake by the roots between the two cultivars. However, Jarjan translocated 49% of the total Cd taken up to the shoots, whereas Nipponbare retained most of the Cd in the roots. In both concentration- and time-dependent experiments, Jarjan showed a superior capacity for root-to-shoot translocation of Cd. These results indicate that the high-Cd-accumulation phenotype in Jarjan results from efficient translocation of Cd from roots to shoots. Genetic analysis using an F(2) population derived from Jarjan and Nipponbare revealed that plants showing high- and low-Cd-accumulation phenotypes segregated in a 1:3 ratio, indicating that high accumulation in Jarjan is controlled by a single recessive gene. Furthermore, we isolated OsHMA3, a gene encoding a tonoplast-localized Cd transporter from Jarjan. The OsHMA3 protein was localized in all roots cells, but the sequence has a mutation leading to loss of function. Therefore, failure to sequester Cd into the root vacuoles by OsHMA3 is probably responsible for high Cd accumulation in Jarjan.

Gene limiting cadmium accumulation in rice.

Intake of toxic cadmium (Cd) from rice caused Itai-itai disease in the past and it is still a threat for human health. Therefore, control of the accumulation of Cd from soil is an important food-safety issue, but the molecular mechanism for the control is unknown. Herein, we report a gene (OsHMA3) responsible for low Cd accumulation in rice that was isolated from a mapping population derived from a cross between a high and low Cd-accumulating cultivar. The gene encodes a transporter belonging to the P(1B)-type ATPase family, but shares low similarity with other members. Heterologous expression in yeast showed that the transporter from the low-Cd cultivar is functional, but the transporter from the high-Cd cultivar had lost its function, probably because of the single amino acid mutation. The transporter is mainly expressed in the tonoplast of root cells at a similar level in both the low and high Cd-accumulating cultivars. Overexpression of the functional gene from the low Cd-accumulating cultivar selectively decreased accumulation of Cd, but not other micronutrients in the grain. Our results indicated that OsHMA3 from the low Cd-accumulating cultivar limits translocation of Cd from the roots to the above-ground tissues by selectively sequestrating Cd into the root vacuoles.

Identification of a novel major quantitative trait locus controlling distribution of Cd between roots and shoots in rice.

Accumulation of Cd in rice grain is a serious concern of food safety since rice as a staple food is a major source of Cd intake in Asian countries. However, the mechanisms controlling Cd accumulation in rice are still poorly understood. Herein, we report both physiological and genetic analysis of two rice cultivars contrasting in Cd accumulation, which were screened from a core collection of rice cultivars. The cultivar Anjana Dhan (Indica) accumulated much higher levels of Cd than Nipponbare (Japonica) in the shoots and grains when grown in both soil and solution culture. A short-term uptake experiment (20 min) showed that Cd uptake by Nipponbare was higher than that by Anjana Dhan. However, the concentration of Cd in the shoot and xylem sap was much higher in Anjana Dhan than in Nipponbare. Of the Cd taken up by the roots, <4% was translocated to the shoots in Nipponbare, compared with 10-25% in Anjana Dhan, indicating a higher root-to-shoot translocation of Cd in the latter. A quantitative trait locus (QTL) analysis for Cd accumulation was performed using an F(2) population derived from Anjana Dhan and Nipponbare. A QTL with large effect for Cd accumulation was detected on the short arm of chromosome 7, explaining 85.6% of the phenotypic variance in the shoot Cd concentration of the F(2) population. High accumulation is likely to be controlled by a single recessive gene. A candidate genomic region was defined to <1.9 Mb by means of substitution mapping.

Further characterization of ferric-phytosiderophore transporters ZmYS1 and HvYS1 in maize and barley.

Roots of some gramineous plants secrete phytosiderophores in response to iron deficiency and take up Fe as a ferric-phytosiderophore complex through the transporter YS1 (Yellow Stripe 1). Here, this transporter in maize (ZmYS1) and barley (HvYS1) was further characterized and compared in terms of expression pattern, diurnal change, and tissue-type specificity of localization. The expression of HvYS1 was specifically induced by Fe deficiency only in barley roots, and increased with the progress of Fe deficiency, whereas ZmYS1 was expressed in maize in the leaf blades and sheaths, crown, and seminal roots, but not in the hypocotyl. HvYS1 expression was not induced by any other metal deficiency. Furthermore, in maize leaf blades, the expression was higher in the young leaf blades showing severe chlorosis than in the old leaf blades showing no chlorosis. The expression of HvYS1 showed a distinct diurnal rhythm, reaching a maximum before the onset of phytosiderophore secretion. In contrast, ZmYS1 did not show such a rhythm in expression. Immunostaining showed that ZmYS1 was localized in the epidermal cells of both crown and lateral roots, with a polar localization at the distal side of the epidermal cells. In maize leaves, ZmYS1 was localized in mesophyll cells, but not epidermal cells. These differences in gene expression pattern and tissue-type specificity of localization suggest that HvYS1 is only involved in primary Fe acquisition by barley roots, whereas ZmYS1 is involved in both primary Fe acquisition and intracellular transport of iron and other metals in maize.

OsFRDL1 is a citrate transporter required for efficient translocation of iron in rice.

Multidrug and toxic compound extrusion (MATE) transporters represent a large family in plants, but their functions are poorly understood. Here, we report the function of a rice (Oryza sativa) MATE gene (Os03g0216700, OsFRDL1), the closest homolog of barley (Hordeum vulgare) HvAACT1 (aluminum [Al]-activated citrate transporter 1), in terms of metal stress (iron [Fe] deficiency and Al toxicity). This gene was mainly expressed in the roots and the expression level was not affected by either Fe deficiency or Al toxicity. Knockout of this gene resulted in leaf chlorosis, lower leaf Fe concentration, higher accumulation of zinc and manganese concentration in the leaves, and precipitation of Fe in the root's stele. The concentration of citrate and ferric iron in the xylem sap was lower in the knockout line compared to the wild-type rice. Heterologous expression of OsFRDL1 in Xenopus oocytes showed transport activity for citrate. Immunostaining showed that OsFRDL1 was localized at the pericycle cells of the roots. On the other hand, there was no difference in the Al-induced secretion of citrate from the roots between the knockout line and the wild-type rice. Taken together, our results indicate that OsFRDL1 is a citrate transporter localized at the pericycle cells, which is necessary for efficient translocation of Fe to the shoot as a Fe-citrate complex.

Characterization of Cd translocation and identification of the Cd form in xylem sap of the Cd-hyperaccumulator Arabidopsis halleri.

Arabidopsis halleri is a Cd hyperaccumulator; however, the mechanisms involved in the root to shoot translocation of Cd are not well understood. In this study, we characterized Cd transfer from the root medium to xylem in this species. Arabidopsis halleri accumulated 1,500 mg kg(-1) Cd in the shoot without growth inhibition. A time-course experiment showed that the release of Cd into the xylem was very rapid; by 2 h exposure to Cd, Cd concentration in the xylem sap was 5-fold higher than that in the external solution. The concentration of Cd in the xylem sap increased linearly with increasing Cd concentration in the external solution. Cd transfer to the xylem was completely inhibited by the metabolic inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cd concentration in the xylem sap was decreased by increasing the concentration of external Zn, but enhanced by Fe deficiency treatment. Analysis with 113Cd-nuclear magnetic resonance (NMR) showed that the chemical shift of 113Cd in the xylem sap was the same as that of Cd(NO3)2. Metal speciation with Geochem-PC also showed that Cd occurred mainly in the free ionic form in the xylem sap. These results suggest that Cd transfer from the root medium to the xylem in A. halleri is an energy-dependent process that is partly shared with Zn and/or Fe transport. Furthermore, Cd is translocated from roots to shoots in inorganic forms.

Mutation in nicotianamine aminotransferase stimulated the Fe(II) acquisition system and led to iron accumulation in rice.

Higher plants acquire iron (Fe) from the rhizosphere through two strategies. Strategy II, employed by graminaceous plants, involves secretion of phytosiderophores (e.g. deoxymugineic acid in rice [Oryza sativa]) by roots to solubilize Fe(III) in soil. In addition to taking up Fe in the form of Fe(III)-phytosiderophore, rice also possesses the strategy I-like system that may absorb Fe(II) directly. Through mutant screening, we isolated a rice mutant that could not grow with Fe(III)-citrate as the sole Fe source, but was able to grow when Fe(II)-EDTA was supplied. Surprisingly, the mutant accumulated more Fe and other divalent metals in roots and shoots than the wild type when both were supplied with EDTA-Fe(II) or grown under water-logged field conditions. Furthermore, the mutant had a significantly higher concentration of Fe in both unpolished and polished grains than the wild type. Using the map-based cloning method, we identified a point mutation in a gene encoding nicotianamine aminotransferase (NAAT1), which was responsible for the mutant phenotype. Because of the loss of function of NAAT1, the mutant failed to produce deoxymugineic acid and could not absorb Fe(III) efficiently. In contrast, nicotianamine, the substrate for NAAT1, accumulated markedly in roots and shoots of the mutant. Microarray analysis showed that the expression of a number of the genes involved in Fe(II) acquisition was greatly stimulated in the naat1 mutant. Our results demonstrate that disruption of deoxymugineic acid biosynthesis can stimulate Fe(II) acquisition and increase iron accumulation in rice.

A specific transporter for iron(III)-phytosiderophore in barley roots.

Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of the iron-chelating phytosiderophore, mugineic acid (MA), and by a specific uptake system for iron(III)-phytosiderophore complexes. We identified a gene specifically encoding an iron-phytosiderophore transporter (HvYS1) in barley, which is the most tolerant species to iron deficiency among graminaceous plants. HvYS1 was predicted to encode a polypeptide of 678 amino acids and to have 72.7% identity with ZmYS1, a first protein identified as an iron(III)-phytosiderophore transporter in maize. Real-time RT-PCR analysis showed that the HvYS1 gene was mainly expressed in the roots, and its expression was enhanced under iron deficiency. In situ hybridization analysis of iron-deficient barley roots revealed that the mRNA of HvYS1 was localized in epidermal root cells. Furthermore, immunohistological staining with anti-HvYS1 polyclonal antibody showed the same localization as the mRNA. HvYS1 functionally complemented yeast strains defective in iron uptake on media containing iron(III)-MA, but not iron-nicotianamine (NA). Expression of HvYS1 in Xenopus oocytes showed strict specificity for both metals and ligands: HvYS1 transports only iron(III) chelated with phytosiderophore. The localization and substrate specificity of HvYS1 is different from those of ZmYS1, indicating that HvYS1 is a specific transporter for iron(III)-phytosiderophore involved in primary iron acquisition from soil in barley roots.

Identification of the form of Cd in the leaves of a superior Cd-accumulating ecotype of Thlaspi caerulescens using 113Cd-NMR.

Thlaspi caerulescens (Ganges ecotype) is a known Cd hyperaccumulator, however, the ligands which coordinate to Cd ions in the leaves have not been identified. In the present study, the chemical form of Cd was investigated by using 113Cd-nuclear magnetic resonance (NMR) spectroscopy. Plants were grown hydroponically with a highly enriched 113Cd stable isotope. Measurements of 113Cd-NMR with intact leaves showed a signal at the chemical shift of around -16 ppm. Crude leaf sap also gave a similar chemical shift. Purification by gel filtration (Sephadex G-10), followed by cationic and anionic exchange chromatography, showed that Cd occurred only in the anionic fraction, which gave the same chemical shift as intact leaves. Further purification of the anionic fraction, combined with 113Cd- and 1H-NMR studies, revealed that only the fraction containing malate showed a chemical shift similar to the intact leaves. These results indicate that Cd was coordinated mainly with malate in the leaves of T. caerulescens. The malate concentration in the leaves was not affected by increasing Cd concentration in the solution, suggesting that malate synthesis is not induced by Cd. Because the Cd-malate complex is relatively weak, we suggest that the complex forms inside the vacuoles as a result of an efficient tonoplast transport of Cd and a constitutively high concentration of malate in the vacuoles, and that the formation of the Cd-malate complex may lead to a decrease of subsequent Cd efflux to the cytoplasm.

Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of Thlaspi caerulescens.

Thlaspi caerulescens (Ganges ecotype) is able to accumulate large concentrations of cadmium (Cd) and zinc (Zn) in the leaves without showing any toxicity, suggesting a strong internal detoxification. The distribution of Cd and Zn in the leaves was investigated in the present study. Although the Cd and Zn concentrations in the epidermal tissues were 2-fold higher than those of mesophyll tissues, 65-70% of total leaf Cd and Zn were distributed in the mesophyll tissues, suggesting that mesophyll is a major storage site of the two metals in the leaves. To examine the subcellular localisation of Cd and Zn in mesophyll tissues, protoplasts and vacuoles were isolated from plants exposed to 50 muM Cd and Zn hydroponically. Pure protoplasts and vacuoles were obtained based on light-microscopic observation and the activities of marker enzymes of cytosol and vacuoles. Of the total Cd and Zn in the mesophyll tissues, 91% and 77%, respectively, were present in the protoplast, and all Cd and 91% Zn in the protoplast were localised in the vacuoles. Furthermore, about 70% and 86% of total Cd and Zn, respectively, in the leaves were extracted in the cell sap, suggesting that most Cd and Zn in the leaves is present in soluble form. These results indicate that internal detoxification of Cd and Zn in Thlaspi caerulescens leaves is achieved by vacuolar compartmentalisation.