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.Contamination of arable soil with cadmium (Cd) is one of the most serious agricultural problems in the world. Crops, particularly irrigated rice (Oryza sativa), are generally suggested as the main source of Cd intake by humans (United Nations Environment Programme, 2008). From the viewpoint of plant nutrition, the dynamics and mechanisms of Cd transition from the soil to the edible parts in the plants should be elucidated.Generally, the process of metal accumulation in plants mainly consists of uptake from the soil by the roots, xylem loading and transport, and distribution between metal sinks in the aerial parts (Clemens et al., 2002). It has been demonstrated that xylem loading and transport but not absorption by the roots is one of the rate-controlling steps for Cd accumulation in the grain of graminaceous plants. Hart et al. (1998) reported that greater Cd accumulation in durum wheat grain than in bread wheat grain was not correlated with the root influx rates of these cultivars. Harris and Taylor (2004) employed two near-isogenic lines of durum wheat that differ in grain Cd accumulation and showed that elevated activity of root-to-shoot transport of Cd was responsible for the higher accumulation of Cd in grain, but Cd uptake by roots was not. Uraguchi et al. (2009) analyzed two rice cultivars that show different levels of Cd accumulation in the grain and concluded that root-to-shoot Cd translocation via xylem is the major process determining the Cd accumulation level in rice grain. Phloem transport has also been considered a key step of Cd translocation to the grain, because xylem transport is directed mainly to the organs of highest transpiration, such as leaves, but not to the sites of highest demand for mineral, such as grains (Marschner, 1995). Cd was detected in the phloem sap of rice collected from leaf sheaths (Tanaka et al., 2003) and from the uppermost internode at the grain-filling stage (Tanaka et al., 2007). Tanaka et al. (2007) also estimated that 91% to 100% of Cd in rice grains is deposited from the phloem. In wheat, it has been observed in a split-root system that Cd fed to one bundle of the roots moved into the other bundle, probably via the phloem (Welch et al., 1999; Page and Feller, 2005), and steam girdling, which stops only phloem transport but not xylem flow, to the peduncle below the ear reduced Cd translocation to the grain (Riesen and Feller, 2005). These results suggest that the xylem loading and transport is the first rate-controlling step of Cd transition from the soil to the grain in graminaceous plants and the phloem transport and unloading into the grain is the last. However, the intermediate steps between the xylem and phloem transport of Cd have not been clarified. In general, it is known that mineral micronutrients are remobilized from senescent leaves to the phloem at the reproductive stage or transferred from the xylem to the phloem directly via transfer cells (Marschner, 1995; Clemens et al., 2002). Therefore, where and how the xylem-to-phloem transfer of Cd occurs is of considerable interest. The main objective of this study was to quantitatively describe the whole route and the time scale of Cd transition from the soil to the grain in rice at the vegetative and reproductive stages.In this study, we also raised a methodological challenge. We employed a positron-emitting tracer imaging system (PETIS), one of the most advanced radiotracer-based imaging methods available today. PETIS provides serial time-course images (i.e. animation) of the two-dimensional distribution of a radiotracer within a living organism without contact. Its principle is the same as that of positron emission tomography, which has been widely used for medical diagnosis, but PETIS was specially designed for studying plants and has been applied to many studies on plant nutrition over the last decade (Uchida et al., 2003; Fujimaki, 2007; Fujimaki et al., 2010). Recently, the transport of metals, including iron (Ishimaru et al., 2006, 2007; Tsukamoto et al., 2009), zinc (Suzuki et al., 2006), and manganese (Tsukamoto et al., 2006), in intact graminaceous plants has been visualized using PETIS. Furthermore, the time course of tracer amounts within any selected region of interest (ROI) on the obtained image can be easily generated and applied for mathematical analyses because PETIS provides highly quantitative images. The rates of photoassimilation and the velocities of phloem transport in intact plants under various environmental conditions have been estimated quantitatively using PETIS (Matsuhashi et al., 2005; Kawachi et al., 2006). However, to our knowledge, no study has been carried out to describe the whole dynamics and kinetics of a substance in an intact plant body by taking full advantage of PETIS, namely noninvasive visualization and quantitative time-course analysis. The second objective of this study was to demonstrate the potential of the latest radiotracer imaging technology for plant physiology.     

Expression in Arabidopsis and cellular localization reveal involvement of rice NRAMP,OsNRAMP1, in arsenic transport and tolerance

Irrigation of paddy fields to arsenic (As) containing groundwater leads to As accumulation in rice grains and causes serious health risk to the people worldwide. To reduce As intake via consumption of contaminated rice grain, identification of the mechanisms for As accumulation and detoxification in rice is a prerequisite. Herein, we report involvement of a member of rice NRAMP (Natural Resistance‐Associated Macrophage Protein) transporter, OsNRAMP1, in As, in addition to cadmium (Cd), accumulation through expression in yeast and Arabidopsis. Expression of OsNRAMP1 in yeast mutant (fet3fet4) rescued iron (Fe) uptake and exhibited enhanced accumulation of As and Cd. Expression of OsNRAMP1 in Arabidopsis provided tolerance with enhanced As and Cd accumulation in root and shoot. Cellular localization revealed that OsNRAMP1 resides on plasma membrane of endodermis and pericycle cells and may assist in xylem loading for root to shoot mobilization. This is the first report demonstrating role of NRAMP in xylem mediated loading and enhanced accumulation of As and Cd in plants. We propose that genetic modification of OsNRAMP1 in rice might be helpful in developing rice with low As and Cd content in grain and minimize the risk of food chain contamination to these toxic metals.     

Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation

Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC–Cd and glutathione–Cd complexes.     

水稻镉积累特性的生理和分子机制研究概述

我国的稻米镉超标对国民身体健康造成严重威胁, 而选育低镉积累的水稻(Oryza sativa)品种是降低稻米镉含量行之有效的策略, 因此有必要了解水稻对镉的积累特性及其生理过程和相关功能基因。该文概述了镉在水稻根部的吸收、木质部中的装载与运输、茎节中的分配、叶片中再分配以及籽粒镉积累等过程的生理和分子机制研究进展, 以期为低镉水稻的选育和安全生产提供理论参考。     

Cadmium distribution in the root tissues of solanaceous plants with contrasting root-to-shoot Cd translocation efficiencies

Root-to-shoot cadmium (Cd) translocation in Solanum torvum is lower than that of the eggplant Solanum melongena; therefore, grafting S. melongena onto S. torvum rootstock can effectively reduce the Cd concentration in eggplant fruits. We hypothesized that Cd transport in S. torvum roots is restricted in the path between the epidermis and xylem vessel; hence, we investigated the Cd distribution in the roots at the micron-scale. Elemental maps of Cd, Zn and Fe accumulation in S. melongena and S. torvum root sections were obtained by synchrotron micro X-ray fluorescence spectrometry. The Cd was localized in both the stele and the epidermis of the S. melongena root cross sections regardless of the distance from the root apex. In S. torvum root sections taken at 30 and 40 mm above the root apex, a higher abundance of Cd was found within the cells of the endodermis and pericycle. The results suggested that the symplastic uptake and xylem loading of Cd in S. torvum roots were restricted, and thereby, the Cd that was unable to be loaded into the xylem accumulated in the endodermis and in the pericycle. Because symplastic uptake differs only slightly between the two species, the difference in xylem loading would explain the comparatively lower Cd concentration in S. torvum shoots.     

Enhanced Sucrose Loading Improves Rice Yield by Increasing Grain Size

Yield in cereals is a function of grain number and size. Sucrose (Suc), the main carbohydrate product of photosynthesis in higher plants, is transported long distances from source leaves to sink organs such as seeds and roots. Here, we report that transgenic rice plants (Oryza sativa) expressing the Arabidopsis (Arabidopsis thaliana) phloem-specific Suc transporter (AtSUC2), which loads Suc into the phloem under control of the phloem protein2 promoter (pPP2), showed an increase in grain yield of up to 16% relative to wild-type plants in field trials. Compared with wild-type plants, pPP2::AtSUC2 plants had larger spikelet hulls and larger and heavier grains. Grain filling was accelerated in the transgenic plants, and more photoassimilate was transported from the leaves to the grain. In addition, microarray analyses revealed that carbohydrate, amino acid, and lipid metabolism was enhanced in the leaves and grain of pPP2::AtSUC2 plants. Thus, enhancing Suc loading represents a promising strategy to improve rice yield to feed the global population.Rice (Oryza sativa) is a staple food for nearly one-half of the global population. Given the rapid growth of the world’s population, there is an urgent need to increase rice yield. Rice yield is a complex trait that is directly associated with grain size, panicle number, and the number of grains per panicle (Xing and Zhang, 2010). Increasing grain size is a prime breeding target, and several genes known to control rice grain size, such as GRAIN SIZE3 (GS3), GS5, GW2 QTL for rice grain width and weight (GW2), GW8, and rice seed width5, have been identified (Fan et al., 2006; Song et al., 2007; Shomura et al., 2008; Li et al., 2011a; Wang et al., 2012). However, our knowledge of the mechanisms that control rice yield is limited. Thus, further improving rice yield remains a challenge for breeders (Sakamoto and Matsuoka, 2008). Identifying and characterizing unique genes or targets that regulate yield traits would improve our understanding of the molecular mechanisms that regulate yield traits and facilitate the breeding of new rice varieties with higher yields.The carbohydrates in rice grains originate from photosynthesis that is carried out predominantly in leaves (sources). Therefore, grain filling and rice yield depend on the efficient transport of carbohydrates from the leaves to seeds (sinks). In most plants, Suc is the main carbohydrate transported long distance in the veins to support the growth and development of roots, flowers, fruits, and seeds (Baker et al., 2012; Braun, 2012). Recently, the entire pathway for the export of Suc from leaves has been elucidated (Baker et al., 2012; Braun, 2012). Suc is synthesized in leaf mesophyll cells and diffuses from cell to cell through plasmodesmata until it reaches the phloem parenchyma cells (Slewinski and Braun, 2010). The SWEET transporters mediate Suc efflux from the phloem parenchyma cells into the apoplast, where Suc is subsequently loaded into the phloem sieve element-companion cell (SE/CC) complexes by Suc transporters (SUTs; Braun and Slewinski, 2009; Ayre, 2011; Chen et al., 2012). The resultant accumulation of Suc in sieve elements produces a hydrostatic pressure gradient that results in the bulk flow of Suc through a conduit of contiguous sieve elements, leading to its arrival and unloading in sink tissues (Lalonde et al., 2004; Baker et al., 2012).Genetic evidence has demonstrated that apoplastic Suc phloem loading is critical for growth, development, and reproduction in Arabidopsis (Arabidopsis thaliana). AtSWEET11 and AtSWEET12 are localized to the plasma membrane of the phloem and are expressed in a subset of phloem parenchyma cells in minor veins. These transporters mediate Suc efflux from phloem parenchyma cells into the apoplast prior to Suc uptake by SE/CC (Chen et al., 2012). The atsweet11 or atsweet12 single mutants exhibit no aberrant phenotypes, possibly due to genetic redundancy. However, atsweet11;12 double mutants are mildly chlorotic and display slower growth and higher levels of starch and sugar accumulation in the leaves than do wild-type plants (Chen et al., 2012). Arabidopsis phloem-specific sucrose transporter (AtSUC2) is a phloem-specific SUT that is expressed specifically in companion cells (Stadler and Sauer, 1996). AtSUC2 plays an essential role in phloem Suc loading and is necessary for efficient Suc transport from source to sink tissues in Arabidopsis (Stadler and Sauer, 1996; Gottwald et al., 2000; Srivastava et al., 2008). The atsuc2 mutants show stunted growth, retarded development, and sterility. Furthermore, these mutants accumulate excess starch in the leaves and fail to transport sugar efficiently to the roots and inflorescences (Gottwald et al., 2000).The proper control of carbohydrate partitioning is fundamental to crop yield (Braun, 2012). It has been reported that increasing sink grain strength by improving assimilate uptake capacity could be a promising approach toward obtaining higher yield. For example, seed-specific overexpression of a potato (Solanum tuberosum) SUT increased Suc uptake and growth rates of developing pea (Pisum sativum) cotyledons (Rosche et al., 2002). In addition, the Suc uptake capacity of grains and storage protein biosynthesis was increased in transgenic wheat (Triticum aestivum) plants expressing the barley (Hordeum vulgare) SUT HvSUT1 under the control of an endosperm-specific promoter (Weichert et al., 2010). Moreover, it was recently found that these transgenic wheat plants had a higher thousand grain weight and grain width and length, as well as a 28% increase in grain yield (Saalbach et al., 2014).Since the carbohydrates in rice grains originate from photosynthesis in source leaves, and carbohydrate partitioning from source leaves to heterotrophic sinks (e.g. seeds) is mediated by Suc transport in plants (Lalonde et al., 2004; Ayre, 2011), enhancing the capacity for Suc transport from leaves to seeds theoretically could increase crop yield. However, until now, enhancing Suc transport from leaves to seeds has not been shown to improve yield (Ainsworth and Bush, 2011).Here, we tested the hypothesis that enhancing Suc transport from leaves to seeds would increase rice yield. We expressed Arabidopsis SUC2 under control of the phloem protein2 promoter (pPP2) in rice and found that enhancing Suc loading did indeed increase rice yield. The pPP2::AtSUC2 plants produced larger grain than the wild type and showed grain yield increases of up to 16% in field trials. Our results suggest that manipulating phloem Suc transport is a useful strategy for increasing grain yield in rice and other cereal crops.     

Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Phloem unloading via the apoplastic pathway is essential for shoot distribution and long-distance translocation of root-synthesized cytokinins from the root to the shoot through the xylem.     

Cadmium translocation and accumulation in developing barley grains

Soil cadmium (Cd) contamination has posed a serious problem for safe food production and become a potential agricultural and environmental hazard worldwide. In order to study the transport of Cd into the developing grains, detached ears of two-rowed barley cv. ZAU 3 were cultured in Cd stressed nutrient solution containing the markers for phloem (rubidium) and xylem (strontium) transport. Cd concentration in each part of detached spikes increased with external Cd levels, and Cd concentration in various organs over the three Cd levels of 0.5, 2, 8 μM Cd on 15-day Cd exposure was in the order: awn > stem > grain > rachis > glume, while the majority of Cd was accumulated in grains with the proportion of 51.0% relative to the total Cd amount in the five parts of detached spikes. Cd accumulation in grains increased not only with external Cd levels but the time of exposure contrast to stem, awn, rachis and glume. Those four parts of detached spike showed increase Cd accumulation for 5 days, followed by sharp decrease till day 10 and increase again after 12.5 days. Awn-removal and stem-girdling markedly decreased Cd concentration in grains, and sucrose or zinc (Zn) addition to the medium and higher relative humidity (RH) also induced dramatic reduction in Cd transport to developing grains. The results indicated that awn, rachis and glume may involve in Cd transport into developing grains, and suggested that Cd redistribution in maturing cereals be considered as an important physiological process influencing the quality of harvested grains. Our results suggested that increasing RH to 90% and Zn addition in the medium at grain filling stage would be beneficial to decrease Cd accumulation in grains.     

Xylem loading process is a critical factor in determining Cd accumulation in the shoots of Solanum melongena and Solanum torvum

Cadmium (Cd) concentration in eggplant (Solanum melongena) fruits can be drastically reduced by grafting them with Solanum torvum rootstock. We thus examined the characteristics of Cd absorption in roots and Cd translocation from roots to shoots between S. melongena and S. torvum over 7 days using a hydroponic culture. Although there is no significant difference in Cd concentration in the roots of S. melongena and S. torvum, Cd concentration in the shoots and xylem sap was higher in S. melongena than in S. torvum. By evaluating symplastic Cd absorption in roots, using enriched isotopes ¹¹³Cd and ¹¹⁴Cd, and measuring the kinetics in xylem loading, we characterized Cd absorption and translocation for S. torvum (low Cd translocation) and S. melongena (high Cd translocation). A concentration-dependent study in roots indicated that K_m values were almost the same for species, but the V_max value was 1.5-fold higher in S. melongena than in S. torvum. A concentration-dependent study in xylem loading indicated that V_max was almost the same, but K_m values were approximately 7-fold higher in S. torvum compared to S. melongena. These results, together, suggest that the affinity for Cd in the xylem loading process is a critical factor for determining the different Cd concentrations in the shoots between both plants under low Cd concentration conditions. In addition, a metabolic inhibitor, carbonyl cyanide-m-chloro-phenyl-hydrazone (CCCP) inhibited Cd absorption and translocation from roots to shoots in both plants. This suggests that Cd absorption in roots and Cd translocation from roots to shoots via the xylem loading process, under low Cd concentration conditions, are partly mediated by an active energy-dependent process in both plants.     

The Arabidopsis Nitrate Transporter NRT1.8 Functions in Nitrate Removal from the Xylem Sap and Mediates Cadmium Tolerance

Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-β-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd²⁺) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd²⁺-sensitive phenotype. Further analyses showed that Cd²⁺ stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd²⁺ tolerance.     

Role of the iron transporter OsNRAMP1 in cadmium uptake and accumulation in rice

The heavy metal cadmium (Cd) is toxic to humans, and its accumulation in rice grains is a major agricultural problem. Rice has seven putative metal transporter NRAMP genes, but microarray analysis showed that only OsNRAMP1 is highly up-regulated by iron (Fe) deficiency. OsNRAMP1 localized to the plasma membrane and transported Cd as well as Fe. OsNRAMP1 expression was observed mainly in roots and was higher in the roots of a high-Cd-accumulating cultivar (Habataki) than in those of a low-Cd-accumulating cultivar (Sasanishiki). The amino acid sequence of OsNRAMP1 in the Sasanishiki and Habataki cultivars was found to be 100% identical. These results suggest that OsNRAMP1 participates in cellular Cd uptake and that the differences observed in Cd accumulation among cultivars are because of differences in OsNRAMP1 expression levels in roots.     

Regulation of Sulfur Nutrition in Wild-Type and Transgenic Poplar Over-Expressing γ-Glutamylcysteine Synthetase in the Cytosol as Affected by Atmospheric H2S

This study with poplar (Populus tremula × Populus alba) cuttings was aimed to test the hypothesis that sulfate uptake is regulated by demand-driven control and that this regulation is mediated by phloem-transported glutathione as a shoot-to-root signal. Therefore, sulfur nutrition was investigated at (a) enhanced sulfate demand in transgenic poplar over-expressing γ-glutamylcysteine (γ-EC) synthetase in the cytosol and (b) reduced sulfate demand during short-term exposure to H₂S. H₂S taken up by the leaves increased cysteine, γ-EC, and glutathione concentrations in leaves, xylem sap, phloem exudate, and roots, both in wild-type and transgenic poplar. The observed reduced xylem loading of sulfate after H₂S exposure of wild-type poplar could well be explained by a higher glutathione concentration in the phloem. In transgenic poplar increased concentrations of glutathione and γ-EC were found not only in leaves, xylem sap, and roots but also in phloem exudate irrespective of H₂S exposure. Despite enhanced phloem allocation of glutathione and its accumulation in the roots, sulfate uptake was strongly enhanced. This finding is contradictory to the hypothesis that glutathione allocated in the phloem reduces sulfate uptake and its transport to the shoot. Correlation analysis provided circumstantial evidence that the sulfate to glutathione ratio in the phloem may control sulfate uptake and loading into the xylem, both when the sulfate demand of the shoot is increased and when it is reduced.     

Role of the node in controlling traffic of cadmium, zinc, and manganese in rice

Heavy metals are transported to rice grains via the phloem. In rice nodes, the diffuse vascular bundles (DVBs), which enclose the enlarged elliptical vascular bundles (EVBs), are connected to the panicle and have a morphological feature that facilitates xylem-to-phloem transfer. To find a mechanism for restricting cadmium (Cd) transport into grains, the distribution of Cd, zinc (Zn), manganese (Mn), and sulphur (S) around the vascular bundles in node I (the node beneath the panicle) of Oryza sativa 'Koshihikari' were compared 1 week after heading. Elemental maps of Cd, Zn, Mn, and S in the vascular bundles of node I were obtained by synchrotron micro-X-ray fluorescence spectrometry and electron probe microanalysis. In addition, Cd K-edge microfocused X-ray absorption near-edge structure analyses were used to identify the elements co-ordinated with Cd. Both Cd and S were mainly distributed in the xylem of the EVB and in the parenchyma cell bridge (PCB) surrounding the EVB. Zn accumulated in the PCB, and Mn accumulated around the protoxylem of the EVB. Cd was co-ordinated mainly with S in the xylem of the EVB, but with both S and O in the phloem of the EVB and in the PCB. The EVB in the node retarded horizontal transport of Cd toward the DVB. By contrast, Zn was first stored in the PCB and then efficiently transferred toward the DVB. Our results provide evidence that transport of Cd, Zn, and Mn is differentially controlled in rice nodes, where vascular bundles are functionally interconnected.     

Grain setting defect1, Encoding a Remorin Protein,Affects the Grain Setting in Rice through Regulating Plasmodesmatal Conductance

Effective grain filling is one of the key determinants of grain setting in rice (Oryza sativa). Grain setting defect1 (GSD1), which encodes a putative remorin protein, was found to affect grain setting in rice. Investigation of the phenotype of a transfer DNA insertion mutant (gsd1-Dominant) with enhanced GSD1 expression revealed abnormalities including a reduced grain setting rate, accumulation of carbohydrates in leaves, and lower soluble sugar content in the phloem exudates. GSD1 was found to be specifically expressed in the plasma membrane and plasmodesmata (PD) of phloem companion cells. Experimental evidence suggests that the phenotype of the gsd1-Dominant mutant is caused by defects in the grain-filling process as a result of the impaired transport of carbohydrates from the photosynthetic site to the phloem. GSD1 functioned in affecting PD conductance by interacting with rice ACTIN1 in association with the PD callose binding protein1. Together, our results suggest that GSD1 may play a role in regulating photoassimilate translocation through the symplastic pathway to impact grain setting in rice.Grain filling, a key determinant of grain yield in rice (Oryza sativa), hinges on the successful translocation of photoassimilates from the leaves to the fertilized reproductive organs through the phloem transport system. Symplastic phloem loading, which is one of the main pathways responsible for the transport of photoassimilates in rice, is mediated by plasmodesmata (PD) that connect phloem companion cells with sieve elements and surrounding parenchyma cells (Kaneko et al., 1980; Chonan et al., 1981; Eom et al., 2012). PD are transverse cell wall channels structured with the cytoplasmic sleeve and the modified endoplasmic reticulum desmotubule between neighboring cells (Maule, 2008). A number of proteins affect the structure and functional performance of the PD, which in turn impacts the cell-to-cell transport of small and large molecules through the PD during plant growth, development, and defense (Cilia and Jackson, 2004; Sagi et al., 2005; Lucas et al., 2009; Simpson et al., 2009; Stonebloom et al., 2009). For example, actin and myosin, which link the desmotubule to the plasma membrane (PM) at the neck region of PD, are believed to play a role in regulating PD permeability by controlling PD aperture (White et al., 1994; Ding et al., 1996; Reichelt et al., 1999). Callose deposition can also impact the size of the PD aperture at the neck region (Radford et al., 1998; Levy et al., 2007) and callose synthase genes such as Glucan Synthase-Like7 (GSL7, also named CalS7), GSL8, and GSL12 have been shown to play a role in regulating symplastic trafficking (Guseman et al., 2010; Barratt et al., 2011; Vatén et al., 2011; Xie et al., 2011). Other proteins that have been shown to impact the structure and function of the PD include glycosylphosphatidylinositol (GPI)-anchored proteins, PD callose binding protein1 (PDCB1), which is also associated with callose deposition (Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN2, which limits the molecular flux through the PD by chitin perception (Faulkner et al., 2013). Changes in PD permeability can have major consequences for the translocation of photoassimilates needed for grain filling in rice. However, the genes and molecular mechanisms underlying the symplastic transport of photoassimilates remain poorly characterized.Remorins are a diverse family of plant-specific proteins with conserved C-terminal sequences and highly variable N-terminal sequences. Remorins can be classified into six distinct phylogenetic groups (Raffaele et al., 2007). The functions of most remorins are unknown, but some members of the family have been shown to be involved in immune response through controlling the cell-to-cell spread of microbes. StREM1.3, a remorin that is located in PM rafts and the PD, was shown to impair the cell-to-cell movement of a plant virus X by binding to Triple Gene Block protein1 (Raffaele et al., 2009). Medicago truncatula symbiotic remorin1 (MtSYMREM1), a remorin located at the PM in Medicago truncatula, was shown to facilitate infection and the release of rhizobial bacteria into the host cytoplasm (Lefebvre et al., 2010). Overexpression of LjSYMREM1, the ortholog of MtSYMREM1 in Lotus japonicus, resulted in increased root nodulation (Lefebvre et al., 2010; Tóth et al., 2012). Although a potential association between remorins and PD permeability has been proposed (Raffaele et al., 2009), the diversity observed across remorins, plus the fact that remorin mutants generated through different approaches fail to show obvious phenotypes (Reymond et al., 1996; Bariola et al., 2004), have made it challenging to characterize the function of remorins in cell-to-cell transport.In this study, we identified a rice transfer DNA (T-DNA) insertion mutant (grain setting defect1-Dominant [gsd1-D]), with a grain setting-deficient phenotype caused by overexpression of GSD1, a remorin gene with unknown function. GSD1 is expressed specifically in phloem companion cells and is localized in the PD and PM. We provide evidence to show that overexpression of GSD1 leads to deficient grain setting in rice, likely as a consequence of reduced sugar transport resulting from decreased PD permeability in phloem companion cells.     

The difference of cadmium accumulation between the indica and japonica subspecies and the mechanism of it

Many studies have shown genotypic differences in Cadmium (Cd) accumulation among rice cultivars, and concentrations in shoots and grains are generally higher in indica rice cultivars than in japonica rice cultivars, but the mechanism remains unknown. The main objective of this study was to investigate differences in heavy metal accumulation between rice subspecies through the analysis of 46 indica cultivars and 30 japonica cultivars. At the seedling stage, the mean Cd concentrations in the shoots of indica subspecies were significantly higher than those in japonica subspecies (1.22-fold), but this pattern was not observed in the roots. At the filling stage, the mean Cd concentrations in the shoots and spikes of indica subspecies were 1.66- and 2.14-fold higher than the respective concentrations in japonica subspecies. At the harvest stage, the mean Cd concentrations in the shoots and brown rice of indica subspecies were 1.61- and 2.27-fold higher than the respective concentrations in japonica subspecies. These results indicate that root-to-shoot and shoot-to-grain translocation, rather than Cd absorption in the roots, may be the key processes that determine the differences in Cd accumulation among rice subspecies. Gene expression analysis revealed that overall, the expression levels of the Cd transporter gene OsNramp1 notably increased (22.46-fold), but the expression levels of OsHMA2, OsHMA3 and OsNRAMP5 were not significantly changed at the seedling stage in the 76 cultivars exposed to Cd; the expression levels of OsNramp1 were positively correlated with the Cd concentrations in spikes at the filling stage. In addition, a significant difference was observed in the expression levels of OsNramp1 between the indica and japonica subspecies, which may explain the higher Cd concentrations in roots but lower Cd concentrations in spikes and brown rice for the japonica subspecies. Together, these results demonstrate that OsNramp1 may be the most important gene among the four selected genes in the promotion of Cd uptake by roots and transfer of Cd into spikes and eventually into brown rice.     

Phytochelatin and cadmium accumulation in wheat

Cadmium (Cd) is a nonessential heavy metal that can be harmful at low concentrations in organisms. Therefore, it is necessary to decrease Cd accumulation in the grains of wheats aimed for human consumption. In response to Cd, higher plants synthesize sulphur-rich peptides, phytochelatins (PCs). PC–heavy metal complexes have been reported to accumulate in the vacuole. Retention of Cd in the root cell vacuoles might influence the symplastic radial Cd transport to the xylem and further transport to the shoot, resulting in genotypic differences in grain Cd accumulation. We have studied PC accumulation in 12-day-old seedlings of two cultivars of spring bread wheat (Triticum aestivum), and two spring durum wheat cultivars (Triticum turgidum var. durum) with different degrees of Cd accumulation in the grains. Shoots and roots were analysed for dry weight, Cd and PC accumulation. There were no significant differences between the species or the varieties in the growth response to Cd, nor in the distributions of PC chain lengths or PC isoforms. At 1 μM external Cd, durum wheat had a higher total Cd uptake than bread wheat, however, the shoot-to-root Cd concentration ratio was higher in bread wheat. When comparing varieties within a species, the high grain Cd accumulators exhibited lower rates of root Cd accumulation, shoot Cd accumulation, and root PC accumulation, but higher shoot-to-root Cd concentration ratios. Intraspecific variation in grain Cd accumulation is apparently not only explained by differential Cd accumulation as such, but rather by a differential plant-internal Cd allocation pattern. However, the higher average grain Cd accumulation in the durum wheats, as compared to the bread wheats, is associated with a higher total Cd accumulation in the plant, rather than with differential plant-internal Cd allocation. The root-internal PC chain length distributions and PC–thiol-to-Cd molar ratios did not significantly differ between species or varieties, suggesting that differential grain Cd accumulation is not due to differential PC-based Cd sequestration in the roots.     

当归根显微结构及其根腐病真菌分布研究

利用徒手切片、石蜡切片和超薄切片及显微摄像的方法,对当归根的显微结构及根腐病致病真菌的分布进行了研究。结果表明:当归的根由周皮和次生维管组织两部分组成,周皮由外向内依次分为木栓层、木栓形成层、栓内层;次生韧皮部占根径的比例在60%以上,主要成分包括筛胞、韧皮薄壁细胞、韧皮纤维和分泌道,薄壁细胞富含淀粉粒等营养物质;次生木质部由导管、木薄壁细胞和木射线组成,木质部呈多元形,木射线和韧皮射线明显。在根的周皮细胞和中柱中均有真菌分布,说明真菌由木栓层、木栓形成层、栓内层依次向里侵入到韧皮薄壁细胞,在薄壁细胞内定殖并形成菌丝结或团块状结构,进而扩展成一定的侵染区域;真菌不仅侵染周皮和韧皮部,而且还进一步侵染木质部并破坏导管。此外,研究还发现,淀粉粒是真菌定殖的主要场所,真菌穿透或缠绕在淀粉粒上,并利用其营养不断地生长与繁殖。