植物对硅的吸收转运机制研究进展

硅(Si)能缓解生物与非生物胁迫对植物的毒害作用,Si的吸收转运是由Si转运蛋白介导的.最近,多个Si转运蛋白(Lsi)基因相继在水稻、大麦和玉米中被克隆出来,并在Si的吸收转运机制方面取得了很大进展.水稻OsLsi在根组织中呈极性分布,OsLsi1定位在根外皮层和内皮层凯氏带细胞外侧质膜,负责将外部溶液中的单硅酸转运到皮层细胞内.OsLsi2定位在凯氏带细胞内侧质膜,在外皮层中负责将Si输出到通气组织质外体中,在内皮层与OsLsi1协同作用将Si转运到中柱中.导管中的Si通过蒸腾流转运到地上部,再由定位在叶鞘和叶片木质部薄壁细胞靠近导管一侧的OsLsi6负责木质部Si的卸载和分配.在大麦和玉米中,ZmLsi1/HvLsi1定位在根表皮和皮层细胞外侧质膜负责Si的吸收,然后Si通过共质体途径被转运到内皮层凯氏带细胞中,再由ZmLsi2/HvLsi2输出转运到中柱中.ZmLsi6在细胞中的定位和活性与OsLsi6相似,推测其可能具有类似的功能,但大麦Lsi6至今未见报道.所以,Si转运机制仍需要进一步研究.     

Tomato roots have a functional silicon influx transporter but not a functional silicon efflux transporter

Silicon (Si) accumulation in shoots differs greatly with plant species, but the molecular mechanisms for this interspecific difference are unknown. Here, we isolated homologous genes of rice Si influx (SlLsi1) and efflux (SlLsi2) transporter genes in tomato (Solanum lycopersicum L.) and functionally characterized these genes. SlLsi1 showed transport activity for Si when expressed in both rice lsi1 mutant and Xenopus laevis oocytes. SlLsi1 was constitutively expressed in the roots. Immunostaining showed that SlLsi1 was localized at the plasma membrane of both root tip and basal region without polarity. Furthermore, overexpression of SlLsi1 in tomato increased Si concentration in the roots and root cell sap but did not alter the Si concentration in the shoots. By contrast, two Lsi2-like proteins did not show efflux transport activity for Si in Xenopus oocytes. However, when functional CsLsi2 from cucumber was expressed in tomato, the Si uptake was significantly increased, resulting in higher Si accumulation in the leaves and enhanced tolerance of the leaves to water deficit and high temperature. Our results suggest that the low Si accumulation in tomato is attributed to the lack of functional Si efflux transporter Lsi2 required for active Si uptake although SlLsi1 is functional.     

Functional Characterization of a Silicon Transporter Gene Implicated in Silicon Distribution in Barley

Silicon (Si) is a beneficial element for plant growth. In barley (Hordeum vulgare), Si uptake by the roots is mainly mediated by a Si channel, Low Silicon1 (HvLsi1), and an efflux transporter, HvLsi2. However, transporters involved in the distribution of Si in the shoots have not been identified. Here, we report the functional characterization of a homolog of HvLsi1, HvLsi6. HvLsi6 showed permeability for Si and localized to the plasma membrane. At the vegetative growth stage, HvLsi6 was expressed in both the roots and shoots. The expression level was unaffected by Si supply. In the roots, HvLsi6 was localized in epidermis and cortex cells of the tips, while in the leaf blades and sheaths, HvLsi6 was only localized at parenchyma cells of vascular bundles. At the reproductive growth stage, high expression of HvLsi6 was also found in the nodes. HvLsi6 in node I was polarly located at the transfer cells surrounding the enlarged vascular bundles toward the numerous xylem vessels. These results suggest that HvLsi6 is involved in Si uptake in the root tips, xylem unloading of Si in leaf blade and sheath, and intervascular transfer of Si in the nodes. Furthermore, HvLsi2 was found to be localized at the parenchyma cell layer adjacent to the transfer cells with opposite polarity of HvLsi6, suggesting that the coupling of HvLsi6 and HvLsi2 is involved in the intervascular transfer of Si at the nodes. Si translocated via the enlarged vascular bundles is unloaded to the transfer cells by HvLsi6, followed by HvLsi2 to reload Si to the diffuse vascular bundles, which are connected to the upper part of the plant, especially the panicles, the ultimate Si sink.Silicon (Si) is a beneficial element for plant growth. It enhances the resistance of plants to various biotic and abiotic stresses (Epstein, 1999; Ma and Takahashi, 2002; Ma and Yamaji, 2006). For example, Si reduces the epidemics of both leaf and panicle blast in rice (Oryza sativa; Datnoff and Rodrigues, 2005) and decreases the incidence of powdery mildew in cucumber (Cucumis sativus), barley (Hordeum vulgare), and wheat (Triticum aestivum; Fauteux et al., 2005). Si also suppresses insect pests such as stem borer (Chilo suppressalis), brown planthopper (Nilaparvata lugens), and rice green leafhopper (Nephotettix cincticeps; Savant et al., 1997). Resistance to the damage by wild rabbit in wheat is also enhanced by an increased amount of Si in leaf tissue (Cotterill et al., 2007). Si is also able to alleviate lodging, drought, and low- and high-temperature stresses (Ma, 2004). The beneficial effects of Si under phosphate deficiency, phosphate excess, and manganese and salt toxicity stresses have been observed in many plants (Ma and Takahashi, 2002). Usually, the more Si that accumulates in the shoots, the greater its effect in enhancing the plant’s response. This is because most effects of Si are expressed through the formation of silica gel, which is deposited on leaves, stems, and other organs of plants (Ma and Yamaji, 2006). Therefore, for the plant to benefit from Si, a high accumulation is required. However, Si accumulation greatly varies with plant species, and this difference has been attributed to the ability of plants to take up Si.Transporters responsible for Si uptake by roots have been identified in several plant species, including barley, maize (Zea mays), pumpkin (Cucurbita moschata), rice, wheat (Ma et al., 2011), and most recently in horsetail (Equisetum arvense; EaNIP3s [for Nod26-like major intrinsic protein3]; Grégoire et al., 2012). Two different types of transporter, Si-permeable channel and efflux transporter, are involved in the Si-uptake process. Low Silicon1 (Lsi1) belongs to a NIP subfamily of aquaporin-like proteins and functions as a Si-permeable channel. Lsi1 in rice is localized in the distal side of root exodermis and endodermis (Ma et al., 2006), but Lsi1 in barley, maize, and pumpkin is localized in the epidermis and cortex (Chiba et al., 2009; Mitani et al., 2009b, 2011). On the other hand, Lsi2 functions as an efflux Si transporter and belongs to a putative anion transporter family without any similarity to Lsi1. Lsi2 in rice is also localized at the root exodermis and endodermis as Lsi1, but it is polarly localized at the proximal side (Ma et al., 2007). By contrast, Lsi2 in barley and maize is localized only to the endodermis of roots. Furthermore, these transporters do not show polar localization in barley and maize (Mitani et al., 2009a). Therefore, Si uptake mediated by Lsi1 and Lsi2 shows different pathways between rice and other plant species (Ma et al., 2011).Following uptake by the roots through Lsi1 and Lsi2, Si is translocated to the aboveground part and distributed in different tissues. Lsi6, a homolog of Lsi1, is involved in xylem unloading of Si in rice (Yamaji et al., 2008). Lsi6 is localized on the adaxial side of the xylem parenchyma cells in the leaf sheaths and leaf blades. Knockout of Lsi6 resulted in altered distribution of Si in the leaf cells. Furthermore, at the reproductive growth stage of rice, Lsi6 is also highly expressed at the nodes (Yamaji and Ma, 2009). At node I below the panicle, Lsi6 is mainly localized at the xylem transfer cells with polarity facing toward the xylem vessel (Yamaji and Ma, 2009). Knockout of Lsi6 decreased Si accumulation in the panicle but increased Si accumulation in the flag leaf. These findings indicate that Lsi6 is also required for the intervascular transfer of Si in rice, transferring Si from the enlarged vascular bundles coming from the roots to the diffuse vascular bundles connected to the panicle.Barley is a Si-accumulating species, although the accumulation extent is lower than that of rice. Transporters responsible for Si uptake in barley roots have been identified (Chiba et al., 2009; Mitani et al., 2009a); however, transporters for Si distribution in aboveground plant tissues are unknown. In this study, we functionally characterized a rice Lsi6 homolog gene in barley, HvLsi6, in terms of transport activity and expression pattern, as well as cellular and subcellular localizations. We found that HvLsi6 is probably involved in Si uptake in the root tip, xylem unloading in the leaf, and intervascular transfer of Si at the nodes in barley. We further found that HvLsi2 was also expressed in the nodes and involved in the intervascular transfer by coupling with HvLsi6.     

Silicon efflux transporters isolated from two pumpkin cultivars contrasting in Si uptake

The accumulation of silicon (Si) differs greatly with plant species and cultivars due to different ability of the roots to take up Si. In Si accumulating plants such as rice, barley and maize, Si uptake is mediated by the influx (Lsi1) and efflux (Lsi2) transporters. Here we report isolation and functional analysis of two Si efflux transporters (CmLsi2-1 and CmLsi2-2) from two pumpkin (Cucurbita moschata Duch.) cultivars contrasting in Si uptake. These cultivars are used for rootstocks of bloom and bloomless cucumber, respectively. Different from mutations in the Si influx transporter CmLsi1, there was no difference in the sequence of either CmLsi2 between two cultivars. Both CmLsi2-1 and CmLsi2-2 showed an efflux transport activity for Si and they were expressed in both the roots and shoots. These results confirm our previous finding that mutation in CmLsi1, but not in CmLsi2-1 and CmLsi2-2 are responsible for bloomless phenotype resulting from low Si uptake.Key words: silicon, efflux transporter, pumpkin, cucumber, bloomSilicon (Si) is the second most abundant elements in earth''s crust.¹ Therefore, all plants rooting in soils contain Si in their tissues. However Si accumulation in the shoot differs greatly among plant species, ranging for 0.1 to 10% of dry weight.^1–3 In higher plants, only Poaceae, Equisetaceae and Cyperaceae show a high Si accumulation.^2,3 Si accumulation also differs with cultivars within a species.^4,5 These differences in Si accumulation have been attributed to the ability of the roots to take up Si.^6,7Genotypic difference in Si accumulation has been used to produce bloomless cucumber (Cucumis sativus L.).⁸ Bloom (white and fine powders) on the surface of cucumber fruits is primarily composed of silica (SiO₂).⁹ However, nowadays, cucumber without bloom (bloomless cucumber) is more popular in Japan due to its more attractive and distinctly shiny appearance. Bloomless cucumber is produced by grafting cucumber on some specific pumpkin (Cucurbita moschata Duch.) cultivars. These pumpkin cultivars used for bloomless cucumber rootstocks have lower silicon accumulation compared with the rootstocks used for producing bloom cucumber.⁹Our study showed that the difference in Si accumulation between bloom and bloomless root stocks of pumpkin cultivars results from different Si uptake by the roots.¹⁰ Si uptake has been demonstrated to be mediated by two different types of transporters (Lsi1 and Lsi2) in rice, barley and maize.^11–15 Lsi1 is an influx transporter of Si, belonging to a NIP subfamily of aquaporin family.^10,11,13,14 This transporter is responsible for transport of Si from external solution to the root cells.¹¹ On the other hand, Lsi2 is an efflux transporter of Si, belonging to putative anion transporter.¹² Lsi2 releases Si from the root cells towards the xylem. Both Lsi1 and Lsi2 are required for Si uptake by the roots.^11,12 To understand the mechanism underlying genotypic difference in Si uptake, we have isolated and functionally characterized an influx Si transporter CmLsi1 from two pumpkin cultivars used for rootstocks of bloomless and bloom cucumber.¹⁰ Sequence analysis showed only two amino acids difference of CmLsi1 between two pumpkin cultivars. However, CmLsi1 from bloom rootstock [CmLsi1(B⁺)] showed transport activity for Si, whereas that from bloomless rootstock [CmLsi1(B⁻)] did not.¹⁰ Furthermore, we found that loss of Si transport activity was caused by one amino acid mutation at the position of 242 (from proline to leucine).¹⁰ This mutation resulted in failure to be localized at the plasma membrane, which is necessary for functioning as an influx transporter. The mutated protein was localized at the ER.¹⁰ Here, we report isolation and expression analysis of Si efflux transporters from two pumpkin cultivars contrasting in Si uptake and accumulation to examine whether Si efflux transporter is also involved in the bloom and bloomless phenotypes.     

High-resolution secondary ion mass spectrometry reveals the contrasting subcellular distribution of arsenic and silicon in rice roots

Rice (Oryza sativa) takes up arsenite mainly through the silicic acid transport pathway. Understanding the uptake and sequestration of arsenic (As) into the rice plant is important for developing strategies to reduce As concentration in rice grain. In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy. Rice plants, both the lsi2 mutant lacking the Si/arsenite efflux transporter Lsi2 and its wild-type cultivar, with or without an iron plaque, were treated with arsenate or arsenite. The formation of iron plaque on the root surface resulted in strong accumulation of As and phosphorous on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. As also accumulated in the vacuoles of some xylem parenchyma cells and in some pericycle cells, particularly in the wild-type mature root zone. Vacuolar accumulation of As is associated with sulfur, suggesting that As may be stored as arsenite-phytochelatin complexes. Si was localized in the cell walls of the endodermal cells with little apparent effect of the Lsi2 mutation on its distribution. This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters.     

Effect of cadmium ion excess over cell structure and functioning of Zea mays and Hordeum vulgare

Zea mays (maize) and Hordeum vulgare (barley) plants were analyzed in order to study the variation in response to Cadmium (Cd) toxicity based on development of leaf symptoms, effect in dry matter production, Cd uptake, lipid peroxidation and effect on cell ultrastructure in leaves and roots. Cd accumulation in roots of Z. mays and H. vulgare was 18–50 times higher than in the aerial parts. Malondialdehyde (MDA) content was more affected in the roots of both Z. mays and H. vulgare than in shoots (60 and 56–51 and 40%, respectively). At ultrastructural level, in Cd treated seedlings, a decline in the vacuolar content of barley roots cells and maize leaf cells was observed. Results corroborate that these gramineous crops can uptake and accumulate substantial amounts of Cd especially in roots. Therefore, H. vulgare and Z. mays could have a phytostabilization potential and thereafter could be tested in phytoremediation technologies.     

<Emphasis Type="Italic">Lsi1</Emphasis>-regulated Cd uptake and phytohormones accumulation in rice seedlings in presence of Si

Silicon (Si) is known for its role in regulating the response of plants to imposed abiotic stresses. Since the stresses generally hinder production of a crop, such as rice, the exploration of the biochemistry and plant physiology relating to the function is of interest. Indeed, recently, there were reports on the function of Lsi1 in regulating the tolerance of rice to cadmium (Cd) stress. This study compared the kinetics of the Cd uptakes in Lemont wild type rice and its transgenic lines exposed to Cd with or without exogenous Si supply. At the same time, changes on the endogenous phytohormones and growth of the rice seedlings were monitored. Genetically, Lsi1 overexpression was found to downregulate K_m and V_max of Cd uptake kinetics in the plants under Cd stress, especially in the presence of Si. On the other hand, Lsi1 RNAi upregulated K_m and V_max regardless whether Si was present or not. It implied that Lsi1 could be capable of regulating Si as well as Cd transports. Under Cd stress, addition of Si reduced the Cd uptake of the rice lines in the order of Lsi1-overexpression line?>?Lemont?>?Lsi1-RNAi line. In addition, it also affected the chlorophyll biosynthesis and dry mass accumulation of the rice plants under Cd stress. Analyses on phytohormones including IAA, GA₃, JA, SA and ABA, as well as physiological functions, of the seedlings further verified the active involvement of Lsi1 in the complex defense system of the plants against Cd stress.     

A Transporter at the Node Responsible for Intervascular Transfer of Silicon in Rice

The concentration of essential mineral nutrients in the edible portion of plants such as grains may affect the nutritional value of these foods, while concentrations of toxic minerals in the plant are matter of food safety. Minerals taken up by the roots from soils are normally redirected at plant nodes before they are finally transported into developing seeds. However, the molecular mechanisms involved in this process have not been identified so far. Herein, we report on a transporter (Lsi6) responsible for the redirection of a plant nutrient at the node. Lsi6 is a silicon transporter in rice (Oryza sativa), and its expression in node I below the panicles is greatly enhanced when the panicle is completely emerged. Lsi6 is mainly localized at the xylem transfer cells located at the outer boundary region of the enlarged large vascular bundles in node I. Knockout of Lsi6 decreased Si accumulation in the panicles but increased Si accumulation in the flag leaf. These results suggest that Lsi6 is a transporter involved in intervascular transfer (i.e., transfer of silicon from the large vascular bundles coming from the roots to the diffuse vascular bundles connected to the panicles). These findings will be useful for selectively enhancing the accumulation of essential nutrients and reducing toxic minerals in the edible portion of cereals.     

Spatial distribution and temporal variation of the rice silicon transporter Lsi1

Rice (Oryza sativa) is a typical silicon (Si) accumulator and requires a large amount of Si for high-yield production. Recently, a gene (Low silicon rice1 [Lsi1]) encoding a Si transporter was identified in rice roots. Here, we characterized Lsi1 in terms of spatial distribution and temporal variation using both physiological and molecular approaches. Results from a multicompartment transport box experiment showed that the major site for Si uptake was located at the basal zone (>10 mm from the root tip) of the roots rather than at the root tips (<10 mm from the root tip). Consistent with the Si uptake pattern, Lsi1 expression and distribution of the Lsi1 protein were found only in the basal zone of roots. In the basal zones of the seminal, crown, and lateral roots, the Lsi1 protein showed a polar localization at the distal side of both the exodermis and endodermis, where the Casparian bands are formed. This indicates that Lsi1 is required for the transport of Si through the cells of the exodermis and endodermis. Expression of Lsi1 displayed a distinct diurnal pattern. Furthermore, expression was transiently enhanced around the heading stage, which coincides with a high Si requirement during this growth stage. Expression was down-regulated by dehydration stress and abscisic acid, suggesting that expression of Lsi1 may be regulated by abscisic acid.     

Physiological,biochemical, and molecular differences in chloroplast synthesis between leaf and corolla of cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. var. chinensis) and rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Cadmium (Cd) pollution is one of the major concerns in the development of sustainable agriculture, particularly for rice production. Silicon (Si) was recently recognized for its ability to mitigate a variety of abiotic stresses including that caused by Cd on rice. However, mechanism of the complex process is still not fully understood. Under Cd-stress, the low Si-influx 1-RNAi transgenic Lemont rice (Lsi1-RNAi line) exhibited an increased Cd-uptake over its counterparts, the wild type and the Lsi1-overexpression transgenic rice (Lsi1-OE line). In contrast, the Lsi1 expression-enhanced Lsi1-OE line showed the greatest Si-uptake among the three lines, with the highest activities on anti-oxidants (such as, superoxide dismutase) and the lowest content of malondialdehyde. Lsi1 also displayed a negative regulation on Low Cd gene and the natural resistance-associated macrophage proteins, Nramp5, indicating its capacity to alleviate Cd-stress on rice. The results obtained by this study suggested that the mitigation of Cd-toxicity on rice by Si might involve functions, such as the inhibition on Cd-uptake and transport and the enhancement on anti-oxidative enzyme activities, as well as the Lsi1-related expression on regulation of Si-uptake in rice. A new avenue might become available for overcoming the rampant pollution that threatens the rice production in China.     

Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants

Background and Aims

Silicon (Si) has been shown to ameliorate the negative influence of cadmium (Cd) on plant growth and development. However, the mechanism of this phenomenon is not fully understood. Here we describe the effect of Si on growth, and uptake and subcellular distribution of Cd in maize plants in relation to the development of root tissues.

Methods

Young maize plants (Zea mays) were cultivated for 10 d hydroponically with 5 or 50 µm Cd and/or 5 mm Si. Growth parameters and the concentrations of Cd and Si were determined in root and shoot by atomic absorption spectrometry or inductively coupled plasma mass spectroscopy. The development of apoplasmic barriers (Casparian bands and suberin lamellae) and vascular tissues in roots were analysed, and the influence of Si on apoplasmic and symplasmic distribution of ¹⁰⁹Cd applied at 34 nm was investigated between root and shoot.

Key Results

Si stimulated the growth of young maize plants exposed to Cd and influenced the development of Casparian bands and suberin lamellae as well as vascular tissues in root. Si did not affect the distribution of apoplasmic and symplasmic Cd in maize roots, but considerably decreased symplasmic and increased apoplasmic concentration of Cd in maize shoots.

Conclusions

Differences in Cd uptake of roots and shoots are probably related to the development of apoplasmic barriers and maturation of vascular tissues in roots. Alleviation of Cd toxicity by Si might be attributed to enhanced binding of Cd to the apoplasmic fraction in maize shoots.     

Genome-wide exploration of silicon (Si) transporter genes, <Emphasis Type="Italic">Lsi1</Emphasis> and <Emphasis Type="Italic">Lsi2</Emphasis> in plants; insights into Si-accumulation status/capacity of plants

Silicon (Si) is a nonessential, beneficial micronutrient for plants. It increases the plant stress tolerance in relation to its accumulation capacity. In this work, root Si transporter genes were characterized in 17 different plants and inferred for their Si-accumulation status. A total of 62 Si transporter genes (31 Lsi1 and 31 Lsi2) were identified in studied plants. Lsi1s were 261–324 residues protein with a MIP family domain whereas Lsi2s were 472–547 residues with a citrate transporter family domain. Lsi1s possessed characteristic sequence features that can be employed as benchmark in prediction of Si-accumulation status/capacity of the plants. Silicic acid selectivity in Lsi1s was associated with two highly conserved NPA (Asn-Pro-Ala) motifs and a Gly-Ser-Gly-Arg (GSGR) ar/R filter. Two NPA regions were present in all Lsi1 members but some Ala substituted with Ser or Val. GSGR filter was only available in the proposed high and moderate Si accumulators. In phylogeny, Lsi1s formed three clusters as low, moderate and high Si accumulators based on tree topology and availability of GSGR filter. Low-accumulators contained filters WIGR, AIGR, FAAR, WVAR and AVAR, high-accumulators only with GSGR filter, and moderate-accumulators mostly with GSGR but some with A/CSGR filters. A positive correlation was also available between sequence homology and Si-accumulation status of the tested plants. Thus, availability of GSGR selectivity filter and sequence homology degree could be used as signatures in prediction of Si-accumulation status in experimentally uncharacterized plants. Moreover, interaction partner and expression profile analyses implicated the involvement of Si transporters in plant stress tolerance.     

Silicon Promotes Exodermal Casparian Band Formation in Si-Accumulating and Si-Excluding Species by Forming Phenol Complexes

We studied the effect of Silicon (Si) on Casparian band (CB) development, chemical composition of the exodermal CB and Si deposition across the root in the Si accumulators rice and maize and the Si non-accumulator onion. Plants were cultivated in nutrient solution with and without Si supply. The CB development was determined in stained root cross-sections. The outer part of the roots containing the exodermis was isolated after enzymatic treatment. The exodermal suberin was transesterified with MeOH/BF₃ and the chemical composition was measured using gas chromatography-mass spectroscopy (GC-MS) and flame ionization detector (GC-FID). Laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) was used to determine the Si deposition across root cross sections. Si promoted CB formation in the roots of Si-accumulator and Si non-accumulator species. The exodermal suberin was decreased in rice and maize due to decreased amounts of aromatic suberin fractions. Si did not affect the concentration of lignin and lignin-like polymers in the outer part of rice, maize and onion roots. The highest Si depositions were found in the tissues containing CB. These data along with literature were used to suggest a mechanism how Si promotes the CB development by forming complexes with phenols.     

Silicon has opposite effects on the accumulation of inorganic and methylated arsenic species in rice

Background and aims

Rice (Oryza sativa) is a main source of human exposure to inorganic arsenic and mitigation measures are needed to decrease As accumulation in this staple crop. It has been shown that silicon decreases the accumulation of arsenite but, unexpectedly, increases the accumulation of dimethylarsinic acid (DMA) in rice grain. The aim of this study was to investigate why Si increases DMA accumulation.

Methods

Pot and incubation experiments were conducted to investigate how the addition of sparingly soluble silicate gel affected As speciation in the soil solution and the accumulation of different As species in rice tissues.

Results

Silicon addition significantly decreased the concentration of inorganic As (mainly arsenite) but increased the concentration of DMA in both the vegetative and reproductive tissues of rice. Silicon increased the concentration of DMA in the soil solution, whereas autoclaving soil decreased DMA concentration. Less DMA was adsorbed by the soil than arsenate and Si addition significantly inhibited DMA adsorption.

Conclusions

Silicon increased DMA accumulation and decreased arsenite accumulation in rice through different mechanisms. Silicic acid released from the silicate gel increased the availability of DMA for rice uptake by inhibiting DMA adsorption on the soil solid phase or by displacing adsorbed DMA. Although silicic acid also increased the concentration of inorganic As in the soil solution, this effect was much smaller than the inhibitory effect of Si on arsenite uptake by rice roots.     

Biallelic and Genome Wide Association Mapping of Germanium Tolerant Loci in Rice (Oryza sativa L.)

Rice plants accumulate high concentrations of silicon. Silicon has been shown to be involved in plant growth, high yield, and mitigating biotic and abiotic stresses. However, it has been demonstrated that inorganic arsenic is taken up by rice through silicon transporters under anaerobic conditions, thus the ability to efficiently take up silicon may be considered either a positive or a negative trait in rice. Germanium is an analogue of silicon that produces brown lesions in shoots and leaves, and germanium toxicity has been used to identify mutants in silicon and arsenic transport. In this study, two different genetic mapping methods were performed to determine the loci involved in germanium sensitivity in rice. Genetic mapping in the biparental cross of Bala × Azucena (an F₆ population) and a genome wide association (GWA) study with 350 accessions from the Rice Diversity Panel 1 were conducted using 15 μM of germanic acid. This identified a number of germanium sensitive loci: some co-localised with previously identified quantitative trait loci (QTL) for tissue silicon or arsenic concentration, none co-localised with Lsi1 or Lsi6, while one single nucleotide polymorphism (SNP) was detected within 200 kb of Lsi2 (these are genes known to transport silicon, whose identity was discovered using germanium toxicity). However, examining candidate genes that are within the genomic region of the loci detected above reveals genes homologous to both Lsi1 and Lsi2, as well as a number of other candidate genes, which are discussed.     

The Rice Aquaporin Lsi1 Mediates Uptake of Methylated Arsenic Species

Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two silicon transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice lsi1 mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild type having 3.5-fold higher V_max than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild type than the lsi1 mutant. Heterologous expression of Lsi1 in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsi1 mediates the uptake of undissociated methylated As in rice roots.Arsenic (As) contamination affects millions of people worldwide, particularly in South Asia where As-contaminated groundwater has been extracted for drinking (Chakraborti et al., 2002; Nordstrom, 2002). Recent studies have shown that foods, especially rice (Oryza sativa), are an important source of inorganic As to populations dependent on a rice diet (Kile et al., 2007; Ohno et al., 2007; Mondal and Polya, 2008). Paddy rice is more efficient than other cereal crops in accumulating As (Williams et al., 2007). This is because anaerobic conditions in submerged paddy soils lead to mobilization of arsenite [As(III); Takahashi et al., 2004; Xu et al., 2008], which is then taken up by rice roots mainly through the highly efficient transport pathway for silicon (Si; Ma et al., 2008). The relatively high accumulation of As in rice is of concern, as it may pose a significant health risk (Zhu et al., 2008; Meharg et al., 2009).A number of As species may be present in soil depending on soil conditions and the history of As contamination. These include arsenate [As(V)], As(III), and methylated As species such as monomethylarsonic acid [MMA(V): CH₃AsO(OH)₂] and dimethylarsinic acid [DMA(V): (CH₃)₂AsO(OH)]. As(V) is the main species in aerobic soils, while As(III) dominates in anaerobic environments such as flooded paddy soils. Both MMA(V) and DMA(V) have been found in paddy soils (Takamatsu et al., 1982), which may have been derived from microbial and algal biomethylation and/or past uses of methylated As compounds. MMA(V), as sodium or calcium salt, and DMA(V), as sodium salt or free acid (also called cacodylic acid), are herbicides widely used for weed control on cotton (Gossypium hirsutum), orchards, and lawns, or as a defoliant of cotton (U.S. Environmental Protection Agency, 2006). Conversion of cotton fields for the production of paddy rice in the United States may be a reason for the high levels of methylated As reported for the U.S. rice (Meharg et al., 2009).The mechanism of As(V) uptake by plants through the phosphate transport system has been well established (for review, see Zhao et al., 2009). In contrast, As(III) is taken up into the cells by aquaglyceroporins in Escherichia coli, yeast (Saccharomyces cerevisiae), and mammalian tissues (for review, see Bhattacharjee and Rosen, 2007). Recent studies have shown that several plant aquaporin channels belonging to the Nodulin 26-like Intrinsic Protein (NIP) subfamily are permeable to As(III) when expressed heterologously in yeast (Bienert et al., 2008; Isayenkov and Maathuis, 2008; Ma et al., 2008). The rice Si transporter Lsi1 (OsNIP2;1; Ma et al., 2006) is also permeable to As(III) when expressed in yeast or Xenopus laevis oocytes (Ma et al., 2008). Furthermore, the lsi1 rice mutant lost 57% of the influx capacity for As(III) compared to the wild type in short-term assays, suggesting that Lsi1 is an important entry route for As(III) (Ma et al., 2008). In rice roots, a second Si transporter, Lsi2, functions as an efflux carrier to transport Si efflux from the exodermis and endodermis cells toward the stele for xylem loading (Ma et al., 2007). This transporter also mediates As(III) efflux; two independent lsi2 mutants had 73% to 91% lower concentrations of As(III) in the xylem sap than their wild types (Ma et al., 2008). The shared uptake pathway between Si (silicic acid) and As(III) (arsenous acid) is consistent with their physiochemical properties; both are present predominantly as undissociated neutral molecules at the normal environmental and physiological pH range (pK_a = 9.2, >99% undissociated at pH ≤ 7.0), and the two molecules have similar sizes.The uptake mechanisms of methylated As species by plant roots are not known. Previous studies showed that both MMA(V) and DMA(V) can be taken up by roots and translocated to shoots in a number of plant species (Marin et al., 1992; Carbonell-Barrachina et al., 1998, 1999; Burló et al., 1999). Marin et al. (1992) found that uptake by rice followed the order of As(III) > MMA(V) > As(V) > DMA (V), although DMA(V) was more efficiently translocated from roots to shoots than other As species. Raab et al. (2007) reported large variations in the absorption and translocation efficiencies for As(V), MMA(V), and DMA(V) among the 46 plant species tested. On average, root absorption of As(V) was 2.5- and 5-times higher than MMA(V) and DMA(V), respectively. The translocation efficiency, defined as the shoot-to-root concentration ratio after 24-h exposure, was highest for DMA(V) (0.8), followed by MMA(V) (0.3) and As(V) (0.09). The concentration-dependent uptake kinetics of MMA(V) in rice roots could be described by the Michaelis-Menten equation, whereas the limited uptake of DMA(V) appeared to be linear in relation to the increasing concentration in the uptake medium (Abedin et al., 2002). Abbas and Meharg (2008) showed that DMA(V) uptake by maize (Zea mays) seedlings was enhanced by more than 10-fold by a pretreatment of phosphorus starvation; this compared with only 2-fold increase in As(V) uptake. They thought that DMA(V) might be taken up by the phosphate transporters, or that phosphorus starvation altered expression of a range of membrane transporters or even membrane permeability itself.In addition to the root uptake of methylated As species, some plants appear to be able to biomethylate As, but the pathway and enzymology remains unclear (Wu et al., 2002; Zhao et al., 2009). In microbes, As methylation follows the Challenger pathway involving repeated steps of As reduction and oxidative methylation (Bentley and Chasteen, 2002). As(V) is first reduced to As(III), which is methylated by S-adenosylmethyltransferase using S-adenosyl-l-Met as the methyl donor. The product of this reaction is pentavalent MMA(V), which is reduced by a reductase to trivalent MMA(III) with thiols (e.g. glutathione). Methylation and reduction steps continue to produce di- and trimethyl As compounds. MMA(III) and DMA(III) are intermediates in the As methylation pathway, which is not very stable (Gong et al., 2001). In rice grain, DMA(V) is the main form of methylated As, and can account for up to 80% of the total As (Zavala et al., 2008; Meharg et al., 2009). In light of the significant presence of methylated As in rice, it is important to elucidate the transport and assimilation pathways of these As species in plants.In this study, we present evidence that MMA(V) and DMA(V) are taken up by rice roots, at least partly, through the NIP aquaporin channel Lsi1, and that this process is strongly pH dependent. We also show that MMA(V) can be reduced to MMA(III) in planta.     

Silicon induces phytochelatin and ROS scavengers facilitating cadmium detoxification in rice

• Cadmium (Cd) is detrimental to crops and the environment. This work examines the natural mechanisms underlying silicon‐ (Si‐)directed Cd detoxification in rice plants.
• The addition of Si to plants under Cd stress caused significant improvements in morphological parameters, chlorophyll score, F_v/F_m and total soluble protein concentration compared to controls, confirming that Si is able to ameliorate Cd‐induced damage in rice plants. This morpho‐physiological evidence was correlated with decreased cell death and electrolyte leakage after Si application.
• The results showed no critical changes in root Cd concentration, while shoot Cd decreased significantly after Si supplementation in comparison with Cd‐stressed rice. Additionally, expression of Cd transporters (OsNRAMP5 and OsHMA2) was significantly down‐regulated while the concentration of phytochelatin, cysteine and glutathione, together with expression of OsPCS1 (phytochelatin synthase) in roots of Cd‐stressed rice was significantly induced when subjected to Si treatment. This confirms that the alleviation of Cd stress is not only limited to the down‐regulation of Cd transporters but also closely related to the phytochelatin‐driven vacuolar storage of Cd in rice roots.
• The enzymatic analysis further revealed the role of SOD and GR enzymes in protecting rice plants from Cd‐induced oxidative harm. These findings suggest a mechanistic basis in rice plants for Si‐mediated mitigation of Cd stress.

     

Deficiency in alcohol dehydrogenase 2 reduces arsenic in rice grains by suppressing silicate transporters

Paddy fields are anaerobic and facilitate arsenite (As(III)) elution from the soil. Paddy-field rice accumulates arsenic (As) in its grains because silicate transporters actively assimilate As(III) during the reproductive stage. Reducing the As level in rice grains is an important challenge for agriculture. Using a forward genetic approach, we isolated a rice (Oryza sativa) mutant, low arsenic line 3 (las3), whose As levels were decreased in aerial tissues, including grains. The low-As phenotype was not observed in young plants before heading (emergence of the panicle). Genetic analyses revealed that a deficiency in alcohol dehydrogenase (ADH) 2 by mutation is responsible for the phenotype. Among the three rice ADH paralogues, ADH2 was the most efficiently produced in root tissue under anaerobic conditions. In wild-type (WT), silicon and As concentrations in aerial tissues increased with growth. However, the increase was suppressed in las3 during the reproductive stage. Accordingly, the gene expression of two silicate transporters, Lsi1 and Lsi2, was increased in WT around the time of heading, whereas the increase was suppressed in las3. These results indicate that the low-As phenotype in las3 is due to silicate transporter suppression. Measurement of intracellular pH by ³¹P-nuclear magnetic resonance revealed intracellular acidification of las3 roots under hypoxia, suggesting that silicate transporter suppression in las3 might arise from an intracellular pH decrease, which is known to be facilitated by a deficiency in ADH activity under anaerobic conditions. This study provides valuable insight into reducing As levels in rice grains.

Deficiency in alcohol dehydrogenase suppresses arsenite uptake via silicate transporters and reduces arsenic levels in rice grains.     

水稻硅转运蛋白研究进展

硅是促进水稻生长和维持持续生产的重要营养元素,它有助于提高植物抗病虫害、抗倒伏以及抗生物和非生物胁迫的能力。硅能改善水稻的形态结构,提高产量和品质,还可以提高氮、磷利用率,减轻一些重金属的毒害作用。综述了水稻硅吸收运输有关的输入转运蛋白Lsi1、输出转运蛋白Lsi2和运输蛋白Lsi6表达和功能。同时,对这些转运蛋白的应用前景进行了展望。