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.
silicon; efflux transporter; pumpkin; cucumber; bloom
Silicon (Si) is the most abundant minerals in soil and exerts beneficial effects on plant growth by alleviating various stresses. The transport of Si from soil to the panicles is mediated by different transporters. Lsi1, belonging to a NIP group of the aquaporin family, is responsible for the uptake of Si from soil into the root cells in both dicots and monocots although its expression patterns and cellular localization differ with plant species. The subsequent transport of Si out of the root cells towards the stele is medicated by an active efflux transporter, Lsi2. Lsi1 and Lsi2 are polarly localized at the distal and proximal sides, respectively, of both exodermis and endodermis in rice root. Silicon in the xylem sap is presented in the form of monosilicic acid and is unloaded by Lsi6, a homolog of Lsi1 in rice. Lsi6 is also involved in the inter-vascular transfer of Si at the node, which is necessary for preferential Si distribution to the panicles.
localization; distribution; uptake; silicon; transporter
Nrat1 is a plasma membrane-localized aluminum transporter recently identified in rice, which is a member of Nramp family. Here, we further characterized this transporter in terms of transport substrate specificity. Heterologous assay in yeast showed that Al transport activity by Nrat1 was unaffected by the presence of high concentration of Ca, but significantly inhibited by trivalent ions including Yb and Ga, analogs of Al. Knockout of Nrat1 did not affect the uptake of Cd and Mn in rice. On the other hand, overexpression of Nrat1 led to enhanced Al uptake by rice roots compared with wild-type rice, but did not affect Cd uptake. These results provide further evidence that unlike other Nramp members, Nrat1 is an influx transporter for trivalent Al ion.
aluminum; transporter; rice; substrate specificity
Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl−-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.
In plants, ascorbate is synthesized in the mitochondria yet plays essential roles as an antioxidant in the chloroplast. Here, Miyaji et al. show that AtPHT4;4 is a chloroplast envelope ascorbate transporter and suggest it is required for dissipation of excess energy under light stress.
AIM: To explore the relationship between the level of proinsulin with cardiovascular risk factors and sleep snoring.
METHODS: Based on the random stratified sampling principle, 1 193 Chinese residents in Pizhou City, Jiangsu Province (530 males and 663 females, aged 35-59 years with an average age of 46.69 years) were recruited. Their sleep snoring habits were investigated. Biotin-avidin based double mAbs ELISA was used to detect specific insulin and proinsulin, and a risk factor score was established to evaluate the individuals according to the number of their risk factors.
RESULTS: The results of Spearman correlation analysis and covariate ANOVA analysis after age and sex were controlled, indicated that not only the level of proinsulin (r = 0.156, P = 0.000, F = 5.980 P = 0.000), but also cardiovascular risk factors score (r = 0.194, P = 0.000, F = 11.135, P = 0.000) significantly associated with the frequency of sleep snoring, and the significant relationship between true insulin and frequency of sleep snoring was only shown in the covariate ANOVA analysis (F = 2.868, P = 0.022). The result of multivariate stepwise logistic regression after age, sex, body mass index, waist circumference and true insulin were controlled showed that proinsulin (division by interval of quartile) was an independent risk factor for sleep snoring (OR = 1.220, 95%CI: 1.085-1.373, P = 0.001).
CONCLUSION: The interaction of cardiovascular risk factors clustering, high proinsulin level and sleep breathing disorder may be a syndrome, which has not been recognized in human beings so far.
True insulin; Proinsulin; Snoring; Epidemiology
AIM: To investigate the association between true insulin and proinsulin and clustering of cardiovascular risk factors.
METHODS: Based on the random stratified sampling principles, 1196 Chinese people (533 males and 663 females, aged 35-59 years with an average age of 46.69 years) were recruited. Biotin-avidin based double monoclonal antibody ELISA method was used to detect the true insulin and proinsulin, and a risk factor score was set to evaluate individuals according to the number of risk factors.
RESULTS: The median (quartile range) of true insulin and proinsulin was 4.91 mIu/L (3.01-7.09 mIu/L) and 3.49 pmol/L (2.14-5.68 pmol/L) respectively, and the true insulin level of female subjects was significantly higher than that of male subjects (P = 0.000), but the level of proinsulin displayed no significant difference between males and females (P = 0.566). The results of covariate ANOVA after age and sex were controlled showed that subjects with any of the risk factors had a significantly higher true insulin level (P = 0.002 for hypercholesterolemia, P = 0.021 for high low-density lipoprotein cholesterol, P = 0.003 for low high-density lipoprotein cholesterol, and P = 0.000 for other risk factors) and proinsulin level (P = 0.001 for low high-density lipoprotein cholesterol, and P = 0.000 for other risk factors) than those with no risk factors. Furthermore, subjects with higher risk factor scores had a higher true insulin and proinsulin level than those with lower risk factor scores (P = 0.000). The multiple linear regression models showed that true insulin and proinsulin were significantly related to cardiovascular risk factor scores respectively (P = 0.000).
CONCLUSION: True insulin and proinsulin are significantly associated with the clustering of cardiovascular risk factors.
True insulin; Proinsulin; Cardiovascular diseases
Overexpression of a tonoplast-localized transporter, OsHMA3, enhanced Cd tolerance and selectively reduced Cd accumulation in the shoots, but shoot Zn level was maintained by up-regulating genes involved in Zn uptake/translocation.
As a member of the heavy metal ATPase (HMA) family, OsHMA3 is a tonoplast-localized transporter for Cd in the roots of rice (Oryza sativa). Overexpression of OsHMA3 selectively reduces Cd accumulation in the grain. Further characterization in the present study revealed that overexpression of OsHMA3 also enhances the tolerance to toxic Cd. The growth of both the roots and shoots was similar in the absence of Cd between an OsHMA3-overexpressed line and vector control, but the Cd-inhibited growth was significantly alleviated in the OsHMA3-overexpressed line. The overexpressed line showed higher Cd concentration in the roots, but lower Cd concentration in the shoots compared with the wild-type rice and vector control line, indicating that overexpression of OsHMA3 enhanced vacuolar sequestration of Cd in the roots. The Zn concentration in the roots of the OsHMA3-overexpressed line was constantly higher than that of vector control, but the Zn concentration in the shoots was similar between the overexpressed line and vector control. Five transporter genes belonging to the ZIP family were constitutively up-regulated in the OsHMA3-overexpressed line. These results suggest that shoot Zn level was maintained by up-regulating these genes involved in the Zn uptake/translocation. Taken together, overexpression of OsHMA3 is an efficient way to reduce Cd accumulation in the grain and to enhance Cd tolerance in rice.
OsHMA3; Cd; overexpression; Oryza sativa; Zn transporter; tolerance.
Different expression of Nrat1, a gene encoding a plasma-membrane-localized Al transporter, is partially responsible for genotypic difference in Al tolerance in rice.
Although rice (Oryza sativa) is the most Al-tolerant species among small-grain cereal crops, there is wide genotypic variation in its tolerance to Al toxicity. A number of quantitative trait loci (QTLs) for Al tolerance have been detected, but the responsible genes have not been identified. By using chromosome segment substitution lines, this work found that Nrat1, a gene encoding an Al transporter, is responsible for a QTL previously detected on chromosome 2. Substitution of the chromosome segment containing Nrat1 from Koshihikari (Al-tolerant variety) by that from Kasalath (Al-sensitive variety) decreased Nrat1 expression and Al uptake and tolerance, but increased binding of Al to the cell wall. Nrat1 in Kasalath showed tissue localization similar to Koshihikari in the roots. Although Koshihikari and Kasalath differed in four amino acids in Nrat1 protein, Nrat1 from Kasalath also showed transport activity for Al. Analysis with site-directed mutagenesis revealed that these differences did not affect the Al-transport activity much. Furthermore, there was no correlation between Al tolerance and the open-reading-frame sequence differences in other rice varieties. On the other hand, there was good correlation between Nrat1 expression and Al tolerance; however, sequence comparison of the promoter region up to 2.1kb did not give a clear difference between the Al-tolerant and -sensitive varieties. Taken together, these results indicate that differential expression of Nrat1 is responsible for the QTL for Al tolerance on chromosome 2, although the mechanism controlling Nrat1 expression remains to be examined.
Al QTL; Al tolerance; expression; genotypic difference; Nrat1; Oryza sativa.
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.
CDF family; manganese tolerance; OsMTP8.1; rice; transporter; vacuole
Rice (Oryza sativa) is the most aluminum (Al)-tolerant crop among small-grain cereals, but the mechanism underlying its high Al resistance is still not well understood. To understand the mechanisms underlying high Al-tolerance, we performed a comparative genome-wide transcriptional analysis by comparing expression profiling between the Al-tolerance cultivar (Koshihikari) and an Al-sensitive mutant star1 (SENSITIVE TO AL RHIZOTOXICITY 1) in both the root tips and the basal roots. Exposure to 20 µM AlCl3 for 6 h resulted in up-regulation (higher than 3-fold) of 213 and 2015 genes including 185 common genes in the root tips of wild-type and the mutant, respectively. On the other hand, in the basal root, genes up-regulated by Al were 126 and 2419 including 76 common genes in the wild-type and the mutant, respectively. These results indicate that Al-response genes are not only restricted to the root tips, but also in the basal root region. Analysis with genes up- or down-regulated only in the wild-type reveals that there are other mechanisms for Al-tolerance except for a known transcription factor ART1-regulated one in rice. These mechanisms are related to nitrogen assimilation, secondary metabolite synthesis, cell-wall synthesis and ethylene synthesis. Although the exact roles of these putative tolerance genes remain to be examined, our data provide a platform for further work on Al-tolerance in rice.
Background and Aims
Brachypodium distachyon is a temperate grass with a small stature, rapid life cycle and completely sequenced genome that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In Zea mays, Yellow Stripe1 (ZmYS1) is the transporter responsible for the uptake of Fe(III)–PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. The aim of this study is to establish brachypodium as a model system to study Fe homeostasis in grasses, identify YSL proteins in brachypodium and maize, and analyse their expression profiles in brachypodium in response to Fe deficiency.
The YSL family of proteins in brachypodium and maize were identified based on sequence similarity to ZmYS1. Expression patterns of the brachypodium YSL genes (BdYSL genes) were determined by quantitative RT–PCR under Fe-deficient and Fe-sufficient conditions. The types of PS secreted, and secretion pattern of PS in brachypodium were analysed by high-performance liquid chromatography.
Eighteen YSL family members in maize and 19 members in brachypodium were identified. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade. The Fe status of the plant can regulate expression of brachypodium YSL genes in both shoots and roots. 3-Hydroxy-2′-deoxymugineic acid (HDMA) is the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated.
PS secretion by brachypodium parallels that of related crop species such as barley and wheat. A single grass species-specific YSL clade is present, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. Finally, the Fe-responsive expression profiles of several YSLs suggest roles in Fe homeostasis.
Brachypodium distachyon; Zea mays; iron homeostasis; phytosiderophore; nicotianamine; Yellow Stripe-Like; YSL; YS1
The plant cuticle, a cutin matrix embedded with and covered by wax, seals the aerial organ's surface to protect the plant against uncontrolled water loss. The cutin matrix is essential for the cuticle to function as a barrier to water loss. Recently, we identified from wild barley a drought supersensitive mutant, eibi1, which is caused by a defective cutin matrix as the result of the loss of function of HvABCG31, an ABCG full transporter. Here, we report that eibi1 epidermal cells contain lipid-like droplets, which are supposed to consist of cutin monomers that have not been transported out of the cells. The eibi1 cuticle is fragile due to a defective cutin matrix. The rice ortholog of the EIBI1 gene has a similar pattern of expression, young shoot but not flag leaf blade, as the barley gene. The model of the function of Eibi1 is discussed. The HvABCG31 full transporter functions in the export of cutin components and contributed to land plant colonization, hence also to terrestrial life evolution.
ABC transporter; cuticle; cuticular wax; drought resistance; inclusion
Nodulin-26-like intrinsic proteins (NIPs) of the aquaporin family are involved in the transport of diverse solutes, but the mechanisms controlling the selectivity of transport substrates are poorly understood. The purpose of this study was to investigate how the aromatic/arginine (ar/R) selectivity filter influences the substrate selectivity of two NIP aquaporins; the silicic acid (Si) transporter OsLsi1 (OsNIP2;1) from rice and the boric acid (B) transporter AtNIP5;1 from Arabidopsis; both proteins are also permeable to arsenite. Native and site-directed mutagenized variants of the two genes were expressed in Xenopus oocytes and the transport activities for Si, B, arsenite, and water were assayed. Substitution of the amino acid at the ar/R second helix (H2) position of OsLsi1 did not affect the transport activities for Si, B, and arsenite, but that at the H5 position resulted in a total loss of Si and B transport activities and a partial loss of arsenite transport activity. Conversely, changes of the AtNIP5;1 ar/R selectivity filter and the NPA motifs to the OsLsi1 type did not result in a gain of Si transport activity. B transport activity was partially lost in the H5 mutant but unaffected in the H2 mutant of AtNIP5;1. In contrast, both the single and double mutations at the H2 and/or H5 positions of AtNIP5;1 did not affect arsenite transport activity. The results reveal that the residue at the H5 position of the ar/R filter of both OsLsi1 and AtNIP5;1 plays a key role in the permeability to Si and B, but there is a relatively low selectivity for arsenite.
Arsenic; boron; NIP aquaporins; selectivity; silicon; substrate
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.
Barley; expression pattern; iron; localization; maize; phytosiderophore; transporter
Maize (Zea mays L.) shows a high accumulation of silicon (Si), but transporters involved in the uptake and distribution have not been identified. In the present study, we isolated two genes (ZmLsi1 and ZmLsi6), which are homologous to rice influx Si transporter OsLsi1. Heterologous expression in Xenopus laevis oocytes showed that both ZmLsi1 and ZmLsi6 are permeable to silicic acid. ZmLsi1 was mainly expressed in the roots. By contrast, ZmLsi6 was expressed more in the leaf sheaths and blades. Different from OsLsi1, the expression level of both ZmLsi1 and ZmLsi6 was unaffected by Si supply. Immunostaining showed that ZmLsi1 was localized on the plasma membrane of the distal side of root epidermal and hypodermal cells in the seminal and crown roots, and also in cortex cells in lateral roots. In the shoots, ZmLsi6 was found in the xylem parenchyma cells that are adjacent to the vessels in both leaf sheaths and leaf blades. ZmLsi6 in the leaf sheaths and blades also exhibited polar localization on the side facing towards the vessel. Taken together, it can be concluded that ZmLsi1 is an influx transporter of Si, which is responsible for the transport of Si from the external solution to the root cells and that ZmLsi6 mainly functions as a Si transporter for xylem unloading.
Influx; Maize; NIP; Root; Silicon; Transporter
Background and Aims
Ethylene diamine tetraacetic acid (EDTA)-assisted phytoremediation has been developed to clean up lead (Pb)-contaminated soil; however, the mechanism responsible for the uptake of EDTA–Pb complex is not well understood. In this study, the accumulation process of Pb from EDTA–Pb is characterized in comparison to ionic Pb [Pb(NO3)2] in sorghum (Sorghum bicolor).
Sorghum seedlings were exposed to a 0·5 mm CaCl2 (pH 5·0) solution containing 0, 1 mm Pb(NO3)2 or EDTA–Pb complexes at a molar ratio of 1:0·5, 1:1, 1:2 and 1:4 (Pb:EDTA). The root elongation of sorghum at different ratios of Pb:EDTA was measured. Xylem sap was collected after the stem was severed at different times. The concentration of Pb in the shoots and roots were determined by an atomic absorption spectrometer. In addition, the roots were stained with Fluostain I for observation of the root structure.
Lead accumulation in the shoots of the plants exposed to EDTA–Pb at 1:1 ratio was only one-fifth of that exposed to ionic Pb at the same concentration. Lead accumulation decreased when transpiration was suppressed. The concentration of Pb in the xylem sap from the EDTA–Pb-treated plants was about 1/25 000 of that in the external solution. Root elongation was severely inhibited by ionic Pb, but not by EDTA–Pb at a 1:1 ratio. Root staining showed that a physiological barrier was damaged in the roots exposed to ionic Pb, but not in the roots exposed to EDTA–Pb.
All these results suggest that sorghum roots are inefficient in uptake of EDTA-chelated Pb and that enhanced Pb accumulation from ionic Pb was attributed to the damaged structure of the roots.
Complex; EDTA; form; Pb; phytoremediation; uptake system; sorghum (Sorghum bicolor)
• Background and Aims Rice (Oryza sativa) is an aquatic plant with a characteristic of forming iron plaque on its root surfaces. It is considered to be the most Al-tolerant species among the cereal crops. The objective of this study was to determine the effects of root surface iron plaque on Al translocation, accumulation and the change of physiological responses under Al stress in rice in the presence of iron plaque.
• Methods The japonica variety rice, Koshihikari, was used in this study and was grown hydroponically in a growth chamber. Iron plaque was induced by exposing the rice roots to 30 mg L−1 ferrous iron either as Fe(II)-EDTA in nutrient solution (6 d, Method I) or as FeSO4 in water solution (12 h, Method II). Organic acid in root exudates was retained in the anion-exchange resin and eluted with 2 m HCl, then analysed by high-performance liquid chromatography (HPLC) after proper pre-treatment. Fe and Al in iron plaque were extracted with DCB (dithionite–citrate–bicarbonate) solution.
• Key Results and Conclusions Both methods (I and II) could induce the formation of iron plaque on rice root surfaces. The amounts of DCB-extractable Fe and Al on root surfaces were much higher in the presence of iron plaque than in the absence of iron plaque. Al contents in root tips were significantly decreased with iron plaque; translocation of Al from roots to shoots was significantly reduced with iron plaque. Al-induced secretion of citrate was observed and iron plaque could greatly depress this citrate secretion. These results suggested that iron plaque on rice root surfaces can be a sink to sequester Al onto the root surfaces and Fe ions can pre-saturate Al-binding sites in root tips, which protects the rice root tips from suffering Al stress to a certain extent.
Rice (Oryza sativa); formation of iron plaque; root surface; Fe(II)-EDTA; aluminium (Al) stress; citrate secretion; DCB (dithionite–citrate–bicarbonate); root tip; Al translocation
Originating from the Fertile Crescent in the Middle East, barley has now been cultivated widely on different soil types including acid soils, where aluminium toxicity is a major limiting factor. Here we show that the adaptation of barley to acid soils is achieved by the modification of a single gene (HvAACT1) encoding a citrate transporter. We find that the primary function of this protein is to release citrate from the root pericycle cells to the xylem to facilitate the translocation of iron from roots to shoots. However, a 1-kb insertion in the upstream of the HvAACT1 coding region occurring only in the Al-tolerant accessions, enhances its expression and alters the location of expression to the root tips. The altered HvAACT1 has an important role in detoxifying aluminium by secreting citrate to the rhizosphere. Thus, the insertion of a 1-kb sequence in the HvAACT1 upstream enables barley to adapt to acidic soils.
Barley is an important food crop that has been adapted to grow on acidic soils that often contain toxic soluble aluminium. In this study, an insertion in the upstream region of a citrate transporter is shown to confer resistance of barley to aluminium toxicity and is found in aluminium-tolerant barley accessions.