HvLsi1 is a silicon influx transporter in barley

Most plants accumulate silicon in their bodies, and this is thought to be important for resistance against biotic and abiotic stresses; however, the molecular mechanisms for Si uptake and accumulation are poorly understood. Here, we describe an Si influx transporter, HvLsi1, in barley. This protein is homologous to rice influx transporter OsLsi1 with 81% identity, and belongs to a Nod26-like major intrinsic protein sub-family of aquaporins. Heterologous expression in both Xenopus laevis oocytes and a rice mutant defective in Si uptake showed that HvLsi1 has transport activity for silicic acid. Expression of HvLsi1 was detected specifically in the basal root, and the expression level was not affected by Si supply. There was a weak correlation between Si uptake and the expression level of HvLsi1 in eight cultivars tested. In the seminal roots, HvLsi1 is localized on the plasma membrane on the distal side of epidermal and cortical cells. HvLsi1 is also located in lateral roots on the plasma membrane of hypodermal cells. These cell-type specificity of localization and expression patterns of HvLsi1 are different from those of OsLsi1. These observations indicate that HvLsi1 is a silicon influx transporter that is involved in radial transport of Si through the epidermal and cortical layers of the basal roots of barley.     

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.     

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.     

Isolation and functional characterization of an influx silicon transporter in two pumpkin cultivars contrasting in silicon accumulation

A high accumulation of silicon (Si) is required for overcoming abiotic and biotic stresses, but the molecular mechanisms of Si uptake, especially in dicotyledonous species, is poorly understood. Herein, we report the identification of an influx transporter of Si in two Cucurbita moschata (pumpkin) cultivars greatly differing in Si accumulation, which are used for the rootstocks of bloom and bloomless Cucumis sativus (cucumber), respectively. Heterogeneous expression in both Xenopus oocytes and rice mutant defective in Si uptake showed that the influx transporter from the bloom pumpkin rootstock can transport Si, whereas that from the bloomless rootstock cannot. Analysis with site-directed mutagenesis showed that, among the two amino acid residues differing between the two types of rootstocks, only changing a proline to a leucine at position 242 results in the loss of Si transport activity. Furthermore, all pumpkin cultivars for bloomless rootstocks tested have this mutation. The transporter is localized in all cells of the roots, and investigation of the subcellular localization with different approaches consistently showed that the influx Si transporter from the bloom pumpkin rootstock was localized at the plasma membrane, whereas the one from the bloomless rootstock was localized at the endoplasmic reticulum. Taken together, our results indicate that the difference in Si uptake between two pumpkin cultivars is probably the result of allelic variation in one amino acid residue of the Si influx transporter, which affects the subcellular localization and subsequent transport of Si from the external solution to the root cells.     

Distinct expression and function of three ammonium transporter genes (OsAMT1;1-1;3) in rice

To study the regulation of ammonium uptake into rice roots, three ammonium transporter genes (OsAMT1;1, 1;2 and 1;3; Oryza sativa ammonium transporter) were isolated and examined. OsAMT1s belong to AMT1 family, containing 11 putative transmembrane-spanning domains. Southern blot analysis and screening of the rice genome database confirmed that with OsAMT1;1-1;3 the complete AMT1 family of rice had been isolated. Heterologous expression of OsAMT1s in the yeast Saccharomyces cerevisiae mutant 31019b showed that all three OsAMT1s exhibit ammonium transport activity. Northern blot analysis showed a distinct expression pattern for the three genes; more constitutive expression in shoots and roots for OsAMT1;1, root-specific and ammonium-inducible expression for OsAMT1;2, and root-specific and nitrogen-derepressible expression for OsAMT1;3. In situ mRNA detection revealed that OsAMT1;2 is expressed in the central cylinder and cell surface of root tips. This gene expression analysis revealed a distinct nitrogen-dependent regulation for AMTs in rice, differing from that in tomato or ARABIDOPSIS:     

Identification of maize silicon influx transporters

Maize (Zea mays L.) shows a high accumulation of silicon (Si),but transporters involved in the uptake and distribution havenot been identified. In the present study, we isolated two genes(ZmLsi1 and ZmLsi6), which are homologous to rice influx Sitransporter OsLsi1. Heterologous expression in Xenopus laevisoocytes showed that both ZmLsi1 and ZmLsi6 are permeable tosilicic acid. ZmLsi1 was mainly expressed in the roots. By contrast,ZmLsi6 was expressed more in the leaf sheaths and blades. Differentfrom OsLsi1, the expression level of both ZmLsi1 and ZmLsi6was unaffected by Si supply. Immunostaining showed that ZmLsi1was localized on the plasma membrane of the distal side of rootepidermal and hypodermal cells in the seminal and crown roots,and also in cortex cells in lateral roots. In the shoots, ZmLsi6was found in the xylem parenchyma cells that are adjacent tothe vessels in both leaf sheaths and leaf blades. ZmLsi6 inthe leaf sheaths and blades also exhibited polar localizationon the side facing towards the vessel. Taken together, it canbe concluded that ZmLsi1 is an influx transporter of Si, whichis responsible for the transport of Si from the external solutionto the root cells and that ZmLsi6 mainly functions as a Si transporterfor xylem unloading.     

Putrescine alleviates aluminum toxicity in rice (Oryza sativa) by reducing cell wall Al contents in an ethylene‐dependent manner

Aluminum (Al³⁺) toxicity in acidic soils limits crop productivity worldwide. In this study, we found that putrescine (PUT) significantly alleviates Al toxicity in rice roots. The addition of 0.1 mM PUT promoted root elongation and reduced the Al content in the root apices of Nipponbare (Nip) and Kasalath (Kas) rice under Al toxicity conditions. Exogenous treatment with PUT reduced the cell wall Al content by reducing polysaccharide (pectin and hemicellulose) levels and pectin methylesterase (PME) activity in roots and decreased the translocation of Al from the external environment to the cytoplasm by downregulating the expression of OsNRAT1, which responsible to encode an Al transporter protein Nrat1 (Nramp aluminum transporter 1). The addition of PUT under Al toxicity conditions significantly inhibited ethylene emissions and suppressed the expression of genes involved in ethylene biosynthesis. Treatment with the ethylene precursor 1‐aminocylopropane‐1‐carboxylic acid (ACC) significantly improved ethylene emission, inhibited root elongation, increased the Al accumulation in root tips and the root cell wall, and increased cell wall pectin and hemicellulose contents in both rice cultivars under Al toxicity conditions. The ethylene biosynthesis antagonist aminoethoxyvinylglycine (AVG, inhibitor of the ACC synthase) had the opposite effect and reduced PME activity. Together, our results show that PUT decreases the cell wall Al contents by suppressing ethylene emissions and decreases the symplastic Al levels by downregulating OsNRAT1 in rice.     

Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11‐controlled root development

Potassium (K) deficiency in plants confines root growth and decreases root‐to‐shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL‐related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low‐K‐enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11‐regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K‐deficiency‐enhanced expression of several RR genes encoding type‐A cytokinin‐responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K‐deficient soil increased total K uptake by 72% and grain yield by 24%–32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.     

Biochemical and molecular changes in rice seedlings (Oryza sativa L.) to cope with chromium stress

Chromium (Cr) is very toxic to both humans and plants. This investigation aimed to understand the physiological and molecular responses of rice seedlings to Cr stress. Cr toxicity did not significantly affect morphological features and Cr accumulation in roots and shoots in Pokkali but not in BRRI 51, although there was a reduction in chlorophyll concentration in leaves of both genotypes. These results imply that Pokkali has mechanisms to cope with Cr supplementation. We therefore performed quantitative real‐time PCR on the expression pattern of two chelator genes, OsPCS1 and OsMT1, but there were no significant changes in expression in roots and shoots of Pokkali and BRRI 51 following Cr stress. This suggests that there was no metal sequestration following heavy metal stress in roots of these genotypes. Moreover, no expression of two heavy metal transporter genes, OsHMA3 and OsNRAMP1, was induced after Cr stress in roots and shoots, suggesting that these transporter genes are not induced by Cr stress or might not be involved in Cr uptake in rice. We also performed a targeted study on the effect of Cr on Fe uptake mechanisms. Our studies showed a consistent reduction in Fe uptake, Fe reductase activity and expression of Fe‐related genes (OsFRO1 and OsIRT1) under Cr stress in both roots and leaves of Pokkali. In contrast, these parameters and genes were significantly increased in Cr‐sensitive BRRI 51 under Cr stress. The results confirm that limiting Fe uptake through the down‐regulation of Fe reductase and Fe transporter genes is the main strategy of Cr‐tolerant Pokkali to cope with Cr stress. Finally, increased CAT, POD and GR activity and elevated glutathione and proline synthesis might provide strong antioxidant defence against Cr stress in Pokkali. Taken together, our findings reveal that Cr stress tolerance in rice (Pokkali) is not related to metal sequestration but is associated with reduced Fe transport and increased antioxidant defence.     

A high activity zinc transporter OsZIP9 mediates zinc uptake in rice

Zinc (Zn) is an essential micronutrient for most organisms including humans, and Zn deficiency is widespread in human populations, particularly in underdeveloped regions. Cereals such as rice (Oryza sativa) are the major dietary source of Zn for most people. However, the molecular mechanism underlying Zn uptake in rice is still not fully understood. Here, we report that a member of the ZIP (ZRT, IRT‐like protein) family, OsZIP9, contributes to Zn uptake in rice. It was expressed in the epidermal and exodermal cells of lateral roots, localized in the plasma membrane and induced during Zn deficiency. Yeast‐expressed OsZIP9 showed much higher Zn influx transport activity than other rice ZIP proteins in a wide range of Zn concentrations. OsZIP9 knockout rice plants showed a significant reduction in growth at low Zn concentrations, but could be rescued by a high Zn supply. Compared with the wild type, accumulation of Zn in root, shoot and grain was much lower in knockout lines, particularly with a low supply of Zn under both hydroponic and paddy soil conditions. OsZIP9 also showed Co uptake activity. Natural variation of OsZIP9 expression level is highly associated with Zn content in milled grain among rice varieties in the germplasm collection. Taken together, these results show that OsZIP9 is an important influx transporter responsible for the take up of Zn and Co from external media into root cells.     

Interactive effects of potassium and sodium on root growth and expression of K/Na transporter genes in rice

Sufficient supply of potassium (K) can alleviate the adverse effects of excess sodium (Na) on plant growth. However, it remains unclear if such a beneficial function is related to regulation of root growth and/or expression of K/Na transporters. Herein we report the responses of a rice cultivar, which was pretreated with normal nutrient solution for 1 month, to three levels of Na (0, 25, and 100 mM) without or with supply of K for 9 days. High Na (100 mM) significantly decreased plant growth, root activity, and total K uptake, and increased biomass ratio of roots to shoots. Short-term removal of K supply (9 days) did not affect root morphology and biomass ratio of roots to shoots, but decreased root activity of seedlings grown in high Na solution. K deficiency increased uptake of Na and transport of K from roots to shoots. Moreover, expression of OsHAK1, a putative K transporter gene, was upregulated by low Na (25 mM) and downregulated by high Na (100 mM) in roots. In leaves, its expression was suppressed by the Na treatments when K supply was maintained. Expression of OsHKT2;1, which encodes a protein that acts mainly as a Na transporter, was downregulated by high Na, but was enhanced by K deficiency both in roots and leaves. Expression of five other putative K/Na transporter or Na⁺/H⁺ genes, OsHKT1;1, OsHKT1;2, OsHKT2;3, OsNHX1, and OsSOS1, was not affected by the treatments. The results suggest that OsHAK1 and OsHKT2;1 were involved in the interactive effects of K and Na on their uptake and distribution in rice. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A multidrug and toxic compound extrusion (MATE) transporter modulates auxin levels in root to regulate root development and promotes aluminium tolerance

MATE (multidrug and toxic compound extrusion) transporters play multiple roles in plants including detoxification, secondary metabolite transport, aluminium (Al) tolerance, and disease resistance. Here we identify and characterize the role of the Arabidopsis MATE transporter DETOXIFICATION30. AtDTX30 regulates auxin homeostasis in Arabidopsis roots to modulate root development and Al-tolerance. DTX30 is primarily expressed in roots and localizes to the plasma membrane of root epidermal cells including root hairs. dtx30 mutants exhibit reduced elongation of the primary root, root hairs, and lateral roots. The mutant seedlings accumulate more auxin in their root tips indicating role of DTX30 in maintaining auxin homeostasis in the root. Al induces DTX30 expression and promotes its localization to the distal transition zone. dtx30 seedlings accumulate more Al in their roots but are hyposensitive to Al-mediated rhizotoxicity perhaps due to saturation in root growth inhibition. Increase in expression of ethylene and auxin biosynthesis genes in presence of Al is absent in dtx30. The mutants exude less citrate under Al conditions, which might be due to misregulation of AtSTOP1 and the citrate transporter AtMATE. In conclusion, DTX30 modulates auxin levels in root to regulate root development and in the presence of Al indirectly modulates citrate exudation to promote Al tolerance.     

Oryza sativa Lysine‐Histidine‐type Transporter 1 functions in root uptake and root‐to‐shoot allocation of amino acids in rice

In agricultural soils, amino acids can represent vital nitrogen (N) sources for crop growth and yield. However, the molecular mechanisms underlying amino acid uptake and allocation are poorly understood in crop plants. This study shows that rice (Oryza sativa L.) roots can acquire aspartate at soil concentration, and that japonica subspecies take up this acidic amino acid 1.5‐fold more efficiently than indica subspecies. Genetic association analyses with 68 representative japonica or indica germplasms identified rice Lysine‐Histidine‐type Transporter 1 (OsLHT1) as a candidate gene associated with the aspartate uptake trait. When expressed in yeast, OsLHT1 supported cell growth on a broad spectrum of amino acids, and effectively transported aspartate, asparagine and glutamate. OsLHT1 is localized throughout the rice root, including root hairs, epidermis, cortex and stele, and to the leaf vasculature. Knockout of OsLHT1 in japonica resulted in reduced root uptake of amino acids. Furthermore, in ¹⁵N‐amino acid‐fed mutants versus wild‐type, a higher percentage of ¹⁵N remained in roots instead of being allocated to the shoot. ¹⁵N‐ammonium uptake and subsequently the delivery of root‐synthesized amino acids to Oslht1 shoots were also significantly decreased, which was accompanied by reduced shoot growth. These results together provide evidence that OsLHT1 functions in both root uptake and root to shoot allocation of a broad spectrum of amino acids in rice.     

A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high‐affinity nitrate influx in Arabidopsis thaliana

In plants that have been deprived of nitrate for a significant length of time, a constitutive high‐affinity nitrate transport system (cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of ¹³NO₃^– influx measurements and Blue Native polyacrylamide gel electrophoresis we examined the role of AtNRT2.5 in cHATS in wild type (WT) and various T‐DNA mutants of Arabidopsis thaliana. We demonstrate that AtNRT2.5 is predominantly expressed in roots of nitrate‐deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63% compared with WT in roots of Atnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high‐affinity transporter encoded by AtNRT2.1/AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low K_m for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment.