Takehiro Kamiya

NIP1;1, an Aquaporin Homolog, Determines the Arsenite Sensitivity of Arabidopsis thaliana *□

Arsenite [As(III)] is highly toxic to organisms, including plants. Very recently, transporters in rice responsible for As(III) transport have been described (Ma, J. F., Yamaji, N., Mitani, N., Xu, X. Y., Su, Y. H., McGrath, S. P., and Zhao, F. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 9931–9935), but little is known about As(III) tolerance. In this study, three independent As(III)-tolerant mutants were isolated from ethyl methanesulfonate-mutagenized M2 seeds of Arabidopsis thaliana. All three mutants carried independent mutations in Nodulin 26-like intrinsic protein 1;1 (NIP1;1), a homolog of an aquaporin. Two independent transgenic lines carrying T-DNA in NIP1;1 were highly tolerant to As(III), establishing that NIP1;1 is the causal gene of As(III) tolerance. Because an aquaglyceroporin is able to transport As(III), we measured As(III) transport activity. When expressed in Xenopus oocytes, NIP1;1 was capable of transporting As(III). As content in the mutant plants was 30% lower than in wild-type plants. Promoter β-glucuronidase and real-time PCR analysis showed that NIP1;1 is highly expressed in roots, and GFP-NIP1;1 is localized to the plasma membrane. These data show that NIP1;1 is involved in As(III) uptake into roots and that disruption of NIP1;1 function confers As(III) tolerance to plants. NIP1;2 and NIP5;1, closely related homologs of NIP1;1, were also permeable to As(III). Although the disruption of these genes reduced the As content in plants, As(III) tolerance was not observed in nip1;2 and nip5;1 mutants. This indicates that As(III) tolerance cannot be simply explained by decreased As contents in plants.

Low-affinity cation transporter (OsLCT1) regulates cadmium transport into rice grains

Accumulation of cadmium (Cd) in rice (Oryza sativa L.) grains poses a potential health problem, especially in Asia. Most Cd in rice grains accumulates through phloem transport, but the molecular mechanism of this transport has not been revealed. In this study, we identified a rice Cd transporter, OsLCT1, involved in Cd transport to the grains. OsLCT1-GFP was localized at the plasma membrane in plant cells, and OsLCT1 showed Cd efflux activity in yeast. In rice plants, strong OsLCT1 expression was observed in leaf blades and nodes during the reproductive stage. In the uppermost node, OsLCT1 transcripts were detected around large vascular bundles and in diffuse vascular bundles. RNAi-mediated knockdown of OsLCT1 did not affect xylem-mediated Cd transport but reduced phloem-mediated Cd transport. The knockdown plants of OsLCT1 accumulated approximately half as much Cd in the grains as did the control plants. The content of other metals in rice grains and plant growth were not negatively affected by OsLCT1 suppression. These results suggest that OsLCT1 functions at the nodes in Cd transport into grains and that in a standard japonica cultivar, the regulation of OsLCT1 enables the generation of “low-Cd rice” without negative effects on agronomical traits. These findings identify a transporter gene for phloem Cd transport in plants.

Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root.

Significance The endodermis acts as a “second skin” in plant roots by providing the cellular control necessary for the selective entry of water and mineral nutrients into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks diffusion across the endodermis in the cell wall. This junction is composed of a fine band of lignin, the polymer that gives wood its strength. Here, we characterize a dirigent protein (from Latin, dirigere: to guide or align) as playing a vital role in the patterning of lignin in the Casparian strip, identifying a new component of the molecular machinery that builds Casparian strips. The endodermis acts as a “second skin” in plant roots by providing the cellular control necessary for the selective entry of water and solutes into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks extracellular diffusion across the endodermis. This junction is composed of lignin that is polymerized by oxidative coupling of monolignols through the action of a NADPH oxidase and peroxidases. Casparian strip domain proteins (CASPs) correctly position this biosynthetic machinery by forming a protein scaffold in the plasma membrane at the site where the Casparian strip forms. Here, we show that the dirigent-domain containing protein, enhanced suberin1 (ESB1), is part of this machinery, playing an essential role in the correct formation of Casparian strips. ESB1 is localized to Casparian strips in a CASP-dependent manner, and in the absence of ESB1, disordered and defective Casparian strips are formed. In addition, loss of ESB1 disrupts the localization of the CASP1 protein at the casparian strip domain, suggesting a reciprocal requirement for both ESB1 and CASPs in forming the casparian strip domain

A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects

The endodermis represents the main barrier to extracellular diffusion in plant roots, and it is central to current models of plant nutrient uptake. Despite this, little is known about the genes setting up this endodermal barrier. In this study, we report the identification and characterization of a strong barrier mutant, schengen3 (sgn3). We observe a surprising ability of the mutant to maintain nutrient homeostasis, but demonstrate a major defect in maintaining sufficient levels of the macronutrient potassium. We show that SGN3/GASSHO1 is a receptor-like kinase that is necessary for localizing CASPARIAN STRIP DOMAIN PROTEINS (CASPs)—major players of endodermal differentiation—into an uninterrupted, ring-like domain. SGN3 appears to localize into a broader band, embedding growing CASP microdomains. The discovery of SGN3 strongly advances our ability to interrogate mechanisms of plant nutrient homeostasis and provides a novel actor for localized microdomain formation at the endodermal plasma membrane. DOI: http://dx.doi.org/10.7554/eLife.03115.001

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

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

The MYB36 transcription factor orchestrates Casparian strip formation

Significance Casparian strips play a critical role in sealing endodermal cells in the root to block uncontrolled extracellular uptake of nutrients and water. Building Casparian strips requires the construction of extracellular lignin structures that encircle cells within the cell wall and that are anchored to the plasma membranes of adjacent cells to form tight seals between them. The transcription factor we have discovered, and the set of genes it regulates, now provides us with the detailed “parts list” necessary to build Casparian strips. This finding has clear implications for better understanding the nature of tight cellular junctions in biology and also has practical implications of agricultural, offering the potential for improved water and nutrient use efficiencies and enhanced resistance to abiotic stresses. The endodermis in roots acts as a selectivity filter for nutrient and water transport essential for growth and development. This selectivity is enabled by the formation of lignin-based Casparian strips. Casparian strip formation is initiated by the localization of the Casparian strip domain proteins (CASPs) in the plasma membrane, at the site where the Casparian strip will form. Localized CASPs recruit Peroxidase 64 (PER64), a Respiratory Burst Oxidase Homolog F, and Enhanced Suberin 1 (ESB1), a dirigent-like protein, to assemble the lignin polymerization machinery. However, the factors that control both expression of the genes encoding this biosynthetic machinery and its localization to the Casparian strip formation site remain unknown. Here, we identify the transcription factor, MYB36, essential for Casparian strip formation. MYB36 directly and positively regulates the expression of the Casparian strip genes CASP1, PER64, and ESB1. Casparian strips are absent in plants lacking a functional MYB36 and are replaced by ectopic lignin-like material in the corners of endodermal cells. The barrier function of Casparian strips in these plants is also disrupted. Significantly, ectopic expression of MYB36 in the cortex is sufficient to reprogram these cells to start expressing CASP1–GFP, correctly localize the CASP1–GFP protein to form a Casparian strip domain, and deposit a Casparian strip-like structure in the cell wall at this location. These results demonstrate that MYB36 is controlling expression of the machinery required to locally polymerize lignin in a fine band in the cell wall for the formation of the Casparian strip.

Expressing ScACR3 in Rice Enhanced Arsenite Efflux and Reduced Arsenic Accumulation in Rice Grains

Arsenic (As) accumulation in rice grain poses a serious health risk to populations with high rice consumption. Extrusion of arsenite [As(III)] by ScAcr3p is the major arsenic detoxification mechanism in Saccharomyces cerevisiae. However, ScAcr3p homolog is absent in higher plants, including rice. In this study, ScACR3 was introduced into rice and expressed under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. In the transgenic lines, As concentrations in shoots and roots were about 30% lower than in the wild type, while the As translocation factors were similar between transgenic lines and the wild type. The roots of transgenic plants exhibited significantly higher As efflux activities than those of the wild type. Within 24 h exposure to 10 μM arsenate [As(V)], roots of ScACR3-expressing plants extruded 80% of absorbed As(V) to the external solution as As(III), while roots of the wild type extruded 50% of absorbed As(V). Additionally, by exposing the As-containing rice plants to an As-lacking solution for 24 h, about 30% of the total As derived from pre-treatment was extruded to the external solution by ScACR3-expressing plants, while about 15% of As was extruded by wild-type plants. Importantly, ScACR3 expression significantly reduced As accumulation in rice straws and grains. When grown in flooded soil irrigated with As(III)-containing water, the As concentration in husk and brown rice of the transgenic lines was reduced by 30 and 20%, respectively, compared with the wild type. This study reports a potential strategy to reduce As accumulation in the food chain by expressing heterologous genes in crops.

Arabidopsis NIP1;1 Transports Antimonite and Determines Antimonite Sensitivity

Antimony (Sb) is toxic to organisms including plants. Although it is not essential to organisms, plants take up Sb from the environment. In this study, we identified an antimonite [Sb(III)] transporter from Arabidopsis thaliana. We examined the Sb(III) tolerance of the disruption mutant plants of arsenite [As(III)] transporters, nodulin 26-like intrinsic proteins (NIPs), since Sb(III) is similar to As(III) in structure. One of the mutants, nip1;1, showed Sb(III) tolerance and accumulated less Sb. Furthermore, yeast expressing NIP1;1 accumulated twice as much Sb as control. These data indicate that NIP1;1 transports Sb(III) and determines the Sb(III) sensitivity of A. thaliana.

Phosphate deficiency signaling pathway is a target of arsenate and phosphate transporter OsPT1 is involved in As accumulation in shoots of rice

Abstract Arsenate [As(V)] is toxic to organisms, and phosphate (Pi) transporter can mediate As(V) uptake due to their similarity in chemical structure. In the rice (Oriza sativa L.) genome, 13 Pi transporter genes (OsPTs) are present. Their response to As(V) and contribution to As(V) accumulation are unknown. We determined absolute mRNA amount of OsPTs in rice seedlings and OsPT1, OsPT2, OsPT4, and OsPT8 were quantified by real-time polymerase chain reaction (PCR). OsPT2, OsPT4, and OsPT8 were highly induced by Pi deficiency, while OsPT1 was not. In accordance with Pi deficiency response, OsPT2, OsPT4, and OsPT8 induction by Pi deficiency were severely suppressed by As(V). Those data suggest that As(V) affects Pi deficiency signaling in rice. To examine the As(V) transport activity of OsPT1 in planta, we obtained T-DNA mutant of OsPT1 (ospt1). The transcript expression level of OsPT1 in ospt1 was reduced by 70% in shoots and 50% in roots compared to those in the wild-type (WT), and arsenic (As) concentrations in shoots were reduced by 60% compared to WT. We further overexpressed OsPT1-GFP in rice. Overexpression lines showed higher As accumulation in shoots compared to wild-type. OsPT1-GFP is localized to plasma membrane. These results indicate that OsPT1 is involved in As(V) uptake from soil or apoplast.

Homeostasis of the structurally important micronutrients, B and Si

This review focuses on recent advances in understanding the transport mechanisms of two elements, B and Si in plants. Both are present as noncharged molecules in soil solution as boric acid and silicic acid. Both function in apoplast: pectic polysaccharides crosslinked with borate and polymers of hydrated silica are important for the physical strength of plant cells. In recent years, molecular genetics revealed analogous transport systems of B and Si. Combinations of NIP channels and exporters localized to distal and proximal sides, allow efficient trans-cellular transport of the nutrients. Polar localization, observed in these transport molecules, is likely to be a key to regulate directional transport of nutrients.