Junpei Takano

Arabidopsis boron transporter for xylem loading.

Boron deficiency hampers the productivity of 132 crops in more than 80 countries. Boron is essential in higher plants primarily for maintaining the integrity of cell walls and is also beneficial and might be essential in animals and in yeast. Understanding the molecular mechanism(s) of boron transport is crucial for alleviating boron deficiency. Here we describe the molecular identification of boron transporters in biological systems. The Arabidopsis thaliana mutant bor1-1 is sensitive to boron deficiency. Uptake studies indicated that xylem loading is the key step for boron accumulation in shoots with a low external boron supply and that the bor1-1 mutant is defective in this process. Positional cloning identified BOR1 as a membrane protein with homology to bicarbonate transporters in animals. Moreover, a fusion protein of BOR1 and green fluorescent protein (GFP) localized to the plasma membrane in transformed cells. The promoter of BOR1 drove GFP expression in root pericycle cells. When expressed in yeast, BOR1 decreased boron concentrations in cells. We show here that BOR1 is an efflux-type boron transporter for xylem loading and is essential for protecting shoots from boron deficiency.

The Arabidopsis Major Intrinsic Protein NIP5;1 Is Essential for Efficient Boron Uptake and Plant Development under Boron Limitation

Boron (B) is essential in plants but often present at low concentrations in the environment. To investigate how plants survive under conditions of B limitation, we conducted a transcriptome analysis and identified NIP5;1, a member of the major intrinsic protein family, as a gene upregulated in B-deficient roots of Arabidopsis thaliana. Promoter–β-glucuronidase fusions indicated that NIP5;1 is strongly upregulated in the root elongation zone and the root hair zone under B limitation, and green fluorescent protein–tagged NIP5;1 proteins localized to the plasma membrane. Expression in Xenopus laevis oocytes demonstrated that NIP5;1 facilitated the transport of boric acid in addition to water. Importantly, two T-DNA insertion lines of NIP5;1 displayed lower boric acid uptake into roots, lower biomass production, and increased sensitivity of root and shoot development to B deficiency. These results identify NIP5;1 as a major plasma membrane boric acid channel crucial for the B uptake required for plant growth and development under B limitation.

Endocytic and Secretory Traffic in Arabidopsis Merge in the Trans-Golgi Network/Early Endosome, an Independent and Highly Dynamic Organelle

This study examines secretory and endocytotic trafficking in Arabidopsis by tracking the movement of a brassinosteroid receptor and a boron exporter through the endomembrane system. Both endocytotic and secretory cargo travel through the trans-Golgi network/early endosome (TGN/EE), and the TGN/EE is shown to be an independent organelle that only transiently associates with the Golgi. Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Polar localization and degradation of Arabidopsis boron transporters through distinct trafficking pathways

Boron (B) is essential for plant growth but is toxic when present in excess. In the roots of Arabidopsis thaliana under B limitation, a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, are required for efficient B uptake and subsequent translocation into the xylem, respectively. However, under high-B conditions, BOR1 activity is repressed through endocytic degradation, presumably to avoid B toxicity. In this study, we investigated the localization of GFP-tagged NIP5;1 and BOR1 expressed under the control of their native promoters. Under B limitation, GFP-NIP5;1 and BOR1-GFP localized preferentially in outer (distal) and inner (proximal) plasma membrane domains, respectively, of various root cells. The polar localization of the boric acid channel and boric acid/borate exporter indicates the radial transport route of B toward the stele. Furthermore, mutational analysis revealed a requirement of tyrosine residues, in a probable cytoplasmic loop region of BOR1, for polar localization in various cells of the meristem and elongation zone. The same tyrosine residues were also required for vacuolar targeting upon high B supply. The present study of BOR1 and NIP5;1 demonstrates the importance of selective endocytic trafficking in polar localization and degradation of plant nutrient transporters for radial transport and homeostasis of plant mineral nutrients.

NIP6;1 Is a Boric Acid Channel for Preferential Transport of Boron to Growing Shoot Tissues in Arabidopsis

Boron (B) in soil is taken up by roots through NIP5;1, a boric acid channel, and is loaded into the xylem by BOR1, a borate exporter. Here, the function of Arabidopsis thaliana NIP6;1, the most similar gene to NIP5;1, was studied. NIP6;1 facilitates the rapid permeation of boric acid across the membrane but is completely impermeable to water. NIP6;1 transcript accumulation is elevated in response to B deprivation in shoots but not in roots. NIP6;1 promoter–β-glucuronidase is predominantly expressed in nodal regions of shoots, especially the phloem region of vascular tissues. Three independently identified T-DNA insertion lines for the NIP6;1 gene exhibited reduced expansion of young rosette leaves only under low-B conditions. B concentrations are reduced in young rosette leaves but not in the old leaves of these mutants. Taken together, these data strongly suggest that NIP6;1 is a boric acid channel required for proper distribution of boric acid, particularly among young developing shoot tissues. We propose that NIP6;1 is involved in xylem–phloem transfer of boric acid at the nodal regions and that the water-tight property of NIP6;1 is important for this function. It is proposed that during evolution, NIP5;1 and NIP6;1 were diversified in terms of both the specificity of their expression in plant tissues and their water permeation properties, while maintaining their ability to be induced under low B and their boric acid transport activities.

Plants Tolerant of High Boron Levels

Reduced crop productivity due to soils containing toxic levels of boron (B) is a worldwide problem in food production. It is estimated that up to 17% of the barley yield losses in southern Australia are caused by B toxicity. We found that the expression of AtBOR4, an Arabidopsis paralog of BOR1, the first identified boron transporter gene, generates plants that are tolerant of high B levels. BOR4 is a polarly localized borate exporter that enhances B efflux from roots. The present study is a foundation for the improvement of crop productivity in soils containing excess B, which are distributed in arid areas of the world.

Boron transport mechanisms: collaboration of channels and transporters

Boron (B) is an essential element for plants, but is also toxic when present in excess. B deficiency and toxicity are both major agricultural problems worldwide, and elucidating the molecular mechanisms of B transport should allow us to develop technology to alleviate B deficiency and toxicity problems. Recent milestones include the identification of a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, from Arabidopsis thaliana. Both proteins were shown to be required for plant growth under B limitation. In addition, BOR1 homologs are required for B homeostasis in mammalian cells and B-toxicity tolerance in yeast and plants. Here, we discuss how transgenic approaches show promise for generating crops that are tolerant of B deficiency and toxicity.

An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil

Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the Km value of MOT1 for molybdate is ≈20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil.

High Boron-induced Ubiquitination Regulates Vacuolar Sorting of the BOR1 Borate Transporter in Arabidopsis thaliana

Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles.

Adaptation of Root Function by Nutrient-Induced Plasticity of Endodermal Differentiation

Plant roots forage the soil for minerals whose concentrations can be orders of magnitude away from those required for plant cell function. Selective uptake in multicellular organisms critically requires epithelia with extracellular diffusion barriers. In plants, such a barrier is provided by the endodermis and its Casparian strips--cell wall impregnations analogous to animal tight and adherens junctions. Interestingly, the endodermis undergoes secondary differentiation, becoming coated with hydrophobic suberin, presumably switching from an actively absorbing to a protective epithelium. Here, we show that suberization responds to a wide range of nutrient stresses, mediated by the stress hormones abscisic acid and ethylene. We reveal a striking ability of the root to not only regulate synthesis of suberin, but also selectively degrade it in response to ethylene. Finally, we demonstrate that changes in suberization constitute physiologically relevant, adaptive responses, pointing to a pivotal role of the endodermal membrane in nutrient homeostasis.