Kyoko Miwa

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.

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.

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 in plants: co-ordinated regulation of transporters

BACKGROUND The essentiality of boron (B) for plant growth was established > 85 years ago. In the last decade, it has been revealed that one of the physiological roles of B is cross-linking the pectic polysaccharide rhamnogalacturonan II in primary cell walls. Borate cross-linking of pectic networks serves both for physical strength of cell walls and for cell adhesion. On the other hand, high concentrations of B are toxic to plant growth. To avoid deficiency and toxicity problems, it is important for plants to maintain their tissue B concentrations within an optimum range by regulating transport processes. Boron transport was long believed to be a passive, unregulated process, but the identification of B transporters has suggested that plants sense and respond to the B conditions and regulate transporters to maintain B homeostasis. SCOPE Transporters responsible for efficient B uptake by roots, xylem loading and B distribution among leaves have been described. These transporters are required under B limitation for efficient acquisition and utilization of B. Transporters important for tolerating high B levels in the environment have also been identified, and these transporters export B from roots back to the soil. Two types of transporters are involved in these processes: NIPs (nodulin-26-like intrinsic proteins), boric acid channels, and BORs, B exporters. It is demonstrated that the expression of genes encoding these transporters is finely regulated in response to B availability in the environment to ensure tissue B homeostasis. Furthermore, plants tolerant to stress produced by low B or high B in the environment can be generated through altered expression of these transporters. CONCLUSIONS The identification of the first B transporter led to the discovery that B transport was a process mediated not only by passive diffusion but also by transporters whose activity was regulated in response to B conditions. Now it is evident that plants sense internal and external B conditions and regulate B transport by modulating the expression and/or accumulation of these transporters. Results obtained in model plants are applicable to other plant species, and such knowledge may be useful in designing plants or crops tolerant to soils containing low or high B.

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.

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.

Cell-Type Specificity of the Expression of Os BOR1, a Rice Efflux Boron Transporter Gene, Is Regulated in Response to Boron Availability for Efficient Boron Uptake and Xylem Loading

We describe a boron (B) transporter, Os BOR1, in rice (Oryza sativa). Os BOR1 is a plasma membrane–localized efflux transporter of B and is required for normal growth of rice plants under conditions of limited B supply (referred to as -B). Disruption of Os BOR1 reduced B uptake and xylem loading of B. The accumulation of Os BOR1 transcripts was higher in roots than that in shoots and was not affected by B deprivation; however, Os BOR1 was detected in the roots of wild-type plants under -B conditions, but not under normal conditions, suggesting regulation of protein accumulation in response to B nutrition. Interestingly, tissue specificity of Os BOR1 expression is affected by B treatment. Transgenic rice plants containing an Os BOR1 promoter–β-glucuronidase (GUS) fusion construct grown with a normal B supply showed the strongest GUS activity in the steles, whereas after 3 d of -B treatment, GUS activity was elevated in the exodermis. After 6 d of -B treatment, GUS activity was again strong in the stele. Our results demonstrate that Os BOR1 is required both for efficient B uptake and for xylem loading of B. Possible roles of the temporal changes in tissue-specific patterns of Os BOR1 expression in response to B condition are discussed.

Highly Boron Deficiency-Tolerant Plants Generated by Enhanced Expression of NIP5;1, a Boric Acid Channel

Boron (B) is an essential element for plants, and B deficiency is a worldwide agricultural problem. In B-deficient areas, B is often supplied as fertilizer, but excess B can be toxic to both plants and animals. Generation of B deficiency-tolerant plants could reduce B fertilizer use. Improved fertility under B-limiting conditions in Arabidopsis thaliana by overexpression of BOR1, a B transporter, has been reported, but the root growth was not improved by the BOR1 overexpression. In this study, we report that enhanced expression of NIP5;1, a boric acid channel for efficient B uptake, resulted in improved root elongation under B-limiting conditions in A. thaliana. An NIP5;1 activation tag line, which has a T-DNA insertion with enhancer sequences near the NIP5;1 gene, showed improved root elongation under B limitation. We generated a construct which mimics the tag line: the cauliflower mosaic virus 35S RNA promoter was inserted at 1,357 bp upstream of the NIP5;1 transcription initiation site. Introduction of this construct into the nip5;1-1 mutant and the BOR1 overexpresser resulted in enhanced expression of NIP5;1 and improved root elongation under low B supply. Furthermore, one of the transgenic lines exhibited improved fertility and short-term B uptake. Our results demonstrate successful improvement of B deficiency tolerance and the potential of enhancing expression of a mineral nutrient channel gene to improve growth under nutrient-limiting conditions.

Roles of BOR2, a Boron Exporter, in Cross Linking of Rhamnogalacturonan II and Root Elongation under Boron Limitation in Arabidopsis

An efflux-type boron transporter facilitates efficient borate cross-linking of rhamnogalacturonan II in cell walls and root cell elongation under boron deficiency. Boron (B) is required for cross linking of the pectic polysaccharide rhamnogalacturonan II (RG-II) and is consequently essential for the maintenance of cell wall structure. Arabidopsis (Arabidopsis thaliana) BOR1 is an efflux B transporter for xylem loading of B. Here, we describe the roles of BOR2, the most similar paralog of BOR1. BOR2 encodes an efflux B transporter localized in plasma membrane and is strongly expressed in lateral root caps and epidermis of elongation zones of roots. Transfer DNA insertion of BOR2 reduced root elongation by 68%, whereas the mutation in BOR1 reduced it by 32% under low B availability (0.1 µm), but the reduction in shoot growth was not as obvious as that in the BOR1 mutant. A double mutant of BOR1 and BOR2 exhibited much more severe growth defects in both roots and shoots under B-limited conditions than the corresponding single mutants. All single and double mutants grew normally under B-sufficient conditions. These results suggest that both BOR1 and BOR2 are required under B limitation and that their roles are, at least in part, different. The total B concentrations in roots of BOR2 mutants were not significantly different from those in wild-type plants, but the proportion of cross-linked RG-II was reduced under low B availability. Such a reduction in RG-II cross linking was not evident in roots of the BOR1 mutant. Thus, we propose that under B-limited conditions, transport of boric acid/borate by BOR2 from symplast to apoplast is required for effective cross linking of RG-II in cell wall and root cell elongation.

Isolation of Arabidopsis thaliana cDNAs That Confer Yeast Boric Acid Tolerance

An Arabidopsis thaliana cDNA library was introduced into a Saccharomyces cerevisiae mutant that lacks ScBOR1 (YNL275W), a boron (B) efflux transporter. Five cDNAs were identified that confer tolerance to high boric acid. The nucleotide sequence analysis identified the clones as a polyadenylate-binding protein, AtPAB2; a ribosomal small subunit protein, AtRPS20B; an RNA-binding protein, AtRBP47c′; and two Myb transcription factors, AtMYB13 and AtMYB68. The expression of these five genes also conferred boric acid tolerance on wild-type yeast. Two yeast genes, ScRPS20 and ScHRB1, that are similar to the isolated clones, were necessary for this boric acid tolerance. The possible roles of these A. thaliana and S. cerevisiae genes in boric acid tolerance are discussed.