Mayuki Tanaka

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.

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.

Boron-dependent degradation of NIP5;1 mRNA for acclimation to excess boron conditions in Arabidopsis.

NIP5;1 encodes a boron channel; this work shows that the 5′ untranslated region of NIP5;1 is required for mRNA accumulation in response to boron deficiency and mRNA degradation in response to high-boron conditions. Boron (B) is an essential plant micronutrient that is toxic at higher levels. NIP5;1 is a boric acid channel required for B uptake and growth under B deficiency. Accumulation of the NIP5;1 transcript is upregulated under B deficiency in Arabidopsis thaliana roots. To elucidate the mechanism of regulation, the 5′ untranslated region (UTR) of NIP5;1 was tested for its ability to confer B-dependent regulation using β-glucuronidase and green fluorescent protein as reporters. This analysis showed that the 5′ UTR was involved in NIP5;1 transcript accumulation in response to B conditions. We also found that high-B conditions trigger NIP5;1 mRNA degradation and that the sequence from +182 to +200 bp in the 5′ UTR is required for this mRNA destabilization. In the nip5;1-1 mutant background, a NIP5;1 complementation construct without the 5′ UTR produced high levels of mRNA accumulation, increased B concentrations in tissues, and reduced growth under high-B conditions. These data suggest that the 5′ UTR controls B-dependent NIP5;1 mRNA degradation and that NIP5;1 mRNA degradation is important for plant acclimation to high-B conditions.

The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation

AUG-stops in the 5′ untranslated regions of Arabidopsis genes, including NIP5;1, which encodes a boron influx transporter, regulate B-dependent mRNA accumulation through ribosome stalling. Upstream open reading frames (uORFs) are often translated ahead of the main ORF of a gene and regulate gene expression, sometimes in a condition-dependent manner, but such a role for the minimum uORF (hereafter referred to as AUG-stop) in living organisms is currently unclear. Here, we show that AUG-stop plays an important role in the boron (B)-dependent regulation of NIP5;1, encoding a boric acid channel required for normal growth under low B conditions in Arabidopsis thaliana. High B enhanced ribosome stalling at AUG-stop, which was accompanied by the suppression of translation and mRNA degradation. This mRNA degradation was promoted by an upstream conserved sequence present near the 5′-edge of the stalled ribosome. Once ribosomes translate a uORF, reinitiation of translation must take place in order for the downstream ORF to be translated. Our results suggest that reinitiation of translation at the downstream NIP5;1 ORF is enhanced under low B conditions. A genome-wide analysis identified two additional B-responsive genes, SKU5 and the transcription factor gene ABS/NGAL1, which were regulated by B-dependent ribosome stalling through AUG-stop. This regulation was reproduced in both plant and animal transient expression and cell-free translation systems. These findings suggest that B-dependent AUG-stop-mediated regulation is common in eukaryotes.

Differential Expression of Three BOR1 Genes Corresponding to Different Genomes in Response to Boron Conditions in Hexaploid Wheat (Triticum aestivum L.)

Boron (B) is an essential micronutrient for plants. Efflux-type B transporters, BORs, have been identified in Arabidopsis thaliana and rice. Here we identified BOR1 genes encoding B efflux transporters, from the hexaploid genome of wheat (Triticum aestivum L.). We cloned three genes closely related to OsBOR1 and named them TaBOR1.1, TaBOR1.2 and TaBOR1.3. All three TaBOR1s showed B efflux activities when expressed in tobacco BY-2 cells. TaBOR1-green fluorescent protein (GFP) fusion proteins were expressed in Arabidopsis leaf cells localized in the plasma membrane. The transcript accumulation patterns of the three genes differ in terms of tissue specificity and B nutrition responses. In roots, transcripts for all three genes accumulated abundantly while in shoots, the TaBOR1.2 transcript is the most abundant, followed by those of TaBOR1.1 and TaBOR1.3. Accumulation of TaBOR1.1 transcript is up-regulated under B deficiency conditions in both roots and shoots. In contrast, TaBOR1.2 transcript accumulation significantly increased in roots under excess B conditions. TaBOR1.3 transcript accumulation was reduced under excess B. Taken together, these results demonstrated that TaBOR1s are the B efflux transporters in wheat and, interestingly, the genes on the A, B and D genomes have different expression patterns.

Molecular Mechanisms of Boron Transport in Plants: Involvement of Arabidopsis NIP5;1 and NIP6;1

Understanding of the molecular mechanisms of boron (B) transport has been greatly advanced in the last decade. BOR1, the first B transporter in living systems, was identified by forward genetics using Arabidopsis mutants. Genes similar to BOR1 have been reported to share different physiological roles in plants. NIPS;1, a member of aquaporins in Arabidopsis, was then identified as a boric acid channel gene responsible for the B uptake into roots. NIP6;1, the most similar gene to NIPS;1, encodes a B channel essential for B distribution to young leaves. In the present chapter, recent advancement of the understanding of molecular mechanisms of B transport and roles of NIP genes are discussed.

Rapid transporter regulation prevents substrate flow traffic jams in boron transport

Nutrient uptake by roots often involves substrate-dependent regulated nutrient transporters. For robust uptake, the system requires a regulatory circuit within cells and a collective, coordinated behaviour across the tissue. A paradigm for such systems is boron uptake, known for its directional transport and homeostasis, as boron is essential for plant growth but toxic at high concentrations. In Arabidopsis thaliana, boron uptake occurs via diffusion facilitators (NIPs) and exporters (BORs), each presenting distinct polarity. Intriguingly, although boron soil concentrations are homogenous and stable, both transporters manifest strikingly swift boron-dependent regulation. Through mathematical modelling, we demonstrate that slower regulation of these transporters leads to physiologically detrimental oscillatory behaviour. Cells become periodically exposed to potentially cytotoxic boron levels, and nutrient throughput to the xylem becomes hampered. We conclude that, while maintaining homeostasis, swift transporter regulation within a polarised tissue context is critical to prevent intrinsic traffic-jam like behaviour of nutrient flow.

Comparison of BOR1-like gene expression in two genotypes with different boron efficiencies in commercial crop plants in Thailand

Abstract In Thailand, boron (B) deficiency in soil is found in the north region where wheat (Triticum aestivum L.), maize (Zea mays L.) and rice (Oryza sativa L.) are promoted cereals. Physiological analysis and genetic variation in B efficiency among plant genotypes have been reported; however, the molecular and genetic mechanisms of low B tolerance remain unclear. In this present study, we investigated the molecular basis of low B tolerance in wheat, maize and rice. Transcript levels of BOR1-like genes, efflux-type B transporters, were compared between B-efficient and B-inefficient genotypes in different organs using quantitative real-time polymerase chain reaction (PCR). The results revealed that the transcript levels of BOR1-like genes are differential between two different genotypes. We found the tendency that transcripts of BOR1-like gene are accumulated to higher levels in B deficiency tolerant cultivar than the sensitive ones in most tested tissues. It is possible that the expression levels of BOR1-like genes are correlated with the B deficiency tolerance in plants. Moreover, BOR1-like genes can be useful as gene expression biomarkers for crop breeding in wheat, maize and rice by selecting appropriate tissues and growth stages.