Boric acid intercepts 80S ribosome migration from AUG-stop by stabilizing eRF1.

In response to environmental changes, cells flexibly and rapidly alter gene expression through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is downregulated in response to an excess amount of boric acid in the environment through upstream open reading frames (uORFs) that consist of only AUG and stop codons. However, the molecular details of how this minimum uORF controls translation of the downstream main ORF in a boric acid-dependent manner have remained unclear. Here, by combining ribosome profiling, translation complex profile sequencing, structural analysis with cryo-electron microscopy and biochemical assays, we show that the 80S ribosome assembled at AUG-stop migrates into the subsequent RNA segment, followed by downstream translation initiation, and that boric acid impedes this process by the stable confinement of eukaryotic release factor 1 on the 80S ribosome on AUG-stop. Our results provide molecular insight into translation regulation by a minimum and environment-responsive uORF.

Involvement of NGATHA-Like 1 Transcription Factor in Boron Transport under Low and High Boron Conditions.

NGATHA-Like 1 (NGAL1) transcription factor has been identified as a gene regulated through AUG-stop-mediated boron (B)-dependent translation stall; however, its function in B response remains unknown. Here, we show that NGAL1 plays an important role in the maintenance of B transport under both low- and high-B conditions in Arabidopsis thaliana. NGAL1 mRNA is accumulated predominantly in shoots in response to B stress. Independent ngal1 mutants carrying transferred DNA (T-DNA) and Ds-transposon insertions exhibit reduced B concentrations in aerial tissues and produce shortened and reduced number of siliques when B supply is limited. Furthermore, the expression of B transporter genes including nodulin 26-like intrinsic protein 6; 1 (NIP6;1), NIP5;1, NIP7;1 and borate exporter 1 (BOR1) is significantly decreased in ngal1 mutants under low-B condition, suggesting that NGAL1 is required for the transcript accumulation of B transporter genes to facilitate B transport and distribution under B limitation. Under high-B condition, ngal1 mutants exhibit reduced growth and increased B concentration in their shoots. The accumulation of BOR4 mRNA, a B transporter required for B efflux to soil, is significantly reduced in roots of ngal1 plants under high-B condition, suggesting that NGAL1 is involved in the upregulation of BOR4 in response to excess B. Together, our results indicate that NGAL1 is involved in the transcriptional regulation of B transporter genes to facilitate B transport and distribution under both low- and high-B conditions.

Three regions of the NIP5;1 promoter are required for expression in different cell types in Arabidopsis thaliana root.

Arabidopsis thaliana NIP5;1, a boric acid diffusion facilitator, is involved in the acquisition of boron (B) from soil for growth under B limitation. AtNIP5;1 is expressed mainly in roots, where its expression is highest in the root cap and elongation zone. Here, we studied the role of the AtNIP5;1 promoter in the expression of this gene in roots. We fused a series of AtNIP5;1 promoter variants with deleted 5'-fragments to the GUS reporter gene and investigated the expression patterns by histochemical staining. We found that three regions of the AtNIP5;1 promoter are required for specific expression in the root cap and elongation zone (-880 to -863 bp from the translation start site), distal side of the differentiation zone (-747 to -722 bp), and basal side of the differentiation zone (-661 and -621 bp). The results suggest that at least three regions of the AtNIP5;1 promoter each confer different cell-type-specific expression.

Global analysis of boron-induced ribosome stalling reveals its effects on translation termination and unique regulation by AUG-stops in Arabidopsis shoots.

We previously reported that ribosome stalling at AUG-stop sequences in the 5'-untranslated region plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. This ribosome stalling is triggered specifically by boric acid, but the mechanisms are unknown. Although upstream open reading frames (uORFs) are known in many cases to regulate translation through peptides encoded by the uORF, AUG-stop stalling does not involve any peptide synthesis. The unique feature of AUG-stops - that termination follows immediately after initiation - suggests a possible effect of boron on the translational process itself. However, the generality of AUG-stop-mediated translational regulation and the effect of boron on translation at the genome scale are not clear. Here, we conducted a ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana shoots. We identified hundreds of translationally regulated genes that function in various biological processes. Under high-boron conditions, transcripts with reduced translation efficiency were rich in uORFs, highlighting the importance of uORF-mediated translational regulation. We found 673 uORFs that had more frequent ribosome association. Moreover, transcripts that were translationally downregulated under high-boron conditions were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Metagene analysis revealed that boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.

Targeted permeabilization of the cell wall and extraction of charged molecules from single cells in intact plant clusters using a focused electric field.

The extraction of cellular contents from plant cells covered with cell walls remains a challenge, as it is physically hindered by the cell wall. We present a new microfluidic approach that leverages an intense pulsed electric field for permeabilizing the cell wall and a focused DC electric field for extracting the cellular contents selectively from a few targeted cells in a cluster of intact plant cells. We coupled the approach with on-chip fluorescence quantification of extracted molecules leveraging isotachophoresis as well as off-chip reverse transcription-quantitative polymerase chain reaction detecting extracted mRNA molecules. Our approach offers a workflow of about 5 min, isolating a cluster of intact plant cells, permeabilizing the cell wall, selectively extracting cytosolic molecules from a few targeted cells in the cluster, and outputting them to off-chip analyses without any enzymatic reactions. We anticipate that this approach will create a new opportunity to explore plant biology through less biased data realized by the rapid extraction of molecules from intact plant clusters.

Boron-dependent regulation of translation through AUGUAA sequence in yeast.

Under high boron (B) conditions, nodulin 26-like intrinsic protein 5;1 (NIP5;1) mRNA, a boric acid channel, is destabilized to avoid excess B entry into roots of Arabidopsis thaliana. In this regulation, the minimum upstream open reading frame (uORF), AUGUAA, in its 5'-untranslated region (5'-UTR) is essential, and high B enhances ribosome stalling at AUGUAA and leads to suppression of translation and mRNA degradation. This B-dependent AUGUAA-mediated regulation occurs also in animal transient expression and reticulocyte lysate translation systems. Thus, uncovering the ubiquitousness of B-dependent unique regulation is important to reveal the evolution of translational regulation. In the present study, we examined the regulation in Saccharomyces cerevisiae. Reporter assay showed that in yeast, carrying ATGTAA in 5'-UTR of NIP5;1 upstream of the reporter gene, the relative reporter activities were reduced significantly under high B conditions compared with control, whereas deletion of ATGTAA abolished such responses. This suggests that AUGUAA mediates B-dependent regulation of translation in Saccharomyces cerevisiae. Moreover, the deletion of ATGTAA resulted in up to 10-fold increase in general reporter activities indicating the suppression effect of AUGUAA on translation of the main ORF. Interestingly, mRNA level of the reporter gene was not affected by B in both yeast cells with and without AUGUAA. This finding reveals that in yeast, unlike the case in plants, mRNA degradation is not associated with AUGUAA regulation. Together, results suggest that B-dependent AUGUAA-mediated translational regulation is common among eukaryotes.

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.

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

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.

OsNIP3;1, a rice boric acid channel, regulates boron distribution and is essential for growth under boron-deficient conditions.

Boron is an essential micronutrient for higher plants. Boron deficiency is an important agricultural issue because it results in loss of yield quality and/or quantity in cereals and other crops. To understand boron transport mechanisms in cereals, we characterized OsNIP3;1, a member of the major intrinsic protein family in rice (Oryza sativa L.), because OsNIP3;1 is the most similar rice gene to the Arabidopsis thaliana boric acid channel genes AtNIP5;1 and AtNIP6;1. Yeast cells expressing OsNIP3;1 imported more boric acid than control cells. GFP-tagged OsNIP3;1 expressed in tobacco BY2 cells was localized to the plasma membrane. The accumulation of OsNIP3;1 transcript increased fivefold in roots within 6 h of the onset of boron starvation, but not in shoots. Promoter-GUS analysis suggested that OsNIP3;1 is expressed mainly in exodermal cells and steles in roots, as well as in cells around the vascular bundles in leaf sheaths and pericycle cells around the xylem in leaf blades. The growth of OsNIP3;1 RNAi plants was impaired under boron limitation. These results indicate that OsNIP3;1 functions as a boric acid channel, and is required for acclimation to boron limitation. Boron distribution among shoot tissues was altered in OsNIP3;1 knockdown plants, especially under boron-deficient conditions. This result demonstrates that OsNIP3;1 regulates boron distribution among shoot tissues, and that the correct boron distribution is crucial for plant growth.

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.

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

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 ß-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.

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.

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.

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 beta-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.

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-beta-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.

Physiological roles and transport mechanisms of boron: perspectives from plants.

Boron, an orphan of the periodic table of the elements, is unique not only in its chemical properties but also in its roles in biology. Its requirement in plants was described more than 80 years ago. Understandings of the molecular basis of the requirement and transport have been advanced greatly in the last decade. This article reviews recent findings of boron function and transport in plants and discusses possible implication to other organisms including humans.