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.

Translational Landscape of a C4 Plant, Sorghum bicolor, Under Normal and Sulfur-Deficient Conditions.

Recent accumulation of genomic and transcriptomic information has facilitated genetic studies. Increasing evidence has demonstrated that translation is an important regulatory step, and the transcriptome does not necessarily reflect the profile of functional protein production. Deep sequencing of ribosome-protected mRNA fragments (ribosome profiling or Ribo-seq) has enabled genome-wide analysis of translation. Sorghum is a C4 cereal important not only as food but also as forage and a bioenergy resource. Its resistance to harsh environments has made it an agriculturally important research subject. Yet genome-wide translational profiles in sorghum are still missing. In this study, we took advantage of Ribo-seq and identified actively translated reading frames throughout the genome. We detected translation of 4,843 main open reading frames (ORFs) annotated in the sorghum reference genome version 3.1 and revealed a number of unannotated translational events. A comparison of the transcriptome and translatome between sorghums grown under normal and sulfur-deficient conditions revealed that gene expression is modulated independently at transcript and translation levels. Our study revealed the translational landscape of sorghum's response to sulfur and provides datasets that could serve as a fundamental resource to extend genetic research on sorghum, including studies on translational regulation.

Regulation, Diversity and Evolution of Boron Transporters in Plants.

Boron (B) is an essential trace element in plants, and borate cross-linking of pectic polysaccharide rhamnogalacturonan-II (RG-II) in cell walls is required for normal cell growth. High concentrations of B are toxic to cells. Therefore, plants need to control B transport to respond to B conditions in the environment. Over the past two decades, genetic analyses of Arabidopsis thaliana have revealed that B transport is governed by two types of membrane transport molecules: NIPs (nodulin-26-like intrinsic proteins), which facilitate boric acid permeation, and BORs, which export borate from cells. In this article, we review recent findings on the (i) regulation at the cell level, (ii) diversity among plant species and (iii) evolution of these B transporters in plants. We first describe the systems regulating these B transporters at the cell level, focusing on the molecular mechanisms underlying the polar localization of proteins and B-dependent expression, as well as their physiological significance in A. thaliana. Then, we examine the presence of homologous genes and characterize the functions of NIPs and BORs in B homeostasis, in a wide range of plant species, including Brassica napus, Oryza sativa and Zea mays. Finally, we discuss the evolutionary aspects of NIPs and BORs as B transporters, and the possible relationship between the diversification of B transport and the occurrence of RG-II in plants. This review considers the sophisticated systems of B transport that are conserved among various plant species, which were established to meet mineral nutrient requirements.

Golgi-localized membrane protein AtTMN1/EMP12 functions in the deposition of rhamnogalacturonan II and I for cell growth in Arabidopsis.

Appropriate pectin deposition in cell walls is important for cell growth in plants. Rhamnogalacturonan II (RG-II) is a portion of pectic polysaccharides; its borate crosslinking is essential for maintenance of pectic networks. However, the overall process of RG-II synthesis is not fully understood. To identify a novel factor for RG-II deposition or dimerization in cell walls, we screened Arabidopsis mutants with altered boron (B)-dependent growth. The mutants exhibited alleviated disorders of primary root and stem elongation, and fertility under low B, but reduced primary root lengths under sufficient B conditions. Altered primary root elongation was associated with cell elongation changes caused by loss of function in AtTMN1 (Transmembrane Nine 1)/EMP12, which encodes a Golgi-localized membrane protein of unknown function that is conserved among eukaryotes. Mutant leaf and root dry weights were lower than those of wild-type plants, regardless of B conditions. In cell walls, AtTMN1 mutations reduced concentrations of B, RG-II specific 2-keto-3-deoxy monosaccharides, and rhamnose largely derived from rhamnogalacturonan I (RG-I), suggesting reduced RG-II and RG-I. Together, our findings demonstrate that AtTMN1 is required for the deposition of RG-II and RG-I for cell growth and suggest that pectin modulates plant growth under low B conditions.

Involvement of boron transporter BOR1 in growth under low boron and high nitrate conditions in Arabidopsis thaliana.

BOR1 is an efflux transporter of boron (B), responsible for loading B into the xylem. It has been reported that nitrate (NO3 - ) concentrations significantly influence B concentrations in leaves and BOR1 mRNA accumulation in roots. Here, to unravel the interactive effects of B and NO3 - on plant growth and the function of BOR1 under the combination of B and NO3 - , seedling growth was analyzed in Col-0 and bor1 mutants. The growth of bor1 mutants was negatively affected by high NO3 - but neither by potassium chloride (KCl) nor ammonium (NH4 + ) under low B conditions, suggesting the involvement of BOR1 in growth under high NO3 - . Mutants of bor2 and bor4 did not exhibit such growth responses, suggesting that this effect was specific to BOR1 among the BORs tested. Under low B conditions, loss of the BOR1 function led to a more significant decrease in B concentrations in the presence of high NO3 - compared to normal NO3 - . Additionally, grafting experiments demonstrated that these effects of NO3 - occurred when BOR1 is absent in roots. High NO3 - treatment elevated BOR1 mRNA accumulation while the BOR1 protein accumulation was downregulated. These apparent opposite responses indicated that the transcriptional and (post-)translational regulations follow different patterns. Our work provides evidence of a novel regulation of BOR1 and another B transport system by both B and NO3 - in an interactive manner.

Boron-Dependent Translational Suppression of the Borate Exporter BOR1 Contributes to the Avoidance of Boron Toxicity.

Boron (B) is an essential element for plants; however, as high B concentrations are toxic, B transport must be tightly regulated. BOR1 is a borate exporter in Arabidopsis (Arabidopsis thaliana) that facilitates B translocation into shoots under B deficiency conditions. When the B supply is sufficient, BOR1 expression is down-regulated by selective degradation of BOR1 protein, while additional BOR1 regulatory mechanisms are proposed to exist. In this study, we identified a novel B-dependent BOR1 translational suppression mechanism. In vivo and in vitro reporter assays demonstrated that BOR1 translation was reduced in a B-dependent manner and that the 5'-untranslated region was both necessary and sufficient for this process. Mutational analysis revealed that multiple upstream open reading frames in the 5'-untranslated region were required for BOR1 translational suppression, and this process depended on the efficiency of translational reinitiation at the BOR1 open reading frame after translation of the upstream open reading frames. To understand the physiological significance of BOR1 regulation, we characterized transgenic plants defective in either one or both of the BOR1 regulation mechanisms. BOR1 translational suppression was induced at higher B concentrations than those triggering BOR1 degradation. Plants lacking both regulation mechanisms exhibited more severe shoot growth reduction under high-B conditions than did plants lacking BOR1 degradation alone, thus demonstrating the importance of BOR1 translational suppression. This study demonstrates that two mechanisms of posttranscriptional BOR1 regulation, each induced under different B concentrations, contribute to the avoidance of B toxicity in plants.

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.

Effects of an advance care planning educational programme intervention on the end-of-life care attitudes of multidisciplinary practitioners at an acute hospital: A pre- and post-study.

BACKGROUND: Advance care planning is a crucial end-of-life care practice. However, an advance care planning educational programme for practitioners in an acute care setting has not yet been established. Consequently, we examined the effects of an advance care planning educational programme in an acute hospital in the hope of achieving increased awareness of end-of-life care. DESIGN: A mixed-methods, pre- and post-design was employed to evaluate the change in attitudes of practitioners post-programme. The intervention programme was conducted thrice over 3 months in 90-min sessions. SETTING/PARTICIPANTS: This study included 85 participants in the baseline assessment working at B acute hospital in Osaka. RESULTS: Participants' scores on the 'Positive attitude for end-of-life care' subscale on the short version of the Frommelt Attitude Toward Care of Dying scale significantly increased after the 6-month intervention. A 'Positive attitude for end-of-life-care' implies that participants would not be afraid to practice end-of-life care. Further, participants' scores on the 'Death relief' subscale of the Death Attitude Inventory also significantly increased. The term 'Death relief' means that death helps in ending suffering. It means participants are not afraid of death. Qualitative results implied that participants believed advance care planning implementation and communicating with patients and patients' families were critical. CONCLUSIONS: Six months post-intervention, participants displayed sustained positive attitudes towards end-of-life care. These results suggest that the present programme was effective at improving practitioners' attitudes towards patients' end-of-life care.

Strategies for optimization of mineral nutrient transport in plants: multilevel regulation of nutrient-dependent dynamics of root architecture and transporter activity.

How do sessile plants cope with irregularities in soil nutrient availability? The uptake of essential minerals from the soil influences plant growth and development. However, most environments do not provide sufficient nutrients; rather nutrient distribution in the soil can be uneven and change temporally according to environmental factors. To maintain mineral nutrient homeostasis in their tissues, plants have evolved sophisticated systems for coping with spatial and temporal variability in soil nutrient concentrations. Among these are mechanisms for modulating root system architecture in response to nutrient availability. This review discusses recent advances in knowledge of the two important strategies for optimizing nutrient uptake and translocation in plants: root architecture modification and transporter expression control in response to nutrient availability. Recent studies have determined (i) nutrient-specific root patterns; (ii) their physiological consequences; and (iii) the molecular mechanisms underlying these modulation systems that operate to facilitate efficient nutrient acquisition. Another mechanism employed by plants in nutrient-heterogeneous soils involves modification of nutrient transport activities in a nutrient concentration-dependent manner. In recent years, considerable progress has been made in characterizing the diverse functions of transporters for specific nutrients; it is now clear that the expression and activities of nutrient transporters are finely regulated in multiple steps at both the transcriptional and post-transcriptional levels for adaptation to a wide range of nutrient conditions.

Roles of BOR2, a boron exporter, in cross linking of rhamnogalacturonan II and root elongation under boron limitation in Arabidopsis.

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.

Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012.

After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.

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.

Mutations in type II Golgi-localized proton pyrophosphatase AVP2;1/VHP2;1 affect pectic polysaccharide rhamnogalacturonan-II and alter root growth under low boron condition in Arabidopsis thaliana.

The essential plant nutrient boron is required for the crosslinking of the pectin polysaccharide, rhamnogalacturonan II (RG-II). The synthesis of the pectic polysaccharides takes place in the Golgi apparatus, acidified by proton pumps. AVP2;1/VHP2;1 is a type II proton pyrophosphatase localized in the Golgi apparatus, which possesses proton pumping activity coupled with pyrophosphate hydrolysis. Its activity and expression patterns have been previously revealed but its role in plants remains unknown. The aim of the present work therefore was to explore the physiological role of AVP2;1 in Arabidopsis thaliana. In the screening of mutants under low boron, a mutant carrying a missense mutation in AVP2;1 was isolated. This mutant showed increased primary root growth under low boron conditions but no significant difference under normal boron condition compared to wild type plants. T-DNA insertion caused similar growth, suggesting that reduced function of AVP2;1 was responsible. Root cell observation revealed an increase in meristematic zone length, cell number in meristem and length of matured cell in avp2;1 mutants compared to wild type under low boron. Calcium concentration was reduced in mutant root cell wall under low boron. RG-II specific sugars also tended to be decreased in mutant root cell wall under low and normal boron conditions. These results suggest that changes in cell wall component by mutations in AVP2;1 may possibly explain the increased root length of mutants under low boron. This supports the idea that AVP2;1 plays a role in pH homoeostasis in Golgi apparatus for pectin synthesis.

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.

Expression of the Arabidopsis borate efflux transporter gene, AtBOR4, in rice affects the xylem loading of boron and tolerance to excess boron.

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.

Highly sensitive in vitro cytokine release assay incorporating high-density preculture.

Immunostimulatory effects of monoclonal antibodies (mAb) through binding to Fcγ receptors (FcγR) on immune cells are a likely cause of cytokine release syndrome. However, it is difficult to detect the potential risk of FcγR-dependent cytokine release associated with mAb in the current standard cytokine release assays (CRA), including the air-drying solid-phase method using human peripheral blood mononuclear cells (PBMC). To increase the sensitivity to detect FcγR-dependent cytokine release due to mAb, a high-density preculture (HDC) method was incorporated into the air-drying solid-phase CRA. Here, PBMC were exposed to panitumumab, trastuzumab, rituximab, or alemtuzumab at 0.1, 0.3, 1, and 3 µg/well for 24 or 48 hr under both non-HDC and HDC conditions. T-cell agonists (anti-CD3 mAb, anti-CD28 super-agonist [SA] mAb) were used as reference mAb. Panitumumab, trastuzumab, rituximab, or alemtuzumab induced cytokine release under both non-HDC and HDC conditions, and cytokine release caused by alemtuzumab was more pronounced under HDC conditions. To investigate FcγR involvement in cytokine release associated with panitumumab, trastuzumab, rituximab, and alemtuzumab, CRA of these four mAb were conducted with anti-FcγRI, -FcγRII, or -FcγRIII F(ab')2 fragments. The results showed cytokine release caused by trastuzumab, rituximab, and alemtuzumab was significantly suppressed by anti-FcγRIII F(ab')2 pretreatment, and slightly reduced by anti-FcγRI or anti-FcγRII pretreatment, indicating these mAb induced FcγR (especially FcγRIII)-dependent cytokine release from PBMC. Cytokine release caused by panitumumab was slightly suppressed by anti-FcγRIII F(ab')2 pretreatment. Anti-CD3 mAb and anti-CD28 SA mAb also induced significant release of cytokines under HDC conditions compared with that under non-HDC conditions. In conclusion, CRA incorporating HDC into the air-drying solid-phase method using human PBMC could sensitively capture the FcγR-dependent cytokine release potential of mAb.

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor.

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.

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.

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.

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.