Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Δ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Δ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Δ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.Boron has been proposed as an important micronutrient in plants and animals. Studies have shown the presence of several genes associated with boron transport and tolerance in plants (18, 25, 27); however, boron transport mechanisms in other organisms, including animals, remain unclear. In plants, boron functions as a cross-linker for rhammogalacturanon II in the cell membrane (9, 14, 21) and also as a structural component in cytoskeleton assembly (1). Arabidopsis thaliana BOR1 was the first gene shown to play a role in boron tolerance (28). Homologs of BOR1 were found in many organisms, including yeasts, plants, and mammals (22, 25, 29). A high level of boron leads to degradation of its own exporter, BOR1, in A. thaliana (27), and A. thaliana BOR1 cannot be used to produce genetically modified plants that grow in soil with high boron levels. However, transgenic plants expressing BOR4, one of six paralogs of BOR1, showed high tolerance to toxic levels of boron (18). Multicopy expression of BOT1, a BOR1 ortholog, provided boron tolerance to barley (25).The yeast Saccharomyces cerevisiae has been used as a model organism for characterization of plant boron tolerance genes (19, 20, 25, 26, 29). While 10 mM boric acid is lethal to Arabidopsis (18), yeast can grow in the presence of 80 mM boron and is considered a boron-tolerant organism (19, 20). Yeast Bor1 was characterized in detail (10). This protein is localized to the plasma membrane and functions as a boric acid exporter (26). The bor1Δ yeast strain overaccumulates boron (20, 28), and cells that overexpress BOR1 have less intracellular boron and show resistance to boron treatment (20). In addition to Bor1, two other proteins, Dur3 and Fps1, have been implicated in boron tolerance in yeast, but their functions are not clear (20). Dur3 is a plasma membrane transporter that plays a role in urea and polyamine transport (5, 31), and Fps1 is a member of the major intrinsic protein family and plays a role in glycerol, acetic acid, arsenite, and antimonite transport (16, 30, 33). Overexpression of FPS1 and DUR3 showed controversial effects on cellular boron levels. While FPS1 expression lowered the protoplasmic boron concentration, DUR3 expression led to a small increase in boron (20).The objective of this study was to identify proteins that are primarily responsible for boron transport in yeast. ATR1 was identified as a boron tolerance gene by screening a yeast DNA expression library. Yeast Atr1 is a member of the DHA2 family of drug-H⁺ antiporters with 14 predicted membrane-spanning segments (7). It was first characterized in a genetic screen as a high-copy-number suppressor of the 3-amino-1,2,4-triazole sensitivity of gcn4Δ mutants (11). It also conferred resistance to the DNA-damaging agent 4-nitroquinoline-N-oxide in a separate genetic screen (17). In this study, we demonstrated that high-copy-number expression of ATR1 conferred extreme resistance to boron and reduced intracellular levels of the element, whereas cells lacking the ATR1 gene were hypersensitive to boron and increased its intracellular levels. We analyzed changes in the global gene expression profile in response to boron and found that ATR1 is the most induced transporter gene. The Atr1-green fluorescent protein (GFP) fusion protein localized to the plasma membrane and vacuole. Taken together, our data show that Atr1 functions as a major boron efflux pump and provides tolerance of the element by pumping boron out of cells.     

植物对硼元素的吸收转运机制

硼是植物生长发育所必需的微量元素,但是在世界范围内,土壤中硼含量过高或者过低都会对植物生长产生影响,是农业生产上的主要问题.近来人们对硼的吸收转运机制的研究取得了突破性进展,鉴定了一些硼的转运通道和转运蛋白,例如：NIP5;1、NIP6;1、BOR1和BOR4,并对它们的转运机制有了一些了解.植物在硼缺少的情况下首先通过转运通道NIP5;1把硼吸收到共质体,然后通过转运蛋白BOR1运入中柱;在高硼毒害时,通过转运蛋白BOR4把过多的硼转出植物体,同时在植物中增加糖醇的含量,过表达BOR1或BOR4都能改变植物对硼含量变化的耐受性.因此,对植物中硼吸收转运机制的研究将有利于人们通过生物学手段提高作物对土壤中硼过高或过低的抗性.     

毛白杨幼苗叶片生长及其光合参数和硼转运蛋白基因对高硼胁迫的响应

该研究采用毛白杨(Populus tomentosa)为试验材料,分析了温室条件下沙培幼苗对短期高硼胁迫(1、5、10 mmol/L硼酸)下的叶片生长、光合参数和硼转运蛋白的响应特征。结果显示:(1)与对照(0.05 mmol/L硼酸)相比,1 mmol/L硼酸处理导致毛白杨幼苗叶片叶绿素荧光参数上调,活性氧含量上升,树苗基部叶片出现少量黑色坏死斑;5 mmol/L硼酸胁迫下,叶片净光合速率、气孔导度和蒸腾速率下调,胞间二氧化碳浓度上升,叶绿素荧光参数和过氧化氢含量进一步上调,超氧阴离子含量较1 mmol/L硼酸胁迫时下调但仍然高于对照,除顶部叶片之外的其他叶片上出现大量坏死斑;10 mmol/L硼酸胁迫下,气体交换参数、叶绿素荧光参数和活性氧含量与5 mmol/L硼酸胁迫时相似,所有叶片均在平行于次级叶脉的方向出现呈带状分布的坏死斑。(2)毛白杨幼苗根和茎硼含量在硼胁迫条件下与对照相比变化幅度较小,而叶片硼含量在5 mmol/L和10 mmol/L硼酸胁迫下比对照显著上升,此时硼转移系数和生物富集系数均维持较高的水平。(3)硼转运蛋白(BOR)基因家族成员中PtoBOR4和PtoBOR8在根中的表达水平随着外界硼浓度的增加呈先上升后下降的趋势;在茎中,PtoBOR3基因下调表达,PtoBOR5上调表达;在叶片中,PtoBOR4表达先上升后下降,而PtoBOR7和PtoBOR8上调表达。研究表明,毛白杨幼苗叶片叶绿素荧光参数、活性氧、气体交换参数及硼转运蛋白基因家族表达对高硼胁迫较为敏感,硼胁迫症状在较短的时间内在叶片上以坏死斑的形式出现,可能与其较强的控制根系硼浓度的能力和向地上部分迅速运输硼的能力有关。     

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.     

硼类化合物在滴眼剂中抑菌效力的探讨

探讨硼类化合物(硼酸、硼砂)在滴眼剂中的抑菌效力。通过实验设计不同浓度的硼酸、硼砂供试品组,测定其抑菌效力。结果表明,在牛磺酸滴眼剂品种中,3组供试品组对真菌组达到抑菌效力的判断标准。但在细菌组中,3组供试品组未达到抑菌效力判断标准。在氯霉素滴眼剂中,白色念珠菌98001、金黄色葡萄球菌26003、铜绿假单胞菌10104组均满足抑菌效力的判断标准,但对大肠埃希菌44102未体现出明显抑菌作用。硼类化合物(硼酸、硼砂)在1 mg/m L剂量下即具有一定的抑菌效力。因此在滴眼剂处方设计时,硼类化合物抑菌效力需被正视,同时需结合配伍注意控制使用剂量。     

Boric acid increases the expression levels of human anion exchanger genes SLC4A2 and SLC4A3

Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 μM; more than 500 μM caused cytotoxicity. The 250 μM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels.     

Roles of ATR1 paralogs YMR279c and YOR378w in boron stress tolerance

Boron is a necessary nutrient for plants and animals, however excess of it causes toxicity. Previously, Atr1 and Arabidopsis Bor1 homolog were identified as the boron efflux pump in yeast, which lower the cytosolic boron concentration and help cells to survive in the presence of toxic amount of boron. In this study, we analyzed ATR1 paralogs, YMR279c and YOR378w, to understand whether they participate in boron stress tolerance in yeast. Even though these genes share homology with ATR1, neither their deletion rendered cells boron sensitive nor their expression was significantly upregulated by boron treatment. However, expression of YMR279, but not YOR378w, from the constitutive GAPDH promoter on a high copy plasmid provided remarkable boron resistance by decreasing intracellular boron levels. Thus our results suggest the presence of a third boron exporter, YMR279c, which functions similar to ATR1 and provides boron resistance in yeast.     

Improved production of ethanol by deleting FPS1 and over-expressing GLT1 in Saccharomyces cerevisiae

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant KAM-3, the FPS1 gene, which encodes a channel protein responsible for glycerol export, was deleted. The mutant KAM-11 had the GLT1 gene (encoding glutamate synthase) placed under the PGK1 promoter while having the FPS1 deletion. Growth rate and biomass concentration remained virtually unchanged with the mutant KAM-11, compared to that of the parent. Over-expression of GLT1 by the PGK1 promoter along with FPS1 deletion resulted in a 14% higher ethanol production and a 30% lower glycerol formation compared to the parental strain under anaerobic fermentation conditions. Furthermore, acetate and pyruvic acid formation was also reduced in order for cells to maintain redox balance.     

Transport-coupled ubiquitination of the borate transporter BOR1 for its boron-dependent degradation

Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.

The borate transporter BOR1 senses the boron concentration through its borate transport activity for K63-linked polyubiquitination and subsequent degradation.     

温度、蔗糖和硼酸对含笑花粉离体萌发的影响

以含笑(Michelia figo)花粉为试验材料,采用花粉人工培养法研究了含笑花粉离体萌发的最适条件。结果表明:蔗糖、硼酸在一定浓度范围内能促进花粉萌发,含笑花粉萌发的最适蔗糖和硼酸分别为50 mg.L-1和5mg.L-1,萌发率分别为21.51%和44.14%;花粉最佳萌发温度为25℃,萌发率达53.33%。正交试验设计结果表明,花粉萌发的最佳组合为70 mg.L-1蔗糖+5 mg.L-1硼酸+28℃,萌发率可达65.33%。     

A molecular framework for the inhibition of Arabidopsis root growth in response to boron toxicity

Boron is an essential micronutrient for plants and is taken up in the form of boric acid (BA). Despite this, a high BA concentration is toxic for the plants, inhibiting root growth and is thus a significant problem in semi-arid areas in the world. In this work, we report the molecular basis for the inhibition of root growth caused by boron. We show that application of BA reduces the size of root meristems, correlating with the inhibition of root growth. The decrease in meristem size is caused by a reduction of cell division. Mitotic cell number significantly decreases and the expression level of key core cell cycle regulators is modulated. The modulation of the cell cycle does not appear to act through cytokinin and auxin signalling. A global expression analysis reveals that boron toxicity induces the expression of genes related with abscisic acid (ABA) signalling, ABA response and cell wall modifications, and represses genes that code for water transporters. These results suggest that boron toxicity produces a reduction of water and BA uptake, triggering a hydric stress response that produces root growth inhibition.     

Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels

To investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8- to 12-fold as compared to wild-type plants), the content of sterols and the levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence-like phenotype and grew less vigorously than wild-type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2-isopentenyladenine (2-iP) recovered the wild-type phenotype. Quantification of endogenous cytokinins demonstrated that FPS1S overexpression specifically reduces the levels of endogenous zeatin-type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The finding that wild-type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.     

Comparative toxicology of borates

Inorganic borates, including boric acid, Na, ammonium, K, and Zn borates generally display low acute toxicity orally, dermally, and by inhalation. They are either not irritant or mild skin and eye irritants. Exceptions owing to physiochemical properties do occur. Longer-term toxicological studies have been reported mainly on boric acid or borax where the properties are generally similar on an equivalent boron (B) basis. The critical effects in several species are male reproductive toxicity and developmental toxicity. The doses that cause these effects are far higher than any levels to which the human population could be exposed. Humans would need to consume daily some 3.3 g of boric acid (or 5.0 g borax) to ingest the same dose level as the lowest animal NOAEL. No effects on fertility were seen in a population of workers exposed to borates or to a population exposed to high environmental borate levels. There is remarkable similarity in the toxicological effects of boric acid and borax across different species. Other inorganic borates that simply dissociate to boric acid are expected to display similar toxicity, whereas those that do not dissociate simply to boric acid may display a different toxicological profile.