Plant Physiology and Biochemistry 42 (2004) 907–912 www.elsevier.com/locate/plaphy
Review
Why boron? Luis Bolaños a,*, Krystyna Lukaszewski b, Ildefonso Bonilla a, Dale Blevins b a
Departamento de Biologia, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain b Plant Science Unit, University of Missouri, Columbia, MO 65211, USA Received 9 June 2004; accepted 17 November 2004 Available online 16 December 2004
Abstract It is now more than 80 years since boron was convincingly demonstrated to be essential for normal growth of higher plants. However, its biochemical role is not well understood at the moment. Several recent reviews propose that B is implicated in three main processes: keeping cell wall structure, maintaining membrane function, and supporting metabolic activities. However, in the absence of conclusive evidence, the primary role of boron in plants remains elusive. Besides plants, growth of specific bacteria, such as heterocystous cyanobacteria and the recently reported actinomycetes of the genus Frankia, requires B, particularly for the stability of the envelopes that control the access of the nitrogenase-poisoning oxygen when they grow under N2-fixing conditions. Likewise, a role for B for animal embryogenesis and other developmental processes is being established. Finally, a new feature of the role of boron comes from signaling mechanisms for communication among bacteria and among legumes and rhizobia leading to N2-fixing symbiosis, and it is possible that new roles for B, based on its special chemistry and its interaction with Ca would appear in the world of signal transduction pathways. In conclusion, the diversity of roles played by B might indicate that either the micronutrient is involved in numerous processes or that its deficiency has a pleiotropic effect. The arising question is why such an element? Since all of the roles clearly established for B are related to its capacity to form diester bridges between cis-hydroxyl-containing molecules, we propose that the main reason for B essentiality is the stabilization of molecules with cis-diol groups turning them effective, irrespectively of their function. © 2004 Elsevier SAS. All rights reserved. Keywords: Animal development; Bacteria signaling; B–Ca relationship; cis-Hydroxyl groups; Molecular linker; Signal transduction; Wall structure
1. Introduction Boron is a member of the semiconductor group of elements and has properties intermediate between metals and non-metals. The boron atom is small with only three valence electrons. The chemistry of boron is unique and, after that of carbon, it might be the most intriguing and complex of any element [22]. Boron, along with other light elements like lithium and berilium, originates from the Big Bang nucleosynthesis or galactic cosmic-ray events [34,40], and its abundance is extremely low: only about 10–9 times that of hydrogen and about 10–6 that of carbon, nitrogen, or oxygen. However, in spite of its low cosmic abundance, boron is widely distributed both in the Earth’s crust (from 5 mg kg–1 in basalts to 100 mg kg–1 in shales) [42] and in the ocean (~4.5 mg l–1) [26].
Abbreviations: FITC, fluorescein-isothiocyanate conjugate. * Corresponding author. Fax: +34 91 497 8344. E-mail address: luis.bolarios@uam.es (L. Bolaños). 0981-9428/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.11.002
The boron requirement for plant growth was first demonstrated in the early 1920s [45], and since then boron has been established as an essential micronutrient for all vascular plants. Boron deficiency causes a plethora of rapid biochemical, physiological, and anatomical aberrations, which, along with the lack of relevant information on boron chemistry, have made determining the primary function of boron in plants one of the most difficult tasks in plant nutrition. While great progress has been made in the last few years, the primary role of boron remains undefined. Ongoing research focuses on boron involvement in three main areas: organization of cell walls, membrane function, and metabolic activities [4,14]. 2. Boron and structures containing molecules with cis-hydroxyl groups At the near-neutral pH found in most biological fluids, boron exists primarily (~96%) as boric acid, B(OH)3, plus a small amount of borate anion B(OH)4–. Both boric acid and borate readily form complexes with a wide variety of sugars
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Fig. 1. Chemical structures of boric acid (A), borate anion (B), and their diol esters (C, D).
and other compounds containing cis-hydroxyl groups (Fig. 1) [27]. Recent identification of the first boron-containing molecules from plants has led to a breakthrough in boron research. Isolation and characterization of rhamnogalacturonan-IIboron (RG-II-B) complexes demonstrated boron cross-link between apiose residues in pectin (Fig. 2), and confirmed in vivo the proposed role of boron in cell wall architecture [24,31,32]. So far, no direct biochemical evidence has been
presented resolving boron action in membranes, but boron linking or binding to hydroxyl-containing constituents, such as phosphoinositides, glycoproteins, and glycolipids, has been proposed to explain the altered membrane composition and transport processes in boron deficient plants [14]. Although for most of the last eight decades boron requirement has been recognized exclusively in plants, recent studies demonstrate a nutritional importance of boron across a broad spectrum of organisms: yeasts [1], animals and human [29]. While the mechanisms of boron action in animals and humans are unknown, a specific function in membranes has been proposed [14,30]. At the opposite end of the biological spectrum, boron essentiality has been established for the growth of specific types of bacteria, such as heterocystous cyanobacteria [12], and actinomycetes of the genus Frankia [9]. Both types of microorganisms require boron for the stability of the envelopes that prevent access of nitrogenase-poisoning oxygen when grown under N2-fixing conditions. As expected, the cells that specifically require boron are the nitrogenase-harboring heterocysts in Anabaena and vesicles in Frankia. Stabilization of these envelopes by boron is intriguing, considering
Fig. 2. Sites of boron attachment in biological structures: plant cell wall boron-rhamnogalacturonan II complex (A), bacterial quorum sensing signaling molecule autoinducer AI-2 (B), phloem boron transport complex with sorbitol (C), and hypothetical models of boron binding with second messenger GMP (D), bacteriohopanetetrol (E) and phosphoinositol IP3 (F).
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their different chemical composition. In the heterocysts, boron is present in an inner laminated layer formed of specific glycolipids. In contrast, in Frankia, boron is found in a multilaminate vesicle wall composed of glycolipids and neutral lipids with a very high proportion of long-chain polyhydroxy fatty acids or alcohols, including the hopanoid bacteriohopanetetrol [2]. All of these components are rich in free diol groups, ideal for bonding with borate (Fig. 2).
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cies, but is produced by a multitude of diverse bacteria, and the gene encoding the AI-2 synthase (luxS) is widely conserved. This raises a possibility that AI-2 might serve as a universal bacterial signal for communication among species [15,47] could also serve as a boron transporter, a way of moving boron in or out of the cell, depending on growth or environmental conditions [16]. It remains unclear at what stage boron binds to the carbohydrate moiety of the AI-2 molecule; in the autoinducer-producing cell, in the surrounding environment, or in the recipient cell [16].
3. Boron and bacteria-plants talking A role for boron has been demonstrated in the establishment of an effective legume–Rhizobium symbiosis [7]. As in plants, boron requirements have been reported for the maintenance of nodule cell wall structure [13]. Furthermore, the micronutrient plays important roles for the correct establishment of the symbiosis. The N2-fixing legume root nodule is the result of genetically determined interactions between rhizobia and the host plant [43]. As a result of molecular signaling, mediated by plant and bacteria derived glycoconjugates most of them rich in cis-diols, the physical and metabolic integration between rhizobia and the host cells becomes progressively more intimate [23]. Boron is required for early symbiont/plant signaling, namely nod-gene activation by root plant exudates and nodule initiation [38]. Moreover, boron is required for infection thread development and nodule invasion [5], due to a role of B as modulator of the interactions between plants derived infection thread matrix glycoproteins and the bacteria cell surface. In the absence of B, the glycoproteins can attach to the cell surface of rhizobia. Therefore, the bacterium can be trapped and unable to interact with the plant cell membrane and hence elicitation of the endocytosis process leading to cell invasion is inhibited. The presence of B (but not Ca, pH changes, salt or high ionic strength) specifically inhibits the in vitro bacteria-matrix glycoprotein attachment and promotes the rhizobial interaction with the plant membrane prior cell invasion. Once Rhizobium is inside the cell B promotes symbiosome (containing N2-fixing bacteroids) development. Specifically, B is needed for the targeting of nodule-specific plant derived glycoproteins [6] that are crucial as signals for bacteroid differentiation into a N2-fixing form [10]. Finally, the recent discovery of a boron-containing bacterial signal molecule, autoinducer AI-2, revealed an unexpected role for boron in bacterial quorum sensing (Fig. 2) [15]. Quorum sensing allows bacterial populations to monitor cell density by means of diffusible pheromones, which accumulate in the extracellular space as the population increases. Higher levels of pheromones activate signaling pathways, leading to coordinated alteration of gene expression throughout the population [16]. AI-2, identified as a furanosyl borate diester, is a novel signaling molecule for both, structure and function. In contrast to the previously known bacterial pheromones, AI-2 is not exclusive to a single spe-
4. Boron (and the B–Ca relationship) in signal transduction and gene expression Another insight into boron function from the perspective of gene expression comes from the area of nitrogen fixation. Boron was found to be essential for bacteria-plant signaling and nod-gene induction in pea [38]. Addition of calcium ameliorated some effects of boron deficiency on nodulation, and increased nodule number [39]. Interaction between calcium and boron has also been reported in pea nodule establishment and development under salt stress [19,20]. Genetic studies of nodulation of Medicago truncatula showed that expression of 60% of the analyzed genes (including genes involved in cell cycle, cell wall assembly, and ribosome biogenesis) was affected by boron deficiency, and, in some cases, overexpression could be reversed by supplemental calcium [36,37]. However, calcium did not reverse either the abnormal cell wall structure of boron deficient nodules or the distribution of pectic polysaccharides immunolocalized by monoclonal antibodies in cell [39]. Calcium association with boron has been observed in plants, bacteria, animals and humans, but the nature of this interaction is still debated. Various types of evidence suggest a role of calcium in stabilizing boron complexes. Pectinassociated calcium in tomato was reduced under boron deficiency [48]. Calcium inhibited decomposition of cell wall RG-II-B dimmers in vitro and in vivo [25,46]. Calcium mediated the recovery of boron deficient, nitrogen-fixing cyanobacteria Anabaena [8], and non-fixing Synechococcus [11], by stabilizing their envelopes. Additionally, the amount of membrane-bound calcium in roots of Vicia faba was down within hours of boron deprivation [28]. Recent studies on the stability of different boron fractions in intact roots suggest that boron cross-linking of RG-II results in a conformation change that creates binding sites for calcium ions, increasing strength of the B-RG-II complex [25]. It has been proposed that the same mechanism could apply to boron complexes in membranes [46]. In plants adequately supplied with boron, as much as 60% of the boron’s total content can be present in soluble form [17,31,33]. However, except for complexes with polyols, little is known about boron binding inside the cell. Considering the calcium effect on boron-responsive genes in Medicago, we envision boron–calcium interplay in signaling events. Well
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known regulatory and/or signaling molecules are potential target for interaction with boron. Nucleic acids and nucleotides, adenylates, guanylates, oxidized nicotinamides, are capable of forming stable boron complexes, as their ribose component has the optimal configuration for borate esterification (Fig. 2). Phosphoinositides are also potential targets for boron binding (Fig. 2), which would place boron in the PI, PIP, PIP2, IP3-modulated signal transduction, thus in the IP3-mediated calcium release from the endoplasmic reticulum or tonoplast into the cytosol, activation of calciumdependent protein kinases, and changes in gene expression. This binding would also affect anchoring of certain proteins to cell membranes. But these are just possibilities. Unfortunately, the very limited information currently available on boron biological complexation does not allow us to speculate which of the boron-attracting molecules are the bona fide target in biological systems, and this is the conundrum of boron research. 5. The problem of discovering boron-dependent biomolecules The development of methods to identify boron ligands among molecules isolated from biological samples is imperative. Capillary electrophoresis has been used to detect, quantify and compare in vitro boron binding by adenylates and nicotinamides [35]. The development of markers that bind to cis-diols in the same manner than borate, such fluoresceinisothiocyanate-boronic-acid conjugate (B-FITC), may be helpful for mapping borate-binding sites in cells [21]. Edelman et al. [18] used immunogold-labeled anti-FITC antibodies and electron microscopy to detect B-FITC binding not only in the cell wall but also in membranes and cytoplasm. Plasma membrane glycoprotein with capacity to bind B could therefore been identified following electrophoresis, western blotting, incubation with B-FITC and detection with antiFITC antibodies. Those techniques that show B-binding capacity, always require artificial enrichment with either borate or bindingsites markers, and cannot be used for in vivo detection and identification of borate diester complexes. The absence of a radioactive isotope makes difficult labeling of such complexes. However, natural boron is a mixture of two stable isotopes, 10B and 11B, and the development of techniques that discriminate between both isotopes appear as the most useful methodology. There are a number of techniques routinely employed for analysis of boron isotopes in materials science matrices, but only a few are sensitive enough to study boron at the trace level [44]. Among nuclear techniques, 11B NMR seems to be a powerful tool not only for detection of borate cross-linked biomolecules, but also for the analysis of the type of borate complex, as recently reported for guar gum [3]. The striking finding by 11B NMR that AI-2 ligand is a furanosyl borate diester [15] opens the possibility that new B complexes can be found in living organisms following application of 11B-NMR to biological samples.
6. Conclusion: boron gets cis-hydroxyl-containing molecules working The cumulative evidence from plant, bacteria, animal, and human experiments, shows boron as a dynamic trace element affecting an exceptionally large number of seemingly unrelated biological functions. The diverse responses to boron deprivation in a broad range of organisms indicate either boron involvement in a broad spectrum of processes, or pleiotropic effects. Our current hypothesis is that the primary role of boron in biological systems is stabilization of molecules with cis-diol groups, independently on their function. It is possible that new roles for B, based on its special chemistry would appear. In fact, last exciting report on this topic proposed that borate minerals could play a crucial role in an early “RNA world” of life on Earth by stabilizing cyclic ribose [41]. The requirement of boron for cross-linking the pectin component RG-II in plant cell walls [31], for vesicle targeting and transmembrane transport in symbiosomes [6], or as a ligand in the cyclic furanosyl bacterial quorum sensing signal AI-2 [15], provides strong evidence for boron function as a “molecular linker”, and supports our hypothesis. Coulthurst et al. [16], referring to the newly found role for boron in bacterial signaling asked, “Why boron?” This question remains relevant in other biological contexts. We conclude that boron chemistry makes it a perfect candidate for atomic diester bridging. Although other atoms, such as phosphorus or sulfur, could form links through diester bridges instead of boron, the resulting configuration would be unstable due to markedly greater electron density of those heavier atoms. At this moment, unraveling the occurrence of boron complexes in biological systems has only begun. While much additional research will be necessary, our model for boron function provides a single unifying explanation for what has previously seemed an incomprehensibly diverse range of biological roles. Although analytical instruments and procedures have steadily improved during the last decade, further progress greatly depends on the development of new methodology with higher capability for analytical imaging of boron isotopes at physiological concentrations in plant tissues.
Acknowledgements This work was supported by MCYT no. BOS2002-04164CO3-02. Ildefonso Bonilla was granted by MECD Programa “Salvador de Madariaga” 2002 for a stay in University of Missouri, Columbia.
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