Functions of Boron in Higher Plants: Recent Advances and Open Questions

Just a few years ago, Marschner (1995[16]) stated in his text “Mineral Nutrition of Higher Plants”, “The role of boron in plant nutrition is still the least understood of all the mineral nutrients and what is known of boron requirement arises mainly from studies of what happens when boron is withheld or resupplied after deficiency”. At that time the primary focus was on the possible role of B in the biosynthesis of cell wall components and cell wall structure. Indirect evidence justifying the emphasis on cell walls comes from the widely accepted belief that B has only an essential function in higher plants. However, recent research results, which indicate that B either stimulates growth or is essential for organisms lacking the cellulose - cell wall typical for higher plants, strongly suggest a broader biological role of B than restricted to the cell wall of higher plants (see for example Bennett et al., 1999[1]; Lanoue et al., 2000[14]; Rowe and Eckhert, 1999[25]). Boron may even have a beneficial function in humans (Nielsen, 2000[18]). It is therefore not surprising that in recent years compelling evidence has been gathered which demonstrates a significant role of B in cell membranes, in addition to novel discoveries in B function in the cell wall. Thus, it was timely to ask two primary research groups in the area of B functions in higher plants at the University of California, Davis, and the University of Hohenheim, respectively, to provide comprehensive updates on recent advances in this important field of plant nutrition (Brown et al., 2002[3]; Dannel et al., 2002[9]).

As pointed out by Brown et al. (2002[3]) the chemical properties of B and its complexes should be better understood. What is known is that B exists in plants primarily as boric acid (B[OH]₃) and that some B occurs as borate (B[OH]₄ ^-) at neutral and alkaline pH. Boron also forms esters and complexes with many different hydroxy compounds. The stability of these B complexes depends on several factors, particularly on the pH. Of special significance to plants are the cell wall rhamnogalacturonan-II B complex and several B-polyol complexes. It is now generally accepted that one of the primary functions of B is associated with the cell wall and involves B complexes with structural cell wall components. Rhamnogalacturonan-II (RG-II) was first identified as the polysaccharide moiety that complexes B by Kobayashi et al. (1996[13]) and O'Neill et al. (1996[20]). In cell walls, B forms dimeric complexes with RG-II (dB-RGII); two chains of monomeric RG-II are cross-linked by a 1 : 2 borate ester with two of the four apiosyl residues of the RG-II side chains (Brown et al., 2002[3]). Formation of the dimeric B complex is accelerated by Ca²⁺, but it is not yet fully understood how Ca²⁺ is associated with this complex and how it interacts with the function of B in the cell wall. It appears that Ca²⁺ and B interact in the wall by stabilizing the pectic network (Matoh and Kobayashi, 1998[17]). Brown et al. (2002[3]) presented a hypothesis for the role of B in cell walls which would explain the function of B in plant growth through its effect on cell wall pore size (see Figure 3 in Brown et al., 2002[3]). Adequate B supply for plant growth would provide optimal pore size for regulation of transport of cell wall precursors and other large molecules, such as proteins. If insufficient B is available to support growth, pore size would increase according to this hypothesis with cell wall growth becoming disrupted. Dannel et al. (2002[9]) also concluded that B complexes RG-II to form a dimer and regulates the pore size of the cell wall, this being a primary function of B. Very recently O'Neill et al. (2001[19]) confirmed that the B cross-linked RG-II dimer in cell walls is essential for turgor-driven cell growth. Work with the L-fucose deficient Arabidopsis thaliana mutant mur 1-1, which contained only half of the RG-II as B cross-linked dimer and exhibited reduced growth, demonstrated that growth depends on the structure and organization of the cell wall pectic polysaccharides.

Much progress has been made in recent years in elucidating mechanisms of B transport through cell membranes which also lead to the recognition of B playing an important role in membrane function. Both passive and active uptake of B by higher plants have been considered (Brown and Shelp, 1997[4]; Hu and Brown, 1997[12]). As highlighted in the reviews by Brown et al. (2002[3]) and Dannel et al. (2002[9]), B transport through the plasma membrane most likely is not solely due to a passive permeation process, as originally proposed by Raven (1980[24]), but at low external B concentrations (< 1 μM) active B transport by means of a membrane carrier is postulated. The first evidence for an active B transport process was presented by Dannel et al. (1997[7]) and Pfeffer et al. (1999[22]). In the latter report, B transport was shown to be sensitive to low temperature and metabolic inhibitors, indicating energy-dependent B transport under conditions of low B supply. At higher external B concentrations that are adequate to meet the B requirement for optimal plant growth, B transport through the membrane is by a passive process, as for example, supported by non-saturable, linear uptake kinetics (see Brown et al., 2002[3]). Brown et al. (2002[3]) and Dannel et al. (2002[9]) now propose three mechanisms by which B is transported through the plasma membrane of plant cells, i.e., (1) passive transmembrane diffusion of B through the lipid bilayer (2) passive, channel-mediated B transport through aquaporin-like channels, (3) energy-dependent, carrier-mediated B transport. Substantial passive B transport occurs through membrane diffusion (Dordas and Brown, 2000[10]). In addition, membrane channel proteins can facilitate transmembrane B transport (Dordas et al., 2000[11]). These authors were able to express certain intrinsic membrane proteins (MIP) in Xenopus oocytes, which resemble non-electrolyte transporting channels in other species. One of these MIP's (PIP1) if expressed lead to a significant increase in the permeability coefficient of boric acid (Pf_b), and this permeability increase could be reversed by HgCl₂. This and other evidence from these researchers indicates the importance of channel-mediated B transport through aquaporin-like channels.

Boron influences several membrane-associated processes. For example, B supply to low-B plants can cause root cell membrane hyperpolarization, stimulation of ATPase and NADH oxidase activity and enhanced ion transport (reviewed by Blevins and Lukaszewski, 1998[2]). Boron may also stabilize the structure of the plasma membrane by complexing membrane constituents (Cakmak et al., 1995[5]). Earlier results by Parr and Loughman (1983[21]) suggested B may link membrane glycoproteins and glycolipids or form complexes with sugars that cause changes in membrane transport. Thus, B appears to play a structural role at the plasma membrane. As summarized by Brown et al. (2002[3]), there is mounting evidence for a considerable variety of membrane constituents (phospholipids, glycoproteins, sugars) to form B complexes. These may be of particular importance for the function of “membrane rafts” which are discrete and physiologically active membrane sub-domains (Simons and Ikonen, 1997[26]; Brown et al., 2002[3], Figure 4). Future research needs to address the significance of these postulated B functions at the plasma membrane for B nutrition integrated in the whole plant.

The subcellular compartmentation of B in plants is an important feature but still poorly understood (Dannel et al., 2002[9]). It is a methodological challenge to analyze B quantitatively in the major plant cell compartments, although the availability of the stable B isotopes ¹⁰B and ¹¹B and their determination by ICP-MS will aid greatly in achieving reliable data on the distribution of B in the cell compartments. Given the chemical properties of B and the high membrane permeability for B, this element is likely to be present in all major cell compartments. Boron compartmentation is influenced by many factors, e.g., plant species and genotypes, plant organ, B supply and B status of the plant (Dannel et al., 2002[9]). Dannel et al. (1995[6], 1998[8]) developed methods for distinguishing between soluble and insoluble B pools. This approach, combined with efflux studies for compartmental analysis of B in sunflower roots (Dannel et al., 2002[9]) allowed these researchers to distinguish between soluble B in the free space, cell wall-bound B and B in the cytoplasmic and vacuolar compartments. (However, see Lohaus et al. [2001[15]] for a critical evaluation of the centrifugation technique for isolation of apoplastic leaf fluid.) The distribution of B depended on external B supply, but in general B expressed as % of total B in the plant was greater in the cell wall than in the other compartments, and intracellularly B in the cytoplasm exceeded the vacuolar B fraction (Dannel et al., 2002[9]).

It is well established that higher plants are very sensitive to low B supply and develop rapidly B deficiency symptoms. Boron deficiency can cause severe yield losses among agriculturally important species. In contrast to low B soils, some agricultural soils contain high concentrations of B that are toxic to most plants. Boron toxicity is often associated with saline soils, particularly in irrigated agricultural areas of the world. Salinity in the soil environment limits the productivity of most agricultural crops, and if saline soils also contain high B concentrations, salinity-B interactions can occur in plants (Pitman and Läuchli, 2002[23]). It is interesting fundamentally as well as of agricultural importance to achieve an improved knowledge of the mechanisms of B toxicity and of salinity - high B interactions in plants (Wimmer et al., 2001[27], 2002[28]). These authors demonstrated that in wheat high external B concentrations caused increased B influx into the cytoplasm where part of the B occurs as borate, which can bind to putative ligands, such as nucleotides and sugars. It is possible that the resulting B complexes in the cytoplasm may primarily be responsible for causing B toxicity symptoms. On the other hand, soluble B was found to increase substantially in the intracellular compartments of leaves at high B supply. Wimmer et al. (2002[28]) concluded that B toxicity in wheat leaves is likely to be caused by an increase in intracellular soluble B. Salinity may interact with high B through induction of the expression of MIP's, which would enhance uptake of water and B and increase inter- and intracellular concentrations of soluble B. Wimmer et al. (2002[28]) proposed that under conditions of B toxicity soluble B might be a better indicator for assessing the B status of the plant than total B content. This hypothesis must now be tested rigorously, using different plant species and a variety of environmental conditions, and better methods to differentiate between soluble and bound B in the major cell compartments.

In this brief review of recent advances on functions of B in higher plants, some answers to old questions were provided but also new open questions and challenges emerged. Particularly emphasized were the areas of B functions in the cell wall and in membranes, mechanisms of B transport through the plasma membrane, subcellular compartmentation of B in the plant and new concepts of B toxicity in plants. The role of B in reproductive growth and development, although not specifically reviewed here, is relevant to the condition of B deficiency under field conditions. However, as stated by Brown et al. (2002[3]), “the unique sensitivity of reproductive structures to B deficiency remains poorly understood”. Except for the latter challenge, significant progress has been made in the last few years in the areas highlighted, which lead to several testable hypotheses and the need for new research. Earlier focus on the role of B complexes in plants is now complemented by the potential significance of soluble B in relation to B toxicity in plants. Clearly, research on the biological function of B has entered an exciting period of discovery and novel insights.

References

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Section Editor: U. Lüttge