Review Article
`
`205
`
`Boron in Plant Biology
`
`P. H. Brown 1, N. Bellaloui 1, M. A. Wimmer 1, E. S. Bassil 1, J. Ruiz 1, H. Hu 1, H. Pfeffer 2, F. Dannel 2,
`and V. Römheld 2
`1 Department of Pomology, University of California, Davis, USA
`2 Institute for Plant Nutrition (330), University of Hohenheim, Stuttgart, Germany
`
`Received: February 11, 2002; Accepted: February 25, 2002
`
`Abstract: The interest of biologists in boron (B) has largely
`been focused on its role in plants for which B was established
`as essential in 1923 (Warington, 1923[153]). Evidence that B has
`a biological role in other organisms was first indicated by the es-
`tablishment of essentiality of B for diatoms (Smyth and Dugger,
`1981[138]) and cyanobacteria (Bonilla et al., 1990[14]; Garcia-Gon-
`zalez et al., 1991[44]; Bonilla et al., 1997[16]). Recently, B was
`shown to stimulate growth in yeast (Bennett et al., 1999[8]) and
`to be essential for zebrafish (Danio rerio) (Eckhert and Rowe,
`1999[33]; Rowe and Eckhert, 1999[121]) and possibly for trout (On-
`corhynchus mykiss) (Eckhert, 1998[32]; Rowe et al., 1998[120]),
`frogs (Xenopus laevis) (Fort et al., 1998[41]) and mouse (Lanoue
`et al., 2000[75]). There is also preliminary evidence to suggest
`that B has at least a beneficial role in humans (Nielsen,
`2000[96]). While research into the role of B in plants has been
`ongoing for 80 years it has only been in the past 5 years that
`the first function of B in plants has been defined. Boron is now
`known to be essential for cell wall structure and function, likely
`through its role as a stabilizer of the cell wall pectic network
`and subsequent regulation of cell wall pore size. A role for B in
`plant cell walls, however, is inadequate to explain all of the ef-
`fects of B deficiency seen in plants. The suggestion that B plays
`a broader role in biology is supported by the discovery that B is
`essential for animals where a cellulose-rich cell wall is not pres-
`ent. Careful consideration of the physical and chemical proper-
`ties of B in biological systems, and of the experimental data
`from both plants and animals suggests that B plays a critical
`role in membrane structure and hence function. Verification of
`B association with membranes would represent an important
`advance in modern biology. For several decades there has been
`uncertainty as to the mechanisms of B uptake and transport
`within plants. This uncertainty has been driven by a lack of ade-
`quate methodology to measure membrane fluxes of B at phy-
`siologically relevant concentrations. Recent experimentation
`provides the first direct measurement of membrane permeabil-
`ity of B and illustrates that passive B permeation contributes
`sufficient B at adequate levels of B supply, but would be inade-
`quate at conditions of marginal B supply. The hypothesis that
`an active, carrier mediated process is involved in B uptake at
`low B supply is supported by research demonstrating that B up-
`take can be stimulated by B deprivation, that uptake rates fol-
`
`Plant biol. 4 (2002) 205 ± 223
` Georg Thieme Verlag Stuttgart ´ New York
`ISSN 1435-8603
`
`low a Michaelis-Menton kinetics, and can be inhibited by appli-
`cation of metabolic inhibitors. Since the mechanisms of ele-
`ment uptake are generally conserved between species, an un-
`derstanding of the processes of B uptake is relevant to studies
`in both plants and animals. The study of B in plant biology has
`progressed markedly in the last decade and we are clearly on
`the cusp of additional, significant discoveries. Research in this
`field will be greatly stimulated by the discovery that B is essen-
`tial for animals, a discovery that will not only encourage the
`participation of a wider cadre of scientists but will refocus the
`efforts of plant biologists toward a determination of roles for B
`outside the plant cell wall. Determination of the function of B
`in biology and of the mechanisms of B uptake in biological sys-
`tems, is essential to our understanding and management of B
`deficiency and toxicity in plants and animals in both agricultu-
`ral and natural environments. Through an analysis of existing
`data and the development of new hypotheses, this review aims
`to provide a vision of the future of research into the biology of
`boron.
`
`Key words: Boron, function, structure, essentiality, membrane,
`uptake, transport, cell wall, reproduction.
`
`Introduction
`
`Because of the rapidity and the wide variety of symptoms that
`occur following B deprivation, determining the primary func-
`tion of B in plants has been one of the greatest challenges in
`plant nutrition (Blevins and Lukaszewski, 1998[10]). Over the
`past decade, however, very significant advances have been
`made in understanding the metabolism of B in plants. Primary
`among these advances has been the determination of the
`chemical form and function of B in plant cell walls, the identi-
`fication of the role of polyhydric alcohols in phloem B trans-
`port and the characterization of the processes of B transport
`across membranes. Each of the major advances in our knowl-
`edge of the biology of B has been strongly grounded in an un-
`derstanding of the physical and chemical characteristics of
`B. Though B is now known to be essential for many organisms,
`it is clear that B plays a uniquely important role in plants.
`Among the essential plant micro-nutrients, B deficiencies oc-
`cur widely and have a significant agronomic impact through-
`out the world (Gupta,1979[49]; Shorrocks, 1997[131]). One signif-
`icant feature of B deficiency that contributes to its importance
`
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`206
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`Plant biol. 4 (2002)
`
`P. H. Brown et al.
`
`in agricultural production is that a deficiency of B inhibits
`growing tissues, specifically reproductive structures, which re-
`present 80 % of the worlds agricultural product. Boron defi-
`ciency creates a wide range of anatomical symptoms including
`the inhibition of apical and extension growth, necrosis of
`terminal buds, cracking and breaking of stems and petioles,
`abortion of flower initials, and shedding of fruits (Mozafar,
`1993[92]; Goldbach, 1997[47]). Boron deficiency also causes
`many physiological, and biochemical changes including, alter-
`ed cell wall structure, altered membrane integrity and func-
`tion, changes in enzyme activity and altered production of a
`wide range of plant metabolites (Goldbach, 1997[47]). The pro-
`found effects of B on meristems reflects the unique role B plays
`in cell growth and is a consequence of the processes that con-
`trol B uptake and transport. It is clear, however, that our
`knowledge of the biology of B is still limited and that we have
`an inadequate understanding to optimally manage B in agri-
`cultural practice.
`
`While a role for B in cell walls has now been well defined, a
`wealth of evidence suggests that B plays a role in the structure
`and function of the plasma membrane of plants. An effect of
`B on membrane structure could occur as a result of B binding
`to phosphoinositides, glycoproteins and glycolipids of mem-
`branes thereby influencing the stability of membrane domains
`with unique biological activity (Parr and Loughman, 1983[103]).
`A role for B in membranes is also supported by findings that B
`deficiency in animals primarily disrupts processes that are
`highly membrane specific or that require synthesis of new
`membranes. In both the African clawed frog (Xenopus laevis)
`(Fort et al., 1998[41]) and the zebrafish (Eckhert and Rowe,
`1999[33]) B is essential for the normal reproduction while in
`mature zebrafish, B deficiency results in dystrophy of photo-
`receptors in the eye (Eckhert and Rowe, 1999[33]). These pro-
`cesses are characterized by a high requirement for membrane
`synthesis. In humans, B affects several membrane specific pro-
`cesses resulting in decreased brain electrical activity, impaired
`cognitive performance, altered concentrations and decreased
`activity of several membrane active hormones (Nielsen,
`2000[96]). A primary role for B in membranes is clearly suggest-
`ed and is consistent with known physical and chemical proper-
`ties of B.
`
`The cumulative evidence that B is essential across a number of
`kingdoms of living organisms represents a major paradigm
`shift and provided the impetus for this review. In the follow-
`ing, we re-examine significant historical observations and re-
`cent advances in the study of B in plants, and interpret these
`findings in light of the discoveries that B plays a role in the
`biology of both plants and animals.
`
`Boron Chemistry
`
`The physical and chemical properties of B and its complexes
`are unique and highly varied. An understanding of these prop-
`erties is essential if we are to predict and interpret the role of B
`in biology (for more complete review see Woods [1996[158]]).
`
`Under physiological conditions, and in the absence of inter-
`action with bio-molecules, B exists as boric acid (B[OH]3) or
`±) (Woods, 1996[158]). Boric acid is a very
`borate anion (B[OH]4
`weak acid, with a pKa of 9.24, and at the pH found in the cyto-
`plasm (pH 7.5), more than 98 % of B would exist in the form of
`
`± (Woods,
`free B(OH)3 and less than 2 % would exist as B(OH)4
`1996[158]). At pH values found in the apoplast (pH 5.5), greater
`than 99.95 % of B would be in the form of B(OH)3 and less than
`±. Boric acid and borate,
`0.05 % would be in the form of B(OH)4
`however, can readily react with many kinds of biological mol-
`ecules and under normal biological conditions available B
`binding molecules will typically exceed the concentration of
`free B. An understanding of B binding reactions is therefore
`central to an understanding of B physiology.
`
`Boric acid forms esters and complexes with a wide variety of
`mono, di- and poly-hydroxy compounds (Woods, 1996[158]).
`These borate esters form and dissociate spontaneously in dy-
`namic pH-dependent equilibrium and with rapid kinetics
`(Friedman et al., 1974[43]). A number of factors affect the sta-
`bility of these B complexes. Increasing pH typically stabilizes
`B complexes, cis-diols are favoured over trans-diols (Boeseken,
`1949[11]); while sugars with a furanoid ring (five-membered
`ring) configuration form more stable complexes than those
`with a pyranoid ring (six-membered ring) (Loomis and Durst,
`1992[81]; Goldbach, 1997[47]; Brown P. H. and Hu H., unpub-
`lished data). The presence and nature of neighboring groups
`in B complexes can have a significant stabilizing effect on B
`complexes. Large substituent groups provide steric protection
`against hydrolysis, while the presence of an adjacent nitrogen
`increases stability by inducing additional H-bonding; and the
`presence of a positively charged substituent group confers
`electrostatic stabilization (Woods, 1996[158]). Thus, the coen-
`zyme NAD+ has a 15-fold greater affinity for B than the reduced
`form NADH since the negative charge of NAD-borate is sta-
`bilized by the positive charge in NAD+ (Smith and Johnson,
`1976[136]). The stability of the borate-rhamnogalacturonan-II
`complex is also greatly enhanced by presence of Ca2+ (Kobaya-
`shi et al., 1999[71]).
`
`Examples of bio-molecules that react strongly with boric acid
`include ribose, apiose (e.g., cell wall), sorbitol and other poly-
`ols (Loomis and Durst, 1992[81]), as well as phenolics and ami-
`no acids, such as serine (Tate and Meister, 1978[143]). Complex
`bio-molecules with predicted B binding capacity include gly-
`coproteins and glycolipids. This has been demonstrated by
`Frantzen et al. (1995[42]) using protein-boronic acid (a boric
`acid analog) affinity column to separate glycohemoglobin from
`non-glycolated hemoglobin. Given the diversity of functional
`groups with which B can bind a wide range of B containing
`bio-molecules are likely present in all biological systems. With
`the exception of the cell wall rhamnogalacturonan-II B com-
`plex identified by Kobayashi et al. (1996[70]) and ONeill et al.
`(1996[102]), and the B-polyol complex identified by Hu et al.
`(1997[59]), the functional significance of these putative biologi-
`cal B complexes has not been determined.
`
`The diversity of factors that influence the stability of B com-
`plexes is so great and our knowledge of biological B complexa-
`tion so poor, that currently it is impossible to predict, with any
`degree of certainty, the binding status of B in biological sys-
`tems. The ubiquitous nature of potential B binding sites has of-
`ten confounded the interpretation of experimental results and
`has resulted in the many purported functions of B that have
`been published over the proceeding 70 years. For example,
`the theoretical capacity of sugars and phenolics to bind B has
`been frequently interpreted as evidence for a role of B in sugar
`and phenol metabolism as pointed out by Goldbach (1997[47]).
`
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`Boron in Plant Biology
`
`Plant biol. 4 (2002) 207
`
`This conclusion has been widely cited even though there is no
`definitive evidence that B-sugar or B-phenol complex for-
`mation has any significant effect on metabolism. Equally pro-
`blematic are studies of B uptake and within plant transport
`that fail to adequately consider the potential effects of B-com-
`plexation on transport kinetics.
`
`Uptake of Boron
`
`As a consequence of its essential role in growing tissues and
`of the inherent phloem immobility of B in most plant species,
`fluctuations in soil B availability can have a profound effect
`on plant growth and productivity. Many species are also sensi-
`tive to high levels of B in soil, and water, and growth inhibi-
`tion as a result of excess B uptake is experienced in many agri-
`cultural regions. Understanding the biology of B uptake by
`plants is therefore critical to the management of B in natural
`and agricultural systems. The past three years have seen a pro-
`gress in our understanding of the mechanisms of B acquisi-
`tion by plants and the factors that govern transmembrane B
`transport.
`
`The subject of B uptake has long been controversial and signif-
`icant evidence supports both the active and passive uptake of B
`in higher plants (Brown and Shelp, 1997[19]; Hu and Brown,
`1997[57]). Hu and Brown (1997[57]) proposed that B uptake, un-
`der conditions of adequate or excessive B supply, is the result
`of passive absorption of undissociated boric acid (B[OH]3). It
`was further hypothesized that B uptake occurred by a non-me-
`tabolic process primarily determined by the B concentration
`outside the root, B-complex formation inside (Brown and Hu,
`1994[18]) or outside of the root, membrane permeability (Ra-
`ven, 1980[115]), translocation of B within the plant (Brown and
`Shelp, 1997[19]) and the transpiration rate. In their analysis Hu
`and Brown (1997[57]) concluded that there is no substantive
`evidence that B uptake occurred through an energy dependent
`process, such as a transport carrier molecule.
`
`Though this conclusion was consistent with the majority of
`available studies, it was incomplete in that it did not provide
`a mechanistic explanation of B uptake and was based largely
`upon the theoretical predictions of membrane permeability
`proposed by Raven (1980[115]), predictions that had not been
`verified in vivo. Furthermore, the conclusion that B uptake
`was a passive process seemed to be at odds with observed dif-
`ferences in B uptake among plant species or cultivars, that can
`be as large as seven-fold when those species are grown under
`identical conditions (Nable et al., 1997[97]). For example, barley
`cultivars ªSahara 3763º and ªSchoonerº accumulated 112 and
`710 mg kg±1 B dry weight in the youngest expanded leaf
`blade, respectively. Such differences in B uptake cannot easily
`be explained through differences in water use, as Passioura
`(1977[104]) reported for wheat that the water use efficiency of
`13 cultivars ranged only from 3.1 to 4 g dry matter kg±1 water.
`
`The apparent contradiction, between in vitro results that sug-
`gest that B uptake is a passive process, and the field results
`that demonstrate significant differences among species and
`genotypes, is difficult to reconcile but is of fundamental im-
`portance to studies of B nutrition. Mechanisms that have been
`postulated to explain this apparent paradox include active up-
`take, exudation of B complexing compounds into the rhizo-
`sphere, species differences in B binding compounds, such as
`
`pectins in the cell walls, physical barriers in the roots, and spe-
`cies differences in membrane permeability (Huang and Gra-
`ham, 1990[60]; Hu and Brown, 1997[57]).
`
`Evidence for passive boron uptake
`
`As early as 1980 Raven (1980[115]) postulated that B uptake
`would occur via passive diffusion since in physiological pH
`range boric acid would have a theoretical lipid permeability
`coefficient of 8 ” 10±6 cm s±1, which is adequate to satisfy the B
`need of the plant. Only very recently have these theoretical
`calculations been verified and the specific mechanisms by
`which B crosses artificial lipid membranes, as well as plant
`and animal membranes, have been tested (Dordas and Brown,
`2000[29]; Dordas et al., 2000[30]).
`
`Using artificial liposomes made of phosphatidylcholine (PC),
`Dordas and Brown (2000[29]) measured the permeability co-
`efficients of boric acid (Pfb), urea, glycerol (non-electrolytes
`of similar size) and water. They calculated the Pfb to be
`4.9 ” 10±6 cm s±1 which closely corresponds to the theoretical
`values estimated from ether-water partitioning coefficients
`and also from the molecular weight and number of H-bonds
`of B (Raven, 1980[115]). When vesicle characteristics, such as
`sterol (STL) composition, type of phospholipids, the presence
`of head groups and length of fatty acid chains were modified,
`Pfb was also affected. With a decreasing PC/STL ratio (from
`100 % PC to 60 % STL), the Pfb was reduced from 5 ” 10±6 to
`1 ” 10±6 cm s±1. These results are consistent with those shown
`previously for other non-electrolytes, where the presence of
`STL in the membrane can substantially reduce membrane
`fluidity, increase mechanical coherence and hence suppresses
`passive transmembrane permeability (Mouritsen et al.,
`1995[91]).
`
`In studies utilizing membranes isolated from squash roots,
`Dordas et al. (2000[30]) determined Pfb to vary from 3.9 ” 10±7
`to 2.4 ” 10±8 cm s±1 in plasma membrane and plasma mem-
`brane-depleted vesicles, respectively. In the charophyte alga,
`Chara corallina, Stangoulis et al. (2001[140]) determined Pfb of
`4.4 ” 10±7 cm s±1 across the combined plasmalemma and tono-
`plast membrane, which is in good agreement with values from
`squash liposomes. These values, however, are 20- to 300-fold
`slower than the permeability predicted by Raven (1980[115]) or
`determined experimentally in artificial liposomes (Dordas and
`Brown, 2000[29]). The lower Pfb found in this study (Dordas et
`al., 2000[30]) compared to that calculated from the ether-water
`partition coefficient can occur because of the effect of lipid
`composition on the membrane properties and permeability.
`Sterols are major components of plant membranes, and are
`known to have a strong effect on the permeability of water
`and non-electrolytes (Schuler et al., 1991[129]; Lande et al.,
`1995[74]). Plant sterols reduce water permeability to an even
`greater extent than cholesterol (Schuler et al., 1991[129]) and
`it is expected that these sterols will have similar effects in re-
`ducing membrane permeability for boric acid or other non-
`electrolytes.
`
`The hypothesis that changes in membrane composition direct-
`ly affect permeability of B and hence plant B uptake, is sup-
`ported by experimentation conducted with mutant lines of
`Arabidopsis thaliana differing in membrane lipid composition
`(Dordas and Brown, 2000[29]). In these experiments, B uptake
`
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`208
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`Plant biol. 4 (2002)
`
`P. H. Brown et al.
`
`varied significantly in a manner that was consistent with re-
`sults from in vitro studies (Dordas and Brown, 2000[29]).
`
`Evidence for active carrier mediated boron uptake
`
`The results of Brown and coworkers (Dordas and Brown,
`2000[29]; Dordas et al., 2000[30]; Stangoulis et al., 2001[140]) ver-
`ify the principle that substantial movement of B occurs
`through passive membrane diffusion and are consistent with
`the theory that B uptake is a passive process. These experi-
`ments also provide a biophysical explanation for observed dif-
`ferences in B uptake among several species and cultivars,
`grown under identical conditions (Hu and Brown, 1997[57];
`Nable et al., 1997[95]). The results also demonstrate that mem-
`brane composition, membrane origin and cultivar differences
`all have a profound impact on the rate of B permeability. The
`presence of substantial membrane permeability does not,
`however, preclude a role for membrane proteins in the facilita-
`tion of transmembrane B movement. Indeed, the permeability
`of water through plant membranes is known to be substantial-
`ly faster than the permeability of B and yet numerous mem-
`brane intrinsic proteins (Aquaporins) are known to be involved
`in water uptake (Weig et al., 1997[154]).
`
`The possible involvement of channel proteins in B transport
`was recently examined by Dordas et al. (2000[30]) and Nuttall
`(2000[97]) utilizing a variety of methods including the applica-
`tion of channel inhibiting mercury and expression of major in-
`trinsic membrane proteins (MIP) in Xenopus laevis oocytes.
`The inhibition of ion transport following application of HgCl2
`is widely interpreted as evidence of the involvement of chan-
`nel proteins in the uptake (Barone et al., 1997[4]). Dordas et al.
`(Dordas and Brown, 2000[29]; Dordas et al., 2000[30]) demon-
`strated that B uptake into squash membrane vesicles was in-
`hibited by HgCl2 and that this effect could be reversed by the
`application of 2-mercaptoethanol. These results were repli-
`cated in in vivo studies conducted with squash and Arabidopsis
`where the presence of HgCl2 reduced B uptake by 35 % (Dordas
`and Brown, 2000[29]).
`
`The hypothesis that channel proteins are involved in B trans-
`port was further supported by experiments in which certain
`MIPs with homology to non-electrolyte transporting channels
`from other species were expressed in Xenopus laevis oocytes
`and B permeability subsequently determined (Dordas et al.,
`2000[30]). The expression of one of the MIPs (PIP1), resulted in
`a 30 % increase in Pfb and this benefit could be reversed with
`the addition of HgCl2. The effectiveness of additional MIPs at
`increasing B transport was verified by Nuttall (2000[97]) who
`demonstrated that expression of PIP1b, PIP2a and PIP2b pro-
`teins in Xenopus oocytes significantly increased the perme-
`ability of the oocyte membrane to boric acid, from below the
`detection limit to 1 ” 10±8 cm s±1. PIP1c did not mediate boric
`acid transport, despite the fact that it increased the water per-
`meability of oocytes, which was interpreted as evidence of
`functional expression of PIP1c proteins in the oocyte plasma-
`lemma.
`
`Prior to 1997 there had been only scattered reports of meta-
`bolically dependent B uptake and many of these experiments
`were technically flawed or could not be reproduced (for review
`see Hu and Brown [1997[57]]). Few, if any, of these early studies
`utilized sufficiently low levels of B supply or included pre-
`treatment under B deficient conditions. All experiments were
`limited by difficulties in B determination in the low concentra-
`tion range and by the lack of a convenient isotope for determi-
`nation of short-term uptake kinetics. Recently, a series of stud-
`ies that address these problems have been conducted by Dan-
`nel, Pfeffer, and Römheld. These experiments contribute sub-
`stantially, to our knowledge, of B uptake and suggest that un-
`der conditions of restricted B supply, B is assimilated through
`a metabolically active, carrier mediated transport process.
`
`The first evidence for the occurrence of an active B transport
`system was presented by Dannel et al. (1997[27]) who observed
`B concentrations in the root cell sap (consisting of cytoplasmic,
`vacuolar and apoplastic solutions) and the xylem exudate of
`sunflower, that were about 22-fold higher than the concentra-
`tion of B in the rooting medium (1 mM B). These results were
`interpreted as evidence of an energy dependent transport
`mechanism capable of accumulating B against a concentration
`gradient. Subsequent studies (Pfeffer et al., 1999[107]) demon-
`strated that the putative concentration mechanism for B is
`suppressed by 24 h of high B supply (100 mM). The energy de-
`pendence of this mechanism was subsequently demonstrated
`by application of the metabolic inhibitors 2,4-dinitrophenol,
`or low root zone temperature which resulted in a suppression
`of the B concentrating mechanism (Pfeffer et al., 1999[107]). This
`suggests that the establishment of a concentration gradient for
`B between external solution and the inside of the cell is direct-
`ly or indirectly dependent on metabolic energy. In a more de-
`tailed study, using stable isotopes and various B concentra-
`tions, Dannel et al. (2000[26]) characterized B uptake in sun-
`flower in greater detail. The results suggest that low B supply
`during plant pre-culture stimulates B uptake, which can be
`eliminated by metabolic inhibition treatments. At higher B
`supplies, uptake then followed a non-saturable linear kinetics
`suggesting that B uptake occurs by two processes, a saturable
`carrier mediated transport at low concentrations and a non-
`saturable diffusion driven process at higher concentrations.
`
`In a series of short-term uptake studies, Pfeffer et al. (2001[109])
`suggested that B uptake at low B supply follows Michaelis-
`Menten kinetics with an apparent Km of 15 mM and Vmax of
`about 31 nmol groot FW±1 h±1. Experiments on B uptake have
`also been conducted by Stangoulis et al. (2001[140]) using the
`charophyte alga Chara corallina. Following at least one day of
`B starvation, B uptake into Chara cells appeared to be a combi-
`nation of a saturable and a linear component, and at 0 to 10 mM
`B supply uptake followed Michaelis-Menten kinetics with an
`apparent Km of about 2 mM and Vmax of about 135 pmol m±2 s±1.
`
`The evidence that substantial B movement can occur through
`diffusion and channel mediated transport is compelling and
`could account for B uptake under conditions of adequate or
`greater B supply. However, this evidence does not address the
`conditions that might exist when B is at or below adequate
`concentrations in the rooting medium, conditions that might
`induce specialized mechanisms for B uptake.
`
`These experiments are the first detailed investigations of B
`uptake at conditions of moderate B supply (< 10 mM) and pro-
`vide evidence that an active B uptake process may function
`in plants. This conclusion, however, must be considered with
`caution since cytoplasmic concentrations of B cannot yet be
`directly determined. The proposed Km for the putative high
`affinity B transporter (15 mM) is also much higher than the
`
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`Boron in Plant Biology
`
`Plant biol. 4 (2002) 209
`
`Table 1 Comparison of maximum potential contribution of passive uptake rate vs. actual relative uptake rate of B in canola and tobacco
`
`Species
`
`Canola
`
`Tobacco
`
`Boron supply
`(mM)
`
`Relative uptake rate
`(nmol ´ g±1 FW d±1)
`
`0.1
`0.1
`0.97
`0.97
`10
`10
`
`10
`10
`
`5
`5
`96
`96
`100
`100
`
`20
`20
`
`Permeability
`coefficient
`(cm ´ s±1)
`
`2.4 ” 10±8**
`3.9 ” 10±7***
`2.4 ” 10±8
`3.9 ” 10±7
`2.4 ” 10±8
`3.9 ” 10±7
`
`2.4 ” 10±8
`3.9 ” 10±7
`
`Root surface area
`(cm2)*
`
`Maximum passive
`permeation rate
`(nmol ´ g±1 FW d±1)
`
`700
`700
`700
`700
`700
`700
`
`700
`700
`
`0.15
`2.4
`1.5
`24
`15
`240
`
`15
`240
`
`Permeability coefficients determined in squash from Dordas et al. (2000[30]).
`* Root surface area is calculated by assuming root hair surface to be 2 ´ 5-fold
`root surface and 1 cm3/g of root.
`
`** Permeability coefficient of plasma membrane-depleted vesicles.
`*** Permeability coefficient of Plasma membrane vesicles.
`
`minimal concentration of B at which plant growth can be
`maintained (Asad et al., 1997[2]) and is significantly higher
`than the Km of other micronutrients required in equivalent
`concentrations by the plant. For example, Km was found to be
`1.5 to 3 mM for Zn in maize and barley, respectively (Veltrup,
`1978[150]; Mullins and Sommers, 1986[94]).
`
`Boron uptake: Conclusions and future directions
`
`One of the primary reasons cited against the occurrence of an
`active (energy dependent) high affinity uptake of B in plants,
`was the prediction by Raven that the theoretical permeability
`of B was such that adequate B uptake would occur by a process
`of passive diffusion through the membrane (Raven, 1980[115]).
`The predictions of Raven can now be verified by determination
`of B permeability coefficients across plant membranes (Dordas
`et al., 2000[30]) and B uptake in plants grown under carefully
`controlled experimental systems (Asad et al., 1997[2]; Bellaloui
`et al., 1999[7]).
`
`In Table 1, we have estimated potential B permeation rates un-
`der a variety of external B concentrations in tobacco and cano-
`la, for which data on plant B demand have been experimental-
`ly determined. These calculations were derived using the
`measured Pfb from the membranes of squash, a species that is
`relatively sensitive to B deficiency. The calculations also pre-
`sume a constant supply of external B and a constant internal
`B concentration of 0 mM, hence this represents the maximal
`potential permeation rates. These data demonstrate that pas-
`sive permeation of B would be adequate to provide the ob-
`served B requirement for both canola and tobacco under nor-
`mal conditions of B supply (e.g., 10 mM B). However, if B is re-
`duced to 1 mM or Pfb is decreased to 2.4 ” 10±8 cm s±1 (as found
`in plasma membrane-depleted membranes) then passive B
`permeation is inadequate to satisfy the B requirements of
`these species.
`
`These calculations support the contention that at low levels of
`B an active B uptake mechanism may exist. These results also
`illustrate the very significant effect that small variations in
`Pfb, external B supply, plant growth rate or root characteristics
`(diameter, length) will have on B uptake. The presence of sub-
`stantial passive permeability under conditions of adequate B
`supply (> 10 mM B), however, does not preclude a role of active
`
`uptake mechanisms in B acquisition at these concentrations.
`Indeed in Chara australis, which has a plasma membrane per-
`meability coefficient for urea that is significantly higher than
`that for boric acid (Wilson and Walker, 1988[156]), the uptake
`of urea is known to be facilitated by a high affinity, electro-
`genic symport with sodium at low concentrations (20 mM)
`(Wilson et al., 1988[155]; Wilson and Walker, 1988[156]; Walker
`et al., 1993[152]).
`
`Evidence to support the occurrence of a saturable, active B up-
`take mechanism is still preliminary, primarily owing to our
`inability to directly measure cytoplasmic B concentrations or
`to directly monitor real time B uptake. These results should,
`therefore, be interpreted with caution and alternative explana-
`tions for the reported results should be considered. In this re-
`gard the unique physical and chemical characteristics of B de-
`scribed above may provide some insight. The uptake kinetics
`of B would be greatly complicated by the abundance of poten-
`tial B complexing molecules in plant cell wall and cytoplasm.
`At low B supply and specifically following a period of B depri-
`vation, many of these potential B complexing molecules would
`not be associated with B. With the addition of B at the com-
`mencement of a short-term uptake period, there would be a
`strong driving force as the unassociated B binding sites be-
`come saturated, effectively reducing the concentration of free
`B and maintaining a driving force for further uptake.
`
`The effect of cytoplasmic B binding molecules on B uptake has
`been demonstrated experimentally in transgenic tobacco
`plants, genetically engineered to produce enhanced concen-
`trations of sorbitol, a B complexing molecule usually not found
`in tobacco (Bellaloui et al., 1999[7]). The production of sorbitol
`in transgenic tobacco significantly increases B uptake, tissue B
`concentration and B transport to meristematic tissue, when
`compared with plants not containing sorbitol (Bellaloui et al.,
`1999[7]). These results suggest that B complexing molecules
`present in the cytoplasm may facilitate B uptake by maintain-
`ing a favourable gradient for B diffusion into the plant. Such a
`complexation driven uptake process would result in uptake
`kinetics that significantly complicate the interpretation of up-
`take data. A more detailed analysis of the quantity and charac-
`teristics