Essay 5.1 Boron Functions in Plants: Looking Beyond the Cell Wall

Essay 5.1 Boron Functions in Plants: Looking Beyond the Cell Wall

Ildefonso Bonilla,1 Dale Blevins,2 and Luis Bolaños1
(1) Depto. Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain; (2) Plant Science Unit, University of Missouri, Columbia, MO, USA

March, 2009

Katherine Warington (1923) is credited with establishing the boron (B) requirement for vascular plants. In her work with broad bean and other legumes, she determined that B must be supplied continuously during the life of the plant. Interestingly, she could not demonstrate a B requirement for the graminacious monocots, barley and rice. Later, Sommer and Lipman (1926), using redistilled water and highly purified chemicals showed a B requirement for six non-leguminous dicots and the graminacous plant, barley. Now plants can be separated into four groups based on their B requirement for growth and development. Lactifers (latex-forming species) have by far the highest B requirement followed by leguminous plants, then the remaining dicots and the lily family of monocots, and finally the lowest B requirements are found in graminacous plants (Mengel and Kirkby, 2001). Importantly, when graminacous plants flower, their B requirement increases to levels equivalent to those of dicots. As will be discussed later, for all species other than lactifers, B requirements can be explained by the amount of pectin (RGII fraction) in cell walls or in pollen tube walls. Why lactifers require so much B remains a mystery.

There are long lists of “postulated roles of B” for higher plant growth and development. Gauch and Dugger (1954) in their thorough review listed 15 potential roles for B in plants, and later Parr and Loughman (1983) listed 10 potential roles. However, many of these “postulated roles” of B can be eliminated as “secondary” by considering the “timing” of the deficiency treatments. Only short term experiments are meaningful when one is trying to establish a major role of B in plants. In fact, now that B has been established as a component of the (RGII) fraction of cell wall pectin, it is much easier to explain many of the “postulated” roles. But the question remains, are there important roles of B beyond (RGII) in cell walls? Again, one must focus on rapid responses to B deficiency.

There are rapid changes in membrane function induced by B deficiencies. Both Pollard et al. (1977) and Tanada (1983) found that B was localized in membranes. Limited phosphorus (P) uptake by Vicia faba roots was restored to normal levels by a 1h pretreatment with B (Robertson and Loughman, 1974). Maize and Vicia faba root uptake of P, Cl, and Rb was restored to 40% of normal within 20 min after B was added to the medium (Pollard, et al. 1977). Both uptake and efflux of P were depressed in B-deficient sunflower roots, but were restored within 1h after addition of B (Goldbach, 1984). In suspension culture cells of carrot and tomato, B treatment repaired a depressed auxin-stimulated ferricyanide-induced proton release within 60 min (Goldbach et al., 1990). Schon et al. (1990) found a hyperpolarization of membranes of sunflower roots within 3 min of B addition to a B-free medium, while an instantaneous stimulation of the plasma membrane NADH oxidase was reported after B addition to low B carrot cell cultures (Barr et al., 1993; Barr and Crane, 1991).

Other rapid chances with B treatment have been recorded in work with pollen tubes. Growth of pollen tubes requires not only rapid cell wall synthesis, but also rapid membrane formation. We now understand that a primary role for B is as a structural component of the RGII fraction of pectin, but B, in smaller quantities, may be a critical component of membranes in pollen tubes. Pollen tube formation depends on rapid fusion of vesicles to form the plasma membrane. Jackson (1984) found very different phase changes in membranes of rapidly growing Petunia pollen tubes when comparing B-sufficient treatments with B-deficient treatments. He used protein secretion from the pollen tube at different temperatures in the presence or absence of B to make those determinations.

Recently, in vivo cross-linking of apiose residues by B in the pectic polysaccharide rhamnogalacturonan II (RGII) has been convincingly demonstrated (O’Neill et al., 2001, 2004), supporting earlier suggestions that the primary function of B in plants is a structural role, related to the stability of the cell wall (Warington, 1923). Boron essentiality for growth of other organisms also has been linked to stability of cell walls and envelopes, diatoms (Smyth and Dugger, 1981), heterocystous cyanobacteria (Bonilla et al., 1990) (Figure 1), or bacteria of the genus Frankia (Bolaños et al., 2002) (Figures 2 and 3). Since most B is associated with cell wall pectin (O’Neill et al., 1996; Bonilla et al., 1997b), the numerous biochemical, physiological, and anatomical effects of B deficiency in plants (Goldbach, 1997; Blevins and Lukaszewski, 1998; and Brown et al., 2002 are excellent reviews) (Figure 4) often have been considered secondary effects. However, reports of abnormal xylem differentiation (Lovatt, 1985) and embryogenesis (as described in Larix by Behrendt and Zoglauer [1996]) together with increasing evidence for B essentiality in organisms without cell walls, including yeast growth (Bennett et al., 1999) or animal embryo development (Rowe and Eckhert, 1999; Lanoue et al., 2000), support a B function beyond cell envelopes.

Figure 1   Heterocysts are specialized cells present in filaments of many species of cyanobacteria grown in the absence of a combined nitrogen source. These cells are capable of aerobically fixing dinitrogen because they maintain the reducing environment required for cyanobacterial nitrogenase activity.

Figure 2   Transmission electron micrographs of Anabaena PCC7119 heterocysts. (A) Control cultures. Arrows indicate heterocyst envelope against to diffusion of oxygen. (B) Heterocyst from cultures grown without boron for 48h, showing disorganization of heterocyst envelope leading to death of the culture.

Figure 3   Scanning electron micrographs of Frankia BCU 110501 demonstrated that the stability of filaments and vesicle envelopes was impaired in absence of boron and, hence, nitrogenase occurrence and nitrogen fixation were also absent. (A) Filaments of Frankia BCU 110501 grown in the presence of boron. (B) Filaments of Frankia BCU 110501 grown in the absence of boron. (C) Vesicles of Frankia BCU 110501 grown in the presence of boron. (D) Vesicles of Frankia BCU 110501 grown in the absence of boron.

Figure 4   Boron deficiency causes many anatomical, physiological, and biochemical changes in plants. (A) Monocotyledons species have very low boron requirements and hardly produce deficiency symptoms during vegetative growth. However the presence of boron is necessary for reproductive growth and high seed yield. Wheat, control and B-deficient conditions. (B) In sugar-beet with severe boron deficiency the young leaves are brown and die, subsequently microbial infections are common in roots. (C) Legumes, under nitrogen-fixing conditions, are very sensitive to deprivation of boron. Pea under control and B-deficient conditions. (D) The narrow concentration range between boron deficiency and toxicity requires special care in the application of boron fertilizers. Pictured is sugar-beet, one of the more tolerant species, in the presence of B, demonstrating the results of B-toxicity when treated with 30 ppm boric acid in culture solution.

The capacity of boric acid and borate to react with hydroxyl groups is considered the key for understanding boron functions (Bolaños et al., 2004a) (Figure 5), and research is now focused on discovery of B-complexes in biological systems. Although analytical instruments and procedures have been improved, the low B concentrations of cell walls make it a difficult challenge. Further progress depends on new methodology with greater analytical capability, and discovery of biological models enriched in B-ligands, to induce B deficiency. The development of markers that bind cis-diols like boronic acids (Bassil et al., 2004) may be helpful for mapping borate-binding sites in cells. Improving techniques to separate the two stable isotopes, 10B and 11B, would be extremely useful methodologies for in vivo detection of borate diester complexes (Thellier et al., 2001; Bishop et al., 2004). Nevertheless, several non-structural molecules able to bind B have been discovered during the last decade. Hu et al. (1997) isolated soluble boron-sorbitol complexes that helped resolve the problem of the phloem mobility of B. Cheng et al. (2002) discovered the B autoinducer, AI-2, a B-containing bacterial signal molecule that induces bacterial quorum sensing by interacting with a proper membrane receptor. Moreover, although only demonstrated to form in vitro, B-adenylate (Ralston and Hunt, 2001) or B-pentose (Ricardo et al., 2004) complexes support the possibility that B may be involved in regulatory or signaling processes, which might encourage boronists to look for other B functions not strictly related with cell walls or envelopes. The fact that most plant B is required for developing tissues rather than for mature organs (Raven, 1980), and that Sommer and Sorokin (1928) described long ago that the first effects of B deficiency appear in meristems, have to be considered as supporting the hypothesis for a role of B in signaling mechanisms during organogenesis.

Figure 5   Chemical structures containing boron. (A) At near-neutral pH, as found in most biological fluids, boron exists primarily as boric acid. At higher pH, boric acid accepts hydroxyl ions from water, thus forming a tetrahedral borate anion. (B) Different compounds containing cis-hidroxyl groups bind with the borate anion. The diversity of roles played by boron might indicate that either it is involved in numerous processes or that its deficiency has a pleiotropic effect. Based on results from the literature, it is likely that the main reason for boron essentiality is the stabilization of molecules with cis-diol groups making them effective, irrespective of their function. It is possible that new roles for boron, based on its special chemistry may appear.

Study of B-complexes implicated in cell-to-cell signaling requires the development of improved technologies, however there are interesting processes in organogenesis affected by B-deficiency that are excellent model systems. One example is the symbiotic interaction between leguminous plants and rhizobia that trigger development of root nodules with specific processes of organogenesis highly regulated by molecular plant-bacteria interactions (Stougaard, 2000).

Throughout each step of root nodule development in leguminous plants, an intense cell wall remodeling takes place (Brewin, 2004), therefore nodules are excellent organs for investigating structural roles of B. Typical symptoms of B-deficiency in root nodules are enlargement and irregular shapes (Figures 6 and 7). Several components of B-deficient nodule cell walls are abnormally assembled, leading to aberrant walls. Bean nodules devoid of B have walls without covalently bound hydroxyproline-/proline-rich glycoproteins (Bonilla et al., 1997a). Assembly of a protein similar to the early extensin-like (Kieliszewski and Lamport, 1994) nodule specific protein (nodulin; ENOD2 gene) is affected by the lack of B and this glycoprotein is absent in walls of the nodule parenchyma cells. Changes in the contents of the cell wall pectin polygalacturonan either as O-methyl esterified or unesterified molecules have also been found (Bonilla et al., 1997b). In nodules of Pisum sativum, abnormal distribution of polygalacturonan and rhamnogalacturonan II has been found under B deficiency (Redondo-Nieto et al., 2003).

Figure 6   Boron deficiency in alfalfa (Medicago sativa) and pea (Pisum sativum L.) causes a decrease in growth of shoots and roots, number of nodules, and alteration in nodule development, leading to an inhibition of nitrogen fixation. (A–E) Effects of boron deficiency on shoot, root and nodule development in alfalfa and pea. (F–H) Three weeks post-inoculation with Rhizobium leguminosarum. In C, E and H, there are some higher magnifications of +B and –B nodules that illustrate differences in development caused by boron deficiency. Nodules developed in the absence of boron are smaller in size and weight than nodules grown with boron. Most nodules from boron-deficient plants appear pale compared to bigger, pink control nodules. This reflects the absence of the oxygen carrier, leghmoglobin, in boron-deficient nodules.

Figure 7   Bean plants grown under boron deprivation show a significant reduction in growth compared with control plants. Both nodule number and fresh weight (50% less after two weeks of treatment) were reduced in B-deficient plants.

Nodules are excellent models for the study of regulation of cell differentiation, especially in inderminate nodules, where cells at different stages of differentiation coexist. Following infection (Figures 8 and 9), endophytic rhizobia are engulfed by plasma membrane (the peribacteroid membrane, PBM) forming an organelle-like compartment, termed the symbiosome. These bacteria, now called bacteroids, proliferate and eventually develop the capacity for nitrogen fixation (Brewin, 1991). Components of the PBM glycocalyx rich in cis-diol groups could be candidates for borate cross-linking. Symbiosome maturation involves gradual differentiation of the PBM and targeting of proteins to the peribacteroid fluid (PBF), involving several plant and bacterial glycoconjugates (Kannenberg and Brewin, 1994) able of cross-linking with borate. Importantly, some of these proteins have been shown to physically associate in vitro (Bolaños et al., 2004b). All of these events in nodule development are affected by B deficiency, therefore the hypothesis of a secondary B effect derived from cell wall perturbation seems to be insufficient (Figure 10). Therefore, characterization of glycolipids and glycoproteins (either secreted or associated with membranes) that are developmentally regulated is important for understanding the symbiotic interaction. Further research should assist in elucidating specific roles of B in cell signaling associated with membrane function and its involvement with secreted glycoproteins. In the last few years, we have discovered that the stability and function of some glycoproteins involved in symbiosome maturation depend on the presence of borate.

Figure 8   Fluorescence microscopy of nodule development in pea plants inoculated with R. leguminosarum 3841 carrying the plasmid pHC60 that constitutively expresses green fluorescenst protein (GFP). Shown are the effects of the boron deficiency on Rhizobium-legume signaling and preinfection events. (A) Curled root hairs (arrows) of pea plants grown with boron, three days after inoculation with Rhizobium leguminosarum 3841. (B) B-deficient pea plants without root hair curling. (C) Infection thread (green fluorescence) reaching the base of the root hair in plants grown in the presence of B. (D) Aborted infection thread (green fluorescence) inside the root hair of a B-deficient plant.

Figure 9   Fluorescence microscopy of nodule development in pea plants inoculated with GFP-expressing R. leguminosarum 3841 cells, showing how boron deficiency inhibited bacterial invasion and proliferation inside the host cells of nodule.

Figure 10   Light micrographs of semi-thin longitudinal sections of three-week-old pea nodules (A, C) and two-week-old bean nodules (B, D) grown in the presence and absence of boron. (A) Control nodule of pea stained with toluidine blue. Cortex and infected zone are well differentiated. (B) Control nodules of bean showing a central tissue with uninfected and infected cells and very few, small intercellular spaces. (C) In boron-deficient conditions, nodule structure is highly disorganized and nodule tissues are not easily distinguishable. Cells are irregularly shaped and infection threads are not completely developed. (D) B-deficient nodules in the central tissue are smaller in size and have irregular shapes (c, cortex; v, vascular bundle; ci, infected cells).

In B deficiency, glycoproteins, like the lectin-line PsNLEC 1 in the PBF, are not correctly glycosylated, suggesting that B is involved either in the glycosylation process or in the stability of the glycosyl-moiety of PsNLEC 1 (Bolaños et al., 2001). Moreover, when B-deficiency occurs, PsNLEC 1 glycoproteins are not secreted into the PBF compartment, leading to aberrant symbiosomes (Figure 11). Other glycoproteins react with anti-RGII antiserum (and therefore possible borate-ligands) that appear associated with the glycocalyx of the PBM of immature symbiosomes and that progressively disappear during symbiosome differentiation (Redondo-Nieto et al., 2007). The stability of these RGII-glycoproteins on the PBM apparently depends on the presence of B, since they were never detected in B-deficient nodules. PsNLEC secretion into the PBF fails in the absence of B, therefore it is likely that the stability of these PBM glycoproteins through interaction with borate is needed for the correct targeting of vesicles during symbiosome development. In addition, B may be needed for stabilization of the interaction of PBM-PsNLEC 1 with the bacterial cell surface that drives bacteroid differentiation. Supporting this hypothesis, RGII-glycoproteins were also found to be associated with plasma membranes of uninfected Pisum sativum root cells, but never detected in B-deficient cells. They were also found in vesicles loaded with pectin polysaccharides during periods when cell growth failed. Therefore, RGII-glycoprotein occurrence could be part of a signal involved in cell to cell communication (either involving plant or plant-bacteria signaling) during growth and differentiation events.

Figure 11   Transmission electron micrographs of pea nodules in the presence (A) and absence (B) of boron. (A) In the presence of boron, bacteroids appear surrounded by a peribacteroid membrane. (B) In B-deficient nodules, the membrane peribacteroid is degraded and only ghost membranes are visible.

B-deficiency leads to development of “tumor-like” undifferentiated root nodules (see Figure 10) and interestingly some studies on animal embryonic development point to a similar role of B during organogenesis in zebrafish (Rowe and Eckhert 1999). Development of zebrafish jaws, pectoral fins, and olfactory organs is preceded by an intense de novo synthesis of membrane-associated glycans (Laughlin et al., 2008). Overall, studies in plants and animals during the last decade can be fitted into a model for understanding B functions based on a role of borate as a stabilizer of glycans involved in cell-to-cell interactions during regulation of development processes.

Finally, it is interesting that in plants with poor B mobility, B deficiency results in necrosis of meristematic tissues, leading to loss of apical dominance, a symptom similar to that of calcium (Ca) deficiency (Smith, 1944; Berry, 2006). Although the traditional functions of Ca in plants are also related to cell wall structure, and membrane structure and function, recent reviews have focused on cytosolic free Ca as one of the most important messengers involved in signal-response coupling (Rudd and Franklin-Tong 2001; Sanders et al., 2002). Our previous data (Torrecilla et al., 2000, 2001, 2004a, and 2004b) suggest that Ca may be involved in early signaling in response to temperature shocks, salinity and osmotic stress, and heterocyst differentiation in cyanobacteria. Moreover, studies on B-Ca interaction in cyanobacteria (Bolaños et al. 1993) and legume nodules clearly demonstrated that both nutrients are required for the maintenance of envelopes in heterocyst (Bonilla et al., 1995) and nodule cell walls (Redondo-Nieto et al., 2003). The B-Ca interaction has also been described for the development of legume-rhizobia interaction and nodule organogenesis (reviewed by Bonilla and Bolaños, 2004). Furthermore, analysis of gene expression during nodule development in Medicago truncatula showed that expression of more than 60% of the analyzed genes was affected by B deficiency, and, in some cases, a supplement of Ca could reverse gene expression to a normal level (Redondo-Nieto et al., 2002). Therefore, it is possible that those genes are regulated by a B-Ca ratio or that B (maybe through its interaction with membrane-associated glycans, as described above) influences entry or liberation of Ca to the cytosol, as seems to occur during the inhibition of cell proliferation of some human cancer lines by application of boric acid (Eckhert et al., 2007). Investigation of the participation of B in the regulation of these genes will also shed new light on the role of B in signal transduction.

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