Use of phenylboronic acids to investigate boron function in plants. Possible role of boron in transvacuolar cytoplasmic strands and cell-to-wall adhesion

Abstract

The only defined physiological role of boron in plants is as a cross-linking molecule involving reversible covalent bonds with cis-diols on either side of borate. Boronic acids, which form the same reversible bonds with cis-diols but cannot cross-link two molecules, were used to selectively disrupt boron function in plants. In cultured tobacco (Nicotiana tabacum cv BY-2) cells, addition of boronic acids caused the disruption of cytoplasmic strands and cell-to-cell wall detachment. The effect of the boronic acids could be relieved by the addition of boron-complexing sugars and was proportional to the boronic acid-binding strength of the sugar. Experiments with germinating petunia (Petunia hybrida) pollen and boronate-affinity chromatography showed that boronic acids and boron compete for the same binding sites. The boronic acids appear to specifically disrupt or prevent borate-dependent cross-links important for the structural integrity of the cell, including the organization of transvacuolar cytoplasmic strands. Boron likely plays a structural role in the plant cytoskeleton. We conclude that boronic acids can be used to rapidly and reversibly induce boron deficiency-like responses and therefore are useful tools for investigating boron function in plants.

Figure 1.

Application of boronic acids causes disassembly of transvaculoar cytoplasmic strands and collapse of the nucleus in cultured tobacco cells. Cells were treated for 2 h with 0.1 mm boron (A and B), 3-NBA (C and D), 3-MBA (E and F), MeBA (G and H). A, C, E, and G, Differential interference contrast images of the same cells (shown in B, D, F, and H), which are fluorescent and result from esterase cleavage of FDA and are viewed using epifluorescence illumination. Arrows indicate particles that stain with DiOC₆(3) (F). V, Vacuoles; N, nuclei; n, nucleoli; CS, cytoplasmic strand. Scale bars, 50 μm.

Figure 2.

Treatment of cultured tobacco cells with boronic acids results in cellular disruption. Cells were treated by adding 0.25 mm of the indicated boronic acids for 4 h before scoring the degree of disruption. Same letters above the stacked bars indicate that disruption is not significantly different between treatments as determined by Tukey's studentized range test (α = 0.05) using SAS (SAS Institute, Cary, NC). Data are from one representative experiment (repeated at least three times) having three replicates of each treatment.

Figure 3.

Boronic acids cause plasma membrane-to-cell wall detachment in cultured tobacco cells. Cells were treated with 0.5 mm boron (A), 3-NBA (B), 3-MBA (C), or 0.1 mm 3-NBA (D and E) for 1 h. Fluorescent images result from Nile red staining of the membrane, viewed using epifluorescence illumination (A–C). For reference, light images of cells pointed by arrows (A–C) are included as insets. D, Light image of nonplasmolyzed cells; E, cells from D that are plasmolyzed. Plasmolysis was induced by adding a 0.3 m CaCl₂ solution to one corner of the coverslip while blotting the other end with filter paper. cw, Cell wall; pm-cw, plasma membrane-cell wall contact; pr, protoplast. Scale bars, 50 μm.

Figure 4.

Sugars that can bind to boronic acids reduce 3-MBA-induced cellular disruption of cultured tobacco cells. Cells were treated with 0.5 mm 3-MBA for 4 h, except in −MBA where no 3-MBA was added. +MBA indicates treatment with 3-MBA and no sugar added. Fru, Glc, glycerol (GLY), sorbitol (SOR), or Suc was added at 100 mm together with 3-MBA. Cells were scored for degree of disruption as described in “Materials and Methods.” Same letters above the stacked bars indicate that disruption was not significantly different between treatments (α = 0.05). Data are from one representative experiment repeated three times having three replicates of each treatment.

Figure 5.

Phenylboronic acid reduces petunia pollen germination. Pollen was considered as germinated if pollen tube length was greater than pollen grain diameter after 1 h of treatment. For each treatment, 400 to 600 pollen grains were counted. Error bars indicate the se of three independent experiments and are not visible when smaller than symbols.

Figure 6.

Boronic acids compete with boron for boron-binding sites but do not substitute for boron in petunia pollen germination. Percentage pollen germination with (A) no PBA added (▵) or 0.25 mm PBA added (▴), or (B) no MeBA added (○) or 1 mm MeBA added (•). C, Pollen germination ratio, defined as germination in presence/germination in absence of PBA (⋄) or MeBA (♦) of data in A and B. BA indicates the boronic acids PBA or MeBA. Pollen was considered as germinated if pollen tube length was greater than pollen grain diameter after 1 h of treatment. Note break in x axis. Data are the average of six experiments. Error bars indicate the se and are not visible when smaller than symbols.

Figure 7.

Phenylboronic acid affinity chromatography of HRP indicates that boronate can disrupt glycoprotein-borate ester linkages. Thirty micrograms of HRP type VI-A in an m-PBA acid affinity column was eluted with 50 mm taurine buffer containing 5 mm Mg²⁺ (pH 8.7) or taurine buffer containing 3-NAA. Arrows indicate a switch of elution buffer containing boron (A) or 3-NBA (B). SOR indicates a switch to buffer containing 50 mm sorbitol. The concentration of ionized borate in 60, 80, and 100 mm boron was 14.4, 19.2, and 24 mm, respectively. The concentration of ionized boronate in 15, 20, and 25 mm 3-NBA was 14.6, 19.4, and 24.3 mm, respectively. Error bars indicate the se of three individual experiments.

Scheme 1.