The boron requirement and cell wall properties of growing and stationary suspension-cultured chenopodium album L. cells

Abstract

Suspension-cultured Chenopodium album L. cells are capable of continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained more enlarged and detached cells, had increased turbidity due to the rupture of a small number of cells, and contained cells with an increased cell wall pore size. These characteristics were reversed by the addition of boric acid (>/=7 &mgr;M) to the boron-deficient cells. C. album cells grown in the presence of 100 &mgr;M boric acid entered the stationary phase when they were not subcultured, and remained viable for at least 3 weeks. The transition from the growth phase to the stationary phase was accompanied by a decrease in the wall pore size. Cells grown without boric acid or with 7 &mgr;M boric acid were not able to reduce their wall pore size at the transition to the stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of wall rupture. The addition of 100 &mgr;M boric acid prevented wall rupture and the wall pore size was reduced to normal values. We conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at growth termination.

Figure 1

Changes in the dry weights of C. album cells during their growth phase and during the transition from the growth phase to the stationary phase. a, Changes in the dry weights of growing cells that were diluted with fresh medium every 2 d (curve 1). Dry weight increase of the cells in transition from the growth phase to the stationary phase (curve 2); before the transition to the stationary phase, the cells had been maintained for 60 passages in the growth phase (100 μm boric acid). The dilution of the cells with fresh medium was at time 0, and at d 2 Suc (2 g/100 mL) was added. b, Maximum dry weights of cells grown continuously with a high frequency of subcultivation (every 2 d) at different boron concentrations. Dry weights (Dry Wt.) were determined before subcultivation. The time in days is the length of time that the cells had been grown continuously at the given boron concentration. The upper x axis is the time for cells grown with 100 and 7 μm boron and the lower x axis is the time for cells grown in the absence of added boron.

Figure 2

Particle-size distribution curves of disaggregated and untreated clusters of growth-phase C. album cells. a, Particle-size distribution curves of suspension-cultured C. album cells after disaggregating treatment without boron (−B) or with 100 μm boron (+B). The particle-size distribution after disaggregating treatment is equivalent to the cell-size distribution. b, Particle-size distribution curves of untreated suspension-cultured C. album cell clusters grown with or without boron. The peak at approximately 45 μm (↓) corresponds to single cells that have separated from cell clusters during their cultivation. Particle size is equivalent to the diameter of a spherical particle and was determined with a laser particle-size analyzer.

Figure 4

Effect of boron concentrations on the cell size and the mean size limits (MSL) of the walls of C. album cells. ▾, Mean size limits ± sd (n = 10). •, Cell size (mode of the cell size distribution) of mechanically disaggregated clusters ± sd (n = 8). Cells were grown continuously in media containing different boric acid concentrations. With the exception of the control, cells were previously grown without boron and, before analysis, subcultivated at least five times in media of the given boron concentrations.

Figure 3

Particle-size distribution profiles of the polydisperse dextran-probing solutions after equilibration with denatured growth-phase cells. Denatured cells derived from cells grown in the presence of 100 μm boron (top) and in the absence of added boron (bottom) were equilibrated for 30 min with the polydisperse dextran-probing solution. The particle-size distribution profile of the dextrans was modified by their partial diffusion into the cell lumina. Untreated (or.) and treated dextran solutions were fractionated by size-exclusion chromatography on a Superdex 200 HR 10/30 column. The eluate was monitored with a polarimetric detector. The dextran concentration (c) is given in arbitrary units. The Stokes' radii of the dextrans were derived from their elution times using a calibration function (Woehlecke and Ehwald, 1995).

Figure 5

Light micrographs of clusters of stationary-phase C. album cells obtained from cultures grown with 100 or 7 μm boric acid. Before the transition to the stationary phase the cells were grown continuously in medium containing 100 μm boron (top) or 7 μm boron (bottom). Scale bar, 50 μm. Cells were photographed 9 d after their last subcultivation.

Figure 6

Effect of boron concentrations on the dry weight, FAV, turbidity, and cell size during the transition of cells from the growth to the stationary phase. Cells grown continuously for 125 passages in the absence of added boron (○), 7 μm boron (▿), or 100 μm boron (•) were not diluted with fresh medium but supplied with Suc (2%, w/v) 2 d after the last subcultivation. a, Increase in dry weight (DWt.) and FAV. b, Turbidity of the filtered medium (A₄₉₀). c, Cell size (mode of the size distribution of the mechanically disaggregated cell clusters). The data are means ± sd obtained from three independent experiments.

Figure 7

Pore-size distribution profiles of the polydisperse dextran-probing solutions after equilibration with denatured stationary cells. Denatured stationary cells derived from cells grown in medium containing 100 μm boron (top) or 7 μm boron (bottom). The pore-size distribution profile of the untreated dextran-probing solution is shown in Figure 3. c, Dextran concentration.

Figure 8

The effect of 100 μm boron on cells grown at 7 μm boron during their transition to the stationary phase. Cells were grown in medium containing 7 μm boron and at time 0 kept without dilution and allowed to reach the stationary phase. Boric acid (100 μm) was added at time 0 or at the times indicated by the arrows. Suc was added at d 2. Cell rupture was followed by measuring the turbidity (A₄₉₀) of the filtered medium. Cell size (mode of the size distribution of the mechanically disaggregated cell clusters) and the mean size limit (MSL) of the walls were measured at d 8. n.d., Not determinable; upper size limit beyond the range of analysis (>9 nm).