Biological activity of reducing-end-derivatized oligogalacturonides in tobacco tissue cultures

Abstract

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspension-cultured N. tabacum cv Xanthi cells, and (d) elicit H2O2 accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.

Figure 1

Structures of G₁₃ and the four reducing-end derivatives of G₁₃ used in this study.

Figure 2

The TCL explants from a representative tobacco morphogenesis bioassay comparing the activities of G₁₂ (top), G₁₃-B (middle), and G₁₃-T (bottom). The explants were incubated in growth medium containing the indicated oligogalacturonide concentrations, with six replicate TCL explants per concentration. The arrows indicate the concentrations at which oligogalacturonides induce a distinct change in the overall morphology of the TCL explants (the morphogenetic switch concentrations).

Figure 3

Plot of the log of G₁₂ concentration versus the average number of flowers induced per TCL explant. The data from 12 experiments are shown, and each data point represents the average number of flowers produced by the six replicate TCL explants used for each treatment in a given experiment. In the absence of G₁₂, no flowers were formed. The line represents the dose-response curve for G₁₂ as determined by least-squares analysis using log concentration.

Figure 4

Representative time courses of the extracellular alkalinization by suspension-cultured cv Samsun cells treated with the indicated concentrations of G₁₂ at time 0.

Figure 5

Effect of the DP (a) and reducing-end derivatization (b) of oligogalacturonides on their ability to induce extracellular alkalinization by suspension-cultured cv Samsun cells. The data were plotted as the alkalinization response of each sample divided by the alkalinization response induced by a saturating concentration of G₁₂ in that experiment (R/R_max). The R/R_max values for the various oligogalacturonides are indicated as follows: G₁₂, ▴; G₁₃, ▪; G₁₅, •; G₉, ♦; G₁₃-T, ⋄; G₁₃-B, □; G₁₃-O, ○; and G₁₃-R, ▵. The data for G₁₂ and G₁₃ are shown on both plots to aid in comparison. The lines represent the linear portion of the dose-response curves as determined by least-squares analysis using log concentration. Separate dose-response curves were generated using combined data from G₁₂ and G₁₃ and combined data from the four reducing-end derivatives of G₁₃ (see text).

Figure 6

Representative time courses for extracellular alkalinization by cv Samsun cells induced by the indicated concentrations of G₁₂ (closed symbols) or G₁₃-T (open symbols). More than a 30-fold higher concentration of G₁₃-T, compared with G₁₂, is required to induce an equivalent alkalinization response.

Figure 7

Representative time courses of extracellular alkalinization by cv Xanthi cells elicited by G₁₃ and of G₁₃-T. All treatments were at 8.7 μm.

Figure 8

Representative time courses of H₂O₂ accumulation in the growth medium of cv Xanthi cells elicited by the addition of G₁₃ and G₁₃-T. These measurements were carried out simultaneously using the same suspension cells as used for the measurement of extracellular alkalinization (Fig. 7). All treatments were at 8.7 μm.