KINETICS OF INTEGRATION OF XYLOGLUCAN INTO THE WALLS OF SUSPENSION-CULTURED ROSE CELLS

To study the kinetics of synthesis, wall-binding and degradation of xyloglucan, we incubated suspension-cultured rose cells for 0.5-24 h in L-[1-H-3]arabinose. > 95% of the [H-3]arabinose was taken up within 2 h. UDP-Pentoses were maximally labelled within 0.5 h and had lost most of their H-3 by 2 h after the addition of [H-3]arabinose. Therefore, the 24 h experiment resembled a pulse-chase regime. The [H-3]xyloglucan formed was fractionated into four cellular pools [detergent-extractable (interpreted as cytoplasmic), and guanidinium thiocyanate-, 0.6 M NaOH- and 6.0 M NaOH-extractable (interpreted as progressively more firmly wall-bound)]; soluble extracellular xyloglucan was collected as a fifth pool. All five pools of xyloglucan had started accumulating H-3 at their respective maximal rates by approximately 0.5 h, indicating that the combined time for uptake of [H-3]arabinose, its conversion to UDP-[H-3]xylose, polysaccharide synthesis, transit through the Golgi vesicular system and tight wall-binding was < 0.5 h. Net degradation of total [H-3]xyloglucan to low-M(r) products appeared to be slight (approximately 1.2 % h-1 between 12-24 h). Cytoplasmic and weakly wall-bound xyloglucan accumulated H-3 at too low a rate, too late, and too stably for the major portion of these fractions to be precursors of the major portion of the firmly wall-bound xyloglucan. However, the firmly wall-bound fraction accumulated H-3 biphasically-50% in the first 2 h and 50% in the next 10 h (cf. for total [H-3]xyloglucan, 72% in the first 2 h and 28% in the next 10 h). Correspondingly, the cytoplasmic and weakly wall-bound fractions lost H-3 after depletion of the UDP-[H-3]pentose pool (from approximately 1.5 and 2 h onwards; half-lives approximately 2 h and approximately 10 h, respectively). These facts indicate that the major population of xyloglucan molecules underwent tight wall-binding very soon after secretion, spending little time in the cytoplasm or in the weakly wall-bound fraction, but that a second population became tightly wall-bound more slowly by gradual transfer from other fractions.