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-[1-3H]arabinose. >95% of the [3H]arabinose was taken up within 2 h. UDP-Pentoses were maximally labelled within 0–5 h and had lost most of their 3H by 2 h after the addition of [3H]arabinose. Therefore, the 24 h experiment resembled a pulse-chase règime. The [3H]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 3H at their respective maximal rates by ∼0·5 h, indicating that the combined time for uptake of [3H]arabinose, its conversion to UDP-[3H]xylose, polysaccharide synthesis, transit through the Golgi vesicular system and tight wall-binding was 0⋅5 h. Net degradation of total [3H]xyloglucan to low-Mr products appeared to be slight (∼ 1·2% h−1 between 12–24 h). Cytoplasmic and weakly wall-bound xyloglucan accumulated 3H 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 wallbound fraction accumulated 3H biphasically—50% in the first 2 h and 50% in the next 10 h (cf. for total [3H]xyloglucan, 72% in the first 2 h and 28% in the next 10 h). Correspondingly, the cytoplasmic and weakly wall-bound fractions lost 3H after depletion of the UDP-[3H]pentose pool (from ∼1·5 and 2 h onwards; half-lives ∼2 h and ∼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.