Cutin:xyloglucan transacylase (CXT) activity covalently links cutin to a plant cell-wall polysaccharide

The shoot epidermal cell wall in land-plants is associated with a polyester, cutin, which controls water loss and possibly organ expansion. Covalent bonds between cutin and its neighbouring cell-wall polysaccharides have long been proposed. However, the lack of biochemical evidence makes cutin-polysaccharide linkages largely conjectural. Here we optimised a portfolio of radiochemical assays to look for cutin-polysaccharide ester bonds in the epidermis of pea epicotyls, ice-plant leaves and tomato fruits, based on the hypothesis that a transacylase remodels cutin in a similar fashion to cutin synthase and cutin:cutin transacylase activities. Through in-situ enzyme assays and chemical degradations coupled with chromatographic analysis of the ³H-labelled products, we observed that among several wall-related oligosaccharides tested, only a xyloglucan oligosaccharide ([³H]XXXGol) could acquire ester-bonds from endogenous cutin, suggesting a cutin:xyloglucan transacylase (CXT). CXT activity was heat-labile, time-dependent, and maximal at near-neutral pH values. In-situ CXT activity peaked in nearly fully expanded tomato fruits and ice-plant leaves. CXT activity positively correlated with organ growth rate, suggesting that it contributes to epidermal integrity during rapid expansion. This study uncovers hitherto unappreciated re-structuring processes in the plant epidermis and provides a step towards the identification of CXT and its engineering for biotechnological applications.