Remodelling cutin by transacylases in the plant epidermis: dynamic processes during growth and response to environmental stress
Based on the discovery of the only known cutin-related GDSL transacylase, cutin synthase (CD1), we hypothesized that cutin can also be re-modelled by transacylases in the same family as CD1 post-synthetically. Cutin was hypothesized to be transiently cut (acyl donor), and then re-form ester bonds with a neighbouring cutin (acyl acceptor) or a cell-wall polysaccharide (acyl acceptor) molecule, in order to allow cell expansion and reinforce cutin strength respectively. These proposed novel transacylase activities were first observed by in- and ex-situ radiochemical assays (i.e. endogenous cutin as the acyl donor; exogenous cutin fatty acid, 16-[3H]hydroxyhexadecanoic acid and xyloglucan oligomer, [3H]XXXGol were models for their polymers as the acyl acceptors) and verified by product analysis via thin-layer chromatography, size exclusion chromatography etc. Moreover, we concluded that these two reactions were catalysed by different transacylases based on their pH optima and responses to growth, as well as temperature change. To test whether CD1 was one of the enzymes involved, we heterologously expressed, purified and assayed it in vitro; however, it did not catalyse either reaction. Environmental stresses were tested for effects on the two transacylase activities: cutin-to-[3H]XXXGol transacylase activity was increased in the first 4 days at 4°C and then decreased; but the cutin-to- [3H]HHA transacylase activity was decreased from the first day. It may suggest that the cutin-to-[3H]XXXGol transacylase activity was involved in resisting short-term cold. Light and humidity did not affect either of the transacylase activities. In conclusion, two novel transacylase activities (renamed as cutin-to-HHA and cutin-to-XGO transacylase activities) were found and their biological significances were studied, providing a breakthrough in cutin biochemistry studies.