13 CO 2 labelling as a tool for elucidating the mechanism of cuticle development A case of Clusia rosea.

•The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. •We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO 2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. •Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. EW differed in composition from IW. The newly synthetized wax was deposited in IW first and later in EW. Both young and mature leaves synthetized IW and EW. The faster dynamics in young leaves was due to lower wax coverage, not a faster synthesis rate. . Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. •We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of Clusia rosea. We discuss the ecophysiological significance of the new insights.