BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF CUTIN SYNTHASE 2 FROM TOMATO (SOLANUM LYCOPERSICUM)

Cutin synthase-like (CUS) proteins comprise a family of GDSL esterase/lipases that catalyze the polymerization of cutin, a major component of plant cuticles. The best characterized example, SLCUS1 from tomato (Solanum lycopersicum), has been shown to polymerize extracellular cutin during fruit expansion and some aspects of its enzymatic activity in vitro have been described. However, fruit from the slcus1 mutant, which does not express SLCUS1, still accumulate a thin layer of polymeric cutin, indicating there are other enzymes or mechanisms that contribute to cutin polymerization in tomato fruit. Here it is reported that SLCUS2, an ortholog of SLCUS1, is also expressed in fruit, albeit to a lesser degree than SLCUS1, and is a likely candidate for contribute to the formation of the polymeric cutin detected in slcus1 fruit. SLCUS2 is predominantly expressed earlier in fruit development than SLCUS1, coincident with cutin deposition, as well as in floral tissues. SLCUS2 was expressed in a heterologous system and mass spectrometric analysis of the products of the enzyme indicated that it catalyzes the polymerization of cutin in vitro. No differences in the products of SLCUS2 and SLCUS1 could be detected using the techniques in this study. Furthermore, no differences in the products upon co-incubation of SLCUS1 and SLCUS2 could be detected. Michaelis-Menten kinetics were determined for SLCUS2 and were found to have comparable kinetics to SLCUS1, with a slower turnover rate. To elucidate the role of the enzyme in planta, null SLCUS2 mutant lines were generated using CRISPR/Cas9 vectors which resulted in a truncated protein. However, no significant changes in polymeric cutin levels, distribution or composition were observed in either the fruits or flowers. While SLCUS2 is able to polymerize cutin, the role of the enzyme in planta could not be determined in this study. Further work may result in determine the in planta role of SLCUS2 including generating double mutants with slcus1 and the use of techniques which can determine architectural differences in the cutin polymer of slcus2 fruits