Despite of a lot of progress in the elucidation of the cutin biosynthetic pathway, little is known about the molecular mechanisms how cutin precursors are transferred from the inside of the cell to the location of polymerization in the cuticle. Several ATP-binding cassette (ABC) transporters have been shown to be involved in cuticle formation. Knockout of PEC1/AtABCG32 in Arabidopsis lead to a reduction in specific hydroxylated cutin monomers, including 10,16 diOH C16:0, suggesting its involvement in cutin precursor export. The chemical nature of the transported precursors and the direct or indirect role of ABCG32 in cutin precursor export remain to be elucidated.
For assessing the chemical nature of the transported precursors, N. benthamiana protoplasts, bearing the AtABCG32 transporter, were used in in vitro transport assays. Two radiolabel monoacylglycerols (MAGs) were used as substrate: 2-mono(C16:0) glycerol and 2-mono(10,16 diOH C16:0) glycerol. Transport assays reveal that only the hydroxylated MAG was exported by AtABCG32 but not the non-hydroxylated one.
Since the main monomer of tomato fruit cuticle is the 10,16 diOH C16:0, we used it as model for a deeper understanding of the function of ABCG32 in cutin formation. We have generated transgenic tomatoes knocked down in the two ABCG32 homologues, i.e. SlABCG32_01 and SlABCG32_02. Transmission electron microscopy revealed that fruit exocarps of SlABCG32-RNAi have a thinner cuticle, as well as alterations in the shape of epidermal cells when compare to wild type. In addition, when analyzed by GC/MS, SlABCG32-RNAi cuticles presented changes in hydroxylated cutin monomer composition consistent with what it was already observed in Arabidopsis pec1 mutant. Moreover, we found a reduction in glycerol content in SlABCG32RNAi fruit cuticles, contrary to what was observed in other tomato cuticle-deficient mutants; gpat6 and cus1. Related to these changes in cutin composition, we found specific modifications in the peak positions of band vibrations typical for aliphatic esters using Fourier Transform Infrared (FTIR-ATR) spectroscopy, suggesting changes in polyester structure.
As RNAi lines presented silencing in the two homologous genes, it is impossible to asset if both genes share the same function. Although, tomato ABCG32_02 was able to rescue partially the cutin composition in flowers of the Arabidopsis pec1 mutant.
All together, these results indicate that ABCG32 is involved in cutin formation. It seems to export hydroxylated C16 MAGs, through the plasma membrane, as suggested by the transport assays. Knocking-down putative ABCG32s in tomato results in thinner fruit cuticle, reduced monomers and glycerol composition, and changes in the polyester structure. However, to clarify the relationship between composition and structure of the cuticle, further studies of SlABCG32-RNAi are still needed.