Synthesis of monomers for the biochemical investigation of plant polyesters

Ignacio Martinez San Segundo

Research output: Book/ReportPh.D. thesis

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Cutin and suberin are some of the most abundant polyesters on Earth, as they are found in all land plants. Both polymers form barriers that protect plants from desiccation and other environmental stresses and present a similar aliphatic composition, in which hydroxy fatty acids, α,ω-dicarboxylic fatty acids and glycerol are the main components. The mechanism of the biosynthesis of cutin and suberin has remained elusive for decades. However, a shred of light was recently cast upon the subject when the CUS family of enzymes was discovered. The essential role of glycerol-3-phosphate acyltransferases (GPAT) in cutin and suberin biosynthesis had previously been established. GPATs which selectively produce 2-monoacylglyceryl esters (2-MAGs) of fatty acid monomers. The most abundant monomer in tomato fruit (Solanum lycopersicum) cutin is 10,16-dihydroxyhexadecanoic acid. Its correspondent 2-MAG, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol (2-MHG) was found in the soluble surface lipids of fruits carrying the cutin deficient 1 (cd1) mutation, which reduces (>95%) the amount of polymerized cutin severely. The cd1 mutation suppresses the formation of the extracellular CUS1. Furthermore, CUS1 have shown in vitro activity towards the polymerization of 2-MHG, further strengthening the hypothesis that cutin biosynthesis is extracellular and occurs through a series of transesterification reactions releasing glycerol. However, some questions remain unanswered, such as the selectivity of CUS1 towards the 2-MAGs of other common cutin or suberin monomers, and whether CUS1 is capable of accepting fatty acid esters other than 2-MAGs as substrates. In the present work, three projects involving the synthesis of cutin and suberin monomer derivatives and their subsequent in vitro oligomerization catalyzed by CUS1 are presented. In the first project, both deuterium and tritium-labelled 2-MHG were successfully synthesized. The deuterium-labelled molecule was used in oligomerization assays together with 9-hydroxy 2-MHG to study the specificity of CUS1 towards the position of the mid-chain hydroxy group. CUS1 showed equal activity towards both monomers, suggesting that CUS1 is likely to participate in the incorporation of both monomers in cutin. Tritium-labelled 2-MHG could potentially be used to monitor transport and location of the monomer in planta. The second project consisted on the synthesis and enzymatic oligomerization of 2-MAG derivatives of three fatty acids commonly found in suberin: behenic acid, ω-hydroxy oleic acid and octadecanedioic acid. The three compounds were successfully synthesized and used as CUS1 substrates in enzymatic assays. CUS1 showed a very limited activity towards the octadecanedioic and ω-hydroxy oleic acid derivatives, and no activity towards the behenic acid 2-MAG. These results suggest that enzymes from the CUS or another similar family could be involved in the biosynthesis of suberin. The third project involved the synthesis of four 2-MHG derivatives, in which the sn-2 glyceryl moiety was substituted by other small alcohols. The subsequent enzymatic assays showed that CUS1 present activity towards fatty acid esters different than 2-MAGs including the product of the migration of the glyceryl moiety to one of the primary hydroxyls, 1-MHG.
Original languageEnglish
PublisherTechnical University of Denmark
Number of pages128
Publication statusPublished - 2018

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