Abstract
While suberin is an insoluble heteropolymer, a number of soluble lipids can be extracted by rapid chloroform dipping of roots. These extracts include esters of saturated long-chain primary alcohols and hydroxycinnamic acids. Such fatty alcohols and hydroxycinnamic acids are also present in suberin. We demonstrate that alkyl coumarates and caffeates, which are the major components of Arabidopsis (Arabidopsis thaliana) root waxes, are present primarily in taproots. Previously we identified ALIPHATIC SUBERIN FERULOYL TRANSFERASE (At5g41040), a HXXXD-type acyltransferase (BAHD family), responsible for incorporation of ferulate into aliphatic suberin of Arabidopsis. However, aliphatic suberin feruloyl transferase mutants were unaffected in alkyl hydroxycinnamate ester root wax composition. Here we identify a closely related gene, At5g63560, responsible for the synthesis of a subset of alkyl hydroxycinnamate esters, the alkyl caffeates. Transgenic plants harboring P(At5g63560)::YFP fusions showed transcriptional activity in suberized tissues. Knockout mutants of At5g63560 were severely reduced in their alkyl caffeate but not alkyl coumarate content. Recombinant At5g63560p had greater acyltransferase activity when presented with caffeoyl-Coenzyme A (CoA) substrate, thus we have named this acyltransferase FATTY ALCOHOL:CAFFEOYL-CoA CAFFEOYL TRANSFERASE. Stress experiments revealed elevated alkyl coumarate content in root waxes of NaCl-treated wild-type and fatty alcohol:caffeoyl-CoA caffeoyl transferase plants. We further demonstrate that FATTY ACYL-CoA REDUCTASEs (FARs) FAR5 (At3g44550), FAR4 (At3g44540), and FAR1 (At5g22500) are required for the synthesis of C18, C20, and C22 alkyl hydroxycinnamates, respectively. Collectively, these results suggest that multiple acyltransferases are utilized for the synthesis of alkyl hydroxycinnamate esters of Arabidopsis root waxes and that FAR1/4/5 provide the fatty alcohols required for alkyl hydroxycinnamate synthesis.
Highlights
While suberin is an insoluble heteropolymer, a number of soluble lipids can be extracted by rapid chloroform dipping of roots
We further demonstrate the involvement of the FATTY ACYL-Coenzyme A (CoA) REDUCTASEs (FARs) FAR1, FAR4, and FAR5 in root wax alkyl hydroxycinnamate synthesis
The reported chain length specificity of each respective FAR was closely reflected in the alkyl hydroxycinnamate waxes of the far1, far4, and far5 mutants (Fig. 8). far5 mutants lacked all C18 alkyl hydroxycinnamates, far4 mutants had reduced levels of C20 alkyl hydroxycinnamates, and far1 mutants had reduced levels of C22 alkyl hydroxycinnamates. These results suggest that FAR1, FAR4, and FAR5 donate the alcohols necessary for alkyl hydroxycinnamate synthesis to FACT for alkyl caffeate synthesis and the yet to be identified acyltransferase(s) required for alkyl coumarate and alkyl ferulate synthesis
Summary
While suberin is an insoluble heteropolymer, a number of soluble lipids can be extracted by rapid chloroform dipping of roots These extracts include esters of saturated long-chain primary alcohols and hydroxycinnamic acids. All three are members of the BAHD family of HXXXD-type acyltransferases (St Pierre and De Luca, 2000; D’Auria, 2006; Tuominen et al, 2011) These genes are primarily involved in the formation of feruloyloxy aliphatics (i.e. ferulate linked to the v-terminus of v-hydroxy fatty acids) in suberin and cutin polymers. We have named this enzyme FATTY ALCOHOL:CAFFEOYL-CoA CAFFEOYL TRANSFERASE (FACT) It is a member of clade Va of the BAHD family of acyltransferases (St Pierre and De Luca, 2000; D’Auria, 2006; Tuominen et al, 2011) that contains ALIPHATIC SUBERIN FERULOYL TRANSFERASE (ASFT) and DEFICIENT IN CUTIN FERULATE (DCF). We describe the effect of salt stress on alkyl hydroxycinnamate composition in roots
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