Functional Characterization of a Hydroxyacid/Alcohol Hydroxycinnamoyl Transferase Produced by the Liverwort Marchantia emarginata

Abstract

The aerial organs of most terrestrial plants are covered by a hydrophobic protective cuticle. The main constituent of the cuticle is the lipid polyester cutin, which is composed of aliphatic and aromatic domains. The aliphatic component is a polyester between fatty acid/alcohol and hydroxycinnamoyl acid. The BAHD/HxxxD family enzymes are central to the synthesis of these polyesters. The nature of this class of enzymes in bryophytes has not been explored to date. Here, a gene encoding a fatty ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (HFT) has been isolated from the liverwort Marchantia emarginata and has been functionally characterized. Experiments based on recombinant protein showed that the enzyme uses ω-hydroxy fatty acids or primary alcohols as its acyl acceptor and various hydroxycinnamoyl-CoAs-preferentially feruloyl-CoA and caffeoyl-CoA-as acyl donors at least in vitro. The transient expression of a MeHFT-GFP fusion transgene in the Nicotiana benthamiana leaf demonstrated that MeHFT is directed to the cytoplasm, suggesting that the feruloylation of cutin monomers takes place there.

Keywords: BAHD; acyltransferase; bryophytes; cutin; ferulate esters.

Figure 1

Sequence alignment of MeHFT with PtFHT from P. trichocarpa (JX515962), StFHT from S. tuberosum (ACS70946), AtASFT (AT5G41040), AtDCF (AT3G48720) and AtFACT (AT5G63560) from A. thaliana. The conserved HxxxD and DFGWG motifs are indicated by red boxing.

Figure 2

A phylogenetic analysis of MeHFT. The Uniprot accession numbers of the proteins included in the analysis are: Populus trichocarpa FHT (JX515962), Solanum tuberosum FHT (ACS70946), A. thaliana ASFT (AT5G41040), Dianthus caryophyllus HCBT (CAB06430), A. thaliana DCF (AT3G48720), A. thaliana FACT (AT5G63560), N. tabacum HQT (CAE46932), Avena sativa HHT (BAC78633), A. thaliana HCT (AT5G48930), N. tabacum HCT (CAD47830).

Figure 3

SDS-PAGE separation of recombinant MeHFT. Lane M: molecular mass standards. Lane 1: culture medium from E coli cells harboring an empty pET32a vector control; lane 2: proteins purified from the culture medium used in lane 1; lane 3: culture medium from E coli cells harboring pET32a-MeHFT; lane 4: proteins purified from the culture medium used in lane 3.

Figure 4

In vitro activity of MeHFT recombinant enzymes. (A) HPLC separation of the reaction products of recombinant MeHFT (the solid blue line) or the negative control (empty vector harboring cells) (the red dashed line) provided with feruloy-CoA and 1-dodecanol; (B) The MS/MS spectrum of the alky ferulate product from (A).

Figure 5

Expression of the p35S::MeHFT-GFP transgene in a transiently transformed N. benthamiana leaf reveals the localization of MeHFT to the cytoplasm. The GFP signal appears green and the chlorophyll auto-fluorescence signal red.