Identification of an Arabidopsis fatty alcohol:caffeoyl-Coenzyme A acyltransferase required for the synthesis of alkyl hydroxycinnamates in root waxes

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.

Figure 1.

Comparison of waxes extracted from Arabidopsis taproots versus remaining roots. Data are presented as the mean of four biological replicates + sd. The inset graph compares total amounts of waxes extracted from taproots versus remaining roots. Alkn, Alkanes; FFAs, free fatty acids; MAGs, monoacylglycerols.

Figure 2.

Analysis of At5g63560 (FACT) promoter activity. Live tissues from plants transformed with P_At5g63560::eYFP were analyzed by CLSM. A, Extended focus image of taproot cross section (approximately 1 cm below rosette) from mature root of transgenic plants showing eYFP expression primarily in the outer circumference of the root (compiled from 33 optical sections, 2.43 μm each). B, Extended focus image of young roots (24 optical sections, 2 μm each). YFP expression is localized to the endodermis. C, Confocal sections of transgenic seeds entering desiccation stage showing YFP expression in the outer integument 1 (oi1) and the chalazal region of the seed coat. Propidium iodide was used to visualize cell walls (red fluorescence). Scale bars = 100 μm (A), 10 μm (B), and 50 μm (C). en, Endodermis; ep, epidermis; oi1, outer integument 1; oi2, outer integument 2.

Figure 3.

Comparison of waxes extracted from mature roots of fact mutant and wild-type (Col-0) Arabidopsis plants. A, Total wax components in each class per g of fresh weight. B, Chain length distribution of individual wax components as mole percent composition. Data are presented as the mean of four biological replicates + sd. Alkn, Alkane; FFAs, free fatty acids; MAGs, monoacylglycerols.

Figure 4.

Suberin monomer composition of fact mutant versus wild-type (Col-0) taproots (periderm). Inset graph shows caffeate content. Data are presented as the mean of four biological replicates + sd. PPs, Phenylpropanoids; FAs, fatty acids; ω-OH FAs, ω-hydroxy fatty acids; DCAs, dicarboxylic acids.

Figure 5.

Caffeate content of seed suberin in fact-1 versus wild-type (Col-0). Data are presented as means of triplicate determinations + sd.

Figure 6.

In vitro formation of dodecyl caffeate by recombinant FACT. A, Partial GC-MS chromatogram of FACT reaction extract showing dodecyl caffeate (cis- and trans-isomers) formed by recombinant FACT and tridecyl ferulate internal standard used for quantification. Black line represents extract from a recombinant FACT-containing reaction. The red line represents an extract from a negative control reaction containing soluble crude protein extracted from nontransgenic (WT) BL21 E. coli cells. B, EI mass spectrum of trans-dodecyl caffeate from FACT reaction extract and dodecyl caffeate molecular structure showing diagnostic ions.

Figure 7.

Substrate specificity of the recombinant FACT enzyme. Coupled cell-free assays were performed using different acyl-CoA donors. Recombinant 4CL was used to generate corresponding CoAs of different phenylpropanoids (i.e. ferulate, coumarate, or caffeate) in vitro via a preincubation step. FACT activity was measured in a second step by the addition of total protein extracts from BL21 E. coli-expressing recombinant FACT or from wild-type (nontransgenic) BL21 cells (WT Crude) and dodecan-1-ol. N.D., Not detected. Data represent the means of quadruplicate assays + sd.

Figure 8.

Root wax alkan-1-ol and alkyl hydroxycinnamate compositions from taproots of far1, far4, far5 mutants, and wild-type (Col-0) plants. Data are presented as the mean of four biological replicates + sd.

Figure 9.

Alkan-1-ol and alkyl hydroxycinnamate composition from roots of 200 mm NaCl-treated wild-type (Col-0) and fact mutant plants. The inset graph shows total wax content, in μg per g fresh weight, for all genotypes and treatments. Data are presented as the mean of four biological replicates + sd.