Identification of an Arabidopsis feruloyl-coenzyme A transferase required for suberin synthesis

Abstract

All plants produce suberin, a lipophilic barrier of the cell wall that controls water and solute fluxes and restricts pathogen infection. It is often described as a heteropolymer comprised of polyaliphatic and polyaromatic domains. Major monomers include omega-hydroxy and alpha,omega-dicarboxylic fatty acids, glycerol, and ferulate. No genes have yet been identified for the aromatic suberin pathway. Here we demonstrate that Arabidopsis (Arabidopsis thaliana) gene AT5G41040, a member of the BAHD family of acyltransferases, is essential for incorporation of ferulate into suberin. In Arabidopsis plants transformed with the AT5G41040 promoter:YFP fusion, reporter expression is localized to cell layers undergoing suberization. Knockout mutants of AT5G41040 show almost complete elimination of suberin-associated ester-linked ferulate. However, the classic lamellar structure of suberin in root periderm of at5g41040 is not disrupted. The reduction in ferulate in at5g41040-knockout seeds is associated with an approximate stoichiometric decrease in aliphatic monomers containing omega-hydroxyl groups. Recombinant AT5G41040p catalyzed acyl transfer from feruloyl-coenzyme A to omega-hydroxyfatty acids and fatty alcohols, demonstrating that the gene encodes a feruloyl transferase. CYP86B1, a cytochrome P450 monooxygenase gene whose transcript levels correlate with AT5G41040 expression, was also investigated. Knockouts and overexpression confirmed CYP86B1 as an oxidase required for the biosynthesis of very-long-chain saturated alpha,omega-bifunctional aliphatic monomers in suberin. The seed suberin composition of cyp86b1 knockout was surprisingly dominated by unsubstituted fatty acids that are incapable of polymeric linkages. Together, these results challenge our current view of suberin structure by questioning both the function of ester-linked ferulate as an essential component and the existence of an extended aliphatic polyester.

Figure 1.

Analysis of ASFT promoter activity in Arabidopsis developing seeds and roots. Living transgenic seeds and roots from plants transformed with Pro_Asft∷eYFP were analyzed by CLSM. A and B, Confocal sections of transgenic seeds entering desiccation stage show YFP expression in the outer integument 1 (oi1) and the chalazal region of the seed coat. C, Magnification of the seed section shown in B. The YFP expression is beneath the columellae of the mucilage cells (oi2). d and E, Extended focus fluorescence images compiled from 26 (D) and 12 (E) optical sections of 2-week-old transgenic roots. YFP expression is localized to the endodermis. F, Extended focus fluorescence image compiled from 11 optical sections of a 5-week-old transgenic root showing YFP fluorescence in peridermal cells. Red fluorescence in the embryo corresponds to chlorophyll; red fluorescence in seed coat columellae and root cell walls is produced by propidium iodide dye. Scale bar = 100 μm (A and D), 50 μm (B), 20 μm (C, E, and F). en, Endodermis; ep, epidermis; pe, peridermis.

Figure 2.

Comparison of lipid polyester monomer composition for wild-type (WT) and asft seeds. A, Individual monomers. Means of triplicate determinations and sd are indicated. Asterisks denote a significant difference from wild type (t test, two-sided P < 0.05). B, Total monomer amounts, with monomers grouped in four classes.

Figure 3.

Root wax composition of wild-type (WT) and asft mutants. Composition of chloroform-extracted root waxes, expressed as mass % respect to total monomer amounts for each line. Means of triplicate determinations and sd are indicated. FAs, Free fatty acids; MAGs, monoacylglycerols; PAs, primary alcohols.

Figure 4.

Transmission electron micrographs of Arabidopsis roots in the later stages of secondary thickening. The fine structure of suberin in the asft-1 knockout mutant (B) is similar to the wild type (A). Samples were treated with hydrogen peroxide before the staining procedure (Heumann, 1990). Scale bar = 100 nm (A), 50 nm (B). CW, Cell wall; PC, peridermal cell; S, suberin.

Figure 5.

Formation of hexadecyl ferulate ester by the recombinant ASFT enzyme in cell-free extracts. Recombinant ASFT protein was incubated with ferulate, ATP, CoASH, recombinant 4-coumarate ligase, and 1-hexadecanol. The GC-MS chromatogram of the extracted product shows a peak at 38.5 min that is absent if any of the above assay components are not present (blank). Insets: EI mass spectrum and molecular structure deduced from the fragmentation pattern of hexadecyl ferulate trimethylsilyl ether. Diagnostic ions: m/z 490, molecular ion; 266, [TMSi-ferulyloxy + H]⁺; 265, [TMSi-ferulyloxy]⁺; 249, [TMSi-feruloyl]⁺.

Figure 6.

Comparison of lipid polyester monomer composition for wild-type and cyp86b1 (at5g23190) seeds. A, Individual monomers. Means of triplicate determinations and sd are indicated. Asterisks denote a significant difference from wild type (WT; t test, two-sided, P < 0.05). B, Total monomer amounts, with monomers grouped into five classes. The cutin designation comprises 1,18-octadecadiene dioate and 9,10-,18-trihydroxyoctadecenoate, which are found in the cutin layers of the seed coat inner integument and the embryo, respectively. The other four classes comprise monomers from the suberized outer integument of the seed coat.

Figure 7.

Changes in cutin content and composition in the stems of the single and coexpressors of GPAT5 and CYP86B1. Means of three experiments and se are shown (8-week-old plants). WT, Wild type.