Unraveling ferulate role in suberin and periderm biology by reverse genetics

Abstract

Plant cell walls are dramatically affected by suberin deposition, becoming an impermeable barrier to water and pathogens. Suberin is a complex layered heteropolymer that comprises both a poly(aliphatic) and a poly(aromatic) lignin-like domain. Current structural models for suberin attribute the crosslinking of aliphatic and aromatic domains within the typical lamellar ultrastructure of the polymer to esterified ferulate. BAHD feruloyl transferases involved in suberin biosynthesis have been recently characterized in Arabidopsis and potato (Solanum tuberosum). In defective mutants, suberin, even lacks most of the esterified ferulate, but maintains the typical lamellar ultrastructure. However, suberized tissues display increased water permeability, in spite of exhibiting a similar lipid load to wild type. Therefore, the role of ferulate in suberin needs to be reconsidered. Moreover, silencing the feruloyl transferase in potato turns the typical smooth skin of cv. Desirée into a rough scabbed skin distinctive of Russet varieties and impairs the normal skin maturation that confers resistance to skinning. Concomitantly to these changes, the skin of silenced potatoes shows an altered profile of soluble phenolics with the emergence of conjugated polyamines.

Recently published reverse genetic studies in Arabidopsis and potato identified two orthologous genes that encode a BAHD feruloyl transferase acting on aliphatics and showed that deficiency in these enzymes produces a decrease in suberin-associated ferulic acid. These results, here discussed, signify an important advance in suberin biochemistry and ultrastructure, providing a valuable new insight into the organization of the suberized tissues and their role in the control of water vapour loss.

The Lipid Barrier in Suberized Tissues

The deposition of suberin into cell walls is a physiological mechanism used by land plants to regulate apoplastic water transport and to restrict infection. Suberization takes place on the periderm of stems, roots and tubers and in a variety of other barrier layers, such as the root endodermis or the seed coat. Suberin is a complex biopolymer made of cross-linked poly(aliphatic) and poly(aromatic) domains (reviewed in refs. ^1–5). The aliphatic domain (aliphatic suberin) consists of a glycerol-based fatty acid derived polyester that, on trans-esterification, releases small amounts of p-hydroxycinnamic acid (mainly ferulic) together with aliphatic monomers and glycerol.⁶ The aromatic domain is a lignin-like polymer of oxydatively cross-linked phenolics.⁷ Generally, but not always, suberin contains a certain amount of soluble lipids or waxes embedded into the aliphatic polymer matrix.^8,9 Suberin and embedded wax are deposited within the primary cell wall to form a secondary wall that usually appears as a lamellar structure of alternating electron-dense (opaque) and electron-translucent (light) bands under the transmission electron microscope (TEM).¹⁰ Current models describing the macromolecular structure of suberin assume that the light lamellae correspond to the fatty acid polyester (aliphatic suberin) and the dense lamellae to the aromatics.^1,3 These models held that ferulic acid cross-links the aliphatic suberin with aromatics, as it may form carboxyl-ester bonds with aliphatic monomers and non-ester radical coupled bonds with phenolics. This is in agreement with partial depolymerization studies found to yield feruloyl esters of ω-hydroxyacids and primary alcohols and a small quantity of a monoferuloylglycerol.¹¹ It is well known that ferulic acid is an important component to enhance rigidity and strength in grass cell walls,^12,13 but also in dicots¹⁴ and gymnosperms.¹⁵ In these cell walls, ferulic acid is linked to glycans by ester bonds and serves as an initiation site for lignification, acting as a system for cross-linking polysaccharides and lignins.^16,17 The role of ferulic acid in suberized cell walls is, however, poorly understood and its function in cross-linking the suberin aliphatic and aromatic domains has not been demonstrated, the macromolecular structure of suberin therefore remaining still ill defined.⁵

Reverse genetic approaches allowed to demonstrate the role of a number of key genes in the biosynthetic pathway of aliphatic suberin, specifically fatty acid elongases,^18,19 ω-hydroxylases^20–22 and a glycerol acyltransferase.^21,23 Knowledge on aromatic suberin is, however, much more incomplete. Recently, the analysis of Arabidopsis knockout mutants proved the involvement of a feruloyl transferase (At5g41040) of the BAHD family in suberin biosynthesis and its deficiency associated with a worsening of the water barrier function in suberized seed coat and root tissues.^24,25 Studies using silenced potato lines confirmed that the putative potato ortholog is also involved in periderm wax biosynthesis, and allowed a further analysis of its role in establishing a proper water barrier.²⁶ Altogether, the characterization of the Arabidopsis and potato deficient mutants demonstrate the importance of suberin aromatic monomers in the water barrier and raises some questions about the role of ferulate in suberin macromolecular organization. Moreover, the analysis of potato RNAi-lines also suggests the potential involvement of feruloyl-CoA derived soluble phenolics, including conjugated polyamines, in maturation and anatomic features of the tuber periderm.

A BAHD Family Feruloyl-CoA Acyltransferase Involved in Suberin

Designated as BAHD in reference to the first four characterized enzymes—BEAT, AHCT, HCBT and DAT—this family of plant acyl-CoA dependent acyltransferases are enzymes that use CoA-thioester donors, including hydroxycinnamoyl-CoAs, in the synthesis of a wide range of natural products.^27,28 Compatible with BAHD enzymes function is the feruloylation of fatty acids, found to be relevant in suberized tissues.⁵ These tissues actually contain ferulate esters of long chain fatty acids as components of the embedded wax fraction, which may act as suberin precursors.^19,29 Moreover, feruloylation activity on fatty ω-hydroxyacids and fatty alcohols was previously detected in protein extracts from wound healing potato discs and other suberizing tissues,^30–32 although nor the proteins neither the genes responsible of this activity have been identified. The first member of the BAHD gene family to be associated with suberin synthesis was a cork oak (Quercus suber) gene isolated from a subtracted phellem (cork) library and annotated as N-hydroxycinnamoyl/benzoyl transferase (HCBT).³³ This gene was strongly upregulated in cork versus wood tissue³³ and its gene expression pattern in cork tissue was similar to that of GPAT and CYP86A1, with confirmed roles in suberin biosynthesis.³⁴

Subsequently, a role in suberin biosynthesis of the Arabidopsis putative ortholog (At5g41040) of this cork gene has been established by two independent studies. On the one side, Gou et al.²⁴ in an attempt to identify BAHD enzymes involved cell wall modification, searched for all putative members of the BAHD family in Arabidopsis and characterized the corresponding T-DNA insertion lines. Reduced levels of cell wall phenolics were detected only in a subset of these lines, a marked deficiency of suberin ferulate being observed in lines carrying a T-DNA insertion in the At5g41040 gene. In turn, Molina et al.²⁵ were able to identify At5g41040 in a screening across microarray data sets for genes showing strong correlation in their gene-expression with the suberin GPAT5 and CYP86A transcripts. Using potato (Solanum tuberosum), a model plant for suberin studies, a gene encoding a BAHD acyltransferase was obtained from a subtracted library of potato phellem tissue in our laboratory.

This potato gene, referred to as FHT for ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase, has close similarity to the cork oak (88–92%) and the Arabidopsis At5g41040 (79%) BAHD acyl transferases. An orthologous relationship among them became feasible in phylogenetic analyses and sequence alignment of the proteins.²⁶ Tissue expression pattern^24,26 and promoter activity studies²⁵ confirmed the BAHD acyltransferases as strong candidates for suberin. The acyl transferase ability of both Arabidopsis and potato enzymes towards aliphatic acceptors was in vitro demonstrated using purified proteins. The Arabidopsis enzyme shows a preference for feruloyl-CoA, but is also capable of using p-coumaroyl CoA as substrate²⁴ and is able to transfer the feruloyl group to primary alcohols of various chain lengths and to ω-hydroxyacids, concretely to the16-hydroxypalmitic and 15-hydroxypentadecanoic acids.^24,25 The potato enzyme, FHT, is able to use feruloyl-CoA as acyl donor and C16:0 ω-hydroxyacid, and primary alcohols as acyl acceptors, specifically 1-dodecanol and 1-tetradecanol.²⁶

Chemical Analysis of Feruloyl Transferase Deficient Mutants

The study of suberin from Arabidopsis and potato feruloyl transferase deficient mutants revealed that the respective enzymes control almost all the ester-linked ferulate in suberin. Transesterification analysis of the suberin fraction revealed marked changes in profile in the respective depolymerised hydrolysates. In the Arabidopsis mutant lines, ferulate and its derivatives were essentially lacking in the seed coat of and were reduced to approximately 20% of wild type levels in roots.²⁴ Concomitantly with these changes, differential modifications in the suberin aliphatic monomers were also observed. Molina et al.²⁵ detected that the amount of hydroxylated monomers (mostly C22 and C24 ω-hydroxy fatty acids) was decreased in a stechiometric proportion with ferulate, while dicarboxylic and a few fatty acids displayed increased levels. In its turn, Gou et al.²⁴ hardly observed any variation in the content of ω-hydroxyacids, but detected an increase in dicarboxylic and fatty acids. Moreover, analysis of the cell wall polysaccharide fraction in root and seed coat tissues of these Arabidopsis mutants did not reveal any reduction of ferulate in this fraction.²⁴

Studies in genetically modified potato periderm agreed with those of the Arabidopsis mutants and provided new data on the changes induced by feruloyl transferase deficiency in the wax fraction.²⁶ Compared with wild type, the suberin from FHT-RNAi tuber periderm yielded upon transesterification much lower quantities of ferulic acid (72% reduction), C18:1 ω-hydroxyacid (76% reduction), and primary alcohols (except C20), while the amounts of fatty acids and dicarboxylic acids increased (with the exception of C30 fatty acid) (Fig. 1A). In the wax fraction, ferulate esters of primary alcohols and alkanes were greatly reduced, while primary alcohols and fatty acids were increased (Fig. 1B). Thus, results in planta agree with those from in vitro studies of the enzyme activity and provide new evidence that suberin and wax biosynthetic pathways share common precursors.

Figure 1.

FHT is responsible of feruloylation of fatty acids derivatives in potato tuber periderm. (A) Aliphatic suberin monomers, which represent the 95% of the periderm lipid load, were released by transesterification from wild type and FHT-RNAi potato wax-free periderm and grouped in substance classes for representation. Note that ferulic acid is reduced in FHT downregulated periderm. (B) Wax compounds, which account only 5% of the periderm lipid load, were released after chloroform:methanol treatment from wild type and FHT-RNAi potato periderm and grouped in substance classes for representation. Note that ferulic acid esters (FE) are reduced when FHT is downregulated. The detected compounds in wax and suberin are the following: alkanes (ALKANE), primary alcohols (PA), fatty acids (FA), ferulate esters of aliphatic compounds (FE), ω-hydroxyacids (ω-HA), α,ω-diacids (α,ω-DA) and ferulic acid (FERULIC).

Ferulate is Required for the Water Barrier but Not for the Lamellar Organization of Suberin

The characterization of feruloyl transferase deficient mutants has led to important progress in the knowledge of the relationship between suberin composition, macromolecular structure and water barrier function. Staining with sudan red showed no differences of coloration in the seed surfaces between the At5g41040 mutant and wild type, thus confirming that the overall lipid load in the mutant was similar to the wild type as found in chemical analyses.²⁴ Staining with the ionic dye tetrazolium salt, however, showed a higher diffusion and an intense coloration in mutant roots and seeds, suggesting an increased permeability.²⁴ Moreover, mutant seeds, when subject to various salt stress conditions, showed a delayed germination compared to wild type.²⁴ Nonetheless, the lamellar suberin ultrastucture was not affected.²⁵

Potato periderm is a more amenable model to water permeability studies, as it can be easily isolated from the tuber using a combination of cellulase and pectinase treatments,³⁵ which makes feasible peridermal permeability measurements by a gravimetric method described by Schönherr and Lendzian³⁶ and Schreiber et al.⁹ Previous experiments using enzymatically isolated potato periderm demonstrated that wax was primarily responsible for the impermeability, as transpiration increased 33–100-fold in wax-free chloroform extracted periderms.^9,35,37 However, it is not yet clear how the wax components seal this polymer.³⁸ A contribution of suberin to periderm permeability was also deduced from measurements of tubers deficient in CYP86A33, a fatty acid ω-hydroxylase involved in the ω-oxidation of hydroxyacids and diacids.²² CYP86A33 downregulated periderm actually displays a 60% reduction in suberin load and shows a deeply altered ultrastructure (Fig. 2B), due to it lacks most of α,ω-diacid monomers and experiences a 3.5-fold increase in transpiration even if its wax load is 2.4-fold greater than in the wild type. In contrast, FHT downregulated periderm, shows a14-fold higher water permeability compared to wild type, although either lipid load or lamellar ultrastructure (Fig. 2C) are largely unaffected, evidencing that defects in permeability are mostly due to the lack of esterified ferulate in suberin and wax.²⁶

Figure 2.

Lamellar ultrastructure in potato tuber periderms genetically modified for suberin. Transmission electron micrographs of suberized cell walls of the phellem (cork) cell layer from wild-type or genetically modified potato tuber periderm. (A) Suberized cell wall from wild-type phellem showing the typical regular ultrastructure of suberin, alternating electron-dense and electron-light lamellae. (B) Suberized cell wall from periderm downregulated for a fatty acid ω-hydroxylase (CYP-86A33) required for the biosynthesis of ω-functionalized monomers, which are the main suberin monomers. The thickness of the suberin wall is reduced by a half and electron dense material forms prominent clumps (arrows) amid of the electron light material. (C) Suberized cell wall from periderm downregulated for a feruloyl transferase (FHT) required for the deposition of suberin and wax esterified ferulate. The cell wall ultrastructure is similar to that of wild type although FHT-RNAi periderm lacks most of the esterified ferulic acid, which, according to the current models of suberin, should play an important role in the regular organization of suberin. Primary wall (PW), suberized secondary wall (SW), tertiary wall (TW), phellem cell (PhC). Scale bar = 300 nm.

Current models for suberin structure attribute the regular organization in electron-light and electron-dense lamellae^1,3 to the formation of an extended aliphatic polyester based on the bifunctionality of ω-hydroxyacids and diacids and glycerol, and assign to ferulic acid a cross-linking role between the aliphatic polyester and aromatics. Moreover, based on depolymerization studies and NMR spectroscopy, the glycerol-α,ω-diacid-glycerol unit is suggested as the base for organization of the translucent lamellae, while polyaromatics will account for opaque lamellae.³ These assumptions are in conflict with the results from suberin of genetically modified plants. The loss of ferulate in feruloyl transferase-deficient periderm has no influence in the regular organization and the insoluble character of the suberin polyester. Thus, current suberin models should be redefined to better reflect current knowledge.

The Skin of FHT Deficient Tuber is a Phenocopy of Potato Russet Varieties

The periderm operates as an efficient external barrier, that protects the potato fleshy parenchyma for extended periods.³⁹ It consists of three layers: the phellem or cork layer that properly constitutes the skin of the tuber wherein suberin is deposited, the phellogen or mother layer and the phelloderm. The phellem is made of strata of tightly packed cork cells, dead at maturity, providing an efficient water barrier. Most potato varieties, such as the cv. Desirée (www.inspection.gc.ca/english/plaveg/potpom/var/desiree/desireee.shtml) used for feruloyl transferase silencing, show a smooth skin, made of 6–8 layers of cork cells, fairly resistant to cracking. Instead, in potato Russet varieties (www.inspection.gc.ca/english/plaveg/potpom/var/russetburbank/russetbe.shtml), the presence of a rough darker heavy netted skin is a natural growth trait.^40,41 In Russet varieties, the phellem is thicker with adhered crashed cork layers in the outer side,^42,43 that look like the scab lesions associated to the common scab disease (Streptomyces sp.).

The most striking feature of FHT-RNAi potatoes was that, instead of a smooth skin typical of cv. Desirée (Fig. 3A), a rough darker scabbed skin is formed, which largely resembles that of Russet varieties (Fig. 3D). Wild type and mutant plants are identical for all morphological characters except for the tuber skin.²⁶ In mutants, the whole tuber surface presents scabby lesions which become more apparent during the post-harvest period as the tubers wrinkle due to the periderm higher water permeability (Fig. 3D). Observed in cross-section, the phellem of the FHT deficient tubers looks more than twice thicker than that of the wild-type, with more cell layers and a less regular cell organization (Fig. 3F). Moreover, FHT deficient tubers obtained from axenic cultured plants developed scabby-like lesions as soil-grown tubers, thus corroborating the genetic origin of these lesions (Fig. 3G–L). In addition, no signs of infection were detected by a careful examination of the lesions under optical and electron microscopy. Noteworthy, periderm sheets isolated by enzymatic treatment from FHT deficient tubers are much heavier and stiffer than wild type and, once dried, break easily.

Figure 3.

Skin phenotype of FHT-RNAi tubers. The skin phenotype of FHT-RNAi and wild type was studied using tubers grown in soil (A–F) and in vitro (G–L). Compared with wild type (A and G), FHT-RNAi tubers show a darker russeted and netted skin surface (D and J). FHT-RNAi soil grown tubers (D) appear much wrinkled after 45-day storage due to the intense water loss compared to wild type (A). Under SEM, the skin surface of both soil b (E) and in vitro (H) grown FHT-RNAi tubers show grid-like cracks and splits that contrast with the smooth skin of wild-type tubers (B and H, respectively). As seen in cross section, FHT-RNAi phellem (F and L) is thicker, with more phellem cell layers and less regular cell organization than wild type (C and I).

However, despite the great similarity of their skin phenotypes, the physiological behavior of native periderm is very different in potatoes from Russet varieties and in FHT-RNAi tubers. In Russet potatoes the periderm completes a normal maturation within the first 2–3 weeks after harvesting, acquiring full water barrier properties and becoming resistant to skinning (phellem excoriation).^42,44,45 In contrast, the maturation process is impaired in FHT-RNAi tubers, the periderm transpiration remaining high and the skin being extremely fragile and susceptible to excoriation (skinning), even after 2 months of storage.²⁶ A proper periderm maturation leading to the skin set is necessary for a good storage life of potatoes.³⁹ The mature potato periderm is characterized by a meristematically inactive phellogen (cork cambium), the acquisition of resistance to skinning during the maturation process being correlated with the thickening and strengthening of the phellogen cell walls.^42,44,46 However, the biochemical and hormonal requirements for periderm maturation and skin set are largely unknown.³⁹

FHT Downregulated Periderm Contains Increased Amounts of Polyamines

Hydroxycinnamic acids, including conjugated amides, are secondary metabolites widely present in plant tissues, which have been tentatively involved in a wide range of plant growth and differentiation processes, although their exact role is not completely understood.^47,48 In potato tuber, these compounds are abundant, mainly in the skin tissue, and have been widely analyzed. The main components are chlorogenic acid, caffeic acid and hydroxycinnamic acid amides.^49–52 Tyramine-derived amides, such as feruloyltyramine and feruloyloctopamine, have been related with potato defence reactions.^53,54 Polyamine-derived amides, such as caffeoyl and feruloyl putrescine, have been related with tuberization^55–58 and with periderm formation during wound healing,^59,60 although the physiological basis of their function remains obscure.

Central to the biosynthesis of hydroxycinnamic acids and derived compounds is feruloyl-CoA, just as it is for ferulate deposition in suberin and wax.¹ Thus, downregulation of suberin feruloyl transferase may result in a digression of the feruloyl-CoA metabolic flux, such that affects the amount and profile of soluble phenolics and, therefore, their putative regulated physiological processes. The characterization by LC-MS of the soluble phenolics in FHT -RNAi periderms actually showed significant changes in their profile.²⁶ As compared with wild type controls, new peaks emerged corresponding to caffeoylputrescine and feruolylputrescine conjugated polyamines. Moreover, although in trace amounts, feruloyltyramine-feruloyltyramine (Grossamide) and feruloyltyramine-feruloyloctopamine dimers also emerged. Thus, changes in these conjugated polyamines can be attributed to a side effect of FHT downregulation on ferulic acid metabolism, being tempting to speculate a possible involvement of these signaling molecules in periderm maturation.

Conclusions

The characterization of Arabidopsis and potato deficient mutants proves the role of BAHD feruloyl transferases in the biosynthesis of ferulate esters in both suberin and suberin embedded wax. In these mutants suberin lacks most esterified ferulate but maintains the typical lamellar ultrastructure. Therefore, in contrary to what current models would predict, ferulate seems not to be necessary or be only marginally required for the regular macromolecular organization of the polymer. Moreover, as suberized tissues in feruloyl transferase mutants show an increase in transpiration while maintaining unchanged the total lipid content and the suberin ultrastructure, a role for ferulate esters in water permeability can be postulated. As a whole, the reverse genetic approaches to suberin biosynthesis emphasize that current model for suberin should be redefined. On the other hand, potatoes deficient in feruloyl transferase experience severe changes in their skin anatomy and maturation process, which may be a consequence of a change in soluble phenolics repertoire by rerouting feruloyl-CoA.

Abbreviations

TEM

transmission electron microscope

SEM

scanning electron microscope

FHT

ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase

GPAT5

glycerol phosphate acyltransferase 5