Suberin Biosynthesis, Assembly, and Regulation

Abstract

Suberin is a specialized cell wall modifying polymer comprising both phenolic-derived and fatty acid-derived monomers, which is deposited in below-ground dermal tissues (epidermis, endodermis, periderm) and above-ground periderm (i.e., bark). Suberized cells are largely impermeable to water and provide a critical protective layer preventing water loss and pathogen infection. The deposition of suberin is part of the skin maturation process of important tuber crops such as potato and can affect storage longevity. Historically, the term "suberin" has been used to describe a polyester of largely aliphatic monomers (fatty acids, ω-hydroxy fatty acids, α,ω-dioic acids, 1-alkanols), hydroxycinnamic acids, and glycerol. However, exhaustive alkaline hydrolysis, which removes esterified aliphatics and phenolics from suberized tissue, reveals a core poly(phenolic) macromolecule, the depolymerization of which yields phenolics not found in the aliphatic polyester. Time course analysis of suberin deposition, at both the transcriptional and metabolite levels, supports a temporal regulation of suberin deposition, with phenolics being polymerized into a poly(phenolic) domain in advance of the bulk of the poly(aliphatics) that characterize suberized cells. In the present review, we summarize the literature describing suberin monomer biosynthesis and speculate on aspects of suberin assembly. In addition, we highlight recent advances in our understanding of how suberization may be regulated, including at the phytohormone, transcription factor, and protein scaffold levels.

Keywords: CASP proteins; abscisic acid; fatty acid metabolism; macromolecular assembly; phenylpropanoid metabolism; suberin; transcription factors.

Figure 1

Comparison of two models of suberin structure. Two models of suberin structure are presented: a two-domain model based on [4] and an integrated model based on [29]. In each case, both phenolic and aliphatic polymers are depicted. The poly(phenolics) are shown as cross-linked by C-C and C-O-C bonds, while the poly(aliphatics) are shown as polyesters cross-linked via glycerol. In the two-domain model, the poly(phenolic) domain is envisioned as integrated into the cell wall, and esterified to the poly(aliphatic) domain via glycerol esters. The characteristic lamellae of suberized cells are proposed to arise from variation in electron density moving through less dense hydrocarbon and more dense areas rich in ester linkages and phenolics. By contrast, the integrated model is shown as repeating layers of poly(phenolic) and poly(aliphatic) components, that give rise to the characteristic lamellae. The degree to which the integrated model is embedded into the cell wall, or how tightly the two polymer types are cross-linked, remains unknown. Several lines of evidence support both models; further targeted experimentation will be required to determine whether one or both exist, or indeed a different arrangement comprises suberin.

Figure 2

Typical suberin phenolic and aliphatic monomers.

Figure 3

Time course of potato tuber wound suberin assembly. Using gene expression and chemical analysis data form wound healing potato tubers, the temporal deposition of the phenolic and aliphatic monomers of suberin is predicted to begin with phenolics in the cell wall (arrange), followed by aliphatics in the space (grey) between the cell wall and plasmalemma. (A) Within 12 hpw, phenolics begin to accumulate in the cell wall and become cross-linked via a peroxidase-mediated process. (B,C) Between 12-36 hpw, phenolics continue to accumulate and become cross-linked within the cell wall. (D) Approx. 36-48 hpw, acyl-hydroxycinnamate esters become cross-linked to the poly(phenolic) matrix, forming a transition between phenolic and aliphatic suberin deposition. (E) After 48 hpw, aliphatic suberin monomers/substructures are delivered to the surface of the cell wall where they are cross-linked to aliphatics at the surface of the cell wall.

Figure 4

Overview of the regulation of suberin biosynthesis and assembly in wound-healing potato tubers. This overview offers a synthesis of findings, and proposes mechanisms of regulation at the levels of monomer biosynthesis, deposition, polymerization and assembly, based on the literature described in this review. Primary metabolic pathways (shaded green) yield precursors and energy molecules that feed into specialized suberin-related metabolic branches. For example, carbohydrate metabolism yields erythrose-4-phosphate and phospho-enol-pyruvate as precursors to the shikimate pathway and production of aromatic amino acids used as precursors for phenolic suberin biosynthesis. Pyruvate and glycolysis-derived glyceraldehyde-3-phosphate are used for the isoprenoid metabolism that yields the phytohormone abscisic acid (ABA) via the carotenoid pathway. Pyruvate is also a substrate for the tricarboxylic acid cycle that yields acetyl-CoA for fatty acid biosynthesis, and results in the generation of 16:0, 18:0 and 18:1 fatty acids that undergo various modifications for aliphatic suberin monomer production. Glycerol-3-phosphate is synthesized from the dehydrogenation of dihydroxyacetone phosphate produced during glycolysis. The biosynthesis of suberin poly(phenolic) domain monomers (dark red) may be regulated by WRKY and MYB TFs. WRKY1 regulates THT and 4CL in relation to phenylpropanoid metabolism in pathogen-infected aerial potato organs. MYB74 and MYB102 may also regulate phenylpropanoid metabolism in potato tuber suberization. ABA regulates the biosynthesis of several key suberin poly(aliphatic) domain monomers (blue) by positively impacting genes involved in their production. NAC103 acts as a transcriptional suppressor of fatty acid and aliphatic suberin-related genes, and is induced by ABA. MYB TFs such as MYB102 and MYB74 may positively regulate aliphatic suberin production. (See Table 1 for key suberin biosynthetic genes and Table 2 for other TFs that regulate phenylpropanoid and/or fatty acid biosynthesis in other species.) It is feasible that potato orthologs of TFs characterized in other species may regulate suberization in wounded tubers. Most phenolic monomers are solely polymerized and incorporated into the SPPD. Feruloyl-CoA can also be conjugated to very long-chain fatty acids (VLCFAs), ω-hydroxy and α,ω-dioic acids, and 1-alkanols to yield ferulate esters, including alkyl ferulates as SPAD-associated soluble wax components. Modified fatty acids can also be esterified to glycerol via the glycerol-3-phosphate acyltransferases (GPATs). Esterified aliphatic constituents are exported by ABCG1. These steps represent a point of convergence between the two major suberin biosynthetic pathways and are labeled as “convergent metabolism” (shaded purple) in the figure. MYB TFs such as MYB102 and MYB74 are putative regulators of the genes encoding convergent metabolic steps. The translocation of aliphatic monomers (alkanes, VLCFAs, modified fatty acids, 1-alkanols) that are not esterified to glycerol or feruloyl-CoA (i.e., soluble components destined for polymerization, or that remain non-polymerized as associated wax) has not been established in potato, but ABCG11 has predicted involvement. Phenolic monomers are thought to undergo a NADPH-dependent oxidase (NOX), superoxide dismutase (SOD), and anionic peroxidase (PRX)-mediated polymerization, whereas SPAD polymerization activities remain uncharacterized. CASP and GDSL-like proteins may play a role in SPAD polymerization. Phenolic suberin associated CASPs may recruit machinery such as NOX, SOD, PRX for polymerization, and influence enzymes involved in the organization of cell wall polysaccharides in a process that may be associated with SPPD deposition via localization of cell wall modifying activities. CASP1-like/CASP8 could regulate the linkage between two domains, their spatial organization, and/or the polymerization and deposition of esterified aliphatics that act as building blocks for SPAD assembly. CASP9 may coordinate aliphatic suberin assembly. GDSLs may act as “suberin synthases” at this stage of assembly, akin to cutin synthases. ABCG1 is required for the export of aliphatic suberin components. ABCG11 is suppressed by NAC103. ABCGs with varied substrate specificities and MYB TFs are likely involved in the export, deposition and polymerization of non-esterified aliphatics, as well as the organization of non-polymerized, soluble waxes. Cell wall modification machinery may be regulated by CASPs (e.g., CASP1B2-like/CASP9) to organize the deposition of polymerized aliphatics between the cell wall and plasma membrane, prior to secondary cell wall formation. Abbreviations: 4CL, 4-coumarate-CoA ligase; ABA, abscisic acid; ABCG1, ATP-binding cassette (ABC) subfamily G transporter 1; ABCG11, ATP-binding cassette (ABC) subfamily G transporter 11; ABCG6, ATP-binding cassette (ABC) subfamily G transporter 6; C3H, p-coumarate 3-hydroxylase; C3’H, p-coumaroyl quinate/shikimate 3’-hydroxylase; C4H, cinnamic acid 4-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; CASP8, Casparian strip membrane domain protein 8; CASP9, Casparian strip membrane domain protein 9; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl CoA reductase; CER, ECERIFERUM; COMT, caffeic acid O-methyltransferase; CYP, cytochrome P450; CYP86A33, cytochrome P450 subfamily 86A 33; CYP86B12, cytochrome P450 subfamily 86B 12; CYTB₅, cytochrome b₅; F5H, ferulate 5-hydroxylase; FA, fatty acid; FAE, fatty acid elongase; GDSL, GDSL domain esterase/lipase; Glycerol-3P, glycerol-3-phosphate; GPAT5, glycerol-3-phosphate acyltransferase 5; GPAT6, glycerol-3-phosphate acyltransferase 6; HCAA, hydroxycinnamic acid amide; HCT, hydroxycinnamate transferase; KAR, β-ketoacyl-ACP reductase; KCS6, β-ketoacyl-CoA reductase 6; LACS, long-chain acyl-CoA synthetase; MYB, MYB family transcription factor; NAC, NAC domain transcription factor (NAM, no apical meristem, ATAF, Arabidopsis transcription activation factor, and CUC, cup-shaped cotyledon); NOX, NADPH-dependent oxidase; PAL, phenylalanine ammonia lyase; PRX, anionic peroxidase; SOD, superoxide dismutase; SPAD, suberin poly(aliphatic) domain; SPPD, suberin poly(phenolic) domain; THT, tyramine hydroxycinnamoyl transferase; VLCFA, very-long-chain fatty acid; WRKY, WRKY domain family transcription factor.