Wound-Induced Metabolism in Potato (Solanum tuberosum) Tubers: Biosynthesis of Aliphatic Domain Monomers

Suberin, a cell specific, wall-associated biopolymer, is formed during normal plant growth and development as well as in response to stress conditions such as wounding. It is characterized by the deposition of both a poly(phenolic) domain (SPPD) in the cell wall and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. Although the monomeric components that comprise the SPPD and SPAD are well known, the biosynthesis and deposition of suberin is poorly understood. Using wound healing potato tubers as a model system, we have tracked the flux of carbon into the aliphatic monomers of the SPAD in a time course fashion. From these analyses, we demonstrate that newly formed fatty acids undergo one of two main metabolic fates during wound-induced suberization: (1) desaturation followed by oxidation to form the 18:1 ω-hydroxy and dioic acids characteristic of potato suberin, and (2) elongation to very long chain fatty acids (C20 to C28), associated with reduction to 1-alkanols, decarboxylation to n-alkanes and minor amounts of hydroxylation. The partitioning of carbon between these two metabolic fates illustrates metabolic regulation during wound healing, and provides insight into the organization of fatty acid metabolism.Key Words: suberin, potato, Solanum tuberosum, carbon flux analysis, abiotic stress     

Metabolite profiling of potato (Solanum tuberosum L.) tubers during wound-induced suberization

Suberin is a specific cell wall-associated biopolymer characterized by the deposition of both a poly(phenolic) domain (SPPD) associated with the cell wall, and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. In planta, suberin functions to prevent plants from desiccation and pathogen attack. Although the chemical identity of the monomeric components of the SPPD and SPAD are well known, their concerted biosynthesis and assembly into the suberin macromolecule is poorly understood. To expand our knowledge of suberin biosynthesis, a GC/MS-based metabolite profiling study was conducted, using wound healing potato (Solanum tuberosum L.) tubers as a model system. A time series of both non-polar and polar metabolite profiles were created, yielding a broad-based, dynamic picture of wound-induced metabolism, including suberization. Principal component analysis revealed a separation of metabolite profiles according to different suberization stages, with clear temporal differences emerging in the non-polar and polar profiles. In the non-polar profiles, suberin-associated aliphatics contributed the most to cluster formation, while a broader range of metabolites (including organic acids, sugars, amino acids and phenylpropanoids) influenced cluster formation amongst polar profiles. Pair-wise correlation analysis revealed strong correlations between known suberin-associated compounds, as well as between suberin-associated compounds and several un-identified metabolites in the profiles. These data may help to identify additional, as yet unknown metabolites associated with suberization process.     

RCN1/OsABCG5, an ATP‐binding cassette (ABC) transporter,is required for hypodermal suberization of roots in rice (Oryza sativa)

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP‐binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP‐RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C₂₈ and C₃₀ fatty acids or ω‐OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.     

A comparison of suberin monomers from the multiseriate exodermis of <Emphasis Type="Italic">Iris germanica</Emphasis> during maturation under differing growth conditions

Iris germanica roots develop a multiseriate exodermis (MEX) in which all mature cells contain suberin lamellae. The location and lipophilic nature of the lamellae contribute to their function in restricting radial water and solute transport. The objective of the current work was to identify and quantify aliphatic suberin monomers, both soluble and insoluble, at specific stages of MEX development and under differing growth conditions, to better understand aliphatic suberin biosynthesis. Roots were grown submerged in hydroponic culture, wherein the maturation of up to three exodermal layers occurred over 21 days. In contrast, when roots were exposed to a humid air gap, MEX maturation was accelerated, occurring within 14 days. The soluble suberin fraction included fatty acids, alkanes, fatty alcohols, and ferulic acid, while the suberin poly(aliphatic) domain (SPAD) included fatty acids, α,ω-dioic acids, ω-OH fatty acids, and ferulic acid. In submerged roots, SPAD deposition increased with each layer, although the composition remained relatively constant, while the composition of soluble components shifted toward increasing alkanes in the innermost layers. Air gap exposure resulted in two significant shifts in suberin composition: nearly double the amount of SPAD monomers across all layers, and almost three times the alkane accumulation in the first layer. The localized and abundant deposition of C18:1 α,ω-dioic and ω-OH fatty acids, along with high accumulation of intercalated alkanes in the first mature exodermal layer of air gap-exposed roots indicate its importance for water retention under drought compared with underlying layers and with entire layers developing under water.     

Abscisic Acid stimulation of suberization : induction of enzymes and deposition of polymeric components and associated waxes in tissue cultures of potato tuber

Effect of abscisic acid (ABA) on suberization of potato (Solanum tuberosum var. Russet-Burbank) tuber tissue culture was studied by measuring deposition of suberin components and the level of certain key enzymes postulated to be involved in suberization. ABA treatment resulted in a 3-fold increase in the polymeric aliphatic components of suberin and a 4-fold increase in the polymeric aromatic components. Hydrocarbons and fatty alcohols, two components characteristic of waxes associated with potato suberin, increased 9- and 5-fold, respectively, as a result of ABA treatment. Thus, the deposition of the polymeric aliphatics and aromatics as well as waxes, all of which have been postulated to be components of suberized cell walls, was markedly stimulated by ABA. ω-Hydroxy-fatty acid dehydrogenase which showed a rather high initial level of activity increased only 60% due to ABA treatment. Phenylalanine ammonia-lyase activity reached a maximum at a 5-fold level after 4 days in the ABA medium, whereas the control showed only a 3-fold increase. ABA treatment also resulted in a dramatic (7-fold) increase in an isozyme of peroxidase which has been specifically associated with suberization. Thus, ABA appears to induce certain key enzymes which are most probably involved in suberization.     

A feruloyl transferase involved in the biosynthesis of suberin and suberin‐associated wax is required for maturation and sealing properties of potato periderm

Suberin and waxes embedded in the suberin polymer are key compounds in the control of transpiration in the tuber periderm of potato (Solanum tuberosum). Suberin is a cell‐wall biopolymer with aliphatic and aromatic domains. The aliphatic suberin consists of a fatty acid polyester with esterified ferulic acid, which is thought to play an important role in cross‐linking to the aromatic domain. In potato, ferulic acid esters are also the main components of periderm wax. How these ferulate esters contribute to the periderm water barrier remains unknown. Here we report on a potato gene encoding a fatty ω‐hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (FHT), and study its molecular and physiological relevance in the tuber periderm by means of a reverse genetic approach. In FHT RNAi periderm, the suberin and its associated wax contained much smaller amounts of ferulate esters, in agreement with the in vitro ability of the FHT enzyme to conjugate ferulic acid with ω‐hydroxyacid and fatty alcohols. FHT down‐regulation did not affect the typical suberin lamellar ultrastructure but had significant effects on the anatomy, sealing properties and maturation of the periderm. The tuber skin became thicker and russeted, water loss was greatly increased, and maturation was prevented. FHT deficiency also induced accumulation of the hydroxycinnamic acid amides feruloyl and caffeoyl putrescine in the periderm. We discuss these results in relation to the role attributed to ferulates in suberin molecular architecture and periderm impermeability.     

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers frominfection and dehydration. Wound-induced suberization and theaccumulation of hydrophobic barriers to reduce water vapourconductance/loss are principal protective wound-healing processes.However, little is known about the cognate mechanisms that effector regulate these processes. The objective of this researchwas to determine the involvement of abscisic acid (ABA) in theregulation of wound-induced suberization and tuber water vapourloss (dehydration). Analysis by liquid chromatography–massspectrometry showed that ABA concentrations varied little throughoutthe tuber, but were slightly higher near the periderm and lowestin the pith. ABA concentrations increase then decrease duringtuber storage. Tuber wounding induced changes in ABA content.ABA content in wound-healing tuber discs decreased after wounding,reached a minimum by 24 h, and then increased from the 3rd tothe 7th day after wounding. Wound-induced ABA accumulationswere reduced by fluridone (FLD); an inhibitor of de novo ABAbiosynthesis. Wound-induced phenylalanine ammonia lyase activitywas slightly reduced and the accumulation of suberin poly(phenolics)and poly(aliphatics) noticeably reduced in FLD-treated tissues.Addition of ABA to the FLD treatment restored phenylalanineammonia lyase activity and suberization, unequivocally indicatingthat endogenous ABA is involved in the regulation of these wound-healingprocesses. Similar experiments showed that endogenous ABA isinvolved in the regulation of water vapour loss, a process linkedto wax accumulation in wound-healing tubers. Rapid reductionof water vapour loss across the wound surface is essential inpreventing desiccation and death of cells at the wound site;live cells are required for suberization. These results unequivocallyshow that endogenous ABA is involved in the regulation of wound-inducedsuberization and the processes that protect surface cells fromwater vapour loss and death by dehydration. Key words: Abscisic acid, poly(aliphatic), poly(phenolic), potato, Solanum tuberosum L., suberin     

Abscisic acid‐mediated modifications of radial apoplastic transport pathway play a key role in cadmium uptake in hyperaccumulator Sedum alfredii

Abscisic acid (ABA) is a key phytohormone underlying plant resistance to toxic metals. However, regulatory effects of ABA on apoplastic transport in roots and consequences for uptake of metal ions are poorly understood. Here, we demonstrate how ABA regulates development of apoplastic barriers in roots of two ecotypes of Sedum alfredii and assess effects on cadmium (Cd) uptake. Under Cd treatment, increased endogenous ABA level was detected in roots of nonhyperaccumulating ecotype (NHE) due to up‐regulated expressions of ABA biosynthesis genes (SaABA2, SaNCED), but no change was observed in hyperaccumulating ecotype (HE). Simultaneously, endodermal Casparian strips (CSs) and suberin lamellae (SL) were deposited closer to root tips of NHE compared with HE. Interestingly, the vessel‐to‐CSs overlap was identified as an ABA‐driven anatomical trait. Results of correlation analyses and exogenous applications of ABA/Abamine indicate that ABA regulates development of both types of apoplastic barriers through promoting activities of phenylalanine ammonialyase, peroxidase, and expressions of suberin‐related genes (SaCYP86A1, SaGPAT5, and SaKCS20). Using scanning ion‐selected electrode technique and PTS tracer confirmed that ABA‐promoted deposition of CSs and SL significantly reduced Cd entrance into root stele. Therefore, maintenance of low ABA levels in HE minimized deposition of apoplastic barriers and allowed maximization of Cd uptake via apoplastic pathway.     

The poly(phenolic) domain of potato suberin: a non-lignin cell wall bio-polymer.

Suberized plant cell walls have three distinguishing features: (1) tissue specificity, (2) a poly(aliphatic) domain and (3) a unique, "lignin-like" poly(phenolic) domain. With respect to the latter, comparisons have often been made to lignin, but the unique phenolic composition of suberized cells yields a unique polymer better designated as a poly(phenolic) domain. Potato tubers that have been induced to suberize through wounding make an excellent model system with which the chemistry, biochemistry and macromolecular assembly of the suberin poly(phenolic) domain can be monitored. For example, wound healing potato tubers have been used to determine the unique hydroxycinnamic acid nature of its poly(phenolic) domain using specific carbon-13 labeling studies and specific chemical degradation techniques (e.g. thioacidolysis). Furthermore, a suberization-associated anionic peroxidase has been purified from suberizing potato tubers and subsequently shown to oxidize hydroxycinnamic acids (and their derivatives) in preference to monolignols, as well as yield an unique polymer in vitro. We have since extended these studies to begin analyzing the macromolecular assembly process leading to the deposition of this suberized tissue specific domain. To this end we have begun to describe an H(2)O(2)-generating system with NAD(P)H-dependent oxidase-like properties that is temporally associated with the formation of potato suberin poly(phenolics) during suberization. Herein we describe our progress to date.     

Fatty acid ω-hydroxylases from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

Key message

Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1.

Abstract

Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.

     

Regulation of a plant aquaporin by a Casparian strip membrane domain protein‐like

The absorption of soil water by roots allows plants to maintain their water status. At the endodermis, water transport can be affected by initial formation of a Casparian strip and further deposition of suberin lamellas and regulated by the function of aquaporins. Four Casparian strip membrane domain protein‐like (CASPL; CASPL1B1, CASPL1B2, CASPL1D1, and CASPL1D2) were previously shown to interact with PIP2;1. The present work shows that CASPL1B1, CASPL1B2, and CASPL1D2 are exclusively expressed in suberized endodermal cells, suggesting a cell‐specific role in suberization and/or water transport regulation. When compared with wild‐type plants, and by contrast to caspl1b1*caspl1b2 double loss of function, caspl1d1*caspl1d2 double mutants showed, in some control or NaCl stress experiments and not upon abscisic acid (ABA) treatment, a weak enlargement of the continuous suberization zone. None of the mutants showed root hydraulic conductivity (Lp_r) phenotype, whether in control, NaCl, or ABA treatment conditions. The data suggest a slight negative role for CASPL1D1 and CASPL1D2 in suberization under control or salt stress conditions, with no major impact on whole root transport functions. At the molecular level, CASPL1B1 was able to physically interact with PIP2;1 and potentially could influence the regulation of aquaporins by acting on their phosphorylated form.     

Effects of NO 3 deficiency and NaCl stress on suberin deposition in rhizo- and hypodermal (RHCW) and endodermal cell walls (ECW) of castor bean (Ricinus communis L.) roots

Castor bean (Ricinus communis L.) plants were hydroponically cultivated to achieve NO₃ deficiency (N starvation), salt stress (addition of 100 mM NaCl), or normal conditions. Endodermal (ECW) and rhizodermal and hypodermal cell walls (RHCW) were isolated enzymatically from roots, and suberin monomers were released by transesterification after solvent extraction. Aromatic and aliphatic suberin monomers were identified and quantified by gas chromatography and mass spectrometry. Between 90 and 95% of the released suberin monomers were linear, long-chain, aliphatic compounds (alcohols, acids, diacids, ω-hydroxy acids and 2-hydroxy acids) with an average chain length of 19 C-atoms. The remainder was an aromatic suberin fraction mainly composed of coumaric and ferulic acid. Suberin amounts were significantly increased in ECW and RHCW in the presence of NaCl. In contrast, N starvation led to significantly reduced levels of suberization in ECW and RHCW. It is concluded that R. communis plants reinforce their apoplastic transport barriers in roots in adaptation to NaCl stress in order to minimize NaCl uptake. Under conditions of N starvation the opposite occurs and plants reduce the suberization of their apoplastic transport barriers to facilitate nutrient uptake form the soil.