©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hydroxycinnamic Acid-derived Polymers Constitute the Polyaromatic Domain of Suberin (*)

(Received for publication, October 18, 1994; and in revised form, January 20, 1995)

Mark A. Bernards (1)(§) Marta L. Lopez (1) Jaroslav Zajicek (2) Norman G. Lewis (1)(¶)

From the  (1)Institute of Biological Chemistry and (2)NMR Spectroscopy Center, Washington State University, Pullman, Washington 99164-6340

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Suberin is an abundant, complex, intractable, plant cell wall polymeric network that forms both protective and wound-healing layers. Its function is, therefore, critical to the survival of all vascular plants. Its chemical structure and biosynthesis are poorly defined, although it is known to consist of both aromatic and aliphatic domains. While the composition of the aliphatic component has been fairly well characterized, that of the phenolic component has not. Using a combination of specific carbon-13 labeling techniques, and in situ solid state C NMR spectroscopic analysis, we now provide the first direct evidence for the nature of the phenolic domain of suberin and report here that it is almost exclusively comprised of a covalently linked, hydroxycinnamic acid-derived polymeric matrix.


INTRODUCTION

Suberin is a complex, intractable, biopolymer found in specialized plant cell walls, where it is laid down between the primary wall and plasmalemma(1, 2) . It is comprised of both aromatic and aliphatic components, a feature which may contribute to the alternating light and dark lamellae observed in transmission electron microscope sections of suberized tissue. It plays a pivotal physiological function in providing a water-impermeable diffusion barrier in dermal tissues of most underground plant organs (e.g. mature roots, tubers, stolons, rhizomes), the periderm (bark) of aerial tissues that undergo secondary thickening, and the Casparian band of the endodermis(2) . Consequently, it is essential for water retention by plants and functions in the overall control of water movement through the apoplastic stream(2) . In this context, the evolutionary development of its biological pathway must have been a critical juncture in the transition of plants from an aquatic to a terrestrial environment, since it provided vascular plants with the ability to cope with a desiccating environment(3) .

In addition to its developmentally regulated biosynthesis, suberin is synthesized and deposited in the walls of cells adjacent to wound sites during wound healing, where it is hypothesized to serve both as a diffusion barrier to water loss and as a physical barrier to opportunistic pathogens(1) . In fact, most of our current knowledge about suberin and the suberization process is derived from wound-induced healing processes. It should be recognized, however, that the molecular mechanism(s) of the induction of suberin biosynthesis (and regulation thereof) remains largely unknown.

A tentative model depicting suberin as an aliphatic polyester network containing a lignin core (i.e. a polymeric matrix of monolignols 1a-1c) interspersed with esterified waxes and hydroxycinnamic acids was first proposed in the early 1980s by Kolattukudy(1) . It emerged out of the most logical reconstruction of compounds identified in the chemical degradation products of suberin-enriched cell wall preparations from wound-healed (i.e. suberized) potato (Solanum tuberosum) tuber disks. For example, saponification, LiAlH(4) reduction, and BF(3)/methanol transesterification of suberin-enriched preparations released mainly L-alkanoic acids, L-alkanols, -hydroxyalkanoic acids, alpha,-alkane-dioic acids, as well as smaller amounts of unsaturated, hydroxylated, and epoxidized aliphatics(4, 5) , and ferulic acid(6, 7, 8) . (These components were presumed to be largely ester-linked since they were readily released from suberin preparations by treatment with alkali.) The lignin core was predicted largely on the basis of alkaline nitrobenzene oxidation (a common method used for lignin detection and characterization), which yielded substituted benzaldehydes considered to be derived from, and characteristic of, lignin(9) .

Recent attempts at providing more direct evidence for a lignin core in suberized tissues have generated conflicting results. Specifically, various research groups have applied techniques developed for detailed lignin analysis to the study of suberized tissues (see below). Thioacidolysis, for example, is a recently developed lignin degradation procedure that releases characteristic trithioethane derivatives of the monolignol 1a-1c building blocks of lignin and, thus, is considered to be more specific and diagnostic than the dated nitrobenzene oxidation technique(10) . Surprisingly, results obtained from the thioacidolytic analysis of suberin-enriched tissues revealed that only minor amounts of ``lignin'' were present in such preparations (i.e. about one-sixth of that predicted by alkaline nitrobenzene oxidation)(7, 8, 10) .

Some attempts to define the aromatic components of suberin through the analysis of organic solvent-soluble metabolites and alkaline hydrolysates obtained from suberized tissues have revealed a rather complicated picture; i.e. they afforded mixtures of feruloyl esters(11, 12) , tyrosine, tyramine, and substituted benzaldehydes/benzoates(8) , caffeic acid (at least in green cotton Gossypium hirsutum L. fibers)(13) , hydroxycinnamoyl amides (14) , and glycerates(13) . The soluble ferulate esters associated with suberized tissue have been suggested to function as suberin precursors (11, 12) , although they could also act as low molecular weight plasticizers in the polymer. The enzyme-catalyzed biosynthesis of certain alkyl ferulates in wound-healing potato tubers has recently been detailed(15) . The presence of phenolic amides in suberized tissue remains intriguing and raises questions about their role in the suberization process (e.g. are they suberin precursors or produced simply as wound-induced antimicrobial compounds?). Whatever case holds, they appear to be synthesized de novo in concert with the initiation of wound-induced suberization(14) .

Methods developed to isolate soluble lignin-derived fragments (i.e. through solvation of extractive-free milled-wood preparations), suitable for characterization by solution state NMR spectroscopy, have also been adapted to suberized tissue (i.e. suberin, like lignin, is an intractable polymer, and there are no methods available to wholly isolate or solubilize it in pure, unaltered form). But when applied to ``suberin'' preparations from the periderm of Quercus suber, Rubus idaeus, and S. tuberosum, the solution state ^1H and C NMR spectra failed to show any resonances characteristic of lignins (16) thereby leaving the question of its constitution again in doubt. Indeed, even natural abundance solid state C NMR spectral analysis of S. tuberosum suberin preparations (17, 18) failed to provide any clarification of its aromatic component; these analyses were further hampered by the fact that the preparations contained at least 50% carbohydrate (17, 18, 19) .

To overcome these difficulties, we have specifically labeled the suberin polymer with carbon-13 at sites designed to clarify the constitution of the polymer through solid state C NMR spectroscopic analysis in situ, as conducted previously for lignin structural determinations(20, 21) . Thus, following uptake and metabolism of [1-C]-, [2-C]-, or [3-C]phenylalanine, the specifically labeled S. tuberosum wound periderm was examined in situ using solid state C NMR spectroscopy. These analyses have revealed that the phenolic domain of suberin is primarily composed of an hydroxycinnamic acid-derived phenolic polymer. Additionally, the chemical shift data for enhanced resonances strongly suggested that a significant amount of covalent cross-linking (other than ester bonds) exists between the phenolic monomers in suberin.


MATERIALS AND METHODS

Chemicals and Instrumentation

All chemicals used were of reagent grade unless otherwise stated. Water was purified through a Barnstead NANOpure II system. L-[U-^14C]Phenylalanine (15 GBq mmol) was purchased from ICN Radiochemicals. L-[1-C]-, [2-C]-, and [3-C]phenylalanine (99 atom % C) were purchased from MSD Isotopes (St. Louis, MO). Carbon-13 cross-polarization-magic angle spinning (CPMAS) (^1)NMR spectra were recorded at 100.6 MHz on a Chemagnetics CMX-400 spectrometer. Both ^1H and C(^1H) solution state NMR spectra were recorded in acetone-d(6) on a Varian VXR-5005 spectrometer at 500 and 125.7 MHz, respectively.

Administration of [C]Phenylalanine

Slices of potato tuber, prepared from surface-sterilized tubers(12) , were administered 200 µl of a fresh, filter-sterilized solution (up to 100 mM) of either L-[^14C]-, natural abundance, [1-C]-, [2-C]-, or [3-C]phenylalanine immediately after slicing (wounding) and allowed to metabolize in the dark at 25 °C for up to 7 days as described previously(12) . Preliminary experiments using L-[^14C]phenylalanine were carried out to establish optimal uptake and incorporation conditions. Subsequently, two independent experiments (each comprising separate administrations of L-natural abundance, [1-C]-, [2-C]-, and [3-C]phenylalanine, respectively, at 100 mM in 200-µl aliquots) were carried out, resulting in essentially identical incorporation and enhancement patterns. Representative data are shown.

Isolation of Potato Wound Periderm

Wound periderm was mechanically removed from 7-day wound-healed tuber slices, frozen in liquid N(2), and ground to a powder. Ground, frozen periderm tissue was extracted for 30 min on ice with buffer (200 mM Tris-HCl, pH 7.8) containing 10 mM each of dithiothreitol, sodium ascorbate, and NaN(3), followed by successive extraction with 95% ethanol (24 h) and CHCl(3)/ethanol (2:1, 24 h) in a Soxhlet apparatus. The resulting extractive free residue was stirred with 90% dimethyl sulfoxide (250 ml g of tissue) for 48 h at room temperature (to remove starch), filtered under gentle suction (Miracloth), washed twice with water (collecting the residue on Miracloth after each wash), boiled in an oxalic acid (45 mM)/NH(3) oxalate (130 mM) solution (250 ml g of tissue) for 1 h (to partially degrade cellulose), collected and washed as above, and finally freeze-dried. The dimethyl sulfoxide/oxalate-treated residue was next incubated with cellulase (5 mgbulletml) and pectolyase (1 mgbulletml) for 16 h in sodium acetate buffer (50 mM, pH 4.0, 40 ml g of freeze-dried residue) at room temperature, and the residue was collected on Miracloth as above and washed with buffer (1times) and water (2times). The resulting residue was further digested with Pronase E (0.5 mgbulletml) for 16 h in sodium phosphate buffer (50 mM, pH 7.5, 40 ml g of freeze-dried residue) at 37 °C, with the residues again collected on Miracloth, washed with buffer (1times) and water (2times), and freeze-dried. Finally, enzyme-treated residues were saponified as described(8) , and the residues were collected on Miracloth as above. Solid state C CPMAS NMR spectra were recorded using from 25 to 100 mg of freeze-dried residues obtained after oxalic acid treatment, Pronase E digestion, and saponification. Sample weights were carefully matched to ensure that reliable difference spectra could be obtained.

Chemical Analysis and Synthesis

Buffer and ethanol solubles were analyzed by high performance liquid chromatography as described (12) . Proteins were quantified by the dye binding method of Bradford (22) . Thioacidolysis was performed as described(10) . Compound 4 was synthesized according to a procedure adapted from (23) .

NMR Spectroscopy

Solid state C CPMAS NMR experiments were performed at ambient temperature (22 °C) using a C-^1H cross-polarization field strength of 48 kHz and a 2-ms contact time. A proton-decoupling of 56 kHz was used during the 68.2-ms acquisition time. Usually, 8,000 to 12,000 scans were collected for each spectrum with 2.5 s recycle time. Samples (25-100 mg) were packed into 5-mm Chemagnetics Pencil zirconia rotors and spun at 8 kHz. Chemical shifts were referenced to those of glycine (24) obtained from an independently recorded spectrum under identical spectrometer settings. Difference spectra were obtained by subtracting the natural abundance resonances recorded for a suberin sample prepared from tuber slices previously administered 100 mM natural abundance L-phenylalanine.


RESULTS AND DISCUSSION

Wound-healing potato (S. tuberosum) tubers have been demonstrated by others to be an excellent model system to study suberization in plants (e.g.(1) ). They suberize quickly and uniformly upon wounding and are amenable to the incorporation of exogenously supplied substrates. Since the deposition of suberin is highly localized and occurs only within the first few cell layers beneath the wound surface, suberin-enriched preparations are, in principle, easily obtained.

Specifically labeled L-[C]phenylalanines were administered as precursors in these experiments, since this amino acid represents the primary source of phenylpropanoids in potatoes (25) and is known to be efficiently incorporated into the periderm of wound healing potato tubers (and hence suberin; (9) ). Importantly, this precursor provides an effective means to distinguish between the potential metabolic products of L-phenylalanine, since protein-bound L-phenylalanine 2, hydroxycinnamic acids (and their derivatives) 3a-3c, the monolignols 1a-1c (and their derivatives) have characteristic and distinct C NMR spectroscopic signals (Fig. S1)(26, 27) . In other words, lignin formation from L-phenylalanine 2 requires its metabolism into various substituted hydroxycinnamic acids 3a-3c, followed by their successive reduction to the monolignols 1a-1c, and subsequent polymerization (reviewed in (28) ). Thus, reduction of the carbonyl carbon of L-phenylalanine 2 into an hydroxymethyl functionality is essential to, and indeed an easily identifiable marker for, the formation of lignin (precursors). Thus, by judicious choice of precursor (e.g.L-[1-C]phenylalanine), the presence or absence of lignin (i.e. polymerized monolignols) in suberized tissues should readily be determined in situ, without having to first degrade the tissue through harsh chemical means.


Scheme 1: Scheme 1Structures of monolignols 1a-1c, protein-bound L-phenylalanine 2, hydroxycinnamates 3a-3c, and possible oxidative coupling products 4-7 of ferulic acid. Solid state C CPMAS NMR chemical shift assignments for 1 (26) and 3 as well as solution state C data for 2(27) , 4, and 5 (31) are shown.



Administration of Phenylalanines to Wound-healing Potato Tubers

Preliminary experiments using L-[^14C]phenylalanine were conducted to optimize conditions of precursor uptake and incorporation. These studies established that the administration of 100 mML-[^14C]phenylalanine (18.3 kBq, 200 µl) per slice, to wound-healing potato tubers, followed by 7 days metabolism, yielded an incorporation of >2% L-[^14C]phenylalanine equivalents into the insoluble (suberized) residue. To ensure that this level of exogenously supplied phenylalanine did not adversely affect tuber metabolism and suberization, endogenous levels of soluble phenolics and proteins were measured in unlabeled (control) tissue, and the values compared to those obtained following administration of 100 mML-phenylalanine. It was found that neither the amount of soluble phenolics (including ferulate esters) (12) nor extractable protein (22) was affected by the addition of exogenously supplied L-phenylalanine (data not shown). Similarly, thioacidolytic analysis (10) of both control and 100 mML-phenylalanine-labeled wound periderms liberated essentially identical amounts of guaiacyl and syringyl products (e.g. 13-16 µmol g extractive free residue) and confirmed that only a minute amount of lignin (if any) was present in the tissue. Lastly, solid state C NMR spectra obtained from either the controls or tissue-administered 100 mML-phenylalanine were identical, again indicating that the administration of 100 mM phenylalanine did not adversely affect metabolism and ultimately suberization.

NMR Spectroscopy of Potato Wound Periderm

Having established satisfactory conditions for the uptake and metabolism of L-phenylalanine, attention was next directed toward the metabolic fate of L-[1-C]phenylalanine in wound-healing (i.e. suberizing) potato tuber disks. Following uptake and metabolism (i.e. 100 mML-[1-C]phenylalanine; 7 days), the suberized wound periderm was excised and subjected to procedures largely aimed at the removal of soluble metabolites and cell wall carbohydrates (see ``Materials and Methods''). Solid state C NMR spectra were recorded at each stage of purification, i.e. following solvent extraction, enzyme digestion, and saponification. Suberin preparations obtained from potato tubers administered 100 mM natural abundance L-phenylalanine were used to subtract natural abundance resonances from the C-enriched preparations.

As can be seen in Fig. 1a, a single large, dominant, enhanced resonance (171.0 ppm) was apparent in the difference spectrum of extractive-free suberin preparations, indicative of carbonyl functionalities (e.g. acids, esters, amides). This result showed that at best only a limited amount of synthesis into hydroxycinnamoyl alcohols ( 63.1 ppm) and, thus lignin, had occurred. That the enhanced signal at 171.0 ppm was not due to L-phenylalanine metabolism into proteinaceous material was revealed by the absence of a characteristic protein-bound phenylalanine resonance at approximately 38 ppm(27) , following metabolism of L-[3-C]phenylalanine (see below). Thus, this spectroscopic data provided the first evidence that the phenolic domain of suberin was not built up from monolignols, as previously speculated, but contained hydroxycinnamic acid derivatives.


Figure 1: Solid state C NMR difference spectra of potato wound periderm following metabolism of L-[1-C]phenylalanine. Spectra were recorded after solvent extraction (including dimethyl sulfoxide and oxalate treatments) (a), enzymatic digestion with cellulase, pectolyase, and Pronase E (b), and saponification (c). See ``Materials and Methods'' for details of sample preparation. Spectra are normalized to the 63.1 ppm resonance in a.



A small resonance at 63.1 ppm, corresponding to an hydroxymethyl functionality (e.g. of lignin) was observed, however, in the 1-C-labeled sample following enzymatic digestion of the 1-C-labeled periderm (Fig. 1b), indicative of traces of lignin in the sample (and thus providing an explanation for the thioacidolytic data described above). Interestingly, this signal persisted when the preparation was saponified (Fig. 1c), even though the 171.0 ppm resonance was substantially reduced (i.e. much of the phenolic polyester nature of suberin was degraded by this treatment, presumably through cleavage of labile ester linkages). Importantly, the persistence of the 171.0 ppm signal after saponification indicated that the phenolic component of suberin was more highly cross-linked than would be predicted for a simple polyester. This could be due to the presence of both stable C-C or C-O cross-links between the monomeric moieties of the phenolic domain, as well as perhaps via stable amide linkages.

Next L-[2-C]- and [3-C]phenylalanine were individually administered to wound healing tubers, and the suberized periderm isolated as before. The labeling pattern observed in the corresponding difference spectra of the 2-C- and 3-Clabeled suberins (Fig. 2, a and b) both confirmed and extended the initial observation that the phenolic domain of suberin contained essentially only hydroxycinnamic acid-derived moieties. For example, the enhanced resonances at 120.5 (C-2) and 142.1 (C-3) ppm are characteristic of olefinic carbons of hydroxycinnamic acids or conjugates thereof. Enhanced resonances were also observed at 84.5 and 55.2 ppm (C-2-enriched suberin; Fig. 2a) and 86.2 and 75.4 ppm (C-3-enriched suberin; Fig. 2b) thereby revealing the presence of covalent linkages other than ester bonds in the polymer (discussed below). As with the 1-C-labeled suberin, saponification did not significantly alter the pattern of signal enhancement (data not shown). However, only tiny resonances corresponding to the olefinic carbons of monolignols 1a-1c (e.g. at 127.9 and 130.7 ppm) (26) were observed, again indicating that minute amounts of lignin, at best, were present in the preparations. Lastly, the absence of an enhanced resonance at approximately 38 ppm in the 3-C-labeled suberin clearly indicated that the suberin preparations were essentially devoid of phenylalanine-containing proteins(27) .


Figure 2: Solid state C NMR difference spectra of enzymedigested suberin preparations obtained from wound-healed potato tubers previously administered either L-[2-C]- (a) or L-[3-C]phenylalanine (b). See ``Materials and Methods'' for details of sample preparation.



Interunit Linkages

The data presented thus far indicates that the phenolic domain of suberin was comprised of hydroxycinnamic acid derivatives. Since there is a growing body of evidence(29, 30) indicating that the formation of suberin may be an H(2)0(2)/peroxidase-mediated polymerization, the coupling products (substructures) within the polymer are likely to be fairly predictable (e.g.4-7, Fig. S1). Consequently, dimers of likely oxidative coupling products of ferulic acid (including some of its esters) were synthesized as needed, and their NMR spectroscopic data were recorded and interpreted using, where possible, appropriate literature assignments(31) . These data were compared to that of the specifically labeled 1-C-, 2-C-, and 3-C-suberins.

For synthetic coupling products 4 and 5 (Fig. S1), the observed side chain carbon resonances were essentially identical with those of the wound-induced potato suberins: i.e. enhanced resonances at 84.5 (C-2) and 75.4 (C-3) ppm (Fig. 2) were consistent with an 8-O-4`-linked compound (e.g.4 in Fig. S1), while those at 55.2 (C-2) and 86.2 (C-3) supported a phenylcoumarin 5 (i.e. 8-5`) type of interunit linkage (Fig. S1). Similarly, the resonances at 171.0 (C-1), 120.5 (C-2), and 142.1 (C-3) ppm ( Fig. 1and Fig. 2) all indicated that suberin contains hydroxycinnamic acid derivatives with intact side chains as in 3a-3c (Fig. S1). These units are presumably linked to the remainder of the matrix either through ester or amide linkages, their aromatic ring carbons, or phenolic hydroxyl moieties. It can therefore be concluded that the aromatic domain of suberin is largely derived from the polymerization of hydroxycinnamic acids and/or their derivatives. More detailed information about likely linkages between aromatic ring carbons (e.g. 5-5` linkages such as in diferulic acid 6) as well as others (e.g. amides) will be the subject of a future report.

Concluding Remarks

Specific labeling of potato wound periderm with L-[1-C]-, [2-C]-, and [3-C]phenylalanine, combined with in situ solid state carbon-13 NMR spectroscopy, has provided the most direct means to date to characterize the phenolic domain of suberin. The resulting data, presented in this paper, establishes that the phenolic domain of suberin is largely comprised of a covalently linked, hydroxycinnamate-derived polymer matrix, with only minute amounts of lignin co-occurring. This finding means that we must now re-evaluate not only the chemical constitution of suberin, but also the sequential stages involved in its assembly.


FOOTNOTES

*
This work was supported by NASA Grant NAGW 3672 and the U.S. Department of Agriculture Grant 91-37103-6638. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: University of Northern British Columbia, Prince George BC, Canada V2N 4Z9.

To whom correspondence and reprint requests should be addressed. Tel.: 509-335-2682; Fax: 509-335-7643.

(^1)
The abbreviation used is: CPMAS, cross-polarization-magic angle spinning.


ACKNOWLEDGEMENTS

We acknowledge the thioacidolytic analysis performed by Koki Fujita and the synthesis of compound 4 by Dr. Alex Chu.


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