(Received for publication, October 18, 1994; and in revised form, January 20, 1995)
From the
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.
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 reduction, and
BF
/methanol transesterification of suberin-enriched
preparations released mainly L-alkanoic acids, L-alkanols,
-hydroxyalkanoic acids,
,
-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 H 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.
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.
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.
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.