J Sci Food Agric 1997, 74, 221228

Ferulic Acid Dehydrodimers in the Cell Walls of Beta vulgaris and their Possible Role in Texture
Keith W Waldron,* Annie Ng, Mary L Parker and Adrian J Parr
Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UK
(Received 20 March 1996 ; revised version received 19 July 1996 ; accepted 20 January 1997)

Abstract : Sugarbeet, a form of Beta vulgaris var vulgaris, fails to soften completely after heating at 100C for several hours. This is due to thermal stability of the cellwall polymers involved in cellcell adhesion. In contrast, beetroot softens within 2530 min due to a relatively rapid increase in the ability of the cells to separate. Information concerning the cellwall polymers responsible for cellcell adhesion was obtained by subjecting sugarbeet and beetroot tissues to a range of chemical and biochemical treatments designed to cleave cellwall chemical bonds selectively. Treatment of sugarbeet tissues with chelating agents, weak base (Na CO , 005 M) or a puried, specic endoxylanase did not facilitate vortex2 induced 3 separation. However, this could be induced after extraction in dilute, cell cold alkali (00501 M KOH) or dilute, hot acid (01 M TFA, 100C). Tissues from beetroot behaved similarly. Furthermore, the cell walls of sugarbeet and beetroot were similar in yield and neutral carbohydrate composition ; the cell wall-galacturonic acid content of beetroot was 50% higher as compared with sugarbeet. They were also rich in ferulic acid (FA) and its derivatives (67 mg g~1 CWM), and exhibited pH-dependent autouorescence which disappeared during alkali-induced cell separation. In sugarbeet, over 20% of the FA was in dimer form. In beetroot, however, the value was only 10%. The main FA dimers were 8-O-4@DiFA and 8,5@DiFA (benzofuran form). The results indicate that the degree of thermal stability of cellcell adhesion and, therefore, texture in Beta vulgaris tissues is related to the degree of FA-cross linking between pectic polysaccharides. Key words : texture, cell walls, Beta vulgaris, sugarbeet, beetroot, ferulic acid dehydrodimers, pectic polysaccharides

INTRODUCTION Sugarbeet and beetroot are both forms of Beta vulgaris var vulgaris (Chenopodiaceae). The mature storage organs are harvested for commercial exploitation, sugarbeet being utilised for sugar production whilst, on a lesser scale, beetroot is cooked and then often pickled prior to consumption as a salad vegetable. A character* To whom correspondence should be addressed.

istic of Beta vulgaris is that the mature storage organ exhibits signicant thermal stability of texture. Sugarbeet tissues show little softening after boiling in water for 1 h ; this can be attributed to a lack of cell separation during cooking (Parker and Waldron 1995). The cellwall chemistry underlying this characteristic is poorly understood. The parenchyma cell walls of Beta vulgaris storage tissues contain signicant quantities of ferulic acid (FA). In sugarbeet, feruloyl groups are ester-linked to O2 of 221

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222 Ara residues of the main core of a-(1-5)-linked arabinan chains. They are also attached to O-6 of Gal residues of the main core of b-(1-4)-linked type I galactan chains (Colquhoun et al 1994) ; Ralet et al 1994). Cellwall FA esters have attracted considerable interest due to their potential for peroxidase-catalysed cross-linking of polysaccharides through the formation of FA dehydrodimers (Biggs and Fry 1987 ; Brett and Waldron 1996). Such cross-linking provides a biochemical mechanism for controlling cellwall mechanical properties in some tissues (Biggs and Fry 1987 ; Carpita and Gibeaut 1993 ; Parker and Waldron 1995). Until recently, the only FA dehydrodimer to have been identied was 5,5@diferulic acid (5,5@DiFA), rst isolated from grass cell walls after saponication (Markwalder and Neukom 1976). The involvement of this dimer in cross-linking of cellwall polymers has been demonstrated by the identication of dimer-cross-linked arabinoxylans in cell walls of bamboo (Ishii 1991). Its physiological signicance has been conrmed in studies of light-inhibition of stem extension in Oryza (Tan et al 1992) and Avena (Miyamoto et al 1994) coleoptiles. Recent investigations have led to the identication and characterisation of additional FA dehydrodimers in a number of plant tissues (Ralph et al 1994 ; Parr et al 1996 ; Waldron et al 1996a, b). Furthermore, the yields of these compounds indicate that phenolic-dehydrodimer cross-linking of cellwall polysaccharides is much greater than previously thought (Ralph et al 1994). Recently, we attributed the thermal stability of texture of Chinese water chestnut storage parenchyma to high levels of these new dimers (Parker and Waldron 1995 ; Parr et al 1996 ; Waldron et al 1996a). Despite the high FA content of the storage tissue cell walls of Beta vulgaris, there is little, if any, denitive information concerning the nature and levels of FA dehydrodimers. In the present paper we report on the presence and levels of several recently characterised ferulic acid dehydrodimers in the cell walls of sugarbeet and beetroot. The ndings are discussed in relation to their cellwall chemistry and thermal stability of texture.

K W W aldron et al glucinol HCl (23 drops of 1% w/v phloroglucin in 95% ethanol on a section followed by the addition of 1 drop of 25% (w/v) HCl) to locate lignin.

Autouorescence of cell walls Autouorescence of raw, cooked and chemically treated sugarbeet and beetroot tissue was examined using a Leitz Ortholux II microscope with a HBO 50 W mercury arc lamp and an exciter and barrier lter combination with transmission of 340380 nm and [430 nm, respectively. The tissues were mounted either in 01 M sodium acetate, pH 45, or in 20 mM NH OH 4 (pH 10) to enhance autouorescence (Parker and Waldron 1995).

Scanning electron microscopy (SEM) Pieces approximately 15 cm long and 075 cm wide, were cut longitudinally or transversely from mature sugarbeet and boiled for 1 h. After cooling, each piece was bent gently until it fractured. The fracture faces were cut away from the underlying tissue and xed in 30 g glutaraldehyde litre~1 in 005 M cacodylate buer (pH 72) for 2 h. The xed tissue was dehydrated in an ethanol series, then transferred to acetone before being critical-point dried using liquid CO . The dry tissues 2 were then mounted, fracture surface upwards, onto aluminium stubs using silver conducting paint. All samples were sputter coated with a layer of gold, approximately 25 nm thick, and examined and photographed in a Stereoscan 360 scanning electron microscope (LEO, Cambridge).

Preparation of cellwall material (CWM) Cellwall material was isolated from the beet tissues using the method developed for Chinese water chestnut (Parker and Waldron 1995).

MATERIALS AND METHODS Plant material Mature sugarbeet and beetroot (Beta vulgaris var vulgaris) were obtained during the 1995 harvest from a local farmer and supplier, respectively. Staining procedures on hand-cut sections Hand-cut sections of sugarbeet and beetroot were examined and photographed unstained or with phloroExtraction of wall-bound ester-linked phenolics Wall-bound phenolics were released by sequential alkaline hydrolysis of isolated CWM as described by Parr et al (1996).

Analysis of phenolics by HPLC Phenolics were detected and quantied by HPLC using an Inertpak ODS2 5 km reverse phase column

Possible role of ferulic acid dehydrodimers in sugarbeet texture (25 cm ] 5 mm : Capital HPLC Ltd, Broxburn, West Lothian, UK) with gradient elution employing progressively increasing methanolacetonitrile levels in 1 mM triuoroacetic acid (TFA) (Waldron et al 1996b). The most suitable gradient prole for separation of wallbound phenolic dehydrodimers and monomers was : Time \ 0 : 90% solvent A, 5% solvent B, 5% solvent C Time \ 25 min : 25% solvent A, 37% solvent B, 37% solvent C (linear gradient) ; Time \ 30 min : 0% solvent A, 50% solvent B, 50% solvent C (exponential gradient) Time \ 45 min : 90% solvent A, 5% solvent B, 5% solvent C (exponential gradient) Time \ 55 min : 90% solvent A, 5% solvent B, 5% solvent C Solvent A is 10% (v/v) aqueous methanol plus TFA to 1 mM, solvent B is 80% (v/v) methanol plus TFA to 1 mM and solvent C is 80% (v/v) acetonitrile plus TFA to 1 mM. Flow rate was 1 ml min~1. All three solvents were sparged with helium. Detection was with a PerkinElmer diode array detector 235 C. Quantitation was by integration of peak areas at 280 nm, with reference to known amounts of pure compounds. The above method facilitated the quantication of the following phenolic moieties : p-hydroxybenzoic acid (pBA) ; vanillic acid (VA) ; p-hydroxybenzaldehyde (pBAL), vanillin (VAN), trans-p-coumaric acid (tCA), trans-ferulic acid (tFA), cis-ferulic acid (cFA), 5,5@diferulic acid (5,5@DiFA) ; 8,5@diferulic acid open form (8,5@DiFA) ; 8,5@diferulic acid dehydrobenzofuran form (8,5@DiFA(B)) ; 8-O-4@diferulic acid (8-O-4@DiFA) ; 8,8@diferulic acid open form (8,8@DiFA) ; 8,8@diferulic acid aryltetralin form (8,8@DiFA(A)).

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tubes vigorously 10 times. The following scores (number of ]) were assigned according to the degree of disruption : 0, each tissue section intact ; 1, each tissue section broken into 35 clumps ; 2, each tissue section broken into 67 clumps ; 3, tissue sections broken into many clumps, some separate cells ; 4, tissue sections disrupted into clumps of approximately 2030 cells or less, many separated cells ; 5, tissue completely disrupted, any clumps less than 510 cells, mostly single separated cells (total VICS). Intermediate values were apportioned if necessary. In some cases, time to total VICS was monitored by testing tissue sections (two tubes of two sections) at 5 min intervals. The maximum time by which total VICS occurred in all tissue sections was recorded. Analysis of carbohydrate composition Cell walls were analysed for their carbohydrate composition as described in Parker and Waldron (1995). Xylanase purication Bioxylanase, a preparation from cultures of T richoderma viride (Biocon, Australia) was puried according to the procedure described by Gibson and McCleary (1987) as modied by Coimbra et al (1995). Xylanase digestion Sections of fresh sugarbeet or beetroot, 4 mm ] 4 mm ] 02 mm (approximately) were incubated with xylanase (400 units each, 1000 units ml~1) in 25 mM sodium acetate buer, pH 50. The mixture was allowed to react at 30C for 18 h. The incubated samples were removed and assessed for VICS in fresh buer. Chemicals Unless otherwise stated, all chemicals were of HPLCgrade purity.

Vortex-induced cell separation (VICS) This was measured as described by Parker and Waldron (1995). Sections of tissue 1 cm ] 1 mm ] 1 mm, were incubated in a number of extractants (10 sections per 50 ml). These included water, H SO , TFA, CDTA, Na CO , KOH, and 2 4 2 3 NaOH. The concentrations, times and temperatures of extractions are given in the results section. After each extraction/treatment, the tendency for cell separation was determined by placing two tissue sections into each of two screw-capped test tubes with 3 ml water, vortexing the tissue sections for 1 min and shaking the RESULTS Morphology The esh of the swollen hypocotyl of sugarbeet and beetroot consists of concentric layers of vascular tissue derived from secondary cambia, separated by wide layers of loosely packed large-celled storage parenchyma (Fig 1). The relatively sparse secondary xylem is lignied, but the associated phloem and small-celled tissue is soft and parenchymatous. When irradiated with

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Fig 1. Transverse section of sugarbeet showing two vascular rings and loosely packed parenchyma. Bar1 mm.

UV light at pH 45, the xylem emits a strong blue autouorescence, whilst the storage parenchyma gives only a very slight blue autouorescence. At pH 10, the parenchyma cell walls show a strong greenish-yellow autouorescence (Fig 2) which contrasts with the blue autouorescence of the xylem. This colour change is consistent with the presence of ferulic acid (Harris and Hartley 1976). Cellcell adhesion To investigate the polymeric and interpolymeric linkages which limit cell separation in sugarbeet and beetroot, sections of tissue were subjected to a variety of extraction procedures designed to cleave selectively cellwall chemical bonds (Parker and Waldron 1995). The tissues were then agitated and assessed for vortexinduced cell separation (VICS). Eect of hot water and hot acid Treatment of fresh sugarbeet tissue sections with hot water (pH 55, 100C for 1 h) resulted in a loss of turgor and rigidity, but had no eect on VICS. Tissue rupture involved cell breakage as in the uncooked control (results not shown). After 15 h at 100C, VICS was minimal ; examination of fractured tissue showed that
Fig 3. SEM of longitudinal fracture through sugarbeet tissue heated at 100C for 15 h. (a) Xylem parenchyma cells are broken open ; (b) loosely packed parenchyma cells separate. Bar150 km.

Fig 2. Autouorescence of sugarbeet at pH 10. Bar50 km.

the parenchyma and vascular cells within the vascular rings were broken open (Fig 3a) whilst the larger parenchyma cells between the rings had separated (Fig 3b). This indicates that in sugarbeet, the thermal stability of cellcell adhesion varies in dierent tissues. In contrast, treatment with 05 M H SO at 100C resulted in sub2 4 stantial VICS in all tissues (vascular and parenchyma) within 15 min, and loss of much of the pH-dependent autouorescence (PDA). In spite of any acid-induced modications to the cellwall polymers, the walls of the separated cells remained intact, even after hydrolysis for up to 2 h (results not shown). Total VICS was also induced within 45 min by treatment in 01 M H SO , 01 2 4 or 005 M TFA at 100C, and after mild acid treatment the walls of the separated cells continued to exhibit PDA (Fig 4). Some of the cells exhibited PDA internally, indicative of soluble phenolics (Fig 4). Extraction of sugarbeet tissue sections with 005 M Na CO , 2 3 20 mM NaBH at 0C overnight followed by neutral4 isation with acetic acid, a procedure which de-esteries the uronyl methyl esters and reduces the potential for b-eliminative degradation, reduced the rate of VICS in 01 M TFA at 100C by only a small amount (Fig 5). Interestingly, pre-soaking the fresh or de-esteried tissue sections in 05 M CaCl for 2 h increased the rate 2 of VICS in the hot, dilute acid (Fig 5).

Possible role of ferulic acid dehydrodimers in sugarbeet texture

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Fig 4. Autouorescence at pH 10 of sugarbeet tissue incubated in 20 mM H SO for 20 min at 100C. Bar50 km. 2 4

yellow in colour, indicative of phenolic components (Parker and Waldron 1995). Separated cells retained their overall integrity, but no longer exhibited PDA. The eect of alkali on cellcell adhesion was investigated further. The CDTA-extracted tissues were subjected to a series of less-severe extractions based on the procedure used to investigate thermal stability of texture in CWC tissues (Parker and Waldron 1995). Extraction in 005, 01 and 025 M KOH for up to 3 h resulted in signicant VICS, particularly in the parenchyma tissues (Table 1). There was little noticeable change in the parenchymal PDA. However, extraction for up to 24 h resulted in both total VICS and loss of PDA. Beetroot tissues behaved in a similar manner. Eect of cellwall degrading enzymes Incubation of sugarbeet tissue with puried endoxylanase had no eect on either VICS or PDA. This contrasts with CWC tissues which underwent total VICS after similar treatment (Parker and Waldron 1995). Carbohydrate composition of CWM CWM was prepared from sugarbeet and beetroot storage tissues by a method designed to remove intracellular components whilst minimising mechanical and chemical damage to the cellwall polymers. The yield of pure CWM was 20 and 18 g kg~1 for sugarbeet and beetroot, respectively, and included parenchyma and vascular tissues. In both beet preparations, the carbohydrate composition was rich in pectic polysaccharides,

Treatment of beetroot tissue sections with hot water (100C) resulted in tissue softening within 25 min, and signicant VICS within 35 min. Hence, beetroot tissues exhibited signicantly less thermal stability of texture compared with sugarbeet. Treatment with 01 M TFA induced total VICS in beetroot tissues within 20 min and, as in sugarbeet, the separated beetroot cells retained a strong wall and internal PDA. Eect of chelating agents and alkali Extraction of sugarbeet tissue sections with CDTA (005 M) at 20C for 16 h or at 100C for 1 h did not induce VICS. In contrast, treatment of tissue sections with degassed 8 M NaOH containing 20 mM NaBH 4 caused complete VICS within 3 h at 20C, and 5 min at 100C. The alkali also caused the tissue to become

Fig 5. Graph showing time-course of VICS of sugarbeet tissue in 005 M TFA, 100C. Symbols : x, fresh control heated in water only ; =, fresh tissue ; K, fresh tissue pre-soaked in 05 M CaCl ; +, fresh tissue pre-treated with cold Na CO (005 M) and 2 neutralised ; ) fresh tissue pre-treated with cold Na 2 CO , neutralised, and pre-soaked in 01 M CaCl .3 2 3 2

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TABLE 1 VICS and PDA of sugarbeet tissues after extraction of cellwall components Cell separation CDTA, 24 h CDTA 100 C, 1 h 005 M KOH, 3 h 01 M KOH, 3 h 025 M KOH, 3 h 005 M KOH, 24 h 01 M KOH, 24 h 025 M KOH, 24 h [[[[[ [[[[[ [[]]] [[]]] []]]] []]]] ]]]]] ]]]]] PDA pH \ 10 ]]]]] ]]]]] ]]]]] []]]] [[[[[ [[[]] [[[[] [[[[[ PDA pH \ 45 [[[[[ [[[[[ [[[[[ [[[[[ [[[[[ [[[[[ [[[[[ [[[[[

K W W aldron et al

as inferred from the levels of uronic acid, Ara and Gal (Table 2). The remaining carbohydrate consisted mainly of glucose which would have been derived mainly from cellulose. There was little hemicellulosic material, Xyl and Man making up only 34% of the cellwall carbohydrate. Approximately 20% of the CWM was unaccounted for ; this may be due to a combination of lignin which contributes to the cell walls of the vascular tissues, and possibly protein, ash and other noncarbohydrate wall components.

Phenolic composition High levels of simple phenolic acids were released from the isolated CWMs by saponication with increasing strengths of alkali. They were identied and quantied by HPLC in conjunction with analysis of their absorbance spectra obtained with a diode-array scanning detector (Waldron et al 1996b). Abbreviations used in Table 3 are dened in the materials and methods section.

TABLE 2 Carbohydrate composition of cell walls of sugarbeet (SB) and beetroot (BR) storage tissues (mol%) Y ield (%CW M) Rha SB BR 20 18 18 14 Fuc 03 03 Ara 309 235 Xyl 21 19 mol% Man 12 17 Gal 63 64 Glc 313 252 Glc1a 25 31 UA 261 397 790 821 T otal (mg g~1)

a Glucose released by hydrolysis in 1 M H SO at 100C 2 4 TABLE 3 Phenolic composition of sugarbeet (SB) and beetroot (BR) CWMs (kg g~1) Extraction conditions 01 M KOH, 1 h SB pBA VA pBAL VAN tCA 8,8@DiFA(A) tFA 8,8@DiFA 8,5@DiFA cFA 5,5@DiFA 8-O-4@DiFA 8,5@DiFA(B) 21 54 35 573 68 0 1597 0 32 407 517 420 341 BR 0 0 0 613 80 0 2725 0 0 333 366 256 239 01 M KOH, 24 h SB 46 0 15 0 0 0 2166 0 50 360 812 166 689 BR 16 32 16 165 68 0 3550 0 459 254 534 864 467 1 M KOH, 24 h SB 85 97 23 179 89 315 303 137 29 238 46 82 51 BR 43 92 22 114 0 288 702 255 0 52 0 0 0 2 M KOH, 24 h SB 25 20 19 56 0 0 38 0 0 4 0 0 0 BR 0 0 14 27 16 0 25 2 0 0 0 0 0 SB 177 171 92 808 157 315 3797 137 111 795 138 594 415 BR 59 124 52 920 164 288 6347 275 459 592 90 342 285 T otal

Possible role of ferulic acid dehydrodimers in sugarbeet texture Sugarbeet Ferulic acid (FA) was the most abundant phenolic component in the CWM, comprising 77% of the total phenolic complement (Table 3). Eighty-three percent of the monomeric FA was in the trans-form. The small quantity of cis-FA may have resulted from light-induced isomerisation, although care had been taken to avoid undue exposure to light. We observed that direct exposure of alkaline extracts to intense light increased the proportion of cis-FA but had no discernable eect on the other components (results not shown). The bulk of the remaining phenolic components consisted of FA dehydrodimers. The most prominant of these were 8-O4@DiFA (94%) and 8,5@DiFA benzofuran form (66%). 8,5@DiFA and the more commonly known 5,5@DiFA were found in smaller quantities (18% and 22%, respectively). 8,8@DiFA and 8,8@DiFA aryltetralin form were present, but only as minor components. Over 20% of the quantied, alkali-soluble wall-bound phenolics comprised FA dehydrodimers, indicating a high degree of polymer cross-linking. A number of other phenolics were also detected. Of these, vanillin was the main-nonFA phenolic, but quantiable levels of p-OH-benzoic acid, vanillic acid, p-OH-benzaldehyde and transcoumaric acid were also present. The FA monomers and dimers were almost totally extracted by 01 M KOH treatment for 1 h and 24 h. Only small amounts of phenolics were extracted by the 1 and 2 M KOH treatments. Interestingly, more than half of the trans-FA remained in the wall after the initial 01 M KOH, 1 h extraction, whilst over 75% of the dehydrodimers were released. This suggests dierences in the ease of saponication of the phenolic esters as reported in Chinese water chestnut (Parr et al 1996 ; Waldron et al 1996a). Beetroot The types of phenolics detected and their ease of extraction were very similar to those in sugarbeet. However, beetroot CWM contained more trans-FA, but approximately half the quantity of FA dehydrodimers. This is discussed further below.

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DISCUSSION Heat-induced cell separation in most vegetables is thought to involve the b-eliminative degradation of the pectic polymers of the middle lamella, thereby reducing the strength of cellcell adhesion (Van-Buren 1979 ; Greve et al 1994 ; Brett and Waldron 1996). In sugarbeet, hot-water treatment results in minimal tissue softening and cell separation, which suggests that the chemistry of cellcell adhesion diers considerably from that in vegetables such as potato which softens rapidly. The resistance of sugarbeet to softening in cold or hot CDTA also indicates that the cross-linking of pectic

polymers via Ca2` is not the limiting factor in cellcell adhesion. As has been shown for Chinese water chestnut (Parker and Waldron 1995), the presence of high levels of ferulic acid dimers able to cross-link cellwall polymers provide an explanation for the thermal stability of texture (TST) exhibited by sugarbeet tissues. This is supported by the observation that, as in Chinese water chestnut (CWC), their removal by gradedextraction in dilute KOH is accompanied by VICS (Parker and Waldron 1995). The ferulic-acid dimers of Beta vulgaris are likely to cross-link pectic polymers via the Ara-rich and possibly Gal-containing neutral side-chains to which most monomeric FA is esteried (Colquhoun et al 1994 ; Ralet et al 1994). The relatively rapid induction of VICS in dilute, hot acid indicates that Ara residues are involved in cell adhesion ; 01 M TFA at 100C preferentially hydrolyses glycosidic linkages involving Ara side-chains because glycosidic linkages between arabinofuranose residues are much more acid labile than those between hexoses (Lindberg et al 1975 ; Fry 1988 ; Parker and Waldron 1995). The attachment of FA and its derivatives to Gal would explain the retention of signicant PDA after treatment in hot, dilute acid. The involvement of cross-linked pectic polysaccharides in TST would also explain why sugarbeet eventually exhibits a small degree of VICS if heated in water at 100C for several hours. Prolonged heating probably results in enough b-eliminative depolymerisation of the highly cross-linked polyuronide components to weaken cell adhesion. This contrasts with tissues of CWC in which the FA dimers are thought to cross-link the more heat-stable arabinoxylan hemicelluloses (Parr et al 1996 ; Waldron et al 1996a). Furthermore, over 40% of the FA in CWC cell walls is in dimer form so that heat-induced cell separation does not occur and thermal-stability of texture is very high. The contrasting susceptibility shown by beet and CWC tissue to pure, specic endoxylanase can also be explained by the dierent cellwall components crosslinked by the FA dimers. In CWC, the cross-linked arabinoxylan is vulnerable to endoxylanase activity and the cells separate : a clear indication of the involvement of the xylan moiety in TST. In sugarbeet, which has a low xylan content (as inferred from the levels of cell wall Xyl), endoxylanase has no eect on cell adhesion. The beetroot exhibited much less thermal stability of texture than sugarbeet. This may reect the relatively lower quantities of wall-bound FA dehydrodimers and a corresponding lower degree of pectic cross-linking. However, other dierences in wall chemistry may also inuence dissolution of the pectic polysaccharides, including the degree of methylation of the uronide component (Brett and Waldron 1996). In addition to the role of the dehydrodimers in conferring thermal stability of texture, their ability to crosslink cellwall polysaccharides is likely to have a

228 profound eect on the mechanical properties of the wall at a cellular level and consequently, at the tissue level. The importance of such phenolic dimers in cellcell adhesion has been highlighted by Kato et al (1994) who obtained evidence for the involvement of cellwall phenolics in cell aggregation in rice cell cultures. They may play a part in the control of cellwall extensibility (Biggs and Fry 1987 ; Kamisaka et al 1990). The presence of cinnamic acid derivatives such as FA has also been associated with a resistance of cell walls to digestion by ruminants (Hartley et al 1992 ; Besle et al 1994). In the underground storage organs of Beta vulgaris, the phenolic moieties may be of importance in protecting against pathogenic invasion (Parker and Waldron 1995). It is clear that 5,5@DiFA forms only a small part (\10%) of the total FA dehydrodimer pool in sugarbeet and beetroot cell walls. This complements the work of Ralph et al (1994) and Parr et al (1996), who identied several dimers in addition to 5,5@DiFA in cell walls of grasses and Chinese water chestnut, respectively. It is also consistent with the results of Wallace and Fry (1994) who demonstrated that peroxidase/H O 2 2 catalysed oxidative coupling of model feruloyl esters in vitro resulted in the formation of a cyclic dimer which exceeded the yield of 5,5@DiFA.

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ACKNOWLEDGEMENT This work was funded by the UK Office of Science and Technology.

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