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Ripening-related changes in raspberry cell wall composition and structure
Phytochemistry 56 (2001) 423±428
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Ripening-related changes in raspberry cell wall composition and structure Derek Stewart *, Peitro P.M. Iannetta, Howard V. Davies Unit of Plant Biochemistry, Department of Cellular and Environmental Physiology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK Received 24 November 1999; received in revised form 24 May 2000
Abstract Cell walls were prepared from the fruit of two cultivars of raspberry at three stages of ripening; green, white and red (ripe). The cultivars, Glen Clova and Glen Prosen, are subjectively classi®ed, at harvest by growers, as soft and ®rm fruit, respectively. The cell walls were analysed for neutral sugar composition, uronic acid content, degree of methyl esteri®cation, lignin and ferulic acid-derived dehydrodimers. Solid-state 13C NMR and diuse re¯ectance infrared (DRIFT) spectra were acquired for the cell wall residues. For both cultivars the progression from green to white produced minimal changes, save for a reduction in pectin. NMR analyses indicated that the solubilized pectin was acetylated. Progression to the red (ripe) stage, in both cultivars, was accompanied by a reduction in the ordered cellulose and a dramatic reduction in pectin content and the degree of methyl-esteri®cation. Signi®cantly, the softer fruit (Glen Clova) exhibited greater reductions in both parameters, implicating increased pectin hydrolysis, as one of the main factors contributing to the dierence in ®rmness between the cultivars. A relative increase in cell wall-associated protein was seen at the red stage. The nature and function of the protein(s) are, as yet unknown. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Rubus; Ripening; Cell-wall; Firmness; Structure
1. Introduction The mechanisms and processes behind the chemical and structural changes in the cell walls of fruit during ripening have been the focus of much research (Brady, 1987; Waldron et al., 1997; Brownleader et al., 1999). However, the ripening of soft fruit, such as strawberry and, in particular, raspberry, has been poorly documented. For raspberry at least, there may be several reasons for this; a limited market size, a short post-harvest shelf-life and, to a lesser extent, the diculty in separating the cell wall from the seed. Recent studies have shown that the inclusion of fresh fruit in the diet improves general health. These improvements arise from an increased consumption of cell wall-related dietary ®bre (Eastwood, 1992; MacDougall et al., 1995) and ingestion of antioxidant plant phenolics (Sarkar et al., 1996), such as the anti-carcinogen, ellagic acid (Maas et al., 1991; Rommel and Wrolstad, 1993). The promotion of raspberries, or for that matter any
* Corresponding author. Tel.: +44-1382-562731; fax: 44-1382-562426. E-mail address: [email protected] (D. Stewart).
other fruit, as being bene®cial for health will mean that more data will be required regarding the composition and structure of the fruit (both walls and contents) during ripening, at harvest and during storage. To this end we studied raspberry and have devised a simple, but eective, method to fractionate the fruit cell walls and seeds. Within the raspberry family there are, like most fruit, varietal dierences with respect to speci®c parameters, especially sweetness and ®rmness. The latter is particularly important since excessive handling leads to bruising and spoilage resulting in unpalatable fruit with a reduced shelf-life. The (bio)chemical basis behind these dierences, in particular ®rmness, have yet to be established. Here we report the changes in cell wall composition and structure, at three stages of ripening, of two cultivars of raspberry, Glen Clova and Prosen, subjectively classi®ed by growers as soft and ®rm, respectively. 2. Materials and methods Two raspberry genotypes, Glen Clova and Prosen, were selected from the breeding program of SCRI. The fruits were sampled from ®eld-grown plants at three
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00410-6
424
D. Stewart et al. / Phytochemistry 56 (2001) 423±428
stages of ripening; green, white and red (ripe). The fruits were homogenized in 20 mM sodium acetate (pH 4.5) containing 0.05% Triton X-100 then poured onto a 0.5 mm sieve. The homogenate was then gently pressed through the sieve using the ¯at bottom of a beaker. The material passing through was collected and re-sieved as before until no seeds remained. The resultant cell wall suspension was then washed twice with distilled water, centrifuged (2000 g) and the pellet was boiled in 80% ethanol for 30 min, washed (2) with neat ethanol, ®ltered and dried at 40 C for 16 h to give a crude cell wall (CCW) preparation. The CCWs were subject to Soxhlet extraction for 16 h with acetone as the solvent. (This removed virtually all residual colour although the red stage CCW did retain a pink tinge.) Following Soxhlet extraction the CCWs were kept in a dessicator until required. Karl±Fischer analysis showed that the CCWs had water contents of 71%. These residues were used for diuse re¯ectance Fourier-transform infra-red (DRIFT) and solid-state NMR spectroscopic analyses as described by Stewart et al. (1997). Brie¯y, Fourier-transform infrared spectroscopy was performed on a Bruker IFS 66 FT-IR spectrometer using a Diuse Re¯ectance FT-IR (DRIFT) cell (Graseby-Specac, Worthington, UK). Two hundred scans were acquired at a resolution of 4 cm 1. Cross Polarisation/Magic Angle Spinning Nuclear Magnetic Resonance (CP/MAS NMR) spectra were acquired on a Chemagnetics 300 MHz NMR spectrometer operating at 75.46 MHz using an air-bearing probe. The spinning rate was 4 kHz, contact time 1 ms, acquisition time 41 ms, sweep width 400 ppm and a delay between pulses of 2 s. Ten thousand transients were accumulated for each sample. Neutral monosaccharides were determined using the phenol±sulphuric method as described by Stewart et al. (1995). The uronic acids were determined using the modi®ed sulfamate/m-hydroxydiphenyl method of Filisetti-Cozzi and Carpita (1991) whilst the degree of methyl esteri®cation was determined by measuring the methanol released following hydrolysis of the ester using the method of Knee (1978). Ester-bound cinnamic acid-derived dehydrodimers were analysed for using reverse phase HPLC following alkaline (1 M NaOH) hydrolysis of the CCWs as described by Grabber et al. (1995). Lignin content was determined using the acetyl bromide/perchloric acid method as described by Iiyama and Wallis (1988) but the levels were low (41 [0.5]%) and are not reported here. All analyses are the mean of triplicates with the exception of the uronic acid and methyl ester data which are the mean of ®ve analyses. Fruit ®rmness was determined by recording the force necessary to penetrate the skin of a raspberry druplet. A penetrometer (Pioden Controls Ltd, UK) with a loadcell (UF1), range 0±100 g was used in conjunction with a motorized platform. The penetrator was a needle
tapered to a 0.5 mm diameter ¯at face and advanced at 0.3 mm s 1 with a full penetration depth of 5 mm. The force, recorded as milli-Newtons (mN), was recorded after each motorized advance. The reading prior to druplet burst was taken to be the ®rmness. 3. Results and discussion The compositional data of the CCWs are shown in Table 1 and the corresponding DRIFT and solid-state NMR spectra in Figs. 1 and 2, respectively. The progression from green to white produced minimal changes in the composition of the CCWs and these were largely restricted to a reduction in the level of pectin (as uronic acid) and the degree of pectin methyl-esteri®cation. The corresponding DRIFT spectra show little, if any, change. This change is restricted to a broadening of the pectic CO2H/CO2 and methyl ester CH3 absorbances centered at 1600±1660 and 1420 cm 1, respectively, in the white stages of both cultivars. This is probably due to cell wall hydrolysis (Iannetta et al., 1999) producing a range of dierent chemical environments thereby broadening the range of absorbance frequencies. There is evidence of reduced level of uronic acid and degree of methyl esteri®cation in the solid-state NMR spectra. There are small but signi®cant reductions in intensity of the pectic acid/ester carbonyl resonance at 176±171 ppm and the associated ester OCH3 at 53 ppm. There are no observable reductions in the pectic carbohydrate signals as these are masked by the overwhelming presence of the other cellulose and non-cellulosic-derived signals. However there is a reduction in the acetyl CH3 resonance at 22 ppm. Where the plant cell wall is concerned the acetyl group is invariably associated with non-cellulosic polysaccharides (NCPs) (Hulleman et al., 1994; Stewart et al., 1995) and since the principal change in NCP has been a reduction in pectin it is likely that the acetyl groups are present on pectin. Acetylated pectins have previously been reported in other fruit (MacDougall et al., 1996). Although the cell wall neutral sugar compositional data showed little change over the progression from green to white the pectin contents and measured ®rmness of both of the cultivars reduced by similar levels of 19 and 12%, and, 31 and 33%, respectively, for Glen Clova and Glen Prosen, respectively (Table 2). It was with the progression from the white to red (ripe) stage that the most signi®cant changes in both composition and cell wall structure occurred and where dierence between cultivars became evident. Both cultivars experienced a net reduction in NCPs (almost entirely accounted for by pectin) of 27 and 24.8% for Glen Clova and Glen Prosen, respectively. (The corresponding reductions on the progression from green to white were 10.0 and 9.7%, respectively.) This suggests
D. Stewart et al. / Phytochemistry 56 (2001) 423±428
425
Table 1 The neutral sugar, uronic acid and methyl esteri®cation content of CCWs prepared from Glen Prosen and Glen Clova raspberries at the green, white and red (ripe) stagesa Rha
Ara
Xyl
Man
Gal
Glc
UA
%Me
Glen Clova Green White Red
0.4 (0.1) 0.3 (0.1) 0.3 (0.1)
2.2 (0.1) 2.2 (0.1) 2.1 (0.1)
23.2 (1.0) 23.5 (0.8) 26.6 (0.4)
0.5 (0.1) 0.5 (0.1) 0.4 (0.1)
1.7 (0.3) 1.2 (0.2) 1.1 (0.1)
0.6 (0.2) 0.7 (0.2) 0.5 (0.1)
26.8 (1.4) 21.6 (1.5) 5.5 (1.1)
39 (5) 31 (6) 7 (4)
Glen Prosen Green White Red
0.4 (0.1) 0.4 (0.1) 0.2 (0.1)
2.9 (0.1) 3.0 (0.1) 2.1 (0.2)
24.1 (0.3) 24.0 (0.2) 24.6 (0.3)
0.7 (0.1) 0.6 (0.1) 0.6 (0.1)
2.7 (0.1) 2.7 (0.1) 2.1 (0.2)
1.3 (0.1) 1.4 (0.2) 1.4 (0.1)
28.0 (1.3) 22.0 (1.1) 9.7 (0.8)
41 (6) 35 (4) 14 (3)
a Figures in parentheses are the standard errors. Non-cellulosic neutral monosaccharide and uronic acid contents are expressed as mg/100 mg cell wall. The non-cellulosic neutral monosaccharide contents are the mean of triplicates whilst. The uronic acid contents and % methyl esteri®cation are the mean of 5 replicates.
that pectin hydrolysis had advanced, at least during the latter stages of ripening, such that the lower molecular weight fragments were lost during preparation of the CCWs. Varietal dierences were evident within the individual neutral monosaccharide composition. Glen Prosen experienced a relative reduction in arabinose and galactose and to a lesser extent, rhamnose. The concurrent reduction of these monosaccharides is often indicative of rhamnogalacturonan degradation and has been reported in other fruit (Gross and Sams, 1984; Huysamer et al., 1997). Glen Clova underwent small reductions in all the neutral monosaccharides with the exception of xylose, the level of which rose signi®cantly. However, by far the most signi®cant change, in both genotypes, was the large reduction in the levels of pectin and degree of methyl ester. These were reduced by 74.5 and 77.4% and 55.9 and 60.0%, respectively for Glen Clova and Glen Prosen. This varietal dierence in the pectin levels may give some clues to the origin of the measured differences in ®rmness. At the red (ripe) stage Glen Prosen was signi®cantly ®rmer (37%) than Glen Clova (Table 2). The ®rmness measurements were done on individual druplets, a complex tissue comprised of hundreds of cells (B. Williamson, personal communication, 2000). It is logical, therefore, to assume that this dierence in ®rmness may be contributed to, at least in part, by cellto-cell adhesion. It is now accepted that there are several factors contributing to cell-to-cell adhesion. Two of these are pectin (and its relative degree of methylation) (Jarvis, 1984; Brady, 1987) while another is the presence of intercellular cinnamic acid-derived bridges in the form of ferulic acid dehydrodimers (Parker and Waldron, 1995; Parr et al., 1996; Waldron et al., 1996). Analyses showed that none of the CCWs analyzed here showed any evidence of having these dimers (data not shown). This suggests that pectin may be one of the key factors determining varietal ®rmness with reduced levels of pectin backbone and methyl ester hydrolysis during ripening in the ®rmer variety, Glen Prosen, leading to a
comparatively greater molecular weight pectic polymer, more able to maintain cell wall cohesion. Indeed it has been previously shown that, at the red (ripe) stage, Glen Clova (softer fruit) had a relatively greater pectin methylesterase (PME) activity than Glen Prosen, thereby allowing polygalacturonase (PG) more substrate to work with leading to a reduced residual uronic acid content (Iannetta et al., 1999). At the red stage there was also evidence of extensive changes in the structure of the cell walls. Their DRIFT spectra are signi®cantly dierent from those obtained from the green and white fruit (Fig. 1). The intensity of the ester CO peak, centred at 1740 cm 1, is almost double that seen in for the other stages, despite a dramatic reduction in the level of pectin methyl ester (OCH3, 1420 cm 1, Table 1). This is may due to digestion by cell wall hydrolases (Iannetta et al., 1999) during ripening with the non-acetylated regions of the cell wall polymers being digested ®rst resulting in a consequential increase in acetylation. The DRIFT spectra also show a signi®cant reduction in crystalline or ``ordered'' cellulose-related absorbances. This was re¯ected in the relative reduction in absorbance at 1100 cm 1 and the increase in intensity and line broadening of the amorphous cellulose-related absorbance at 900 cm 1 (Michell, 1990). (Although xyloglucan can also absorb at this frequency it is reported to accounts for less than 6% of the total cell wall in raspberry; Iannetta et al., 1998.) The progression from green to white had minimal eect on the degree of ordered cellulose save an apparent reduction in the C4 resonance, attributed to crystalline cellulose (Stewart et al., 1997), at 89 ppm with a concomitant increase in the corresponding amorphous cellulose peak at 85 ppm (Fig. 2). Also the C2,3,5 resonances were slightly less sharp, or de®ned, at the resonance maxima (73 ppm). However, the progression to the red (ripe) stage saw an large change in the ordered cellulose resonances, with reductions in the intensity of the C1,4,&6 resonances (65, 89 and 105 ppm) and increases in
426
D. Stewart et al. / Phytochemistry 56 (2001) 423±428
Fig. 2. The solid-state NMR spectra of crude cell wall preparations from green, white and red (ripe) fruit from (a) Glen Clova and (b) Glen Prosen, respectively.
Fig. 1. The DRIFT spectra of crude cell wall preparations from green, white and red (ripe) fruit from (a) Glen Clova and (b) Glen Prosen, respectively.
the corresponding amorphous resonances (63, 85 and 101 ppm) (Earl and Vanderhart, 1981; Attala and VanderHart, 1984). Also, the main C2,3,5 resonances collapsed to give one main peak with an up®eld shoulder. This is in agreement with the reported increase in cellulase
activity on progression from the white to red stage in raspberry fruit (Iannetta et al., 1999). The pectic methyl ester resonance at 53 ppm also reduced in intensity, at the red ripe stage, but the corresponding acid/ester carbonyl resonance centred at 174 ppm was still signi®cant despite a large reduction in uronic acid content (Table 1). This may be due to the digestion of the cell wall by glycosylases resulting in a relative increase in cell wall associated protein. The carbonyl resonance of the (protein) amide bond is also, centred at 174 ppm (Kemp, 1991), and the concurrent relative reduction and increase of pectic and protein carbonyl resonance intensities, respectively, results in a small but signi®cant resonance at 174 ppm. This relative increase in protein was corroborated by the corresponding DRIFT spectra (Fig. 1). At both the red stages, the spectra have signi®cant absorbances at 1650
D. Stewart et al. / Phytochemistry 56 (2001) 423±428 Table 2 The measured ®rmnesses (mN) of druplets from Glen Clova and Glen Prosen at three stages of ripening Genotype Subjective ®rmness (at harvest) Ripening stage Green White Red
Glen Clova
Glen Prosen
Soft
Firm
605 415 133
748 497 210
and 1550 cm 1, regions classically associated with amide (protein) absorbance (Kemp, 1991). Slight additional evidence for this is seen at the higher wavenumber end of the DRIFT spectra (Fig. 1 inset). The water hydration absorbance at 3100±3700 cm 1 is similar in both intensity and line shape for the green and white stages within variety. However, at the red (ripe) stage the absorbance has broadened and the maxima has shifted from 3360 to 3410 cm 1, the region of absorbance for the main N H stretch of secondary amides (including proteins) (Kemp, 1991). Further supportive evidence for the presence of protein in the red (ripe) stage cell walls is seen in the increase in the shoulder of the absorbance centered at 2900 cm 1, a region in which there is an additional, albeit weak, N H stretch reported to be representative of N H hydrogen bonding (Kemp, 1991). The origins of these protein absorbances are, as yet, unclear although one possible explanation may be that during the preparation of the CCWs the cytoplasmic proteins have become associated/complexed with the cell wall phenolics and intractably-bound anthocyanins. The complexation of proteins and phenolics has been well reported (McManus et al., 1985). 4. Conclusion Raspberry appears to follow a classic fruit ripening scenario involving pectin demethylation, hydrolysis and solubilization particularly at the red (ripe) stage. At this latter stage extensive cellulose degradation was evident, as was an apparent relative increase in cell wall bound protein. Hydrolytic reduction of pectin molecular weight and demethylation appears to play a key role in contributing to the dierence in ®rmness between the two genotypes and is likely to contribute to the short shelflife of raspberry. Acknowledgements The authors acknowledge funding from the Scottish Executive Regional Aairs Department (D.S. & H.V.D.) and the Ministry of Agriculture, Food and Fisheries (P.P.M.I.).
427
References Attala, R., VanderHart, D.L., 1984. Native cellulose Ð a composite of 2 distinct crystalline forms. Science 223, 283±285. Brady, C.J., 1987. Fruit ripening. Annu. Rev. Plant Physiol. 38, 155± 178. Brownleader, M.D., Jackson, P., Mobasheri, A., Pantelides, A.T., Sumar, S., 1999. Molecular aspects of cell wall modi®cations during fruit ripening. Crit. Rev. Food Sci. Nutr. 39, 149±164. Earl, W.L., VanderHart, D.L., 1981. Observations by high-resolution C13 nuclear magnetic-resonance of cellulose-I related to morphology and crystal-structure. Macromolecules 14, 570±574. Eastwood, M.A., 1992. The physiological eect of dietary ®ber Ð an update. Annu. Rev. Nutr. 12, 19±35. Filisetti-Cozzi, T.M.C.C., Carpita, N.C., 1991. Measurement of uronic-acids without interference from neutral sugars. Analyt. Biochem. 197, 157±162. Grabber, J.H., Hat®eld, R.D., Ralph, J., Zon, J., Amrhein, N., 1995. Ferulate cross-linking in cell-walls isolated from maize cell-suspensions. Phytochemistry 40, 1077±1082. Gross, K.C., Sams, C.E., 1984. Changes in cell-wall neutral sugar composition during fruit ripening Ð a species survey. Phytochemistry 23, 2457±2461. Hulleman, S.H.D., van Hazendonk, J.M., van Dam, J.E.G., 1994. Determination of crystallinity in native cellulose from higher-plants with diuse-re¯ectance Fourier-transform infrared-spectroscopy. Carbohydr. Res. 261, 163±172. Huysamer, M., Greve, L.C., Labavitch, J.L., 1997. Cell wall metabolism in ripening fruit 8. Cell wall composition and synthetic capacity of two regions of the outer pericarp of mature green and red ripe c.v. Jackpot tomatoes. Physiol. Plantarum 101, 314± 322. Iannetta, P.P.M., Fry, S.C., Stewart, D., McDougall, G.J., Davies, H.V., 1998. Raspberry (Rubus ideaus) fruit cell-wall composition: xyloglucan endotransglycosylase activity and xyloglucan content. Abstracts International Cell wall Meeting, Norwich, UK, 7.15. Iannetta, P.P.M., Van den Berg, J., Wheatley, R., McMillan, G., McNicol, R.J., Davies, H.V., 1999. The role of ethylene and cell wall modifying enzymes in raspberry (Rubus idaeus) fruit ripening. Physiologia Plantarum 105, 338±347. Iiyama, K., Wallis, A.F.A., 1988. An improved acetyl bromide procedure for determining lignin in woods and wood pulps. Wood Sci. Technol. 22, 271±280. Jarvis, M.C., 1984. Structure and properties of pectin gels in plant-cell walls. Plant Cell Environ. 7, 153±164. Kemp, W., 1991. Organic Spectroscopy. Macmillan, London. Knee, M., 1978. Metabolism of polymethylgalacturonate in apple fruit cortical tissue during ripening. Phytochemistry 17, 1257±1264. Maas, J.L., Galleta, G.J., Stoner, G.D., 1991. Ellagic acid, an anticarcinogen in fruits, especially in strawberries Ð a review. Hortscience 26, 10±14. MacDougall, A.J., Needs, P.W., Rigby, N.M., Ring, S.G., 1996. Calcium gelation of pectic polysaccharides isolated from unripe tomato fruit. Carbohydr. Res. 293, 235±249. MacDougall, G.J., Morrison, I.M., Stewart, D., Hillman, J.R., 1995. Plant cell walls as dietary ®bre: range, structure, processing and function. J. Sci. Food. Agric. 70, 133±150. McManus, J.P., Davis, K.G., Beart, J.E., Ganey, S.H., Lilley, T.H., Haslam, E., 1985. Polyphenol interactions 1. Introduction Ð some observations on the reversible complexation of polyphenols with proteins and polysaccharides. J. Chem. Soc. Perkin Trans. II 9, 1429±1438. Michell, A., 1990. 2nd-derivative FT-IR spectra of native celluloses. Carbohydr. Res. 197, 53±60. Parker, M.L., Waldron, K.W., 1995. Texture of chinese water chestnut Ð involvement of cell-wall phenolics. J. Sci. Fd. Agric. 68, 337±346.
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Parr, A.J., Waldron, K.W., Ng, A., Parker, M.L., 1996. The wallbound phenolics of Chinese water chestnut (Eleocharis dulcis). J. Sci. Fd. Agric. 71, 501±507. Rommel, A., Wrolstad, R.E., 1993. Ellagic acid content of red raspberry juice as in¯uenced by cultivar, processing and environmentalfactors. J. Agric. Fd. Chem. 41, 1951±1960. Sarkar, D., Sharma, A., Talkuder, G., 1996. Plant extracts as modulators of genotoxic eects. Bot. Rev. 62, 275±300. Stewart, D., Azzini, A., Hall, A.T., Morrison, I.M., 1997. Sisal ®bres and their constituent non-cellulosic polymers. Industr. Crops 6, 17± 26.
Stewart, D., Wilson, H.M., Hendra, P.J., Morrison, I.M., 1995. A Fourier-transform infrared and Raman study of biochemical and chemical treatments of Oak (Quercus rubra) and Barley (Hordeum vulgare) straw. J. Agric. Fd. Chem. 43, 2219±2225. Waldron, K.W., Ng, A., Parker, M.L., Parr, A.J., 1996. Ferulic acid dehydrodimers in the cell walls of Beta vulgaris and their possible role in texture. J. Sci. Fd. Agric. 74, 221±228. Waldron, K.W., Smith, A.C., Parr, A.J., Ng, A., Parker, M.L., 1997. New approaches to understanding and controlling cell separation in relation to fruit and vegetable texture. Trends Fd. Sci. Technol. 8, 213±221.
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Ripening-related changes in raspberry cell wall composition and structure Derek Stewart *, Peitro P.M. Iannetta, Howard V. Davies Unit of Plant Biochemistry, Department of Cellular and Environmental Physiology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK Received 24 November 1999; received in revised form 24 May 2000
Abstract Cell walls were prepared from the fruit of two cultivars of raspberry at three stages of ripening; green, white and red (ripe). The cultivars, Glen Clova and Glen Prosen, are subjectively classi®ed, at harvest by growers, as soft and ®rm fruit, respectively. The cell walls were analysed for neutral sugar composition, uronic acid content, degree of methyl esteri®cation, lignin and ferulic acid-derived dehydrodimers. Solid-state 13C NMR and diuse re¯ectance infrared (DRIFT) spectra were acquired for the cell wall residues. For both cultivars the progression from green to white produced minimal changes, save for a reduction in pectin. NMR analyses indicated that the solubilized pectin was acetylated. Progression to the red (ripe) stage, in both cultivars, was accompanied by a reduction in the ordered cellulose and a dramatic reduction in pectin content and the degree of methyl-esteri®cation. Signi®cantly, the softer fruit (Glen Clova) exhibited greater reductions in both parameters, implicating increased pectin hydrolysis, as one of the main factors contributing to the dierence in ®rmness between the cultivars. A relative increase in cell wall-associated protein was seen at the red stage. The nature and function of the protein(s) are, as yet unknown. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Rubus; Ripening; Cell-wall; Firmness; Structure
1. Introduction The mechanisms and processes behind the chemical and structural changes in the cell walls of fruit during ripening have been the focus of much research (Brady, 1987; Waldron et al., 1997; Brownleader et al., 1999). However, the ripening of soft fruit, such as strawberry and, in particular, raspberry, has been poorly documented. For raspberry at least, there may be several reasons for this; a limited market size, a short post-harvest shelf-life and, to a lesser extent, the diculty in separating the cell wall from the seed. Recent studies have shown that the inclusion of fresh fruit in the diet improves general health. These improvements arise from an increased consumption of cell wall-related dietary ®bre (Eastwood, 1992; MacDougall et al., 1995) and ingestion of antioxidant plant phenolics (Sarkar et al., 1996), such as the anti-carcinogen, ellagic acid (Maas et al., 1991; Rommel and Wrolstad, 1993). The promotion of raspberries, or for that matter any
* Corresponding author. Tel.: +44-1382-562731; fax: 44-1382-562426. E-mail address: [email protected] (D. Stewart).
other fruit, as being bene®cial for health will mean that more data will be required regarding the composition and structure of the fruit (both walls and contents) during ripening, at harvest and during storage. To this end we studied raspberry and have devised a simple, but eective, method to fractionate the fruit cell walls and seeds. Within the raspberry family there are, like most fruit, varietal dierences with respect to speci®c parameters, especially sweetness and ®rmness. The latter is particularly important since excessive handling leads to bruising and spoilage resulting in unpalatable fruit with a reduced shelf-life. The (bio)chemical basis behind these dierences, in particular ®rmness, have yet to be established. Here we report the changes in cell wall composition and structure, at three stages of ripening, of two cultivars of raspberry, Glen Clova and Prosen, subjectively classi®ed by growers as soft and ®rm, respectively. 2. Materials and methods Two raspberry genotypes, Glen Clova and Prosen, were selected from the breeding program of SCRI. The fruits were sampled from ®eld-grown plants at three
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00410-6
424
D. Stewart et al. / Phytochemistry 56 (2001) 423±428
stages of ripening; green, white and red (ripe). The fruits were homogenized in 20 mM sodium acetate (pH 4.5) containing 0.05% Triton X-100 then poured onto a 0.5 mm sieve. The homogenate was then gently pressed through the sieve using the ¯at bottom of a beaker. The material passing through was collected and re-sieved as before until no seeds remained. The resultant cell wall suspension was then washed twice with distilled water, centrifuged (2000 g) and the pellet was boiled in 80% ethanol for 30 min, washed (2) with neat ethanol, ®ltered and dried at 40 C for 16 h to give a crude cell wall (CCW) preparation. The CCWs were subject to Soxhlet extraction for 16 h with acetone as the solvent. (This removed virtually all residual colour although the red stage CCW did retain a pink tinge.) Following Soxhlet extraction the CCWs were kept in a dessicator until required. Karl±Fischer analysis showed that the CCWs had water contents of 71%. These residues were used for diuse re¯ectance Fourier-transform infra-red (DRIFT) and solid-state NMR spectroscopic analyses as described by Stewart et al. (1997). Brie¯y, Fourier-transform infrared spectroscopy was performed on a Bruker IFS 66 FT-IR spectrometer using a Diuse Re¯ectance FT-IR (DRIFT) cell (Graseby-Specac, Worthington, UK). Two hundred scans were acquired at a resolution of 4 cm 1. Cross Polarisation/Magic Angle Spinning Nuclear Magnetic Resonance (CP/MAS NMR) spectra were acquired on a Chemagnetics 300 MHz NMR spectrometer operating at 75.46 MHz using an air-bearing probe. The spinning rate was 4 kHz, contact time 1 ms, acquisition time 41 ms, sweep width 400 ppm and a delay between pulses of 2 s. Ten thousand transients were accumulated for each sample. Neutral monosaccharides were determined using the phenol±sulphuric method as described by Stewart et al. (1995). The uronic acids were determined using the modi®ed sulfamate/m-hydroxydiphenyl method of Filisetti-Cozzi and Carpita (1991) whilst the degree of methyl esteri®cation was determined by measuring the methanol released following hydrolysis of the ester using the method of Knee (1978). Ester-bound cinnamic acid-derived dehydrodimers were analysed for using reverse phase HPLC following alkaline (1 M NaOH) hydrolysis of the CCWs as described by Grabber et al. (1995). Lignin content was determined using the acetyl bromide/perchloric acid method as described by Iiyama and Wallis (1988) but the levels were low (41 [0.5]%) and are not reported here. All analyses are the mean of triplicates with the exception of the uronic acid and methyl ester data which are the mean of ®ve analyses. Fruit ®rmness was determined by recording the force necessary to penetrate the skin of a raspberry druplet. A penetrometer (Pioden Controls Ltd, UK) with a loadcell (UF1), range 0±100 g was used in conjunction with a motorized platform. The penetrator was a needle
tapered to a 0.5 mm diameter ¯at face and advanced at 0.3 mm s 1 with a full penetration depth of 5 mm. The force, recorded as milli-Newtons (mN), was recorded after each motorized advance. The reading prior to druplet burst was taken to be the ®rmness. 3. Results and discussion The compositional data of the CCWs are shown in Table 1 and the corresponding DRIFT and solid-state NMR spectra in Figs. 1 and 2, respectively. The progression from green to white produced minimal changes in the composition of the CCWs and these were largely restricted to a reduction in the level of pectin (as uronic acid) and the degree of pectin methyl-esteri®cation. The corresponding DRIFT spectra show little, if any, change. This change is restricted to a broadening of the pectic CO2H/CO2 and methyl ester CH3 absorbances centered at 1600±1660 and 1420 cm 1, respectively, in the white stages of both cultivars. This is probably due to cell wall hydrolysis (Iannetta et al., 1999) producing a range of dierent chemical environments thereby broadening the range of absorbance frequencies. There is evidence of reduced level of uronic acid and degree of methyl esteri®cation in the solid-state NMR spectra. There are small but signi®cant reductions in intensity of the pectic acid/ester carbonyl resonance at 176±171 ppm and the associated ester OCH3 at 53 ppm. There are no observable reductions in the pectic carbohydrate signals as these are masked by the overwhelming presence of the other cellulose and non-cellulosic-derived signals. However there is a reduction in the acetyl CH3 resonance at 22 ppm. Where the plant cell wall is concerned the acetyl group is invariably associated with non-cellulosic polysaccharides (NCPs) (Hulleman et al., 1994; Stewart et al., 1995) and since the principal change in NCP has been a reduction in pectin it is likely that the acetyl groups are present on pectin. Acetylated pectins have previously been reported in other fruit (MacDougall et al., 1996). Although the cell wall neutral sugar compositional data showed little change over the progression from green to white the pectin contents and measured ®rmness of both of the cultivars reduced by similar levels of 19 and 12%, and, 31 and 33%, respectively, for Glen Clova and Glen Prosen, respectively (Table 2). It was with the progression from the white to red (ripe) stage that the most signi®cant changes in both composition and cell wall structure occurred and where dierence between cultivars became evident. Both cultivars experienced a net reduction in NCPs (almost entirely accounted for by pectin) of 27 and 24.8% for Glen Clova and Glen Prosen, respectively. (The corresponding reductions on the progression from green to white were 10.0 and 9.7%, respectively.) This suggests
D. Stewart et al. / Phytochemistry 56 (2001) 423±428
425
Table 1 The neutral sugar, uronic acid and methyl esteri®cation content of CCWs prepared from Glen Prosen and Glen Clova raspberries at the green, white and red (ripe) stagesa Rha
Ara
Xyl
Man
Gal
Glc
UA
%Me
Glen Clova Green White Red
0.4 (0.1) 0.3 (0.1) 0.3 (0.1)
2.2 (0.1) 2.2 (0.1) 2.1 (0.1)
23.2 (1.0) 23.5 (0.8) 26.6 (0.4)
0.5 (0.1) 0.5 (0.1) 0.4 (0.1)
1.7 (0.3) 1.2 (0.2) 1.1 (0.1)
0.6 (0.2) 0.7 (0.2) 0.5 (0.1)
26.8 (1.4) 21.6 (1.5) 5.5 (1.1)
39 (5) 31 (6) 7 (4)
Glen Prosen Green White Red
0.4 (0.1) 0.4 (0.1) 0.2 (0.1)
2.9 (0.1) 3.0 (0.1) 2.1 (0.2)
24.1 (0.3) 24.0 (0.2) 24.6 (0.3)
0.7 (0.1) 0.6 (0.1) 0.6 (0.1)
2.7 (0.1) 2.7 (0.1) 2.1 (0.2)
1.3 (0.1) 1.4 (0.2) 1.4 (0.1)
28.0 (1.3) 22.0 (1.1) 9.7 (0.8)
41 (6) 35 (4) 14 (3)
a Figures in parentheses are the standard errors. Non-cellulosic neutral monosaccharide and uronic acid contents are expressed as mg/100 mg cell wall. The non-cellulosic neutral monosaccharide contents are the mean of triplicates whilst. The uronic acid contents and % methyl esteri®cation are the mean of 5 replicates.
that pectin hydrolysis had advanced, at least during the latter stages of ripening, such that the lower molecular weight fragments were lost during preparation of the CCWs. Varietal dierences were evident within the individual neutral monosaccharide composition. Glen Prosen experienced a relative reduction in arabinose and galactose and to a lesser extent, rhamnose. The concurrent reduction of these monosaccharides is often indicative of rhamnogalacturonan degradation and has been reported in other fruit (Gross and Sams, 1984; Huysamer et al., 1997). Glen Clova underwent small reductions in all the neutral monosaccharides with the exception of xylose, the level of which rose signi®cantly. However, by far the most signi®cant change, in both genotypes, was the large reduction in the levels of pectin and degree of methyl ester. These were reduced by 74.5 and 77.4% and 55.9 and 60.0%, respectively for Glen Clova and Glen Prosen. This varietal dierence in the pectin levels may give some clues to the origin of the measured differences in ®rmness. At the red (ripe) stage Glen Prosen was signi®cantly ®rmer (37%) than Glen Clova (Table 2). The ®rmness measurements were done on individual druplets, a complex tissue comprised of hundreds of cells (B. Williamson, personal communication, 2000). It is logical, therefore, to assume that this dierence in ®rmness may be contributed to, at least in part, by cellto-cell adhesion. It is now accepted that there are several factors contributing to cell-to-cell adhesion. Two of these are pectin (and its relative degree of methylation) (Jarvis, 1984; Brady, 1987) while another is the presence of intercellular cinnamic acid-derived bridges in the form of ferulic acid dehydrodimers (Parker and Waldron, 1995; Parr et al., 1996; Waldron et al., 1996). Analyses showed that none of the CCWs analyzed here showed any evidence of having these dimers (data not shown). This suggests that pectin may be one of the key factors determining varietal ®rmness with reduced levels of pectin backbone and methyl ester hydrolysis during ripening in the ®rmer variety, Glen Prosen, leading to a
comparatively greater molecular weight pectic polymer, more able to maintain cell wall cohesion. Indeed it has been previously shown that, at the red (ripe) stage, Glen Clova (softer fruit) had a relatively greater pectin methylesterase (PME) activity than Glen Prosen, thereby allowing polygalacturonase (PG) more substrate to work with leading to a reduced residual uronic acid content (Iannetta et al., 1999). At the red stage there was also evidence of extensive changes in the structure of the cell walls. Their DRIFT spectra are signi®cantly dierent from those obtained from the green and white fruit (Fig. 1). The intensity of the ester CO peak, centred at 1740 cm 1, is almost double that seen in for the other stages, despite a dramatic reduction in the level of pectin methyl ester (OCH3, 1420 cm 1, Table 1). This is may due to digestion by cell wall hydrolases (Iannetta et al., 1999) during ripening with the non-acetylated regions of the cell wall polymers being digested ®rst resulting in a consequential increase in acetylation. The DRIFT spectra also show a signi®cant reduction in crystalline or ``ordered'' cellulose-related absorbances. This was re¯ected in the relative reduction in absorbance at 1100 cm 1 and the increase in intensity and line broadening of the amorphous cellulose-related absorbance at 900 cm 1 (Michell, 1990). (Although xyloglucan can also absorb at this frequency it is reported to accounts for less than 6% of the total cell wall in raspberry; Iannetta et al., 1998.) The progression from green to white had minimal eect on the degree of ordered cellulose save an apparent reduction in the C4 resonance, attributed to crystalline cellulose (Stewart et al., 1997), at 89 ppm with a concomitant increase in the corresponding amorphous cellulose peak at 85 ppm (Fig. 2). Also the C2,3,5 resonances were slightly less sharp, or de®ned, at the resonance maxima (73 ppm). However, the progression to the red (ripe) stage saw an large change in the ordered cellulose resonances, with reductions in the intensity of the C1,4,&6 resonances (65, 89 and 105 ppm) and increases in
426
D. Stewart et al. / Phytochemistry 56 (2001) 423±428
Fig. 2. The solid-state NMR spectra of crude cell wall preparations from green, white and red (ripe) fruit from (a) Glen Clova and (b) Glen Prosen, respectively.
Fig. 1. The DRIFT spectra of crude cell wall preparations from green, white and red (ripe) fruit from (a) Glen Clova and (b) Glen Prosen, respectively.
the corresponding amorphous resonances (63, 85 and 101 ppm) (Earl and Vanderhart, 1981; Attala and VanderHart, 1984). Also, the main C2,3,5 resonances collapsed to give one main peak with an up®eld shoulder. This is in agreement with the reported increase in cellulase
activity on progression from the white to red stage in raspberry fruit (Iannetta et al., 1999). The pectic methyl ester resonance at 53 ppm also reduced in intensity, at the red ripe stage, but the corresponding acid/ester carbonyl resonance centred at 174 ppm was still signi®cant despite a large reduction in uronic acid content (Table 1). This may be due to the digestion of the cell wall by glycosylases resulting in a relative increase in cell wall associated protein. The carbonyl resonance of the (protein) amide bond is also, centred at 174 ppm (Kemp, 1991), and the concurrent relative reduction and increase of pectic and protein carbonyl resonance intensities, respectively, results in a small but signi®cant resonance at 174 ppm. This relative increase in protein was corroborated by the corresponding DRIFT spectra (Fig. 1). At both the red stages, the spectra have signi®cant absorbances at 1650
D. Stewart et al. / Phytochemistry 56 (2001) 423±428 Table 2 The measured ®rmnesses (mN) of druplets from Glen Clova and Glen Prosen at three stages of ripening Genotype Subjective ®rmness (at harvest) Ripening stage Green White Red
Glen Clova
Glen Prosen
Soft
Firm
605 415 133
748 497 210
and 1550 cm 1, regions classically associated with amide (protein) absorbance (Kemp, 1991). Slight additional evidence for this is seen at the higher wavenumber end of the DRIFT spectra (Fig. 1 inset). The water hydration absorbance at 3100±3700 cm 1 is similar in both intensity and line shape for the green and white stages within variety. However, at the red (ripe) stage the absorbance has broadened and the maxima has shifted from 3360 to 3410 cm 1, the region of absorbance for the main N H stretch of secondary amides (including proteins) (Kemp, 1991). Further supportive evidence for the presence of protein in the red (ripe) stage cell walls is seen in the increase in the shoulder of the absorbance centered at 2900 cm 1, a region in which there is an additional, albeit weak, N H stretch reported to be representative of N H hydrogen bonding (Kemp, 1991). The origins of these protein absorbances are, as yet, unclear although one possible explanation may be that during the preparation of the CCWs the cytoplasmic proteins have become associated/complexed with the cell wall phenolics and intractably-bound anthocyanins. The complexation of proteins and phenolics has been well reported (McManus et al., 1985). 4. Conclusion Raspberry appears to follow a classic fruit ripening scenario involving pectin demethylation, hydrolysis and solubilization particularly at the red (ripe) stage. At this latter stage extensive cellulose degradation was evident, as was an apparent relative increase in cell wall bound protein. Hydrolytic reduction of pectin molecular weight and demethylation appears to play a key role in contributing to the dierence in ®rmness between the two genotypes and is likely to contribute to the short shelflife of raspberry. Acknowledgements The authors acknowledge funding from the Scottish Executive Regional Aairs Department (D.S. & H.V.D.) and the Ministry of Agriculture, Food and Fisheries (P.P.M.I.).
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