Journal of the Science of Food and Agriculture J Sci Food Agric 83:1289–1292 (online: 2003)DOI: 10.1002/jsfa.1441

Cell wall composition of vascular andparenchyma tissues in broccoli stemsS Muller,1† WG Jardine,1 BW Evans,1 RJ Vietor,1 CE Snape2‡ and MC Jarvis1∗1Chemistry Department, Glasgow University, Glasgow G12 8QQ, UK2Department of Pure and Applied Chemistry, Strathclyde University, Glasgow G1 1XL, UK

Abstract: Broccoli stems can become tough and stringy owing to excessive development of the vascularring. Thickened cell walls from the vascular ring were isolated and their composition was determined. Theywere derived principally from anatomically recognisable xylem vessels, fibres and tracheids but containedan assemblage of polysaccharides typical of primary cell walls. Their pectin content was particularly highand they contained only 6% lignin as estimated by solid state 13C NMR spectroscopy. They did not differmarkedly in composition from parenchyma cell walls within the same stems. Thus, despite their thicknessand anatomical appearance, these cell walls resembled the walls of non-woody cells in their polymercomposition. 2003 Society of Chemical Industry

Keywords: broccoli; cell wall; pectin; lignin; NMR

INTRODUCTIONUnsatisfactory textural quality of broccoli (Brassicaoleracea L var italica) is connected with toughness of‘stringy’ tissues within the stems.1 The most effectiveapproach to solving this problem will depend on thenature of the tissues in question and the reason for theirtoughness. If existing cell walls become lignified witha consequent increase in their mechanical strength,then preventing lignification will maintain texturalquality. If, on the other hand, differentiation of tissueswithin the stem leads to newly thickened or modifiedcell walls, then a more appropriate target will be thedevelopmental cues for this differentiation.

In the upper region of the flowering stem ofcertain other Brassica spp the vascular ring containsunusual thickened cell walls, which can be isolatedby a sequence of controlled homogenisation andsieving that depends on their resistance to mechanicaldisintegration.2,3 These previous studies provide botha method for preparing the problematic cell wallsfrom broccoli and a parallel for their chemical andanatomical nature, which is the subject of this paper.

MATERIALS AND METHODSPlant materialsBroccoli stems were excised immediately below thelowest flowering branch. The florets were removed and

the epidermis was excised with a razor blade to avoidcontamination of the vascular cell wall preparationwith epidermal fragments.

Cell wall preparationParenchyma and vascular cell walls were preparedseparately by detergent extraction and differentialsieving as described by McCluskey et al,2 withan additional cryo-milling step in liquid nitrogen.Anatomical purity of the cell wall preparations wasassessed by light microscopy after staining withphloroglucinol/HCl.

Chemical analysisNeutral monosaccharides were derivatised to alditolacetates by the method of Englyst and Cummings4

after hydrolysis in triplicate in sealed tubes ineither 1 M H2SO4 at 100 ◦C for 3 h or chilled 72%w/w H2SO4 followed by dilution and heating at100 ◦C for 3 h (Saeman conditions). The alditolacetates were separated by GLC on a DB-225column (15 m × 0.53 mm) with split injection, using a190–220 ◦C gradient programmed at 15 ◦C min−1 andHe (11.3 cm3 min−1) carrier gas. Total carbohydratewas determined by the phenol/H2SO4 method ofDubois et al,5 and uronic acid by the 3-phenylphenolmethod of Blumenkrantz and Asboe-Hansen6 afterdissolution of the cell walls in chilled 95% H2SO4.

∗ Correspondence to: MC Jarvis, Chemistry Department, Glasgow University, Glasgow G12 8QQ, UK†Current address: Institut fur Lebensmittelchemie der Technischen Universitat Carolo-Wilhelmina, D-38106 Braunschweig, Germany‡Current address: School of Chemical, Environmental and Mining Engineering, University of Nottingham, University Park, NottinghamNG7 2RD, UKContract/grant sponsor: BBSRCContract/grant sponsor: EPSRC(Received 14 August 2002; revised version received 21 January 2003; accepted 24 April 2003)

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Galacturonic acid was used as standard, and blankcorrection for neutral sugars was made by omitting3-phenylphenol from the colorimetric reagent. Thedegree of esterification of pectic galacturonans wasdetermined by a titrimetric procedure.7 The contentof non-esterified uronic units determined by titrationwas compared with the uronic acid content determinedcolorimetrically to derive the percentage esterification.Note that this includes any non-methyl galacturonoylesters8 that may have been present.

Solid state NMR experimentsFor one cell wall sample to which water (0.5 cm3 g−1)was added, the high-field cross-polarisation magic-angle spinning (CP-MAS) 13C spectrum was obtainedat 75.4 MHz with a MAS rate of 3 kHz. To allowfor radiofrequency absorption by water protons, theproton radiofrequency field was set at maximumpower (60 kHz) and the 13C field was adjusted tooptimise the Hartmann–Hahn match. The initialproton 90◦ pulse was then re-optimised. The sameproton radiofrequency field strength was maintainedduring decoupling. For the samples examined withoutadded water, the low-field CP-MAS spectrum wasobtained at 25 MHz with a MAS rate of 4 kHz.

RESULTSBy differential sieving, two types of cell wall prepa-ration were isolated from broccoli stems immedi-ately below the florets. The <150 µm (‘parenchyma’)fraction consisted almost entirely of the thin wallsof polyhedral parenchymatous cells, with a verysmall proportion of wall fragments from xylem ves-sels. The >150 µm (‘vascular’) fraction consistedof xylem vessel fragments with helical thickening;thickened walls, with numerous pits, from cellsresembling tracheids and fibres; some clusters ofsieve elements; and small quantities of epidermaland parenchymatous cell wall fragments. Only thehelical thickenings of the vessel walls were consis-tently lignified as assessed by phloroglucinol/HCl

staining, but sporadic lignification was also visi-ble in other tissues, in the more mature sam-ples only, and was largely restricted to the mid-dle lamella.

The composition of the cell walls from a maturesample of broccoli is shown in Table 1. Both cellwall fractions contained large quantities of pectins,with a degree of total esterification greater than80%. The proportion of hemicellulosic polysaccha-rides, as assessed from the percentage of xyloseand non-cellulosic glucose, was rather low in bothcell wall preparations. It is not possible to dis-tinguish xylans from xyloglucans reliably by theirmonosaccharide composition alone, but the quan-tity of non-cellulosic glucose was consistent withthe presence of both polymers in each type of cellwall. The solid state NMR spectrum of the vas-cular fraction obtained with the addition of waterto improve spectral resolution9 (Fig 1 and Table 2)confirmed the presence of methyl-esterified pecticgalacturonans (54, 69, 171 ppm) and also showedthat the cell walls were considerably acetylated(21 ppm).

The solid state NMR conditions used in recordingthe spectrum in Fig 1 are not optimal for detectinglignin. Solid state 13C spectra at lower field, usingeither a relatively long CP contact time or direct 13Cexcitation, are advisable for this purpose. A low-fieldspectrum of the vascular cell walls from an excessivelymature broccoli sample, obtained using CP-MASconditions optimised for wood lignin,10 is shown inFig 2. It demonstrates the presence of a small quantityof typical dicot lignin, with a normal proportion of(β-4)-linked syringyl and guaiacyl residues and noneof the peaks diagnostic for esters of phenolic acids.The lignin content was estimated at 6% from thespectral intensity in the region 135–160 ppm. CP-MAS spectra are only semi-quantitative for ligninbecause of the difference in CP kinetics between ligninand carbohydrate, but when the percentage of lignin isas low as in these cell walls, accepted methods of lignindetermination are subject to a range of interferences.

Table 1. Chemical composition (g kg−1) of parenchyma and secondary xylem cell walls from flowering stems of broccoli. Monosaccharide

composition is based on quantities recovered after 1 M H2SO4 hydrolysis with (‘total’) and without (‘non-cellulosic’) a prehydrolysis step in 72%

H2SO4. Mean and standard error of mean (SEM) of three replicates

Secondary xylem cell walls Parenchyma cell walls

Total Non-cellulosic Total Non-cellulosic

Mean SEM Mean SEM Mean SEM Mean SEM

Rhamnose 16 2 12 0 14 1 14 1Fucose 7 1 6 1 6 1 6 1Arabinose 108 10 112 4 79 12 100 13Xylose 52 3 34 7 44 4 32 4Mannose 27 2 6 0 22 1 6 2Galactose 51 5 54 5 53 5 57 0Glucose 243 15 13 1 231 21 11 2Uronic acid 364 7 315 7% esterification 89.1 0.7 83 0.7

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Cell wall composition of broccoli stem tissues

Figure 1. Solid state 75 MHz 13C NMR spectrum of hydrated broccolivascular cell walls. See Table 2 for peak assignments. The addition ofwater assisted spectral resolution, but these conditions wereunsuitable for recording the contribution of lignin to the spectrum.

Table 2. Assignment of peaks in 13C NMR spectrum of broccoli

vascular cell walls9,10

Chemicalshift (ppm) Assignment

175 C-6 of galacturonan in calcium form173 Carboxyl of acetyl171 C-6 of galacturonan in H or Me form153 C-3/C-5 of ether-linked syringyl units

in lignin148 C-3/C-4 of lignin aromatic rings105 C-1 of cellulose, some hemicelluloses

and β (1–4)-galactan101 C-1, predominantly of galacturonan89 C-4 of crystalline cellulose84 C-4 of crystallite surface cellulose75 General carbohydrate C-2, C-3, C-572 General carbohydrate C-2, C-3, C-569 C-2/C-3 of galacturonan65 C-6 of crystalline cellulose62 C-6 of crystallite surface cellulose and

β (1–4)-galactan56 Lignin methoxyl53 Pectic methoxyl21 CH3 of acetyl

NMR methods too become more difficult at low ligninlevels owing to limited signal/noise, but provided thatinterference from paramagnetic ions is avoided, as inisolated cell walls, the NMR approach may be morereliable.10–13

Figure 2. Low-field (25 MHz) solid state 13C NMR spectrum of drybroccoli vascular cell walls under CP-MAS conditions optimised forquantification of lignin.

DISCUSSIONOn the basis of their anatomy and their positionwithin the stem, the thickened cell walls of broccolimay be assigned to the vascular tissues despite theirlow lignin content and their similarity in compositionto typical primary cell walls of dicots. Stem tissueswith thickened primary cell walls are sometimesdefined as collenchyma, but we do not regard thatdefinition as appropriate here because collenchymahas a different developmental origin, differentiatingfrom parenchymatous cells between the vascular ringand the epidermis,14 and a specialised function inwithstanding tensile but not compressive stresses.15

Vascular cell walls without lignin or with very localisedlignification have been described from alfalfa16 andfrom the upper part of the cauliflower stem (Brassicaoleracea L var botrytis),17 where they were rich in highlymethyl-esterified pectins as described here.18

In the brassicas the characteristics of the vascularcell walls vary widely under developmental control, asindeed does the form of the plant.19 In swede (B napus)the edible part consists principally of unlignifiedsecondary xylem tissue with thin, pectin-rich wallssimilar in composition to typical parenchyma.20 Thethickened vascular cell walls in the upper stemregion of forage kale,3 cauliflower18 and forage rape(Ferguson E et al, unpublished) closely resembles thecell walls of this tissue in broccoli, with less than10% lignin, significant levels of pectic polysaccharidesand both xylan and xyloglucan hemicelluloses incomparable abundance. However, in the floweringstems of oilseed rape (B oleracea)21 and in thin-stemmed, leafy kale varieties3 and Arabidopsis22 thesame tissue has a lignin content of 15–30% and a

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polysaccharide composition dominated by celluloseand xylans as in dicotyledonous wood.

In commercial broccoli the thickened vascular cellwalls are perceived as being tough enough to causeproblems with consumer acceptability. Clearly thesemechanical properties are not the result of theircomposition and must be caused by other featuresof cell wall organisation or simply by the thickness ofthe cell walls.23 Attempts to inhibit lignification aretherefore unlikely to solve the commercial problem. Asolution is more likely to emerge from an improvedunderstanding of the developmental cues that incitethe formation of normal secondary wall layers, andthe modification of these cues that results in thickenedwalls of anomalous composition in brassica plantssuch as broccoli. The vascular ring of brassica stemsis an interesting, exceptionally flexible model systemin which to study how cell wall structure and themechanical properties of plant tissues are related.

ACKNOWLEDGEMENTSThis work was supported by BBSRC and EPSRC. Wethank Dr DC Apperley for executing the high-fieldsolid state NMR experiments.

REFERENCES1 Kahn BA, Shilling PG, Brusewitz GH and McNew RW, Force

to shear the stalk, stalk diameter, and yield of broccoli inresponse to nitrogen-fertilization and within-row spacing. JAm Soc Hort Sci 116:222–227 (1991).

2 McCluskey JG, Allison MJ, Duncan HJ and Jarvis MC, Iso-lation of anatomically defined cell walls from fodder kale,and their contributions to determining the in vitro cellulasedigestibility of the whole plant. J Agric Sci 103:347–352(1984).

3 Wilson WD, Barwick JM, Lomax JA, Jarvis MC and Dun-can HJ, Lignified and non-lignified cell walls from kale. PlantSci 57:83–90 (1988).

4 Englyst HN and Cummings JH, Simplified method for the mea-surement of total non-starch polysaccharides by gas–liquidchromatography of constituent sugars as alditol acetates. Ana-lyst 109:937–942 (1984).

5 Dubois M, Gilles KA, Hamilton JK, Rebers RA and Smith F,Colorimetric method of determination of sugars and relatedsubstances. Anal Chem 28:351–365 (1956).

6 Blumenkrantz N and Asboe-Hansen G, New method forquantitative determination of uronic acids. Anal Biochem54:484–489 (1973).

7 MacKinnon IM, Jardine WG, O’Kennedy N, Renard CMGCand Jarvis MC, Pectic methyl and non-methyl esters in potatocell walls. J Agric Food Chem 50:342–346 (2002).

8 Kim JB and Carpita NC, Changes in esterification of the uronicacid groups of cell wall polysaccharides during elongation ofmaize coleoptiles. Plant Physiol 98:646–653 (1992).

9 Ha MA, Apperley DC and Jarvis MC, Molecular rigidity in dryand hydrated onion cell walls. Plant Physiol 115:593–598(1997).

10 Love GD, Snape CE and Jarvis MC, Determination of thearomatic lignin content in oak wood by quantitative solidstate 13C NMR. Biopolymers 32:1187–1192 (1992).

11 Love GD, Snape CE, Jarvis MC and Morrison IM, Determina-tion of phenolic structures in flax fibre by Solid state 13CNMR. Phytochemistry 35:489–491 (1994).

12 Love GD, Snape CE and Jarvis MC, Comparison of leaf andstem components in barley straw by solid state 13C NMR.Phytochemistry 49:1191–1194 (1998).

13 Reeves JB and Schmidt WF, Solid state 13C NMR analysis offorage and byproduct-derived fiber and lignin residues. Res-olution of some discrepancies among chemical, infrared andpyrolysis–gas chromatography–mass spectroscopic analyses.J Agric Food Chem 42:1462–1468 (1994).

14 Esau K, Plant Anatomy, 2nd edn. Wiley, New York (1965).15 Jarvis MC, Collenchyma, in Encyclopedia of Life Sciences, Vol 4.

Nature Publishing Group, London, pp 708–710 (2001)(http://www.els.net).

16 Engels FM and Jung HG, Alfalfa stem tissues: cell-walldevelopment and lignification. Ann Bot 82:561–568 (1998).

17 Femenia A, Garosi P, Roberts K, Waldron KW, Selvendran RRand Robertson JA, Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassicaoleracea L var botrytis) stems. Planta 205:438–444 (1998).

18 Femenia A, Waldron KW, Robertson JA and Selvendran RR,Composition and structural modification of the cell wallof cauliflower (Brassica oleracea L var botrytis) duringtissue development and plant maturation. Carbohydr Polym39:101–108 (1999).

19 Kieffer M, Fuller MP and Jellings AJ, Explaining curd andspear geometry in broccoli, cauliflower and ‘romanesco’:quantitative variation in activity of primary meristems. Planta206:34–43 (1998).

20 Millard P and Chesson A, Glycosidic linkages of swede cell-walls and their residues recovered from the terminal ileum ofthe pig. Eur J Biochem 142:367–369 (1984).

21 Alexander RH, Gordon AH, Lomax JA and Chesson A, Com-position and rumen degradability of straw from three varietiesof oilseed rape before and after alkali, hydrothermal andoxidative treatment. J Sci Food Agric 41:1–16 (1987).

22 Turner SR and Somerville CR, Collapsed xylem phenotype ofArabidopsis identifies mutants deficient in cellulose depositionin the secondary cell wall. Plant Cell 9:689–701 (1997).

23 Wilson WD, Jarvis MC and Duncan HJ, In-vitro digestibility ofkale (Brassica oleracea) secondary xylem and parenchyma cellwalls and their polysaccharide components. J Sci Food Agric48:9–14 (1989).

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