immunocytochemical localisation ofpara-coumaric acid and feruloyl-arabinose in the cell walls of...

J Sci Food Agric 1998, 78, 373È381

Immunocytochemical Localisation ofpara-Coumaric Acid and Feruloyl-Arabinose inthe Cell Walls of Maize StemCarole Migne� ,1 Ge� rard Prensier,2 Jean-Pierre Utille,3 Pierre Angibeaud,3Agnes Cornu1 and Elisabeth Grenet1*1 Unite� de la Digestion Microbienne, Station de Recherches sur la Nutrition des Herbivores, INRA,Clermont-Ferrand-Theix, 63122 Saint-Genes Champanelle, France2 Universite� Blaise Pascal, Laboratoire de Protistologie Mole� culaire et Cellulaire, URA CNRS 1944,Complexe ScientiÐque des Ce� zeaux, 63177 Aubiere Cedex, France3 Centre de Recherches sur les Macromole� cules Ve� ge� tales, (CNRS), BP 53 X, 38041 Grenoble Cedex,France

(Received 15 July 1997 ; revised version received 26 January 1998 ; accepted 11 March 1998)

Abstract : Two phenolic compounds, p-coumaric acid and feruloyl-arabinose,were localised by immunocytochemistry in the cell walls of the apical internodeof two lines of maize (Co125 and W401) of di†erent digestibility. The compoundswere detected at two stages of cell maturity in the ligniÐed tissues (sclerenchyma,Ðbres and xylem) and in the medullary parenchyma, which, in the samplesstudied, was not ligniÐed. p-Coumaric acid is a phenolic acid associated withlignins, which confer resistance on plant cell walls to microbial degradation inthe rumen. Feruloyl-arabinose is a compound associated with xylans, the prin-cipal hemicelluloses in Gramineae, which are potentially degradable. Labelling ofp-coumaric acid decreased in both maize lines with cell age and as the cell wallsbecame ligniÐed. The mass of lignin deposited in the cell walls masked p-coumaric acid, thereby making it less accessible to the antibodies. There was aninverse relationship in the labelling of p-coumaric acid and feruloyl-arabinose.Feruloyl-arabinose was more heavily labelled as the plant cell walls matured inall the ligniÐed tissues of both maize lines and in the parenchyma of the lessdigestible line. All tissues except the parenchyma were more heavily labelled withboth sera in Co125, the more digestible line. 1998 Society of Chemical(Industry.

J Sci Food Agric 78, 373È381 (1998)

Key words : immunocytochemistry ; plant cell wall ; para-coumaric acid ; feruloyl-arabinose ; maize.

INTRODUCTION

Polysaccharides in plant cell walls are bound to ligninsand phenolic acids by covalent bonds (Hartley andFord 1989 ; Jung 1989). Two types of bonds areinvolved : alkali-labile ester linkages and alkali-resistantether bonds (Hartley 1972). Ferulic acid and p-coumaricacid are the two predominant phenolic acids in the cellwalls of Gramineae. The latter is mainly esteriÐed to

* To whom correspondence should be addressed.

lignins (Jung 1989), whereas esteriÐed ferulic acid is ableto bridge arabinoxylan chains by forming dimers (Jungand Deetz 1993). Two studies (Jacquet et al 1995 ; Ralphet al 1995) have shown that ferulate esters, which arethought to act as initiation sites for ligniÐcation (Briceand Morrison 1982 ; Wallace et al 1991), bridge ligninand polysaccharides. Phenolic acids can be extractedfrom cell walls by alkali or enzyme treatments in theform of monomers, dimers or “FAXXÏ (O-[5-O-(trans-feruloyl)-a-L-arabinofuranosyl]-(1 ] 3)-O-b-D-xylopyr-anosyl-(1 ] 4)-D-xylopyranose) and “PAXXÏ (O-[5-O-

3731998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

374 C Migne� et al

(trans - para - coumaroyl) - a - L - arabinofuranosyl] -(1 ] 3)-O -b -D -xylopyranosyl- (1 ] 4)-D -xylopyranose)(Smith and Hartley 1983 ; Kato and Nevins 1985 ;Mueller-Harvey et al 1986 ; Hartley and Ford 1989).

In young Gramineae, lignin content in the apicalinternode increases with maturity from the baseupwards (Joseleau et al 1976) and, at the same time,there is a rise in p-coumaric acid content. In contrast,there is hardly any variation in ferulic acid content(Jung 1989 ; Scobbie et al 1993) : saponiÐable ferulic acidis present early in the primary cell wall and becomesetheriÐed during growth (Lam et al 1992). The increasein phenolic acid esters in cell walls during growthderives mainly from the rise in p-coumaric acid content(Iiyama et al 1990).

p-Coumaric acid is associated with the indigestiblefraction of forages (Monties 1984). The combined pres-ence of p-coumaric acid and lignins has been cited as areason for the resistance of Gramineae cell walls torumen microbial degradation. The inverse relationbetween p-coumaric acid content and cell wall digest-ibility (Hartley and Jones 1977) could be the result ofthe concomitant increase in p-coumaric acid and lignincontent.

Di†erent microscopy techniques were developed tolocalise and visualise phenolic compounds in plant cellwalls. Staining has been used to evidence di†erent typesof lignin in the tissues (Akin and Burdick 1981 ; Akin etal 1990). More speciÐc techniques such as UV absorp-tion microspectrometry have been used to detect FAXXand PAXX in the cell walls of di†erent tissues (Akin andHartley 1992 ; Goto et al 1992). Immunocytochemistrytechniques have also been developed and antibodieshave been produced against p-coumaric acid (Barry1992) and lignins (Petterson et al 1988 ; Ruel et al 1994).

The aim of the present study was to localise p-coumaric acid and feruloyl-arabinose by immuno-cytochemistry in the cell walls of di†erent tissues of themaize stem. The base and the top of the apical inter-node of the stem of two maize lines of di†erent digest-ibility were used (Migne� et al 1996).

MATERIAL AND METHODS

Plant material

Two lines of maize (Co125 and W401) were grownunder glass by ICI Seeds SES at Tienen, Belgium, andharvested 5 days after anthesis. Sample plants (stemsonly) were transported to the laboratory under dry iceand maintained frozen at [15¡C until required. Theapical internode was divided into three equally long sec-tions. Fragments 5 mm long were sampled half-wayalong the upper and lower sections and used for micros-copy studies.

The 48-h dry matter disappearance in the rumen,measured in nylon bags, of the top and the base of theapical internode was 72É1 and 86É7% for Co125, and60É8 and 84É4% for W401, respectively (Migne� et al1996).

Preparation of samples for transmission electronmicroscopy

Samples were embedded in LR White resin and treatedas described previously (Migne� et al 1994).

Preparation of antigens for immunisation

The synthesised feruloyl-arabinose was etheriÐed atposition 1 of arabinose by 4-thio 6-hydroxy hexanoicacid (Scheme 1) to provide the carboxylic functionrequired for coupling with a protein. Haptens werecoupled with bovine serum albumin (BSA) according tothe method of Catty and Raykundaliac (1988) adaptedas follows. Haptens, BSA and carbodiimide concentra-tions in the reaction mixture were 1É0, 0É5 and2É5 mg ml~1, respectively, and the pH was 7É4 for p-coumaric acid and 6É8 for feruloyl-arabinose. Unboundhaptens and carbodiimide were removed by ultraÐltra-tion (30 min at 2000g) in Centrisart tubes (Sartorius,10 000 D). The resulting solutions were immediatelyused for immunisation.

Scheme 1. Chemical structure of sodium 5-O-(trans-feruloyl)-b-L-arabinofuranosyl-4-thio-6-hydroxy-hexanoate (a) andpara-coumaric acid (b).

Control of coupling

The coupling of p-coumaric acid with the BSA waschecked by HPLC on a C18 silica column (25 cm,10 mm, Merck), eluted by a gradient of 6È100% meth-anol in water in the presence of phosphoric acid A(2&).

Immunocytochemical localisation of p-coumaric acid and feruloyl-arabinose 375

control solution of p-coumaric acid (1 mg ml~1 in PBS)and the ultraÐltered solution containing the uncoupledantigen (originally 1 mg ml~1 in PBS) were injected.The yield of p-coumaric acid coupling to BSA wasabout 40%.

The coupling of feruloyl-arabinose was checked bythin layer chromatography on silica plate, eluted byethyl acetate and read under UV light. The antigenalone, ferulic acid, BSA alone and BSAÈferuloyl-arabinose coupling medium were chromatographed.Compared to the solvent, the of ferulic acid was 0É23Rfand that of feruloyl-arabinose was 0É37. BSA alone didnot move from the deposition site. In the couplingmedium, the intensity of the feruloyl-arabinose spotstrongly decreased compared to what was observedaccompanying BSA. This conÐrms that coupling didoccur.

Immunisation

Pre-immune blood was sampled from three rabbits(New Zealand breed). The rabbits were immunised byintradermic injections in the back of p-coumaric acidÈBSA and FreundÏs complete adjuvant (Sigma) emulsion.Hapten concentration was 0É5 mg ml~1 of emulsion.Booster injections of antigens and FreundÏs incompleteadjuvant emulsion were given every 4 weeks. Blood wassampled 8 days after each booster. The blood sampleswere left to coagulate 1 h at 37¡C. After overnight exu-dation at 4¡C, they were centrifuged at 4200g for 20 minto obtain the immune sera. The sera were tested at dilu-tions of 1/10, 1/20, 1/40, 1/80 and 1/160. Immunisationwith p-coumaric acid was performed over 4 months.Three rabbits of the same breed were likewise immu-nised with feruloyl-arabinose.

The Ðrst anti-p-coumaric acid antibodies weredetected 9 weeks after the beginning of immunisation,whereas anti feruloyl-arabinose antibodies wereobtained at the Ðfth week. The serum of the 13th week,diluted to 1/40, was used for p-coumaric acid labelling,and the serum of the 9th week, diluted to 1/20, forferuloyl-arabinose labelling.

Immunocytochemical procedure

ControlsImmunolabelling was performed on ultrathin sectionsof maize with pre-immune sera and with protein Aalone.

T ests of depletion of antibodies by antigensEach antigen was coupled with the BSA as describedabove. Immune sera were diluted to 1/40 and incubatedwith their corresponding antigen (1 mg ml~1) for 1 h at37¡C and then overnight at 4¡C. This technique wasused to check for a possible cross-reactivity between thetwo antigens.

Dot immunoblottingThe antigens were coupled with another carrier protein,keyhole limpets hemocyanin (KLH, Sigma), to preventthe anti-BSA antibodies in the immune sera from react-ing with the carrier protein.

The control was performed on an immobilon mem-brane (Millipore), previously made permeable by meth-anol treatment. p-Coumaric acid, feruloyl-arabinose andother molecules similar in structure, such as ferulic,vanillic, syringic and sinapic acids coupled with KLH,were deposited on a membrane and incubated for 1 h.The non-speciÐc sites were then saturated with a solu-tion of 5% skimmed milk in PBS bu†er for 1 h atambient temperature with constant shaking. Theimmune sera were diluted to 1/100 in a solution of 1%milk/PBS placed on the membrane and incubated for1 h. Peroxidase-coupled goat secondary anti-rabbit IgGantibodies (Promega) diluted to 1/1000 were used in thesolution of 1% milk/PBS. The antigen speciÐc antibodyreaction was revealed by the addition ofdiaminobenzidine (DAB), a substrate of peroxidase,which in the presence of hydrogen peroxide stainsbrownish.

The pre-immune sera and secondary antibodies alonewere checked. The two total sera were also tested onL-arabinose, arabinoxylan and arabinogalactan. Eachserum was depleted by its own antigen and by ara-binogalactan. Anti p-coumaric acid and anti-feruloyl-arabinose sera were also depleted by feruloyl-arabinoseand p-coumaric acid, respectively. The two depletedsera were also tested on all the molecules referred to inthis paragraph.

Immunocytochemical labelling of p-coumaric acid andferuloyl-arabinoseLabelling was performed on ultrathin sections of maizeas previously described (Migne� et al 1994), except thatovalbumin was replaced in the saturating solution bygelatin (Merck). The sera were tested at dilutions of1/10, 1/20, 1/40, 1/80 and 1/160. The anti-p-coumaricacid serum was used after depletion by feruloyl-arabinose and the anti-feruloyl-arabinose after deple-tion by p-coumaric acid.

Quantitative analysis of immunocytochemical labelling

QuantiÐcation of gold particles was done on negativeÐlms at a magniÐcation of 19 500 with a semi-automaticimage analysis system (Ernst-Leitz-Wetzlar ASM 68K).The results were expressed in numbers of particles percm2 negative Ðlm, ie per 0É263 lm2 cell wall. Each mea-surement was repeated 10 times, and the means andstandard deviations were calculated.

Statistical analysis

Variance analysis of individual results after data trans-formation ()(x ] 1)) was done by the GLM procedure


(SAS 1985), and the di†erence between the labelling ofthe cell walls was tested by means comparison with theDuncan (1955) test when the F test was signiÐcant(P\ 0É05). The data of each serum were processedseparately.

RESULTS

Immune sera speciÐcity

No signiÐcant labelling was observed either on ultrathinsections or in dot blot tests with the pre-immune sera(Fig 1), the antigen-saturated immune sera and theprotein-A colloidal gold control alone. The anti-p-cou-maric acid and feruloyl-arabinose total sera recognisedtheir own antigen : cross-reactivity with the other mol-ecules tested, in particular with the phenolic acids, wasslight. Depletion of the anti-p-coumaric acid serum byferuloyl-arabinose in no way modiÐed the responseobtained on maize sections with the total anti-p-cou-maric acid serum. Nor did depletion of the anti-feruloyl-arabinose by p-coumaric acid.

Immunodetection of p-coumaric acid and feruloyl-arabinose in the cell walls of Co125 and W401 maize

Immunodetection of p-coumaric acidFigures 2È9 show the four tissues in the base and top ofthe internode of maize W401. They were chosen to illus-

Fig 1. No signiÐcant labelling of the cell wall of sclerenchymain the base of the internode of maize W401 is observed withpre-immune serum: pw, primary wall ; sw, secondary wall ; t,

tricellular junction ; ]33 000 ; bar\ 0É5 lm.

Fig 2. Labelling in maize W401 with anti-para-coumaric acidserum. Labelling of the cell wall of sclerenchyma in the top ofthe internode : pw, primary wall ; sw, secondary wall ; t, tri-

cellular junction ; ]33 000 ; bar\ 0É5 lm.

trate the labelling. In most cases, cytoplasm wasdamaged by freezing the fresh samples.

In both lines the ligniÐed tissues (sclerenchyma, Ðbressurrounding the vascular bundles and xylem) were more

Fig 3. Labelling in maize W401 with anti-para-coumaric acidserum. Labelling of the cell wall of sclerenchyma in the base ofthe internode is greater than in the top and localised mainly in

the secondary cell wall ; abbreviations as in Fig 2 ; ]33 000.


Fig 4. Labelling in maize W401 with anti-para-coumaric acidserum. Labelling of the cell wall of Ðbres in the top of the

internode ; abbreviations as in Fig 3 ; ]33 000.

heavily labelled (P\ 0É05) than the inner parenchyma,both in the top and in the base of the internode (Table1). In all four tissues studied, labelling was signiÐcantlyheavier (P\ 0É05) in the base than in the top. The mostdensely labelled tissues in the two lines, both in the topand in the base, were the Ðbres.

In line W401, the ligniÐed tissues were less heavilylabelled (P\ 0É05) than in line Co125, the more digest-ible line. However, labelling of the parenchyma wastwice as great as in maize Co125 (P\ 0É05) both in thebase and in the top. In addition, the di†erences in thedensity of labelling between the base and the top were

Fig 5. Labelling in maize W401 with anti-para-coumaric acidserum. Labelling of the cell wall of Ðbres in the base of theinternode increases compared to the top ; abbreviations as in

Fig 4 ; ]33 000.

signiÐcantly greater (P\ 0É05) in W401 than in lineCo125.

Immunodetection of feruloyl-arabinoseIn line Co125, the four tissues studied were moredensely labelled (P\ 0É05) in the top than in the base ofthe internode (Table 2, Figs 10È13). The di†erence inlabelling intensity between the top and the base wasgreatest in the xylem. In the parenchyma it was not sig-niÐcant.

In line W401, the sclerenchyma was the most heavilylabelled (P\ 0É05) of the four tissues in the top of the

TABLE 1para-Coumaric acid labelling in sclerenchyma (S), Ðbres (F), xylem (X) and par-

enchyma (P) in maize lines Co125 and W401 (number of particles per cm2)a

T issues Co125 W 401

T op Base T op Base

Mean SD Mean SD Mean SD Mean SD

S 11É1e 1É6 17É7b 2É9 6É5g 1É2 15É1c,d 2É7F 12É6e 2É0 20É2a 3É4 8É0f,g 0É9 17É2b,c 2É9X 8É8f 1É7 15É2c,d 2É9 6É3g 1É4 12É5e 1É5P 1É5i 0É4 4É2h 1É5 2É7h 0É6 8É4f 3É5

a Mean values with di†erent superscript letters were signiÐcantly di†erentP\ 0É05).


Fig 6. Labelling in maize W401 with anti-para-coumaric acidserum. The density of gold particles in the cell wall of xylem inthe top of the internode is weak. Abbreviations : pw, primarywall ; sw, secondary wall ; t, tricellular junction ; ]33 000 ;

bar \ 0É5 lm.

internode (Table 2), and labelling was greater in the topthan in the base. Labelling of the Ðbres was signiÐcantly(P\ 0É05) but only slightly denser in the top than in thebase. In the xylem and parenchyma of W401, labellingwas not signiÐcantly di†erent (P\ 0É05) between thebase and the top. In the top of line W401, the lessdigestible line, the tissues were less heavily labelled(P\ 0É05) than in line Co125.

DISCUSSION

The speciÐcity of the sera was checked using di†erentmolecules capable of producing cross-reactions. As anti-

Fig 7. Labelling in maize W401 with anti-para-coumaric acidserum. Labelling of the cell wall of xylem in the base of theinternode is greater than in the top ; abbreviations as in Fig 6 ;

]33 000.

arabinogalactan antibodies could have originated fromthe injection of FreundÏs complete adjuvant, dot immu-noblotting was performed with the sera depleted by ara-binogalactan. The resulting responses corresponded to abackground. Because of the presence of BSA in thecoupled antigens, the sera may have recognised someprotein material such as “hydroxyprolin-rich glycopro-teinsÏ. Since the two sera obtained by the same tech-nique yielded di†erent results, labelling was consideredto be slight.

All the tissues studied were labelled by the two anti-sera, which shows the p-coumaric acid and feruloyl-arabinose were present in varying amounts in all thecell walls (He and Terashima 1989). The parenchyma

TABLE 2Feruloyl-arabinose labelling in sclerenchyma (S), Ðbres (F), xylem (X) and par-

enchyma (P) in maize lines Co125 and W401 (number of particles per cm2)a

T issues Co125 W 401

T op Base T op Base

Mean SD Mean SD Mean SD Mean SD

S 9É7a 1É6 6É9b 1É8 8É0b 1É8 3É2e,f 1É1F 8É0b 1É3 5É5c 1É3 5É1c,d 1É3 3É7e,f 0É6X 10É1a 1É8 2É7f 0É3 2É9e,f 1É0 2É7f 0É8P 4É3c,d,e 1É2 3É2e,f 1É1 3É1e,f 1É1 4É0d,e,f 1É1

a Mean values with di†erent superscript letters are signiÐcantly di†erent (P\ 0É05).


Fig 8. Labelling in maize W401 with anti-para-coumaric acidserum. Labelling of the cell wall of parenchyma in the top of

the internode is slight ; abbreviations as in Fig 6 ; ]33 000.

Fig 9. Labelling in maize W401 with anti-para-coumaric acidserum. Labelling of the cell wall of parenchyma in the base of

the internode is notably greater than in the top ; ]33 000.

Fig 10. Labelling in maize Co125 with anti feruloyl-arabinoseserum. Labelling of the cell wall of sclerenchyma in the top ofthe internode ; the cell wall of sclerenchyma is notably lessthick than in maize W401 : pw, primary wall ; sw, secondary

wall ; t, tricellular junction ; ]33 000 ; bar\ 0É5 lm.

Fig 11. Labelling in maize Co125 with anti feruloyl-arabinoseserum. Labelling of the cell wall of sclerenchyma in the base ofthe internode is greater than in the top ; abbreviation as in Fig

10 ; ]33 000.


Fig 12. Labelling in maize Co125 with anti feruloyl-arabinoseserum. Labelling of the cell wall of Ðbres in the top of the

internode ; abbreviations as in Fig 10 ; ]33 000.

Fig 13. Labelling in maize Co125 with anti feruloyl-arabinoseserum. Labelling of the cell wall of Ðbres in the base of theinternode increases compared to the top ; abbreviations as in

Fig 10 ; ]33 000.

was labelled in the two lines whatever its position in theinternode. Lignin-speciÐc staining showed that it wasnot ligniÐed (Migne� et al 1996). The present labellingresults, in agreement with those of Harris and Hartley(1976) and Barry (1992), showed the presence of phenol-ic compounds in the parenchyma.

With anti-p-coumaric acid serum, the tissues at thebase (youngest stage) were the most heavily labelled.Hence, p-coumaric acid is accessible to the antibodies inthe young tissues, whose cell walls are not at this stageligniÐed, as observed previously on wheat straw (Barry1992). Lignin and p-coumaric acid contents increasewith maturity (Scobbie et al 1993), and so it is likelythat p-coumaric acid in the top was masked by lignins(in sclerenchyma, Ðbres and xylem). Terashima et al(1988) showed that ligniÐcation begins in the cellcorners and then spreads to the primary cell wall. Withanti-p-coumaric acid serum, the cell corners of the scle-renchyma and Ðbre walls were labelled in the base ofthe internode but not in the top. This, too, has beenobserved on wheat straw (Barry 1992).

The di†erences between the two lines are highlightedby the variations with maturity in the labelling offeruloyl-arabinose. Ferulic acid content increased fromthe base of the internode upwards in Co125 but not inW401 (Scobbie et al 1993). In line Co125, labelling ofthe feruloyl-arabinose increased in density from thebase of the internode upwards. In contrast, in lineW401, except in the sclerenchyma, little change wasobserved in labelling of feruloyl-arabinose from the baseof the internode upwards.

The more digestible line, Co125, was more intenselylabelled than W401 with both sera except in the par-enchyma. Thus, p-coumaric acid and feruloyl-arabinosewere more accessible to antibodies in Co125 and prob-ably to microbial enzymes. p-Coumaric acid is associ-ated with lignin fractions that are particularly stronginhibitors of digestibility (Cornu et al 1994), and ferulicacid plays a key role in the bridging of lignins and poly-saccharides. Lignin content is not in itself sufficient toexplain variations in digestibility, which depend greatlyon intermolecular bonds. Although it is sometimes diffi-cult to interpret the results of lignin model DHP(dehydrierungs polymerizat) immunolabelling (Ruel etal 1994), as for example, in G-DHPs, which have a dif-ferent structure from that of G lignins in vivo, it wouldnevertheless be interesting to perform this immuno-labelling along with that of p-coumaric acid andferuloyl-arabinose. Immunocytochemistry resultssuggest that p-coumaric acid and feruloyl-arabinosehave separate functions in the cell walls, since their dis-tribution in the tissues and their fate during cell matu-ration in the cell walls are both quite di†erent. Thismethod, which allows a quantiÐcation of the results,could provide new information on the digestibility ofthe cell walls of di†erent tissues and be useful in thestudy of rumen cell wall degradation.


ACKNOWLEDGEMENTS

The authors thank M Vincenot for printing of the pho-tographs and Ms Tache� for technical assistance.

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immunocytochemical localisation ofpara-coumaric acid and feruloyl-arabinose in the cell walls of...

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