carnation (dianthus petals' - plant physiologycreased in quantity during development of...

Plant Physiol. (1991) 95, 853-8600032-0889/91/95/0853/08/$01 .00/0

Received for publication September 14, 1990Accepted November 16, 1990

Endoglycanase-Catalyzed Degradation ofHemicelluloses during Development of

Carnation (Dianthus caryophyllus L.) Petals'

Nicolaas C. de Vetten, Donald J. Huber*, and Kenneth C. GrossVegetable Crops Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida

32611 (N.C.de V., D.J.H.); and U.S. Department of Agriculture-Agricultural Research Service,Horticultural Crops Quality Laboratory, Beltsville, Maryland 20705 (K.C.G.)

ABSTRACT

Large molecular-size hemicelluloses, including xyloglucan, de-creased in quantity during development of carnation (Dianthuscaryophyllus L. cv White Sim) petals, along with a relative in-crease in polymers with an average size of 10 kilodaltons. Anenzyme extract from senescing petal tissue depolymerized thelarge molecular-size hemicelluloses in a pattern similar to thatoccurring in vivo during petal development. The products gener-ated in vitro were composed of polymeric and monomeric com-ponents, the latter consisting primarily of xylose, galactose, andglucose. The 10 kilodalton hemicelluloses were resistant to invitro enzymic hydrolysis. Glycosyl-linkage composition of thelarge molecular-size polymers provided evidence for the pres-ence of xyloglucan with smaller amounts of arabinoxylan andarabinan. The 10 kilodalton polymers were enriched in mannosyland 4-linked glucosyl residues, presumably derived from gluco-mannan. During petal development or enzymic hydrolysis, nochange was observed in the relative glycosyl-linkage compositionof the large molecular-size hemicelluloses. The in vitro activity ofcarnation petal enzymes active toward native hemicellulosesincreased threefold at the onset of senescence and declinedslightly thereafter. Gel chromatography revealed 23 and 12 kilo-dalton proteins with hemicellulase activity. The enzymes hydro-lyzed the large molecular-size hemicelluloses extensively andwithout formation of monomers. Endoxylanase activity was de-tected in the partially purified enzyme preparation. Xyloglucanwas depolymerized in the absence of cellulase activity, suggest-ing the presence of a xyloglucan-specific glucanase. Thesedata indicate that the hemicellulose molecular-size changes ob-served during development of carnation petals are due, in part,to the enzymic depolymerization of large molecular-sizehemicelluloses.

Senescence of flower petals is characterized by a series ofwell-described physiological and biochemical changes (3).Many of these features, including alterations in ethylenesynthesis, respiration rate and membrane permeability aresimilar to those noted to occur in ripening fruit. Similaritiesbetween the two organ types have also been observed instudies of cell wall (7). Increases in soluble pectins, net loss ofcertain noncellulosic neutral sugars, and changes in hemicel-

Florida Agricultural Experiment Station Journal Series No. R-01142.

853

lulose MS2 distribution, all characteristics of various ripeningfruits, are also a feature of expanding and senescing carnationpetals (7).

In particular, the hemicelluloses appeared to be depolymer-ized to the same extent as has been observed in ripening fruitincluding tomato (16, 18), strawberry (17), hot pepper (1 1),and muskmelon (21). The hemicellulose changes are observedprimarily as alterations in MS distribution, with large MSpolymers becoming less prevalent as ripening proceeds. Huber(16) reported that the relative amount of small MS hemicel-luloses increased during ripening and suggested that theymight represent a population of newly synthesized, yet mod-ified polymers. Evidence for this possibility was provided byMitcham et al. (22), who demonstrated continued hemicel-lulose synthesis throughout ripening of tomato fruit, with alarge increase noted during the stages of prominent changesin MS distribution. Using glycosyl-linkage analysis of tomatofruit hemicellulosic fractions, Tong and Gross (31) proposedthat small MS polymers containing mannosyl and glucosylresidues were synthesized de novo. Although the synthesis ofsmall MS hemicelluloses appears to contribute to the hemi-cellulose changes, McCollum et al. (21) presented data indi-cating that the disappearance of large MS polymers may bedue to enzymic hydrolysis.Enzymic catabolism of hemicelluloses has been observed

in other organ types, and in particular those undergoingelongation growth. In graminaceous monocotyledons, mixed-linkage glucans are hydrolyzed by endo- and exo-f-D-glucan-ases (19). In elongating dicotyledons, attention has focusedon the depolymerization of xyloglucan and arabinogalactan(14, 25). The enzyme able to degrade xyloglucan has beenreported to be an endo-3-D-glucanase (EC 3.2.1.4)(14, 15, 20).The present study was undertaken to obtain information

on hemicellulose metabolism of senescing carnation petals.The mechanism appears to involve, in part, multienzymicdepolymerization of relatively large MS polymers.

MATERIAL AND METHODS

Plant Material

Carnation (Dianthus caryophyllius L. cv White Sim) flowerswere obtained from Rocky Mountain Wholesale Florist,

2 Abbreviations: MS. molecular-size: MG, mature-green.

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Plant Physiol. Vol. 95, 1991

Commerce City, CO. Flowers were harvested at the whitecross stage (outer whorl of petals reflexing). Upon arrival inGainesville, the stems of the flowers were trimmed to 40 cmand placed in solutions containing 58 mm sucrose and 0.56mM 8-hydroxyquinoline citrate. The flowers were maintainedat 20°C, 40 to 60% RH, and 12 W/m2 cool-white fluorescentlight with a 12-h photoperiod. Flower development was dis-tinguished in terms of five morphological stages as definedpreviously (7): preblooming (stage I), blooming (stage II),onset of wilting (stage III), wilted (stage IV), and completelywilted (stage V). Petals were harvested from flowers at theabove stages and stored in sealed polyethylene bags at -30°C.

Mature-green tomatoes (Lycopersicon esculentum Mill. cvSunny) were obtained from the IFAS Gulf Coast Researchand Education Center at Bradenton, FL. Fruit were surface-sterilized with 100 ppm NaOCl, rinsed, and sectioned; outerpericarp tissue was excised and stored at -300C.

Extraction of Camation Petal Protein

Frozen petal tissue (5-15 g) was homogenized in 60 to 70mL of cold (40C) 40 mm Na-acetate (pH 5.0) containing afinal concentration of 1.0 M NaCl for 5 min in a SorvallOmni-mixer set at maximum speed. After a 4-h incubationon ice, the homogenate was filtered through two layers ofMiracloth and centrifuged at 25,400g for 20 min. The super-natant was brought to 80% saturation with solid (NH4)2SO4and maintained for 12 to 16 h at 1 °C. After centrifugation at25,400g for 20 min, the pellet was resuspended in 3 mL 30mm Na-acetate (pH 4.5) containing 150 mM NaCl, and de-salted on a bed (14 x 1.4 cm) of Sephadex G-25-300 (Sigma)eluted with the same buffer. Fractions containing proteinwere pooled and stored at -300C.

Partial Purification of Carnation Petal Hemicellulases

Approximately 20 mg of protein (stage III carnation petals)in 5 mL 10 mm Na-acetate (pH 6.0) containing 150 mM NaClwere applied to a bed (70 x 2.5 cm) of Ultrogel AcA 44 (LKB)and eluted with Na-acetate buffer. Fractions of 3.5 mL werecollected and individually analyzed for hemicellulase, glycan-ase, and glycosidase activities as described below. Protein wasmonitored at 280 nm. The AcA 44 column was calibratedusing blue dextran, bovine serum albumin (67,000 mol wt),egg albumin (45,000 mol wt), carbonic anhydrase (29,000mol wt), and Cyt c (12,500 mol wt) (Sigma Chemical Co.).

Enzyme Assay

Hemicellulase activity in the Ultrogel AcA 44 chromatog-raphy fractions was assayed by measuring the hydrolysis ofhemicelluloses isolated from MG tomato pericarp cell wall.Reaction mixtures containing 250 ,uL of each fraction alongwith 1 mg glucose equivalents (8) of tomato hemicellulosesin 1 mL 30 mm Na-acetate (pH 4.5) containing 150 mm NaClwere incubated for 60 min at 37°C. Activity was measuredreductometrically using the Nelson-Somogyi method (24).The production of reducing equivalents from hemicellulosesincubated with 300 ,ug of protein (BSA equivalents [28]) waslinear with respect to incubation time up to 1.5 h (data not

shown). Other reaction conditions are as indicated in thefigure legends.The activity of selected glycanases in the column fractions

was measured using laminarin (U.S. Biochemical Corp.,Cleveland, OH), xylan (larchwood, Sigma), and arabinogalac-tan (larchwood, Sigma). One-half milliliter of substrate (1 mg)in 25 mm Na-acetate (pH 4.5) containing 150 mm NaCl and250 ,uL from each fraction were incubated for 1 h at 37°C.Reducing equivalents were measured with the Nelson-Somo-gyi procedure (24) using glucose as standard.

Glycosidase activity was measured employing p-nitro-phenol D-glycosides (Sigma). Reaction mixtures containing100 gL of substrate (25 mM) and 100 gsL of the columnfractions in a total volume of 1 mL of 25 mm Na-acetate (pH4.5) containing 150 mM NaCl were incubated for 30 min at37°C. Reactions were terminated by adding 2 mL of 200 mMsodium carbonate. The concentration of free p-nitrophenolwas determined at 400 nm using p-nitrophenol as standard.Heat-inactivated protein controls were used in all assays.

Isolation of Cell Wall and Hemicelluloses

Cell walls were prepared from carnation petals and MGtomatoes using a procedure described previously (7). Hemi-cellulose B (4 A alkali-soluble) was isolated as described byHuber and Nevins (19) and modified by De Vetten andHuber (7).

Sephacryl S-300 Chromatography of Hemicellulose B

Hemicellulose B (2 mg glucose equivalents [8]) was appliedto a bed (60 x 1.5 cm) of Sephacryl S-300 (Pharmacia LKBBiotechnology, Uppsala, Sweden) and eluted with 25 mM Na-acetate (pH 5.0) containing 100 mM NaCl and 3 mMNa2EDTA. Fractions of 2 mL were collected and analyzedfor total carbohydrate using the phenol-sulfuric procedure (8)and for xyloglucan using Kooiman's iodine staining techniqueas described by Nishitani and Masuda (26). The presence ofoligomers and monomers was determined using a Bio-Gel P-2 (-400 mesh) column eluted with 25 mm Na-acetate (pH4.5) containing 50 mM NaCl and 3 mM Na2EDTA at 44°C.Fractions of 1 mL were collected and analyzed for totalcarbohydrate (8).

Linkage Analysis of Hemicellulose B

For glycosyl-linkage analysis, 1 to 1.5 mg (glucose equiva-lents) of each sample were per O-methylated according toHakamori (12) using modifications developed by Carpita (5)and York et al. (32). In addition, butyllithium was used ratherthan potassium-methyl sulfinyl carbanion (2). The per-O-methylated polymers were purified using a Sep-Pak C18 car-tridge (23) and hydrolyzed in 2 N TFA for 60 min at 120°C.After evaporation of the TFA under N2, the per-O-methylatedsugars were reduced and acetylated according to the methodof Blakeney et al. (1).

Partially methylated alditol acetates were identified by rel-ative retention time on a 0.25-mm x 30-m Restek Rtx-5dimethyl 5% diphenyl polysiloxane cross-bonded SE-54 cap-illary column and subsequent mass spectral analysis of frag-

854 DE VETTEN ET AL.



HEMICELLULOSE DEGRADATION IN DEVELOPING CARNATION PETALS

mentation patterns after electron impact mass spectrometry.Separations were carried out with a Hewlett Packard 5890 gaschromatograph coupled to a 5970A mass selective detectorinterfaced to a Series 9000-236 computer system. The GCconditions were as follows: injection size, 1 ,IL; injection porttemperature, 250°C; flow rate, He at 1 mL/min; detectortemperature, 250°C. The oven temperature was programmedfrom 140 to 200°C at 1°C/min, then to 230°C at 10°C/minwhere it was held for 10 min. Quantitation was done using aHewlett Packard 5880 GC-FID based on the effective carbonresponse as determined by Sweet et al. (29).

RESULTS

Developmental Changes in Hemicelluloses

Sephacryl S-300 profiles of hemicellulose B (4 N alkali-soluble) extracted from carnation petals of different stages ofdevelopment are shown in Figure 1. During petal develop-ment, a trend of decreasing quantities of large MS polymers,here defined as those not retained on Sephacryl S-300, and arelative increase in polymers retained by the gel was observed.The latter polymers eluted as a rather symmetrical populationwith an average apparent MS of 10 kD. The major changesin hemicellulose distribution occurred predominantly duringpetal expansion (stages I and II) and early senescence (stagesIII and IV). Hemicelluloses from stage V petal tissue (datanot shown) showed no change relative to stage IV (Fig. ID).The change in MS distribution of hemicelluloses was accom-panied by a reduction in the MS of xyloglucan (Fig. 1).

Enzymic Hydrolysis of Hemicelluloses

In an examination of hemicellulose modification duringmuskmelon softening, McCollum et al. (21) suggested thatthe loss of large MS polymers was due, in part, to enzymicdegradation. Data presented in Figure 2 provide clear evi-dence for the presence, in carnation petals, of enzymes withthe capacity to degrade hemicelluloses. Hemicelluloses, fol-

0%

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0)

.0)4:

0.6

0.A

02

0.6

OA

0.2

A+

7,t40 60 80 100 40 60 80 100

Elution volume (ml)

Figure 1. Sephacryl S-300 profiles of hemicellulose B extracted fromcarnation petals at various stages of development. (0), Total carbo-hydrate, (0), xyloglucan. A, Stage I; B, stage 11; C, stage IlIl; D, stageIV. The arrows in Sephacryl S-300 profile A index the elution volumeof, left to right, Dextran 2000, dextrans of MS 70 kD, 40 kD, 10 kD,and glucose.

0

co

(0)

0.6

OA

Q2

06

0.4

D Q2:

iA B

I

410 60 80100 410 60 80 100Elution volume (ml)

Figure 2. Sephacryl S-300 distribution of hemicellulose B followingtreatment with a stage IV enzyme extract. Two milligrams of hemi-cellulose B in 25 mm Na-acetate (pH 4.5) containing 150 mm NaCI,0.02% sodium azide, and 1 mg of protein (BSA equivalents [28]) in atotal volume of 2 mL were incubated for various periods at 300C.The reaction mixtures were heat-inactivated and applied to a Seph-acryl column. Carnation petal hemicellulose B reacted for 48 h withheat-inactivated protein (A), reacted with active protein for periods of5 (B), 24 (C), or 48 (D) h. Calibration of the column was as describedfor Figure 1. (0), Total carbohydrate; (0), xyloglucan.

lowing 5, 24, and 48 h of incubation with enzyme extracts,showed a gradual decrease of large MS polymers and a cor-responding appearance ofsmall MS products, eluting predom-inantly in the inclusion region of the gel (Fig. 2). The 10 kDpolymers were resistant to enzymic hydrolysis. However, pro-longed incubation (up to 5 d) with the enzyme preparationresulted in further degradation of the large MS polymers anda significant reduction in the amount of 10 kD polymers (datanot shown). Xyloglucan was depolymerized to some extentby the enzyme preparation and a 15% loss ofiodine-detectablexyloglucan was observed. Our previous work demonstratedthat carnation petal hemicelluloses were amylase insensitive(7). Disregarding the region in which hydrolysis productseluted (90-100 mL), the enzymic modification of hemicellu-loses closely resembled the changes occurring during petaldevelopment (Figs. 1 and 2).

Bio-Gel P-2 chromatography of the Sephacryl S-300 inclu-sion peak (Fig. 2D, volume 94-104 mL) disclosed the presenceof a polymeric component (DP 2 10), monosaccharide and adiverse array of oligosaccharides. Monomer accounted for 35to 40% of the P-2 profile (Fig. 3) and included xylose (35%),galactose (25%), glucose (21%), and smaller quantities ofarabinose (9%), mannose (3%), and fucose (6%).The in vitro production of monomers implicates exo-gly-

canase activity. A number of exo-glycanases have been shownto be sensitive to mercurials (6). Thus, the reaction mixturewas incubated for 48 h in the presence of 100 ,M HgCl2 in anattempt to inhibit exo-glycanase activity. In the presence ofmercury, the large MS polymers decreased in quantity to thesame extent as was observed in the absence of the inhibitor;however, monomer production was reduced nearly 30% anda portion of the hydrolysis products accumulated in the 10kD region of the Sephacryl profile (data not shown). Glycosylcomposition analysis of the monomers produced in the pres-

855




E

0.40)

't 0.3.l l

0.1

50 60 70 80 90 100

Elution volume (ml)

Figure 3. Biogel P-2 profile of the products formed from carnationhemicellulose B following incubation with an enzyme extract fromstage IV petals. One milligram of enzymically generated products(derived from Fig. 2D, volume 94-104 ml) in a volume of 1 mL was

applied to a water-jacketed Bio-Gel P-2 column (-400 mesh, 1.5 x

60 cm) operated at 440C. One milliliter fractions were collected and0.5 mL samples analyzed for total carbohydrate using the phenol-sulfuric method (8). The arrows index the elution positions of bluedextran, stachyose, raffinose, cellobiose, and glucose used for col-umn calibration.

ence of mercury disclosed that they were free of galactose.Mercury concentration up to 1 mM did not further reducemonomer formation. Dithiothreitol at a concentration of 10mm had no effect on hydrolysis.

Linkage Analysis of Hemicellulose Fractions

Previous studies demonstrated that compositional differ-ences between the large MS hemicelluloses and the 10 kDpopulation in carnation hemicellulose were minor (7). In viewof the fact that their susceptibility to enzymic degradationdiffered dramatically, it seemed likely that the two polymergroups possessed markedly different linkage characteristics.Glycosyl-linkage analysis of the large MS hemicelluloses dem-onstrated predominant proportions of 4- and 4,6-linked glu-cosyl and terminal-linked xylosyl residues (Table I), typicalof dicot xyloglucans (27 and references therein). This fractionalso contained 4-linked xylosyl and t-arabinosyl residues char-acteristic of arabinoxylan and some arabinans (27). Theamount of xyloglucan was substantially lower in the 10 kDfraction. This hemicellulosic fraction was enriched in poly-mers containing mannosyl and 4-glucosyl residues, presum-

ably glucomannan (31). During petal development and invitro enzymic hydrolysis, proportions of linkages of the largeMS polymers remained fairly constant. Little change in the10 kD polymers was observed during petal development,whereas after enzymolysis, a noticeable relative increase in 4-linked glucosyl and 4-mannosyl residues was detected.

Table I. Glycosyl-Linkage Composition of Carnation Hemicellulose BTwo milligrams of hemicellulose B were subjected to methylation analysis as described in "Materials

and Methods." For analysis of the Sephacryl S-300-separated hemicellulosic fractions, the void (V, 40-64 mL) and retained (R, 66-86 mL) regions were individually combined. Samples analyzed were: stageI (Fig. 1 A), stage IV (Fig. 1 D), and the 48 h enzyme digest (Fig. 2D). Values are in mol % and are themean of two separate experiments.

IV 48 hSugar Linkage

V R V R V R

t-Araa 1.7 13.6 2.2 6.4 1.3 9.25-Arab 4.8 5.6 9.0 7.4 10.6 2.93,5-Ara 1.4 1.7 2.0 1.2 4.3 NDCT-Xyl 23.2 3.0 18.7 3.1 19.8 0.74-Xyl 14.7 11.1 16.3 15.4 10.6 4.42,4-Xyl ND trd ND 2.6 ND ND2,4-Man ND 6.1 ND 4.3 ND 6.14-Man ND 14.5 1.9 13.8 ND 20.84,6-Man ND ND ND 9.7 ND 8.62-GIc ND tr ND ND ND 0.64-Glc 11.3 23.7 9.4 21.2 10.6 30.74,6-Glc 40.0 9.9 34.7 9.0 41.0 9.2t-Gal 4.0 9.3 4.2 6.3 1.9 5.13,6-Gal ND 1.1 ND ND ND 1.8

a t-Ara, Nonreducing terminal arabinosyl unit. b 5-Linked arabinosyl unit deduced from the 1,4,5-tri-O-acetyl-2,3-di-O-methyl-arabinitol derivative. c Not detected. d Trace amounts less than0.5%.





Characterization and Partial Purification of theHemicellulases

Hemicellulase activity was measured reductometricallyusing MG tomato fruit hemicelluloses. Tomato fruit hemi-celluloses were employed because of the ease of obtaining thequantities needed in these studies. We first determined thatthe carnation protein extracts degraded tomato fruit hemicel-luloses in a manner qualitatively similar to that observed withcarnation petal hemicelluloses (data not shown). This obser-vation is consistent with the apparent compositional andstructural similarities between hemicelluloses from the twoorgan types (7, 18, 31).Using an enzyme extract from stage IV carnation petals,

hydrolysis of the polysaccharides showed a broad pH range,with an apparent optimum at pH 4.5 (Fig. 4). The effect ofselected metal ions on enzyme activity was investigated afterincubation for 1 h at 37°C. Sodium at 150 mm was slightlystimulatory (9%); higher concentrations were inhibitory. Cal-cium (1 mM) and Hg2" (100 yM) reduced the activity by 13and 40%, respectively.Changes in hemicellulase activity during petal development

assayed under conditions as determined above (150 mM NaCl[pH 4.5]), are shown in Figure 5. The activity was relativelylow and remained constant during stages I and II. A threefoldincrease occurred between stages II and III (onset of senes-cence), and declined slightly during subsequent stages ofdevelopment.To characterize the enzymes involved in the hydrolysis of

the large MS hemicelluloses, we employed molecular sievechromatography to obtain partially purified enzyme(s). Arepresentative AcA 44 profile of protein from stage III petaltissue is presented in Figure 6. Assays using tomato fruithemicelluloses revealed the presence of two peaks of hemicel-lulase activity with apparent MS of 23 and 12 kD. Each peakwas analyzed further for activity against a number of polysac-charide and p-nitrophenyl-D-glycoside substrates. The 23 kDpeak showed high activity against laminarin, xylan, and thenitrophenyl substrates f-mannoside and 3-xyloside (data notshown). Of these, the xylanase and p-mannosidase activitieswere congruent with hemicellulase activity. Bio-Gel P-2 chro-matography of xylan digests (profile not shown) revealed a

,-

1.2w0

cL 0.8

I.C0

E3.0 41.0 5.0 6.0 70 8.0

pH

Figure 4. pH dependency of hemicellulase activity from carnationpetals. One milligram of MG tomato hemicellulose B was incubatedwith 300 ,g of protein (BSA equivalents [28]) in 1 mL of 50 mm Na-citrate-phosphate buffer. After 1 h the reaction was terminated andproducts measured reductometrically (24).

.

e 1.2wfi0

.- 0.A, P

E:L

I[

I 11 III IV V

.

302020

10 7.-

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Stages

Figure 5. Changes in hemicellulase activity during carnation petaldevelopment. One milligram of MG tomato hemicellulose B wasincubated with 300 Ag of carnation petal protein (BSA equivalents[28]) in a total volume of 1 mL 25 mm Na-acetate (pH 4.5) containing150 mM NaCI. After 1 h, reducing sugars were measured reducto-metrically. Products are expressed as glucose equivalents releasedper mg protein * h-1 (U) and per flower * h-1 (0). Values are meansof three experiments. SE did not exceed 5%.

series of oligomers and small quantities of monomer, provid-ing evidence for an endoxylanase. 3-Galactosidase and 3-glucosidase were associated with the excluded, large MS pro-teins. The 12 kD peak contained activity toward arabinoga-lactan and a-galactoside (data not shown). Carnation hemi-cellulose B treated with fractions from the 23 and 12 kDpeaks, alone and in combination, yielded the profiles shownin Figure 7. The profiles are indicative of hydrolysis of thelarge MS hemicellulosic polymers in an endo-fashion. Bothreactions involved the formation of 10 kD MS products. Bio-Gel P-2 chromatography revealed that relatively small quan-tities of monosaccharides were generated (data not shown).Treatment of hemicelluloses with both enzyme fractions re-sulted in a synergistic effect on hydrolysis (Fig. 7D), but againmonomers constituted a minor product component (data notshown).

DISCUSSION

In a previous study, we demonstrated that carnation petalhemicelluloses exhibit a marked shift in MS distributionduring development (7). The pattern by which this MS shiftoccurred was very similar to those which have been noted fornumerous species of ripening fruit (1 1, 16-18, 21). The sig-nificance of these hemicellulose changes in fruits and carna-tion petals is unknown. A study comparing tomato fruitgenotypes varying in intrinsic firmness suggests that the hem-icellulose changes are not a major determinant of firmnessdifferences (18). That similar changes occur in carnationpetals, which do not soften, is consistent with this contention,and suggests instead that the hemicellulose MS shift reflects asenescence-related alteration in the balance of cell wall ana-bolism/catabolism.The results of the present study show that the decrease in

large MS hemicelluloses in carnation petals is enzyme me-diated. The 10 kD fraction, although similar in neutral sugarcomposition (7) was not derived from the large MS polymersand, in fact, was quite resistant to enzymic degradation.

857




,,- 0.11

°' 0.3003

, 0.2

< 0.1

/~~I ~~~~0.20

111~~~~~0

120 200 300 400Elution volume (ml)

Figure 6. Ultrogel AcA 44 profile of protein derived from stage IlIlcarnation petals. Approximately 20 mg of protein were applied to a

bed (70 x 2.5 cm) of Ultrogel AcA 44 and eluted with 10 mm Na-acetate (pH 6.0) containing 150 mm NaCI. Fractions of 3.5 mL were

collected. (-- - -0), Protein; ( -*), hemicellulase activity, deter-mined as described in "Materials and Methods." V,, exclusion limit;Vi, inclusion limit.

Glycosyl-linkage analysis indicated that the large MS hemi-celluloses were comprised primarily of xyloglucan (approxi-mately 70% of the large MS hemicellulose), with smallerquantities of arabinoxylan and arabinans. The 10 kD fractioncontained predominantly mannosyl and 4-glucosyl units, pre-

sumably glucomannan. This fraction also contained smallquantities of xyloglucan, arabinoxylan, and arabinans.

Depolymerization of xyloglucan was apparent during petaldevelopment (Fig. 1). The isolated crude (Fig. 2) and partiallypurified (Fig. 6) enzyme extracts were able to degrade xylog-lucan in a manner similar to or more extensive, respectively,than that observed to occur in vivo during petal development.Xyloglucan consists of a :-1,4-linked glucan backbone, sub-stituted through carbon 6 with a-D-xylopyranose and termi-nated with a-L-fucopyranose and #-D-galactopyranose resi-dues (14). In pea and soybean tissue, xyloglucan is hydrolyzedby cellulase (endo- 1,4-fl-D-glucanase) into hepta- and nona-

saccharide (15) or into comparatively large fragments (20),respectively. These fragments can be degraded further intomonosaccharides by various glycosidases (14). Cellulase was

not detected in carnation petals by viscometric and reducto-metric assays even after prolonged incubation periods. Addi-tionally, cellulase purified from avocado fruit had limitedcapacity to hydrolyze carnation xyloglucan (data not shown),an observation confirming results obtained by Hatfield andNevins (13). These authors demonstrated restricted activityof avocado cellulase toward soybean hypocotyl xyloglucan.Also, studies on tomato fruit hemicelluloses revealed thatcellulase likely does not participate in the senescence-relatedmetabolism of hemicelluloses in this fruit (16).We propose that the xyloglucan in carnation petals was

hydrolyzed by a xyloglucan-specific glucanase. An enzymewith similar catalytic specificity was reported in germinatingnasturtium seeds (9). These glucanases must require the con-

formational/structural features of the side chains for substraterecognition and/or catalysis.The monomeric component recovered after digestion with

unpurified protein was rich in xylose, galactose, and glucose,constituting evidence for further hydrolysis of xyloglucan.The glycosidases a-D-xylosidase, f-D-galactosidase, and f-D-

glucosidase are likely participants in this hydrolysis (14).

While a-D-xylosidase was not measured, the latter two gly-cosidases were detected in the protein isolated from carnationpetals. Upon purification on Ultrogel AcA 44, these enzymeswere well resolved from the endo-hemicellulases. It is clearthat these enzymes, if involved in the hydrolysis of xyloglucanor other hemicelluloses, are greatly dependent on initial hy-drolysis by distinct endo-hydrolases. Gel-chromatography re-solved endo-hydrolases with limited capacity for monomerproduction, and other glycosidases which alone exhibited nocapacity to depolymerize hemicelluloses. Whether the latterenzymes represent authentic hemicellulolytic exo-enzymes or

0.6 A

0A

0.2

~0.6B

-'O 0.6SL *A

0

co0.2

~0.6c0

co 0.2

0Q6 D

OL-I

02

-10 60 80 100Elution volume (ml)

Figure 7. Sephacryl S-300 distribution of hemicellulose B followingtreatment with partially purified carnation hemicellulase. Two millilitersof hemicellulose and 100 ,ug of protein in a total reaction volume of 1mL containing 25 mm Na-acetate (pH 4.5), 150 mm NaCI, and 0.02%sodium azide were incubated for 48 h at 300C. Hemicellulose Btreated with (A) heat-inactivated protein, (B) 100 jig protein of 23 kDpeak hemicellulase (recovered from Ultrogel AcA 44, 266-301 mL,see Fig. 6), (C) 100 MAg protein of 12 kD peak hemicellulase (recoveredfrom Ultrogel AcA 44, 315-336 mL), (D) 50 ug 23 kD peak hemicel-lulase and 50 Mg 12 kD peak hemicellulase. Reactions were termi-nated by boiling and the samples applied to a Sephacryl column. (@),Total carbohydrate; (0), xyloglucan.





perhaps cell wall or cytosolic proteins with other cellular rolesis unknown. In terms of function, the glycosidases in generalare a poorly understood class of enzymes (6).

Glycosyl-linkage analysis in combination with gel-filtrationdata provide evidence that arabinoxylan, representing 18% ofthe large MS hemicelluloses, was depolymerized during petaldevelopment and during in vitro enzyme digestion. Enzymo-lysis of arabinoxylan might involve degradation by endoxy-lanase to produce oligosaccharides, followed by the action ofa-arabinosidase and ,B-xylosidase to produce monosaccharides(30). Endoxylanase activity was detected in the crude andpartially purified protein prepared from carnation petal tissue.The 1O kD hemicelluloses consisted predominantly of glu-

comannan. This observation is in agreement with the workofTong and Gross (31). These authors observed that the smallMS hemicellulosic fraction isolated from tomato fruit cellwall contained mostly mannosyl and 4-linked glucosyl unitsand that their relative content increased during ripening. Indeveloping carnation petals, no such increase in mannosyland glucosyl residues was detected (7; Table I). Additionally,the small MS hemicelluloses derived from muskmelon fruitcontained only 1% mannose and changed little during ripen-ing (21). These observations imply that there is no generalrelationship between the MS shift of hemicelluloses and thede novo synthesis of glucomannan as proposed for ripeningtomato fruit (31).Upon enzymic digestion, a relative increase in glucoman-

nan was noted in the 10 kD fraction (Table I). This enzymi-cally induced increase would appear to be inconsistent withthe linkage characteristics of the 10 kD fraction of senescentcarnation petals, which showed no evidence of an increase inglucomannan. This discrepancy is possibly explained by theenzymically induced loss in the xyloglucan and arabinoxylancomponents in the 10 kD fraction. Why a similar hydrolysisof these polymers would not occur in vivo is not known.The lack of glucomannan hydrolysis is not surprising inview of the fact that this polymer is resistant to enzymicdegradation (10).As indicated, the function of hemicellulose changes in

carnation petals in particular and in senescing organs ingeneral is not well understood. It is becoming apparent,however, that both hydrolytic and synthetic reactions contrib-ute to the overall redistribution in polymer size. A loss insynthetic capacity might partially explain the decrease in largeMS polymers in carnation petals. Evidence for this is theobservation that the initial loss in large MS polymers occurredbetween stages I and II (Fig. 1), during which time enzymeactivity remained constant (Fig. 5). Thereafter, the three- tofourfold increase in hemicellulase activity indicates that en-hanced depolymerization became a dominant factor. Regard-ing this trend, we did not determine whether this increaseinvolved a synchronous rise in all relevant enzymes or perhapsthe appearance of functionally unique proteins. It has beenestablished that a number ofnew gene products appear duringcarnation petal senescence (3). Altered gene expression is alsoa common feature of ripening fruit (4 and references therein)and in these instances the proteins produced are often thosewith either known or putative functions in the cell wall. Itwould seem that alterations in cell wall metabolism represent

yet another biochemical parallel between senescing fruit andpetal organs.

ACKNOWLEDGMENTS

The authors would like to thank Rocky Mountain WholesaleFlorist, Commerce City, CO, for providing the plant material andMr. J. Norman Livsey for his technical assistance in the methylationanalysis.

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