alpha galact coffea_marraccini_2005

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Original article

Biochemical and molecular characterizationof-D-galactosidase from coffee beans

Pierre Marraccini a,1, W. John Rogers a, Victoria Caillet a, Alain Deshayes a,Dominique Granato b, Franoise Lausanne a, Sylvianne Lechat a,

David Pridmore b, Vincent Ptiard a,*a Nestl Research Center, 101, avenue Gustave Eiffel, B.P. 9716, 37097 Tours cedex 2, France

b

Nestl Research Center, Vers-Chez-les-Blancs, 1000 Lausanne 26, Switzerland

Received 6April 2005; received in revised form 20 June 2005; accepted 18 August 2005

Available online 04 October 2005

Abstract

-D-Galactosidase (-Gal; EC 3.2.1.22) is one of three principal enzymes involved in the modification or degradation of plant cell wallgalactomannans. In the present paper it is shown that -galactosidase activities in field-grown coffee beans are variable amongst cultivars ofthe two species investigated (Coffea arabica and C. canephora var. Robusta). Higher activities were found in Arabica cultivars. Using beansfrom greenhouse-cultivated C. arabica as a model, we showed that -Gal activity was undetectable in the bean perispem tissue, but increasedgradually during the endosperm development, to reach a peak at approximately 30 weeks after flowering (WAF) which coincided with the

hardening of the endosperm. -Gal-specific transcripts detected at 22 and 27 WAF accompanied the peak of-Gal activity, but were reducedto be undetectable in mature beans at 30 WAF, while -Gal activity still persisted. Two isoforms were distinguished in 2-DE profiles of crudeprotein extracts by N-terminal sequencing analysis. Analysis of two-dimensional gel electrophoresis profiles demonstrated that both isoformsaccumulated in a linear fashion throughout grain maturation. -Gal activity was also observed to increase to high levels during in vitrogermination of coffee beans suggesting an important function of this enzyme in this process. -Gal cDNA sequences from Arabica andRobusta were sequenced and their deduced proteins appeared to be very similar, differing by only eight amino acids. Southern-blot analysissuggests that the enzyme was encoded by at least two genes in C. arabica that could explain the existence of the two isoforms identified in2-DE profiles. 2005 Published by Elsevier SAS.

Keywords: a-Galactosidase; Bean development; Cell wall polysaccharide; Coffea arabica; Coffea canephora; Galactomannan; Germination

1. Introduction

The polysaccharide fraction of green coffee beans repre-sents about half of the dry weight [4]. Among these polysac-charides, mannans predominate (50%), followed by ara-binogalactan (30%), cellulose (15%) and pectines (5%). The

mannans consist of a (14)-b-linked mannan chain thatcouldbe substituted at O-6 with single galactose residues to givegalactomannans (GMs). The degree of GM galactose substi-tution varies widely in nature and influences the water solu-bility of the polymer [7,34]. For example, pure unsubstitutedmannans form insoluble polymers, as observed in Ivory nuts(Phytelephas macrocarpa), while highly substituted GMs pos-sessing Gal/Man ratios of up to 1:2, as is the case for Guar(Cyamopsis tetragonoloba) gum, are able to form viscoussuspension in water. In the case of coffee, the literature reportsGal/Man ratios of between 1:130 [3] and 1:30 [17]. The lattervalue was confirmed recently by a detailed chemical analysisof GMs extracted at regular stages of coffee bean develop-ment [45]. In this study, it was shown that the GM Gal/Manratio is high (between 1:2 and 1:7) in earliest developmental

Abbreviations: DAI, days after imbibition; GM, galactomannan; 2-DE,two dimensional electrophoresis; UTR, untranslated region; WAF, weeksafter flowering.

* Corresponding author. Fax: +33 2 47 49 14 14.E-mail address: [email protected] (V. Ptiard).

1

Present address: CIRAD UMR PIA 1096/IAPAR AMG Laboratory ofPlant Biotechnology, Rodovia Celso Garcia Cid Km 375, CP 481, 86001-970 Londrina Paran, Brazil.

Plant Physiology and Biochemistry 43 (2005) 909920

www.elsevier.com/locate/plaphy

0981-9428/$ - see front matter 2005 Published by Elsevier SAS.doi:10.1016/j.plaphy.2005.08.010


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stages of endosperm (i.e. at 11 weeks after flowering (WAF))and decreases (between 1:7 and 1:40) during further the matu-ration of this tissue. Changes of GM Gal/Man ratios occurwhen the maternal perisperm tissue is gradually displaced by

the endosperm [47,48], a phenomenon that is accompaniedby the hardening of the endosperm [7,8]. Based on theseobservations, the authors suggested that galactose removalfrom the primary synthetic product could be dependent on-D-galactosidase activity.-Gal occurs widely in microorganisms, plants and ani-

mals [1,13]. In plant species the hydrolase usually actstogether with (14)-b-mannan endohydrolases (endo-b-mannanase) (EC 3.2.1.78) andb-mannosidases (EC 3.2.1.25)to degrade GMs, mainly during germination of plant seeds[46]. However, high activity of-Gal has also been reportedin dry carob seeds [27] as well as during seed development ofSenna occidentalis [15]. The enzyme from coffee beans was

one of the first -Gal to be partially purified and biochemi-cally characterized [9,12,19,21,22]. In these initial reports,this enzyme was described as occurring as two isoforms (Iand II) having different molecular weights (28 and 36.5 kDa),different activity pH optima (5.3and 6.3) and isoelectricpoints(pI).

Furthermore, the enzyme has attracted attention in the fieldof biotechnology due to its capacity to remove the terminalgalactose units (a 13 linked) from the blood group B cellsurface carbohydrate moiety of glycoprotein complexes, thusgenerating type O red blood cells [51]. -Gal from microbialsources (A. niger, S. carlsbergensis and E. coli) was foundnot able to play this function [40]. For this reason, cDNA

sequences coding the coffee bean a-Gal are now available inthe literature [52] or in international patents [25], and therecombinant enzyme has been expressed in a number of cellculture systems [53,54,57]. Based on these analyses, it is sug-gested that the mature active enzyme is composed of363 amino acids, with an estimated molecular weight ofapproximately 40 kDa, and that it may be synthesized as apre-proenzyme of 420 residues. More recently, site-directedmutation experiments permitted to identify amino acids resi-dues essential for the activity of coffee bean -Gal[31,32,55,56]. However, the species or cultivars of coffeeplants used in these publications have not always been speci-

fied.In this article, we present data of-Gal activities in beansof various coffee species cultivated under different condi-tions and results of gene expression studies during coffee beandevelopment, as well as in other plant tissues. We also iden-tified -Gal in protein extracts of mature (40 WAF) beansanalyzed by 2-DE and compared-Gal cDNA sequences fromcommercial Coffea arabica (Arabica) and C. canephora(Robusta) cultivars.

2. Results

2.1. Optimum conditions for-galactosidase activity assays

Activity of both the commercial Boehringer Mannheimpreparation and coffee grain protein extracts was tested for

optimum pH of the reaction and for tolerance to certain elec-trophoretic reagents substance (data not shown). The pH opti-mum was found to be broad with a peak at 7.5. Tolerance ofthe activity to the presence of SDS, Triton-X 100 and glycine

was high. Tricine was less tolerated, although 75% activitywas still retained in the presence of a concentration of 50 mM.

2.2. -Galactosidase activity in grains

of different cultivars and sources

Activity was measured during maturation of grains (beans)harvested from trees cultivated either in field (Fig. 1A) or ingreenhouse (Fig. 1B). In all of cases,-Gal enzymatic activi-ties always appeared extremely low or undetectable in thefirst stages of the development where the perisperm tissuepredominated.After that,-Gal enzymatic activities increasedgradually to reached a peak usually observed before the har-vesting stage for grains collected in the field or earlier peri-carp reddening stage for grains collected from greenhouse-grown trees.-Gal enzymatic activities measured in grains of the Ara-

bica cultivar Caturra T2308 and the cultivar Dormilon of C.

Fig. 1. a-Galactosidase activities during maturation of coffee grains harves-ted in cultivars grown in greenhouse and in field conditions. (A) Grains har-vested in 1995 from field grown plants were from C. arabica cv. CaturraT2308 (n) and CRM (e), or from C. canephora ROM (*) and Dormilon

(C). (B) Grains from plants grown in greenhouse were from C. arabica cv.Caturra T2308 (harvested in 1994) (M) and from C. canephora (harvestedin 1993) 4FPRC8 x ?3484/6 (D) or 43484/6 x ?FPRC8 (m).

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canephora cultivated in the field, remained relatively stableup to the harvest, whereas they decreased in the latest matu-ration stages of grains from CRM (Arabica) and ROM(Robusta). In addition, levels of-Gal activity in ROM cul-

tivar were close to those detected in grains collected fromgreenhouse-grown trees.Fruits harvested in greenhouse were characterized by

overly long maturation periods compared to those harvestedin the field. -Gal activities detected from two C. canephoracrosses(4FPRC/8 ?3484/6 and43484/6 ?FPRC/8) andfrom Arabica cv. Caturra T2308 appeared amongst the low-est measured (Fig. 1B). After reaching a peak before the red-dening phase of the pericarp, -Gal activity declined gradu-ally up to the maturation stage of the cherries. Theseobservations were confirmed during 2 consecutive years ingrains from greenhouse-grown plants (data not shown).

To analyze the variability of-Gal activities encountered

in coffee, measurements were performed in grains of differ-ent species and cultivars originated from several countries(Table 1). The range of -Gal activities varied from0.26 nkat mg per protein for the clone 197 of Robusta culti-vated in IvoryCoast to 2.63 nkat mg per protein forthe CaturraT2308 cultivar of Arabica from Ecuador. Three Arabica cul-tivars (T2308, RM and commercial) had the highest activi-ties, five to six times higher than -Gal activities of otherfield-grown plants of Arabica. The only Arabusta (ARM)examined and one Robusta cultivar (C. canephora cv.Dormilon) had intermediate activities, respectively, with

0.89 and 1.1 nkat mg per protein at 30 WAF. -Gal activitiesmeasured in various Robusta coffee plants always appearedlower than thosein Arabica, ranging from 0.26 to 0.75 nkat mgper protein.

2.3. Expression of-galactosidase gene during grain

maturation

Accumulation of-Gal-specific mRNA was measured ingrains from greenhouse-grown Caturra T2308, and com-pared to the evolution of activity in separated tissues (i.e.perisperm and endosperm) measured in the same material(Fig. 3). Even though-Gal activities measured in beans har-vested from greenhouse-grown Caturra T2308 were lowerthan those measured in beans of the same clone cultivated inthe field, this activity was specifically detected in the en-

dosperm but not never observed in the perisperm (maternal)tissue (Fig. 3A). This was confirmed by a Northern blotexperiment, demonstrating the appearance of-Gal-specifictranscripts (around 1.2 kb) during the early stages of en-dosperm development (at 22 WAF), with highest expressionat approximately 27 WAF when enzyme activity was stillincreasing (Fig. 3B). At the peak of-Gal activity (around36 WAF), specific transcripts were still present but barelydetectable. In mature beans (40 WAF in our greenhouse con-ditions), -Gal activity was still high while no more tran-scripts were observed (data not shown).

2.4. -Galactosidase activity during germination

-Gal activities were also measured during germinationof grains from Caturra T2308 cultivated in the field or in thegreenhouse (Fig. 2). Very low activity was measured in thenewly imbibed seeds, but it increased gradually during thegermination, up to maximum of 2.3 and 3.66 nkat mg perprotein at 42 days after imbibition (DAI) for grains harvested

Table 1

Preliminary comparisonsof-Galactivity (nkat mg1protein)in coffee beans(endosperm) from various origins and species.Activities represent the meanof at least three separate extractions from the same harvest batch.(ARM = Arabusta Rojo Micropropagated; RM = Rojo Micropropagated;F = field-grown plants; GH = greenhouse-grown plants; M = mature har-vest stage without specified WAF)

Species/cultivar Activity Observations

C. arabica cv. Caturra T2308 2.63 Quito F24 WAF

C. arabica cv. Caturra T2308 0.42 Tours GH33 WAF

C. arabica cv. Caturra T2308 0.35 Tours GH3740 WAF

C. arabica cv. Caturra Commercial 1.75 Quito F24 WAF

C. arabica cv. Typica 0.48 Quito F30 WAF

C. arabica cv. Typica 0.45 Tours GH37

40 WAFC. arabica cv. Typica 0.54 Mxico FM

C. arabica cv. Pacas 0.58 Quito FM

C. arabica cv. Bourbon 0.48 Mxico FM

C. arabica cv. Garnica F3 0.49 Mxico FM

C. arabica cv. Caturra RM 1.5 Quito24 WAF

C. arabusta cv. ARM 0.88 Quito F24 WAF

C. arabusta cv. ARM 0.89 Quito F30 WAF

C. canephora C126 0.31 Abidjan FM




C. canephora cv. Dormilon 1.1 Quito F30 WAF

C. canephora cv. ROM 0.34 Quito F24 WAF

C. canephora ?FPRC/8 x 43484 0.48 Tours GH49 WAF

C. canephora ?3484 x 4FPRC/8 0.53 Tours GH39 WAF

Fig. 2. -Galactosidase activities during coffee grain maturation and germi-nation. Grains were harvested from cloned C. arabica cv. Caturra T2308 cul-tivated either in the field (n) or in greenhouse (M). Germination proceeded

under in vitro conditions. Time scale is indicated in WAF for bean matura-tion and in DAI for the germination. The radicule length is also indicated(C).

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from the field and greenhouse, respectively. For greenhouse-grown grains, which had lower activities during their matu-

ration,-Gal activity measured during their germination pre-sented the highest increase compared to that observed forfield-collected beans.

2.5. -Galactosidase activity and expression

in coffee tissues

Activities were also analyzed in different organs of agreenhouse-grown C. arabica cv. Caturra T2308, and com-pared to those measured in separated tissues (pericarp,perisperm and endosperm) from coffee cherries (Table 2).

Highest -Gal activities were always observed in endospermof fruits, whatever their origin, and to a lower extend in zygoticembryos. These observations were very well correlated withlevel of-Gal-specific products amplified by RT-PCR in thesame tissues (Fig. 4). However, -Gal activities were muchlower in other tissues analyzed, like roots and leaves forexample, and closeto the limits of detection in the perisperm.Part of these observations was confirmed by RT-PCR experi-ment (Fig. 4), particularly in roots and leaves forwhich-Gal-specific products amplified appeared very low. Finally, levelsof-Gal gene expression in stems and flowers were veryweak.

2.6. Isoelectric localization of-galactosidase

The -Gal commercial preparation was analyzed by SDS-PAGE and IPG-2-DE (Fig. 5A). A single band representingthe -Gal enzyme was visible by SDS-PAGE. When ana-lyzed by 2-DE, this band appeared to be formed by at leasttwo isoforms with an estimated MW of 40 kDa and pI ofapproximately 5.5 and 5.7. For both proteins, N-terminalsequences were identified (LANGLGLTPP) and were iden-tical. The analysis of these peptides revealed that they corre-sponded to the first amino acids of the N-terminal region of

other coffee-Gal protein sequences (Fig.7, positions 5867).The commercial preparation also contained Bovine Serum

Albumin at 66 kDa, and three contaminating proteins (namedUP for unidentified proteins), which were also subjected toN-terminal sequencing. UP 1 and 2 shared identicalN-terminals up to the first fifteen amino acids (GGGEN-LFQGKKVVD[S]), and separated into three isomers each.Internal sequences were also obtained (R/KGNDA;R/KGLSTXAQXVNQXXVS, X is for unidentified aminoacid residue). No sequences could be obtained for UP 3. Noconvincing homology of these sequences could be found inpublic protein databases and these proteins remained un-known.

The serum containing anti--Gal antibodies was used totest a crude protein extract of mature (40 WAF) coffee ( C.

Fig. 3. -Galactosidase activities in perisperm (C) and endosperm (M) tis-sues separated from C. arabica cv. Caturra T2308 grains grown in green-house (A). Tests of activity represent the mean of at least three separateextractions. (B) Total RNAs extracted at different weeks after flowering(WAF) from beans of the same plant were probed with the CaGAL1 cDNA.(C) Total RNA stained by ethidium bromide. Numbers indicated at the bot-tom of each figures indicate the maturation time expressed in WAF.

Table 2-D-Galactosidase activity (nkat mg1 protein) of in various tissues fromcloned C. arabica cv. Caturra T2308 grown in greenhouse (GH) or in thefield (F). Activities represent the mean of at least three separate extractionsfrom the same harvest batch

Plant source Tissue Activity

C. arabica cv. CaturraT2308 Quito F

Mature endosperm(30 WAF)

2.56

Mature pericarp (30 WAF) 0.21

C. arabica cv. CaturraT2308 Tours GH

Mature endosperm (3740 WAF)

0.52

Mature pericarp(3740 WAF)

0.31

Perisperm (1217 WAF) 8.103

Leaves 0.12

Roots 0.17

Zygotic embryo 0.41

Fig. 4. Expression ofCaGAL1 in different tissues ofC. arabica cv. CaturraT2308 grown in greenhouse. RT-PCR products specific of the -Gal trans-cripts are presented in the upper panel. Amplification products of the cons-titutively expressed CaSUI1 gene [18] were used as a control and are pre-sented in the lower panel. RNA samples used are from young leaves(length < 2 cm) (lane 1), medium leaves (length 210 cm) (lane 2), largeleaves(length > 10 cm) (lane 3),open flowers (lane 4),stems(lane 5), flowerbuds (lane 6), roots (lane 7), grains at 27 WAF (lane 8) and zygotic embryos(lane 9).



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arabica) beans separated by IPG-2-DE (Fig. 5B). Thiswestern-blotting experiment allowed highlighting two -Galisoforms with MW and pIs similar to those observed in thecommercial preparation. Using this information about thelocalization of-Gal isoforms in 2-DE gel, correspondingproteins were identified in a silver-stained 2-DE gel (Fig. 5C) and their nature was confirmed by N-terminal amino acid

sequencing (data not shown). The same approach also per-mitted to localize and identify in the 2-DE gel, all the UPsproteins encountered in the commercial -Gal preparation.All of them appeared to be very abundant in theprotein extractof mature (40 WAF) coffee beans, which in part permitted toexplain their presence as contaminants in the commercialpreparation.

2.7. Analysis of the coffee -galactosidase cDNAs

The CaGAL1 and CcGAL1 cDNAs were compared withthe two other coffee -Gal cDNA sequences already pub-

lished [25,52]. Nucleic acid alignments showed that all thesesequences were highly homologous both in UTR and cDNAcoding regions (Fig. 6). One of the few differences con-cerned the adenosine in position 172, which was not observedin the CaGAL1 cDNA as well as in the sequence reported byIvy and Clements [25], but which was present in the Arabicasequence [52]. For this latter sequence, the -Gal codingsequence should begin at the ATG-198. However, if we con-sider that the A-172 does not exist, the ATG-72 of CaGAL1cDNA should be used instead of the internal ATG-198 (seesequence 3 in Figs. 6 and 7), leading to a -Gal precursor of420 amino acids with a molecular mass of 46 kDa and a pI of7.14. Analogies with other pre-proenzymes suggest the exist-ence of a protease cleavage site between amino acids resi-dues Ala-22 and Ser-23, delimiting a secretion peptide signal

of 22 residues and a proenzyme -Gal form of 398 aminoacids corresponding to the residues 23420 with an esti-mated molecular mass of 43.4 kDa. Further processing wouldtake place by endogenous peptidases to remove the peptidecorresponding to amino acid residues 2357, leading the Leu-58 at the extremity of the mature and active-Gal enzyme asidentified in the commercial preparation, as well as in mature

grains C. arabica cv. Caturra T2308 at 40 WAF (Fig. 5).Of the 24-nucleic differences observed between the four

coffee -Gal cDNA coding regions (Fig. 6), only 10 wouldbe expected to modify the amino acid composition of thetranslated proteins withoutaffecting their length (Fig. 7). Eightof these amino acid differences were observed betweenCaGAL1 and CcGAL1 proteinswhich present together 97.8%identity at the amino acid level. Surprisingly, the Ca-GAL1 protein appeared less homologous to the Arabicasequence [52] than the CcGAL1 protein, respectively, with98.4 and 99.5% of identity. However, theoretical pIs ofCaGAL1 and CcGAL1 mature enzymes are identical, close

to 5.7, whereas those of the two other-Gal (sequences 1 and2 in Fig. 7) were estimated to 5.88.Screening of the mature CaGAL1 against protein data-

base confirmed i ts strong similarity with other-galactosidases of dicotyledonous plants, members of thefamily 27 of glycosyl hydrolases [23]. For example, higherscores of amino acid identities of 81.2%, 80.7%, 80.4%,78.8% and 78.1% were observed, respectively, for kidneybean(Phaseolus vulgaris: AAA73964), sunflower (Helianthusannuus: AB092594), soybean (Glycine max: AAA73963),tomato (Lycopersicon esculentum: AAF04591) and guar (C.tetragonoloba: CAA32772). These identity scoresfall to 55%with Clostridium josui (BAB83765), which presents the high-est identity within prokaryotic -Gal sequences. Similarresults were observed for the mature CcGAL1 protein.

Fig. 5. 2-DE gel electrophoresis analysis of coffee grain a-galactosidases. (A) The commercial preparation of-Gal was analyzed by IPG-2-DE andproteins were identified by N-terminal sequencing. The SDS-PAGE of the same preparation was inserted in the left part of the graph. UP represents unidentifiedproteins. (B) Proteins extracted from mature (40 WAF) C. arabica cv. Caturra T2308 grains were separated by 2-DE gel, blotted and tested by western-blottingusing polyclonal antibodies raised against the commercial -Gal. The two -Gal isoforms identified are marked by a black arrow. (C) The same coffee proteinextract was separated by 2-DE and silver-stained. White arrows and circles indicate the protein spots (a Gal and UP) analyzed by N-terminal sequencing.Whitearrows localize a and b arms of coffee 11S-storage proteins. For each gel, molecular weight markers (in kDa) are indicated in the left and a pH scale in thebottom.



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2.8. Southern hybridization analysis

Arabica genomic DNA was single-digested with DraI,double-digested by DraI/ScaI and then hybridized with theentire CaGAL1 cDNA (Fig. 8). Several bands were observedin both patterns although no restriction sites were present inthe CaGAL1 cDNA. Strong hybridizations were detected at

1.15, 1.3 and 1.5 kb as well as faint signals of 0.3, 2.4, 3 and3.5 kb for genomic DNA digested by DraI. These two latterbands were no more detected in the double digestionDraI/ScaI, probably leading to the fragment of 2.8 kb. Othersmaller fragments (near 0.75 kb)were generated by the doubledigestion, part of them coming from the cleavage of the1.5 and 2.4 kb detected after the DraI digestion. In addition,

Fig. 6. Alignments of-Gal cDNA sequences. Nucleic acid sequences are from Ivy and Clements [25] (1, AR366583), Zhu and Goldstein [52] (2, L27992) orobtained from C. arabica cv. Caturra T2308 (3: CaGAL1, AJ877911) or from C. canephora (4461 X ?197) (4: CcGAL1, AJ877912). Stars highlightdifferences. Arrows indicate the position and the orientation of the primers. Putative start (ATG) and stop (TGA) codons are in bold. Nucleic acid numbering is

indicated and refers to the sequence 1.

Fig. 7. Alignments of-Gal proteins sequences from Ivy and Clements [25] (1, AAQ77888), Zhu and Goldstein [52] (2, AAA33022) or obtained from C.arabica cv. Caturra T2308 (3, CAI47559) or from C. canephora (4461 X ?197) (4, CAI47560). Amino acid differences are indicated by stars. The putativeN-glycosylation site NIS (193-195) is underlined. Amino acid residues Trp-73, Tyr-150 and Asp-329 are indicated by black vertical arrows. The putativeprotease cleavage site is indicated by a white arrow. The residue Leu-58, corresponding to the N-terminal sequence of-Gal found in mature grain is doubleunderlined. Amino acid numbering is indicated and refers to the CaGAL1 protein (sequence 3).



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we recently amplified, from the Arabica genome, aBETA100/BETA101 PCR fragment of around 5 kb contain-ing the entire CaGAL1 gene (data not shown). Together withthe sequence information deduced from the CaGAL1 cDNA,all these data indicated the presence of at least two copies ofthe -Gal gene in the Arabica coffee genome.

3. Discussion

One of the objectives of this work was to increase ourknowledge on coffee-galactosidase, which is thought to playa key role in the metabolism of GMs [45]. Together with otherglycosidases (i.e. b-galactosidase, -mannosidase, N-acetyl-b-D-glucosiminidase),-Gal activity was previously reportedin entire ripening fruits of C. arabica [19]. In the presentwork, we clearly demonstrated that no activity was observedduring the first few weeks of coffee fruit development, whenthe perisperm (maternal) tissue predominated. During the sec-ond part of coffee cherry development, -Gal enzymaticactivities were detected both in the pericarp (also referred asthe pulp) and in the endosperm, with a higher activity in thelatter tissue. As a model, we studied the evolution of-Galenzymatic activity in endosperm of coffee fruits grown ingreenhouse conditions and showed that it was undetectable

at 20 WAF, but reaching a maximum at around 36 WAF. Ouranalysis clearly showed that this increase of activity over-lapped quite well the accumulation of-Gal-specific tran-scripts observed between 22 and 27 WAF, which also corre-

sponded to the rapid expansion and hardening of theendosperm. This demonstrated that the -Gal gene expres-sion was temporally and spatially regulated during coffee fruitdevelopment, as also observed for the csp1 gene coding for11S-storage proteins [47]. To confirm this pattern, it wouldhave been interesting to follow -Gal gene expression in theendosperm from fruits ofC. arabica cv. Caturra T2308 har-vested from field grown plants where higher levels of enzy-matic activity were measured (Fig. 1A). Unfortunately, evenif these fruits were shipped in dry ice, all our attempts toextract their RNAs failed, probably because fruits were notfrozen in liquid nitrogen immediately after their harvest, acondition that in our hands always appeared critical to extractintact RNAs from various coffee tissues (Caillet and Marrac-cini, unpublished observations).

Using polyclonal antibodies against the commercialenzyme and N-terminal sequencing, at least two well-separated and abundant-Gal isoformswere detected in beansat the optimal stage of harvest (40 WAF), with equal molecu-lar weights (near 39.5 kDa) but differing by their pI (5.5 and5.7). Other observations reporting the presence of-Galisoenzymes of close molecular mass were also made for plantseeds ofP. vulgaris [14] and Glycine max L. [20] for example.To clarify the origin of cDNA sequences already available inthe literature and patents [25,52,53], but also to see if the-Gal pIs could be explained by subtle changes at the aminoacid level of-Gal isoforms, full-length cDNA sequenceswere cloned from fruits of well identified C. arabica (Ara-bica) and C. canephora (Robusta) coffee plants. The analysisof the CaGAL1 cDNA sequence suggested that coffee -Galwas probably synthesized as a pre-proenzyme containing ahydrophobic signal peptide of 22 amino acids. BecauseN-terminal sequencing of the -Gal revealed that the matureprotein began with Leu-58 amino acid residue, this clearlydemonstrated that residues 2357 should constituted a pro-sequence that should be removed during the trafficking of-Gal protein in the cell, as previously suggested [25]. By

2-DE, the molecular weights of the mature -Gal isoformswere estimated close to 39.5 kDa, which agreed with the theo-retical protein of 363 amino acid residues deduced from bothCaGAL1 and CcGAL1 cDNA sequences.

Thepresence of a signal peptide in the pre-proenzymeformof-Gal is interesting because this should allow it to be trans-portedinto theendoplasmic reticulum, and thence in cell wallswhere mannans accumulate. This should also permit the tar-geting of the -Gal into proteins bodies like it was shown incotyledons of soybean and lupin [24,39,43] and also inendosperm and embryo of date palm [11]. The possible extra-cellular localization of coffee -Gal is another point of par-ticular interest because it should be a prerequisite to maintainthe high Gal/Man ratio of newly synthesized GMs and con-sequently their relative solubility, in order to permit their intra-

Fig. 8. Southern-blot ofC. arabica DNA digested with DraI (Dr) and ScaI

(Sc) restriction enzymes and hybridized with the CaGAL1 cDNA (sequence3 in Fig. 6). Labeled molecular markers (M in kbp) are also presented.



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cellular trafficking. In this model, the hardening of endospermshould be directly controlled by the -Gal activity, throughthe removal of galactosyl residues from mannan chains, lead-ing to the deposition of these polysaccharides in cell walls as

proposed previously [2,45]. Immunolocalization and cell frac-tionation experiments should be required to confirm the cel-lular localization of-Gal isoforms in coffee.

The comparison of Arabica (CaGAL1) and Robusta(CcGAL1) -Gal cDNA sequences also revealed few differ-ences, some of them leading to amino acid changes between-Gal proteins of the two coffee species. However, none ofthese differences implicated the Trp-73 and Tyr-150 aminoacid residues, which were shown to be essential for coffee-Gal activity [32,55,56], or the Asp-329 that was suggestedto play a role in the active site of the guar enzyme [41]. Thetheoretical pIs of-Gal proteins deduced from Arabica andRobusta cDNAs were identical (close to 5.7), and could be

related to the -Gal isoform of similar pI identified by 2-DEgel electrophoresis, both in mature beans and in the commer-cial preparation. Concerning the -Gal isoform with a lowerpI (5.5), we showed that its N-terminal amino acid sequencewas identical to the former, therefore excluding the possibil-ity of post-translational modifications in this N-terminal part,to explain the different pIs observed. The existence of post-translational modifications in the C-terminus region of thisprotein, i.e. like the removal of few amino acids, also seemsunlikely because the Trp-Pro-Gln carboxyl terminal extrem-ity was shown to be critical for the -Gal enzymatic activity[31]. Finally, the involvement of a glycosylation process, forexample at the putative N-glycosylation NIS site (position193195, Fig. 7), was also highly improbable, as -Gal puri-fied from coffee beans did not bind to ConA Sepharose [53].

Altogether, these observations highly argued for the pres-ence of two copies of-Gal-encoding genes in C. arabica,as also suggested by the Southern blot experiment (Fig. 8).Such a situation was also observed in other plants, like inArabidopsis thaliana where two putative genes coding forthe acidic -Gals were present, one on the chromosome 3(At3g56310) and the other on chromosome 5 (At5g08370).The number of-Gal-coding genes seems to be higher inrice(Oryza sativa cv. japonica) where four copies were found,two of them on the chromosome 7 (OJ1409_C08.26 and

OSJNBb0062P14.111) and the other on the chromosome 10(OSJNBa0041P03.8 and OSJNBa0051D19.18). Theseexamples contrasted with the situation observed in tomato(L. esculentum) where-Gal seems to be encoded by a uniquegene [16]. Because C. arabica is amphidiploid, resulting froma natural cross between diploid species C. eugenoides and C.canephora [29], it is possible that the two -Gal-encodinggenes we detected in Arabica genome, represented indepen-dent genes coming from each parent rather than two isoformsof the same gene that could be due to recent gene duplica-tion. The screening of available coffee BAC libraries fromRobusta [30] andArabica [37] with the present-Gal cDNAswould be very useful to solve this question.

Even when using the same cultivar (CaturraT2308) ofAra-bica, our analysis also demonstrated that grains from

greenhouse-grown plants had lower-Gal activities than thosefrom plants grown in the field. We did not investigate whatwere the reasons of these differences, but they could be prob-ably linked to variationsof environmental parameters between

field and greenhouse conditions, as those concerning tem-perature and light intensity received by the plants. Forexample, plants grown in sub tropical regions under full-sun field conditions receive a photosyntheticphoton flux den-sity (PPFD) normally varying from 1500 to 2500 E s1 m2

at noon, respectively, for winter and summer season [50].However, plants grown in greenhouse received a constantPPFD of 300 E s1 m2 throughout the year, therefore mim-icking coffee plant grown in shade condition. These envi-ronmental parameters were reported to have great impacts oncoffee fruit development, like delayed maturation and in-creased fruit weight and bean size [36,50]. In addition, werecently observed slight reduction of sucrose synthase activ-ity levels in endospermof coffee plants cultivated under shad-ing in comparison to full-sun growing conditions (Ger-omel, Mazzafera and Marraccini, unpubl. obs.). Such aphenomenon could also occurred for-Gal enzymatic activi-ties in order to explain the lower levels in greenhouse.

The present work also showed that-Galactivities in seedsof reverse crosses of greenhouse-grown C. canephora weresimilar, which suggested the absence of a maternal effecton the control of-Gal enzymatic activity. We observed atendency of higher -Gal activities in beans of Arabica thanin those of Robusta, which needs to be confirmed before tobe considered as a general rule. However, it is interesting to

note that -Gal activities detected in Caturra and ROM vari-eties (Fig. 1) were inversely correlated with the ratios ofunbranched to branched mannose previously analyzed in thewater-soluble GMs of the same varieties [17].

Finally, -Gal activities followed during the in vitro ger-mination of coffee beans of C. arabica cv. Caturra T2308,harvested either from the field or in greenhouse, showed acontinuous increase during germination, leading to a maxi-mum (3.66 nkat mg per protein) near 40 DAI. Even if theactivity was no longer followed after this time, the profileobtained clearly differed from that previously reported forendo-b-mannanase activity during germination, which

reached a peak at 28 DAI and declined drastically after thistime [33]. Because the expression of the CaGAL1 gene wasnot tested during the germination, we do not know if the largeincrease of-Gal activity measured came from the activationof pre-existing CaGAL1 protein in green beans and/or fromde novo synthesis, through the activation of transcriptionaland/or transcriptional processes. Like described in other plants[13,20], the expression of other types of-Gal, could alsooccurred in coffee (Buckeridge, personal communication).During this experiment, -Gal activity measured just afterthe harvest of mature beans of field-grown Caturra(2.56 nkat mg per protein at 30 WAF) was higher that activityof the same beans at the beginning of the germination test(0.15 nkat mg per protein at DAI). Such a difference was dif-ficult to explain but could be attributed to problems of ship-



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ments and/or storage of the samples. However, this did notaffect the efficiency of germination of the beans nor the-Galactivity that presented a great increase during the germina-tion, reinforcing the importance of this enzyme during this

process. While major substrates of this enzyme in the coffeegrain were still not fully clarified, the -Gal activity couldalso be linked to the degradation of raffinose family of oli-gosaccharides (RFOs) and galactosyl cyclitols as proposedby Obendorf [39]. In that model, -Gal synthesized duringseed development is targeted to protein bodies and their sub-strates (RFOs and galactosyl cyclitols) are accumulated inthe cytosol [35,44]. During germination, these oligosaccha-rides are transported from the cytosol to the protein bodiesthat become a vacuole for their hydrolysis [6,39]. RFOs wereidentified in mature coffee beans [10,48] and therefore couldbe used as substrates for-galactosidase during germination.Interestingly, Rogers et al. [48] showed that Robusta variet-ies accumulated stachyose to equal amounts to those mea-sured in Arabica, but seems to contain reduced raffinose lev-els in comparison to Arabica, as also mentioned byChabrillange et al. [10]. Because -Gal activity removes thegalactosyl from the stachyose to give raffinose oligosaccha-ride, low levels of stachyose observed in Robusta could berelated to the lower -Gal activity in this specie compared toArabica. The broad range of substrates used by the coffee-Gal [12], and its capacity to perform transglycosylation[26,49] also argue for a central role of this enzyme in thereorganization of coffee cell walls. In that sense, the knowl-edge acquired here about the-Gal-encoding gene now opens

the way for the search in coffee plants with down-regulated-Gal expression through different techniques includingbreeding programs managed by marker assisted selection, tillgenetic modification as recently shown in petunia [38].

4. Methods

4.1. Plant material and conditions of germination

Greenhouse-grown coffee trees were maintained at a day-time temperature of approximately 25 C (24 h variation

between 20 and 27 C), 70% humidity and 16 h photoperiod(light intensity of 300 E s1 m2). For measurement of-Galactivity, fruits ofC. canephora were obtained following cross-pollination by hand of the cultivars FPRC/8 and 3484/6.-GalcDNAs were obtained from grains resulting from hand-crossed Robusta cultivars 4461 X ?197 and from a self-pollinated tree ofC. arabica L. cv. Caturra T2308. The sametree was also used to analyze -Gal activities and expressionin different organs (leaves, root, flowers) and in separatedgrain tissues for maturation studies. In that case, the en-dosperm was separated from all maternal fruit layers includ-ing the locules (parchment) and the maternal perisperm (theremnant of which is also called silver skin in mature beans).

Fruit samples from various varieties of field-grown C. ara-bica and C. canephora were from Equator (Nestl R&D,

Quito Equator). C. arabica CRM was a Caturra Rojo cul-tivar propagated by micro-cutting. The Caturra T2308 culti-var was a cloned material identical to that analyzed from thegreenhouse. The Robusta Dormilon and ROM were well

known commercial varieties. Harvests were overseen byNestl workers (Quito Equator). Other samples of mature (red)fruits were obtained from other sources (Nestl R&DIvoryCoast and Nestl-Mexico).

All greenhouse-grown fruits and tissue samples were fro-zen in liquid nitrogen immediately following harvesting andstored at 85 C until use. Samples from other countries wereshipped on dry ice and stored under the same conditions.

For in vitro germination assays, grains were dehulled, sur-face sterilized by stirring in a solution of calcium hypochlo-rite 6% w/v, TEEPOL 0.1% for 1 h, rinsed four times in dis-tilled H2O, and put individually into sterile plugged tubes,which allowed gaseous exchange, containing a 2 cm bed of

agar (0.7%). Germination period is expressed as DAI.

4.2. Preparation of crude enzyme extracts

Plant material was ground in liquid nitrogen and extractedin ice-cold enzyme extraction buffer (glycerol 10% v/v,sodium metabisulfite 10 mM, EDTA 5 mM, MOPS (NaOH)40 mM, pH 6.5) at an approximate ratio of 20 mg l1. Themixture was stirred on ice for 20 min, centrifuged 30 min. at12,000 g, aliquoted and stored at 85 C until use.

4.3. Assay of-D-galactosidase

-Gal activity was detected spectrophotometrically withthe substrate p-nitrophenyl--D-galactopyranoside (pNGP).The reaction mixture contained 200 l pNGP 100 mM inMcIlvains buffer (citric acid 100 mMNa2HPO4 200 mM,pH 6.5) up to 1 ml final volume, with enzyme extract asrequired. The reaction was maintained at 26 C and startedwith the addition of enzyme. At the times indicated, one vol-ume of reaction mixture was added to 4 volumes of stop solu-tion (Na2CO3NaHCO3 100 mM, pH 10.2) and absorptionwas read at k = 405 nm. Appearance of nitrophenyl was cal-culated using molar extinction coefficient e = 18300 (spe-cific for pH 10.2) and converted to nkat mg1 protein. All

assays were performed in triplicate and the results expressedas means. Total protein was measured in samples extracted inaqueous buffer by the method of Bradford [5].

4.4. SDS-polyacrylamide electrophoresis

SDS-PAGE was performed according to Laemmli [28].Under reducing, denaturing conditions, grains were groundin liquid nitrogen and proteins extracted directly in buffer(urea 8 M, SDS 1%, b-mercaptoethanol 5%). Following cen-trifugation (13,000 g for 5 min) samples were stored at85 C until use. Samples were denaturated (95 C for 4 min)before loading. The gels were stained with either colloidalCoomassie Blue or silver methods as described previously in[47].



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4.5. Protein separation and N-terminal sequencing

Methods for protein extraction from grains, sample prepa-ration, first and second dimension electrophoresis, gel stain-

ing, transfer of proteins to PVDF membrane, and subsequentN-terminal microsequencing, wasperformed as described pre-viously in [47].

4.6. Antibody production

Anti--Gal polyclonal antibodies were raised against-Galpurified from the commercial preparation (Boehringer Man-nheim, cat. 105 023). The commercial solution was dilutedfour times with 50 mM phosphate buffer, pH 7.5, and appliedto a D10 column (Amersham Bioscience, Sephadex G-25M).After elution with NH4SO4, fractions were monitored by

absorption at 280 nm and those containing proteins were thenseparated by FPLC on a Mono Q column (equipments andcolumns from Amersham Bioscience). Eluted fractions weremonitored by absorption at 280 nm, by the measurement of-Gal activity, and by SDS-PAGE. For antibody production,10 mice were injected subcutaneously two times at 1 weekintervals with0.05mg of purifiedenzyme previously absorbedon alum. Third and fourth injections were made in PBS 15 and30 days later. The final bleeding was made 5 days after thelast injection.

4.7. Cloning and sequencing of coffee grain

-D-galactosidase cDNAs

The 27 WAF cDNA library from Caturra T2308 [47] wastested by PCR using the primers BETA100 (5-TGCTCCACAAAGCAGTGGCAATT-3 ) and BETA101 (5-ATTTATTGACTTAATCTCTTCAA-3) deduced from the-Gal cDNA sequence previously cloned [25] The reactionwas performed with Pfu DNA polymerase (Stratagene) inappropriate buffer, 0.2 mM of each dNTP and 0.25 M ofeach oligonucleotide. Denaturation, annealing and extensiontemperatures were 94 C for 30 s, 46 C for 30 s and 72 Cfor 3 min, respectively. This cycle was repeated 30 times in aBiomed thermocycler (Braun). The CaGAL1 cDNA (C. ara-

bica -galactosidase-encoding cDNA, AJ877911) obtainedwas cloned and double-strand sequenced (Licor automaticsequencer, Nestl Research Center, Lausanne, Switzerland)(Fig. 6, sequence 3). The CcGAL1 cDNA (C. canephora-Gal-encoding cDNA, AJ877912) was cloned by RT-PCRbecause no cDNA library from this specie was available.TotalRNA was extracted as described elsewhere in [47] from20 grainsat 30 WAF, obtained by cross-pollination of the vari-eties4461X?197.Approximately 10 ng of total RNA weretreated by the Access RT-PCR kit (Promega) using BETA1(5-ATGGTGAAGTCTCCAGG-3) and BETA3 (5-TCACTGTGGGGTTAGGA-3) also deduced from avail-able sequences [25,52,53], with PCR conditions identical tothose used for the amplification of CaGAL1 cDNA. TheCcGAL1 cDNA obtained was polished by Pfu polymerase,

cloned into pCR-Script (SK+) and sequenced (Fig. 6, se-quence 4).

4.8. Analysis of gene expression by Northern blotting

and RT-PCR

Total RNA (10 g) was extracted from grains ranging from6 to 35 WAF and analyzed by Northern blotting as describedelsewhere in [33]. The probe corresponded to the CaGAL1cDNA that was labeled with 32P-dCTP. Even loading of RNAsamples was controlled by equal abundance of 18S and 26SrDNA (Fig. 3C).

RT-PCR reactions were performed using 1 g of total RNAaccording to the manufacturers recommendation (AccessRT-PCR kit-Promega). -Galactosidase transcripts wereamplified using PCR conditions used to amplify the CaGAL1

cDNA. RT-PCR conditions for CaSUI1 were described pre-viously in [18]. PCR products were analyzed by electrophore-sis on agarose gel stained with ethidium bromide (Fig. 4).

4.9. Southern-blot analysis

Genomic DNA was extracted from fresh coffee leaves ofC. arabica Et29 x Ca5 as described previously in [42]. DNA(10 g) was digested with restriction enzymes and hybrid-ized with the 32P-labeled CaGAL1 cDNA as described beforein [33]. Membranes were washed at 65 C twice in 2 SSC,0.1% (w/v) SDS and twice in 0.1 SSC, 0.1% (w/v) SDS,

and exposed to Hyperfilm MP (Amersham Pharmacia Bio-tech) at 80 C.

Acknowledgements

We thank Milton Alvarez and other workers at OrecaoQuito-Equator for supplying coffee fruit samples for thiswork. We are also grateful to Drs. L.G. Vieira (IAPAR-Londrina PR, Brazil) and M.S. Buckeridge (Inst. Botany-SoPaulo SP, Brazil) for discussions and critical reading of themanuscript.

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