Plant Physiology and Biochemistry 43 (2005) 909–920

www.elsevier.com/locate/plaphy

Original article

Biochemical and molecular characterization

of ␣-D-galactosidase from coffee beans
Pierre Marraccini a,1, W. John Rogers a, Victoria Caillet a, Alain Deshayes a,
Dominique Granato b, Françoise Lausanne a, Sylvianne Lechat a,
David Pridmore b, Vincent Pétiard a,*
a
Nestlé Research Center, 101, avenue Gustave Eiffel, B.P. 9716, 37097 Tours cedex 2, France
b
Nestlé Research Center, Vers-Chez-les-Blancs, 1000 Lausanne 26, Switzerland
Received 6 April 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 wall
galactomannans. In the present paper it is shown that ␣-galactosidase activities in field-grown coffee beans are variable amongst cultivars of
the two species investigated (Coffea arabica and C. canephora var. Robusta). Higher activities were found in Arabica cultivars. Using beans
from greenhouse-cultivated C. arabica as a model, we showed that ␣-Gal activity was undetectable in the bean perispem tissue, but increased
gradually 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 reduced
to be undetectable in mature beans at 30 WAF, while ␣-Gal activity still persisted. Two isoforms were distinguished in 2-DE profiles of crude
protein extracts by N-terminal sequencing analysis. Analysis of two-dimensional gel electrophoresis profiles demonstrated that both isoforms
accumulated in a linear fashion throughout grain maturation. ␣-Gal activity was also observed to increase to high levels during in vitro
germination of coffee beans suggesting an important function of this enzyme in this process. ␣-Gal cDNA sequences from Arabica and
Robusta were sequenced and their deduced proteins appeared to be very similar, differing by only eight amino acids. Southern-blot analysis
suggests that the enzyme was encoded by at least two genes in C. arabica that could explain the existence of the two isoforms identified in
2-DE profiles.
© 2005 Published by Elsevier SAS.

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

1. Introduction mannans consist of a (1→4)-b-linked mannan chain that could

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
Abbreviations: DAI, days after imbibition; GM, galactomannan; 2-DE,
two dimensional electrophoresis; UTR, untranslated region; WAF, weeks
after flowering.
* Corresponding author. Fax: +33 2 47 49 14 14.
E-mail address: vincent.petiard@rdto.nestle.com (V. Pétiard).
1
Present address: CIRAD UMR PIA 1096/IAPAR AMG Laboratory of
Plant Biotechnology, Rodovia Celso Garcia Cid Km 375, CP 481, 86001-
970 Londrina Paraná, Brazil.
0981-9428/$ - see front matter © 2005 Published by Elsevier SAS.
doi:10.1016/j.plaphy.2005.08.010
be substituted at O-6 with single galactose residues to give
galactomannans (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 unsubstituted
mannans 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 viscous
suspension in water. In the case of coffee, the literature reports
Gal/Man ratios of between 1:130 [3] and 1:30 [17]. The latter
value was confirmed recently by a detailed chemical analysis
of GMs extracted at regular stages of coffee bean develop-
ment [45]. In this study, it was shown that the GM Gal/Man
ratio is high (between 1:2 and 1:7) in earliest developmental
910 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920

stages of endosperm (i.e. at 11 weeks after flowering (WAF)) optimum pH of the reaction and for tolerance to certain elec-
and decreases (between 1:7 and 1:40) during further the matu- trophoretic reagents substance (data not shown). The pH opti-
ration of this tissue. Changes of GM Gal/Man ratios occur mum was found to be broad with a peak at 7.5. Tolerance of
when the maternal perisperm tissue is gradually displaced by the activity to the presence of SDS, Triton-X 100 and glycine
the endosperm [47,48], a phenomenon that is accompanied was high. Tricine was less tolerated, although 75% activity
by the hardening of the endosperm [7,8]. Based on these was still retained in the presence of a concentration of 50 mM.
observations, the authors suggested that galactose removal
from the primary synthetic product could be dependent on 2.2. ␣-Galactosidase activity in grains
␣-D-galactosidase activity. of different cultivars and sources
␣-Gal occurs widely in microorganisms, plants and ani-
mals [1,13]. In plant species the hydrolase usually acts Activity was measured during maturation of grains (beans)
together with (1→4)-b-mannan endohydrolases (endo-b- harvested from trees cultivated either in field (Fig. 1A) or in
mannanase) (EC 3.2.1.78) and b-mannosidases (EC 3.2.1.25) greenhouse (Fig. 1B). In all of cases, ␣-Gal enzymatic activi-
to degrade GMs, mainly during germination of plant seeds ties always appeared extremely low or undetectable in the
[46]. However, high activity of ␣-Gal has also been reported first stages of the development where the perisperm tissue
in dry carob seeds [27] as well as during seed development of predominated. After that, ␣-Gal enzymatic activities increased
Senna occidentalis [15]. The enzyme from coffee beans was gradually to reached a peak usually observed before the har-
one of the first ␣-Gal to be partially purified and biochemi- vesting stage for grains collected in the field or earlier peri-
cally characterized [9,12,19,21,22]. In these initial reports, carp reddening stage for grains collected from greenhouse-
this enzyme was described as occurring as two isoforms (I
grown trees.
and II) having different molecular weights (28 and 36.5 kDa),
␣-Gal enzymatic activities measured in grains of the Ara-
different activity pH optima (5.3 and 6.3) and isoelectric points
bica cultivar Caturra T2308 and the cultivar Dormilon of C.
(pI).
Furthermore, the enzyme has attracted attention in the field
of biotechnology due to its capacity to remove the terminal
galactose units (a 1→3 linked) from the blood group B cell
surface carbohydrate moiety of glycoprotein complexes, thus
generating type O red blood cells [51]. ␣-Gal from microbial
sources (A. niger, S. carlsbergensis and E. coli) was found
not able to play this function [40]. For this reason, cDNA
sequences coding the coffee bean a-Gal are now available in
the literature [52] or in international patents [25], and the
recombinant enzyme has been expressed in a number of cell
culture systems [53,54,57]. Based on these analyses, it is sug-
gested that the mature active enzyme is composed of
363 amino acids, with an estimated molecular weight of
approximately 40 kDa, and that it may be synthesized as a
pre-proenzyme of 420 residues. More recently, site-directed
mutation 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 coffee
plants used in these publications have not always been speci-
fied.
In this article, we present data of ␣-Gal activities in beans
of various coffee species cultivated under different condi-
tions and results of gene expression studies during coffee bean
development, as well as in other plant tissues. We also iden-
tified ␣-Gal in protein extracts of mature (40 WAF) beans
analyzed by 2-DE and compared ␣-Gal cDNA sequences from
commercial Coffea arabica (Arabica) and C. canephora
(Robusta) cultivars.
Fig. 1. a-Galactosidase activities during maturation of coffee grains harves-
2. Results 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. Caturra
2.1. Optimum conditions for ␣-galactosidase activity assays T2308 (n) and CRM (e), or from C. canephora ROM (*) and Dormilon
(C). (B) Grains from plants grown in greenhouse were from C. arabica cv.
Activity of both the commercial Boehringer Mannheim Caturra T2308 (harvested in 1994) (M) and from C. canephora (harvested
preparation and coffee grain protein extracts was tested for in 1993) 4FPRC8 x ?3484/6 (D) or 43484/6 x ?FPRC8 (m).
 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920 911

canephora cultivated in the field, remained relatively stable
up 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 from
greenhouse-grown trees.
Fruits harvested in greenhouse were characterized by
overly long maturation periods compared to those harvested
in the field. ␣-Gal activities detected from two C. canephora
crosses (4FPRC/8 × ?3484/6 and 43484/6 × ?FPRC/8) and
from 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. These
observations were confirmed during 2 consecutive years in
grains 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 from
0.26 nkat mg per protein for the clone 197 of Robusta culti-
vated in Ivory Coast to 2.63 nkat mg per protein for the Caturra
T2308 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 other
field-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 activities
measured in various Robusta coffee plants always appeared
lower than those in Arabica, ranging from 0.26 to 0.75 nkat mg
per protein.
2.3. Expression of ␣-galactosidase gene during grain
maturation
Accumulation of ␣-Gal-specific mRNA was measured in
grains 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 lower
than those measured in beans of the same clone cultivated in
the 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 blot
experiment, demonstrating the appearance of ␣-Gal-specific
transcripts (around 1.2 kb) during the early stages of en-
dosperm development (at 22 WAF), with highest expression
at approximately 27 WAF when enzyme activity was still
increasing (Fig. 3B). At the peak of ␣-Gal activity (around
36 WAF), specific transcripts were still present but barely
detectable. 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).

Table 1 2.4. ␣-Galactosidase activity during germination

Preliminary comparisons of ␣-Gal activity (nkat mg–1 protein) in coffee beans
(endosperm) from various origins and species. Activities represent the mean
of at least three separate extractions from the same harvest batch. ␣-Gal activities were also measured during germination
(ARM = Arabusta Rojo Micropropagated; RM = Rojo Micropropagated; of grains from Caturra T2308 cultivated in the field or in the
F = field-grown plants; GH = greenhouse-grown plants; M = mature har- greenhouse (Fig. 2). Very low activity was measured in the
vest stage without specified WAF)
newly imbibed seeds, but it increased gradually during the
Species/cultivar Activity Observations germination, up to maximum of 2.3 and 3.66 nkat mg per
C. arabica cv. Caturra T2308 2.63 Quito F–24 WAF
protein at 42 days after imbibition (DAI) for grains harvested
C. arabica cv. Caturra T2308 0.42 Tours GH–33 WAF
C. arabica cv. Caturra T2308 0.35 Tours GH–37–
40 WAF
C. arabica cv. Caturra ‘Commercial’ 1.75 Quito F–24 WAF
C. arabica cv. Typica 0.48 Quito F–30 WAF
C. arabica cv. Typica 0.45 Tours GH–37–
40 WAF
C. arabica cv. Typica 0.54 México F–M
C. arabica cv. Pacas 0.58 Quito F–M
C. arabica cv. Bourbon 0.48 México F–M
C. arabica cv. Garnica F3 0.49 México F–M
C. arabica cv. Caturra RM 1.5 Quito–24 WAF
C. arabusta cv. ARM 0.88 Quito F–24 WAF
C. arabusta cv. ARM 0.89 Quito F–30 WAF
C. canephora C126 0.31 Abidjan F–M
C. canephora C461 0.53 Abidjan F–M
C. canephora C182 0.75 Abidjan F–M
C. canephora C197 0.26 Abidjan F–M Fig. 2. ␣-Galactosidase activities during coffee grain maturation and germi-
nation. Grains were harvested from cloned C. arabica cv. Caturra T2308 cul-
C. canephora cv. Dormilon 1.1 Quito F–30 WAF
tivated either in the field (n) or in greenhouse (M). Germination proceeded
C. canephora cv. ROM 0.34 Quito F–24 WAF
under in vitro conditions. Time scale is indicated in WAF for bean matura-
C. canephora ?FPRC/8 x 43484 0.48 Tours GH–49 WAF
tion and in DAI for the germination. The radicule length is also indicated
C. canephora ?3484 x 4FPRC/8 0.53 Tours GH–39 WAF (C).
912 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920

Fig. 4. Expression of CaGAL1 in different tissues of C. arabica cv. Caturra

T2308 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 2–10 cm) (lane 2), large
leaves (length > 10 cm) (lane 3), open flowers (lane 4), stems (lane 5), flower
buds (lane 6), roots (lane 7), grains at 27 WAF (lane 8) and zygotic embryos
(lane 9).

Highest ␣-Gal activities were always observed in endosperm

of fruits, whatever their origin, and to a lower extend in zygotic
embryos. These observations were very well correlated with
Fig. 3. ␣-Galactosidase activities in perisperm (C) and endosperm (M) tis- level of ␣-Gal-specific products amplified by RT-PCR in the
sues separated from C. arabica cv. Caturra T2308 grains grown in green- same tissues (Fig. 4). However, ␣-Gal activities were much
house (A). Tests of activity represent the mean of at least three separate lower in other tissues analyzed, like roots and leaves for
extractions. (B) Total RNAs extracted at different weeks after flowering
(WAF) from beans of the same plant were probed with the CaGAL1 cDNA.
example, and close to the limits of detection in the perisperm.
(C) Total RNA stained by ethidium bromide. Numbers indicated at the bot- Part of these observations was confirmed by RT-PCR experi-
tom of each figures indicate the maturation time expressed in WAF. ment (Fig. 4), particularly in roots and leaves for which ␣-Gal-
specific products amplified appeared very low. Finally, levels
from the field and greenhouse, respectively. For greenhouse- of ␣-Gal gene expression in stems and flowers were very
grown grains, which had lower activities during their matu- weak.
ration, ␣-Gal activity measured during their germination pre-
sented the highest increase compared to that observed for 2.6. Isoelectric localization of ␣-galactosidase
field-collected beans.
The ␣-Gal commercial preparation was analyzed by SDS-
2.5. ␣-Galactosidase activity and expression PAGE and IPG-2-DE (Fig. 5A). A single band representing
in coffee tissues the ␣-Gal enzyme was visible by SDS-PAGE. When ana-
lyzed by 2-DE, this band appeared to be formed by at least
Activities were also analyzed in different organs of a two isoforms with an estimated MW of 40 kDa and pI of
greenhouse-grown C. arabica cv. Caturra T2308, and com- approximately 5.5 and 5.7. For both proteins, N-terminal
pared to those measured in separated tissues (pericarp, sequences were identified (LANGLGLTPP) and were iden-
perisperm and endosperm) from coffee cherries (Table 2). tical. The analysis of these peptides revealed that they corre-
sponded to the first amino acids of the N-terminal region of
Table 2
␣-D-Galactosidase activity (nkat mg–1 protein) of in various tissues from
other coffee ␣-Gal protein sequences (Fig. 7, positions 58–67).
cloned C. arabica cv. Caturra T2308 grown in greenhouse (GH) or in the The commercial preparation also contained Bovine Serum
field (F). Activities represent the mean of at least three separate extractions Albumin at 66 kDa, and three contaminating proteins (named
from the same harvest batch UP for unidentified proteins), which were also subjected to
Plant source Tissue Activity N-terminal sequencing. UP 1 and 2 shared identical
C. arabica cv. Caturra Mature endosperm 2.56 N-terminals up to the first fifteen amino acids (GGGEN-
T2308 Quito F (30 WAF) LFQGKKVVD[S]), and separated into three isomers each.
″ Mature pericarp (30 WAF) 0.21
Internal sequences were also obtained (R/KGNDA;
C. arabica cv. Caturra Mature endosperm (37– 0.52
T2308 Tours GH 40 WAF)
R/KGLSTXAQXVNQXXVS, X is for unidentified amino
″ Mature pericarp 0.31 acid residue). No sequences could be obtained for UP 3. No
(37–40 WAF) convincing homology of these sequences could be found in
″ Perisperm (12–17 WAF) 8.10−3 public protein databases and these proteins remained un-
″ Leaves 0.12 known.
″ Roots 0.17 The serum containing anti-␣-Gal antibodies was used to
″ Zygotic embryo 0.41 test a crude protein extract of mature (40 WAF) coffee (C.
 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920 913

Fig. 5. 2-DE gel electrophoresis analysis of coffee grain a-galactosidases. (A) The commercial preparation of ␣-Gal was analyzed by IPG-2-DE and
proteins were identified by N-terminal sequencing. The SDS-PAGE of the same preparation was inserted in the left part of the graph. UP represents unidentified
proteins. (B) Proteins extracted from mature (40 WAF) C. arabica cv. Caturra T2308 grains were separated by 2-DE gel, blotted and tested by western-blotting
using polyclonal antibodies raised against the commercial ␣-Gal. The two ␣-Gal isoforms identified are marked by a black arrow. (C) The same coffee protein
extract 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. White
arrows 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 the
bottom.

arabica) beans separated by IPG-2-DE (Fig. 5B). This
western-blotting experiment allowed highlighting two ␣-Gal
isoforms with MW and pIs similar to those observed in the
commercial preparation. Using this information about the
localization of ␣-Gal isoforms in 2-DE gel, corresponding
proteins were identified in a silver-stained 2-DE gel (Fig. 5
C) 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 UPs
proteins encountered in the commercial ␣-Gal preparation.
All of them appeared to be very abundant in the protein extract
of mature (40 WAF) coffee beans, which in part permitted to
explain their presence as contaminants in the commercial
preparation.
2.7. Analysis of the coffee ␣-galactosidase cDNAs
The CaGAL1 and CcGAL1 cDNAs were compared with
the two other coffee ␣-Gal cDNA sequences already pub-
lished [25,52]. Nucleic acid alignments showed that all these
sequences were highly homologous both in UTR and cDNA
coding regions (Fig. 6). One of the few differences con-
cerned the adenosine in position 172, which was not observed
in the CaGAL1 cDNA as well as in the sequence reported by
Ivy and Clements [25], but which was present in the Arabica
sequence [52]. For this latter sequence, the ␣-Gal coding
sequence should begin at the ATG-198. However, if we con-
sider that the A-172 does not exist, the ATG-72 of CaGAL1
cDNA should be used instead of the internal ATG-198 (see
sequence 3 in Figs. 6 and 7), leading to a ␣-Gal precursor of
420 amino acids with a molecular mass of 46 kDa and a pI of
7.14. 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 amino
acids corresponding to the residues 23–420 with an esti-
mated molecular mass of 43.4 kDa. Further processing would
take place by endogenous peptidases to remove the peptide
corresponding to amino acid residues 23–57, leading the Leu-
58 at the extremity of the mature and active ␣-Gal enzyme as
identified 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 would
be expected to modify the amino acid composition of the
translated proteins without affecting their length (Fig. 7). Eight
of these amino acid differences were observed between
CaGAL1 and CcGAL1 proteins which present together 97.8%
identity at the amino acid level. Surprisingly, the Ca-
GAL1 protein appeared less homologous to the Arabica
sequence [52] than the CcGAL1 protein, respectively, with
98.4 and 99.5% of identity. However, theoretical pIs of
CaGAL1 and CcGAL1 mature enzymes are identical, close
to 5.7, whereas those of the two other ␣-Gal (sequences 1 and
2 in Fig. 7) were estimated to 5.88.
Screening of the mature CaGAL1 against protein data-
base confirmed its strong similarity with other
␣-galactosidases of dicotyledonous plants, members of the
family 27 of glycosyl hydrolases [23]. For example, higher
scores of amino acid identities of 81.2%, 80.7%, 80.4%,
78.8% and 78.1% were observed, respectively, for kidney bean
(Phaseolus vulgaris: AAA73964), sunflower (Helianthus
annuus: AB092594), soybean (Glycine max: AAA73963),
tomato (Lycopersicon esculentum: AAF04591) and guar (C.
tetragonoloba: CAA32772). These identity scores fall to 55%
with Clostridium josui (BAB83765), which presents the high-
est identity within prokaryotic ␣-Gal sequences. Similar
results were observed for the mature CcGAL1 protein.
914 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920

Fig. 6. Alignments of ␣-Gal cDNA sequences. Nucleic acid sequences are from Ivy and Clements [25] (1, AR366583), Zhu and Goldstein [52] (2, L27992) or
obtained from C. arabica cv. Caturra T2308 (3: CaGAL1, AJ877911) or from C. canephora (4461 X ?197) (4: CcGAL1, AJ877912). Stars highlight
differences. 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 putative
N-glycosylation site NIS (193-195) is underlined. Amino acid residues Trp-73, Tyr-150 and Asp-329 are indicated by black vertical arrows. The putative
protease 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 double
underlined. Amino acid numbering is indicated and refers to the CaGAL1 protein (sequence 3).

2.8. Southern hybridization analysis
Arabica genomic DNA was single-digested with DraI,
double-digested by DraI/ScaI and then hybridized with the
entire CaGAL1 cDNA (Fig. 8). Several bands were observed
in both patterns although no restriction sites were present in
the 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 and
3.5 kb for genomic DNA digested by DraI. These two latter
bands were no more detected in the double digestion
DraI/ScaI, probably leading to the fragment of 2.8 kb. Other
smaller fragments (near 0.75 kb) were generated by the double
digestion, part of them coming from the cleavage of the
1.5 and 2.4 kb detected after the DraI digestion. In addition,
 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920
Fig. 8. Southern-blot of C. arabica DNA digested with DraI (Dr) and ScaI
(Sc) restriction enzymes and hybridized with the CaGAL1 cDNA (sequence
3 in Fig. 6). Labeled molecular markers (M in kbp) are also presented.
we recently amplified, from the Arabica genome, a
BETA100/BETA101 PCR fragment of around 5 kb contain-
ing the entire CaGAL1 gene (data not shown). Together with
the sequence information deduced from the CaGAL1 cDNA,
all these data indicated the presence of at least two copies of
the ␣-Gal gene in the Arabica coffee genome.
3. Discussion
One of the objectives of this work was to increase our
knowledge on coffee ␣-galactosidase, which is thought to play
a key role in the metabolism of GMs [45]. Together with other
glycosidases (i.e. b-galactosidase, ␣-mannosidase, N-acetyl-
b-D-glucosiminidase), ␣-Gal activity was previously reported
in entire ripening fruits of C. arabica [19]. In the present
work, we clearly demonstrated that no activity was observed
during the first few weeks of coffee fruit development, when
the perisperm (maternal) tissue predominated. During the sec-
ond part of coffee cherry development, ␣-Gal enzymatic
activities were detected both in the pericarp (also referred as
the pulp) and in the endosperm, with a higher activity in the
latter tissue. As a model, we studied the evolution of ␣-Gal
enzymatic activity in endosperm of coffee fruits grown in
greenhouse conditions and showed that it was undetectable
915
at 20 WAF, but reaching a maximum at around 36 WAF. Our
analysis 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 the
endosperm. This demonstrated that the ␣-Gal gene expres-
sion was temporally and spatially regulated during coffee fruit
development, as also observed for the csp1 gene coding for
11S-storage proteins [47]. To confirm this pattern, it would
have been interesting to follow ␣-Gal gene expression in the
endosperm from fruits of C. arabica cv. Caturra T2308 har-
vested from field grown plants where higher levels of enzy-
matic activity were measured (Fig. 1A). Unfortunately, even
if these fruits were shipped in dry ice, all our attempts to
extract their RNAs failed, probably because fruits were not
frozen in liquid nitrogen immediately after their harvest, a
condition that in our hands always appeared critical to extract
intact RNAs from various coffee tissues (Caillet and Marrac-
cini, unpublished observations).
Using polyclonal antibodies against the commercial
enzyme and N-terminal sequencing, at least two well-
separated and abundant ␣-Gal isoforms were detected in beans
at the optimal stage of harvest (40 WAF), with equal molecu-
lar weights (near 39.5 kDa) but differing by their pI (5.5 and
5.7). Other observations reporting the presence of ␣-Gal
isoenzymes of close molecular mass were also made for plant
seeds of P. vulgaris [14] and Glycine max L. [20] for example.
To clarify the origin of cDNA sequences already available in
the literature and patents [25,52,53], but also to see if the
␣-Gal pIs could be explained by subtle changes at the amino
acid level of ␣-Gal isoforms, full-length cDNA sequences
were cloned from fruits of well identified C. arabica (Ara-
bica) and C. canephora (Robusta) coffee plants. The analysis
of the CaGAL1 cDNA sequence suggested that coffee ␣-Gal
was probably synthesized as a pre-proenzyme containing a
hydrophobic signal peptide of 22 amino acids. Because
N-terminal sequencing of the ␣-Gal revealed that the mature
protein began with Leu-58 amino acid residue, this clearly
demonstrated that residues 23–57 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 isoforms
were estimated close to 39.5 kDa, which agreed with the theo-
retical protein of 363 amino acid residues deduced from both
CaGAL1 and CcGAL1 cDNA sequences.
The presence of a signal peptide in the pre-proenzyme form
of ␣-Gal is interesting because this should allow it to be trans-
ported into the endoplasmic reticulum, and thence in cell walls
where mannans accumulate. This should also permit the tar-
geting of the ␣-Gal into proteins bodies like it was shown in
cotyledons of soybean and lupin [24,39,43] and also in
endosperm 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 maintain
the high Gal/Man ratio of newly synthesized GMs and con-
sequently their relative solubility, in order to permit their intra-
916 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920
cellular trafficking. In this model, the hardening of endosperm
should be directly controlled by the ␣-Gal activity, through
the 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 of
these differences implicated the Trp-73 and Tyr-150 amino
acid residues, which were shown to be essential for coffee
␣-Gal activity [32,55,56], or the Asp-329 that was suggested
to play a role in the active site of the guar enzyme [41]. The
theoretical pIs of ␣-Gal proteins deduced from Arabica and
Robusta cDNAs were identical (close to 5.7), and could be
related to the ␣-Gal isoform of similar pI identified by 2-DE
gel electrophoresis, both in mature beans and in the commer-
cial preparation. Concerning the ␣-Gal isoform with a lower
pI (5.5), we showed that its N-terminal amino acid sequence
was 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 this
protein, i.e. like the removal of few amino acids, also seems
unlikely 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, for
example at the putative N-glycosylation NIS site (position
193–195, 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 in
Arabidopsis thaliana where two putative genes coding for
the 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 in
rice (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). These
examples contrasted with the situation observed in tomato
(L. esculentum) where ␣-Gal seems to be encoded by a unique
gene [16]. Because C. arabica is amphidiploid, resulting from
a natural cross between diploid species C. eugenoides and C.
canephora [29], it is possible that the two ␣-Gal-encoding
genes we detected in Arabica genome, represented indepen-
dent genes coming from each parent rather than two isoforms
of the same gene that could be due to recent gene duplica-
tion. The screening of available coffee BAC libraries from
Robusta [30] and Arabica [37] with the present ␣-Gal cDNAs
would be very useful to solve this question.
Even when using the same cultivar (Caturra T2308) of Ara-
bica, our analysis also demonstrated that grains from
greenhouse-grown plants had lower ␣-Gal activities than those
from plants grown in the field. We did not investigate what
were the reasons of these differences, but they could be prob-
ably linked to variations of environmental parameters between
field and greenhouse conditions, as those concerning tem-
perature and light intensity received by the plants. For
example, plants grown in sub tropical regions under “full-
sun” field conditions receive a photosynthetic photon flux den-
sity (PPFD) normally varying from 1500 to 2500 µE s–1 m–2
at noon, respectively, for winter and summer season [50].
However, plants grown in greenhouse received a constant
PPFD of 300 µE s–1 m–2 throughout the year, therefore mim-
icking coffee plant grown in “shade” condition. These envi-
ronmental parameters were reported to have great impacts on
coffee fruit development, like delayed maturation and in-
creased fruit weight and bean size [36,50]. In addition, we
recently observed slight reduction of sucrose synthase activ-
ity levels in endosperm of coffee plants cultivated under shad-
ing in comparison to “full-sun” growing conditions (Ger-
omel, Mazzafera and Marraccini, unpubl. obs.). Such a
phenomenon could also occurred for ␣-Gal enzymatic activi-
ties in order to explain the lower levels in greenhouse.
The present work also showed that ␣-Gal activities in seeds
of reverse crosses of greenhouse-grown C. canephora were
similar, which suggested the absence of a “maternal effect”
on the control of ␣-Gal enzymatic activity. We observed a
tendency of higher ␣-Gal activities in beans of Arabica than
in those of Robusta, which needs to be confirmed before to
be 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 of
unbranched to branched mannose previously analyzed in the
water-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 a
continuous increase during germination, leading to a maxi-
mum (3.66 nkat mg per protein) near 40 DAI. Even if the
activity was no longer followed after this time, the profile
obtained clearly differed from that previously reported for
endo-b-mannanase activity during germination, which
reached a peak at 28 DAI and declined drastically after this
time [33]. Because the expression of the CaGAL1 gene was
not tested during the germination, we do not know if the large
increase of ␣-Gal activity measured came from the activation
of pre-existing CaGAL1 protein in green beans and/or from
de novo synthesis, through the activation of transcriptional
and/or transcriptional processes. Like described in other plants
[13,20], the expression of other types of ␣-Gal, could also
occurred in coffee (Buckeridge, personal communication).
During this experiment, ␣-Gal activity measured just after
the harvest of mature beans of field-grown Caturra
(2.56 nkat mg per protein at 30 WAF) was higher that activity
of 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-
 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920
ments and/or storage of the samples. However, this did not
affect the efficiency of germination of the beans nor the ␣-Gal
activity 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 coffee
grain were still not fully clarified, the ␣-Gal activity could
also be linked to the degradation of raffinose family of oli-
gosaccharides (RFOs) and galactosyl cyclitols as proposed
by Obendorf [39]. In that model, ␣-Gal synthesized during
seed development is targeted to protein bodies and their sub-
strates (RFOs and galactosyl cyclitols) are accumulated in
the cytosol [35,44]. During germination, these oligosaccha-
rides are transported from the cytosol to the protein bodies
that become a vacuole for their hydrolysis [6,39]. RFOs were
identified in mature coffee beans [10,48] and therefore could
be 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 by
Chabrillange et al. [10]. Because ␣-Gal activity removes the
galactosyl from the stachyose to give raffinose oligosaccha-
ride, low levels of stachyose observed in Robusta could be
related to the lower ␣-Gal activity in this specie compared to
Arabica. 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 the
reorganization 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 including
breeding programs managed by marker assisted selection, till
genetic 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 µE s–1 m–2). For measurement of ␣-Gal
activity, fruits of C. canephora were obtained following cross-
pollination by hand of the cultivars FPRC/8 and 3484/6. ␣-Gal
cDNAs were obtained from grains resulting from hand-
crossed Robusta cultivars 4461 X ?197 and from a self-
pollinated tree of C. arabica L. cv. Caturra T2308. The same
tree was also used to analyze ␣-Gal activities and expression
in different organs (leaves, root, flowers) and in separated
grain 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 (the
remnant 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 (Nestlé R&D,
917
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 the
greenhouse. The Robusta Dormilon and ROM were well
known commercial varieties. Harvests were overseen by
Nestlé workers (Quito Equator). Other samples of mature (red)
fruits were obtained from other sources (Nestlé R&D—Ivory
Coast and Nestlé-Mexico).
All greenhouse-grown fruits and tissue samples were fro-
zen in liquid nitrogen immediately following harvesting and
stored at –85 °C until use. Samples from other countries were
shipped 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 extracted
in 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 µl–1. The
mixture was stirred on ice for 20 min, centrifuged 30 min. at
12,000 g, aliquoted and stored at –85 °C until use.
4.3. Assay of ␣-D-galactosidase
␣-Gal activity was detected spectrophotometrically with
the substrate p-nitrophenyl-␣-D-galactopyranoside (pNGP).
The reaction mixture contained 200 µl pNGP 100 mM in
McIlvain’s buffer (citric acid 100 mM–Na2HPO4 200 mM,
pH 6.5) up to 1 ml final volume, with enzyme extract as
required. The reaction was maintained at 26 °C and started
with the addition of enzyme. At the times indicated, one vol-
ume of reaction mixture was added to 4 volumes of stop solu-
tion (Na2CO3–NaHCO3 100 mM, pH 10.2) and absorption
was 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 mg–1 protein. All
assays were performed in triplicate and the results expressed
as means. Total protein was measured in samples extracted in
aqueous 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 ground
in 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 at
–85 °C until use. Samples were denaturated (95 °C for 4 min)
before loading. The gels were stained with either colloidal
Coomassie Blue or silver methods as described previously in
[47].
918 P. Marraccini et al. / Plant Physiology and Biochemistry 43 (2005) 909–920
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 subsequent
N-terminal microsequencing, was performed as described pre-
viously in [47].
4.6. Antibody production
Anti-␣-Gal polyclonal antibodies were raised against ␣-Gal
purified from the commercial preparation (Boehringer Man-
nheim, cat. 105 023). The commercial solution was diluted
four times with 50 mM phosphate buffer, pH 7.5, and applied
to 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 then
separated by FPLC on a Mono Q column (equipments and
columns from Amersham Bioscience). Eluted fractions were
monitored 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 week
intervals with 0.05 mg of purified enzyme previously absorbed
on alum. Third and fourth injections were made in PBS 15 and
30 days later. The final bleeding was made 5 days after the
last injection.
4.7. Cloning and sequencing of coffee grain
␣-D-galactosidase cDNAs
The 27 WAF cDNA library from Caturra T2308 [47] was
tested by PCR using the primers BETA100 (5′-
TGCTCCACAAAGCAGTGGCAATT-3′) and BETA101 (5′-
ATTTATTGACTTAATCTCTTCAA-3′) deduced from the
␣-Gal cDNA sequence previously cloned [25] The reaction
was performed with Pfu DNA polymerase (Stratagene) in
appropriate buffer, 0.2 mM of each dNTP and 0.25 µM of
each oligonucleotide. Denaturation, annealing and extension
temperatures were 94 °C for 30 s, 46 °C for 30 s and 72 °C
for 3 min, respectively. This cycle was repeated 30 times in a
Biomed thermocycler (Braun). The CaGAL1 cDNA (C. ara-
bica ␣-galactosidase-encoding cDNA, AJ877911) obtained
was cloned and double-strand sequenced (Licor automatic
sequencer, Nestlé Research Center, Lausanne, Switzerland)
(Fig. 6, sequence 3). The CcGAL1 cDNA (C. canephora
␣-Gal-encoding cDNA, AJ877912) was cloned by RT-PCR
because no cDNA library from this specie was available. Total
RNA was extracted as described elsewhere in [47] from
20 grains at 30 WAF, obtained by cross-pollination of the vari-
eties 4461 X ?197. Approximately 10 ng of total RNA were
treated 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 to
those used for the amplification of CaGAL1 cDNA. The
CcGAL1 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 µg) was extracted from grains ranging from
6 to 35 WAF and analyzed by Northern blotting as described
elsewhere in [33]. The probe corresponded to the CaGAL1
cDNA that was labeled with 32P-dCTP. Even loading of RNA
samples was controlled by equal abundance of 18S and 26S
rDNA (Fig. 3C).
RT-PCR reactions were performed using 1 µg of total RNA
according to the manufacturer’s recommendation (Access
RT-PCR kit-Promega). ␣-Galactosidase transcripts were
amplified 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 of
C. arabica Et29 x Ca5 as described previously in [42]. DNA
(10 µg) was digested with restriction enzymes and hybrid-
ized with the 32P-labeled CaGAL1 cDNA as described before
in [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 Orecao
Quito-Equator for supplying coffee fruit samples for this
work. We are also grateful to Drs. L.G. Vieira (IAPAR-
Londrina PR, Brazil) and M.S. Buckeridge (Inst. Botany-São
Paulo SP, Brazil) for discussions and critical reading of the
manuscript.
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