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咖啡中绿原酸CYP合成 CYP98A3536
咖啡中绿原酸CYP合成 CYP98A3536
DOI 10.1007/s11103-007-9141-3
Received: 6 June 2006 / Accepted: 24 January 2007 / Published online: 27 February 2007
Springer Science+Business Media B.V. 2007
Abstract Chlorogenic acid (5-CQA) is one of the ma- metabolized p-coumaroyl shikimate at similar rates, but
jor soluble phenolic compounds that is accumulated in that only one hydroxylates the chlorogenic acid pre-
coffee green beans. With other hydroxycinnamoyl qui- cursor p-coumaroyl quinate. CYP98A35 appears to be
nic acids (HQAs), this compound is accumulated in the first C3¢H capable of metabolising p-coumaroyl
particular in green beans of the cultivated species Coffea quinate and p-coumaroyl shikimate with the same effi-
canephora. Recent work has indicated that the biosyn- ciency. We studied the expression patterns of both genes
thesis of 5-CQA can be catalyzed by a cytochrome P450 on 4-month old C. canephora plants and found higher
enzyme, CYP98A3 from Arabidopsis. Two full-length transcript levels in young and in highly vascularized or-
cDNA clones (CYP98A35 and CYP98A36) that encode gans for both genes. Gene expression and HQA content
putative p-coumaroylester 3¢-hydroxylases (C3¢H) were seemed to be correlated in these organs. Histolocaliza-
isolated from C. canephora cDNA libraries. Re- tion and immunolocalization studies revealed similar
combinant protein expression in yeast showed that both tissue localization for caffeoyl quinic acids and p-cou-
maroylester 3¢-hydroxylases. The results indicated that
HQA biosynthesis and accumulation occurred mainly in
V. Mahesh N. Chabrillange J. Bustamante the shoot tip and in the phloem of the vascular bundles.
M. Noirot S. Hamon A. de Kochko The lack of correlation between gene expression and
C. Campa (&)
HQA content observed in some organs is discussed in
Laboratoire de Génomique et Qualité du café, IRD, UMR
1097 DGPC, 911 Avenue Agropolis, BP 64501, 34394 terms of transport and accumulation mechanisms.
Montpellier cedex 5, France
e-mail: campa@mpl.ird.fr Keywords Caffeoyl quinic acids Chlorogenic acid
Coffea canephora Cytochrome P450 hydroxylase
R. Million-Rousseau P. Ullmann D. Werck-Reichhart
Department of Plant Stress Response, Institute of Plant
Molecular Biology, CNRS-UPR 2357, Université Louis
Pasteur, 28 rue Goethe, 67083 Strasbourg, France Abbreviations
CQA caffeoyl quinic acid
L. Mondolot
Laboratoire de Botanique, Phytochimie et Mycologie, 5-CQA chlorogenic acid
UMR 5175 CEFE-CNRS, Faculté de Pharmacie, 15 av. HQA hydroxycinnamoyl quinic acid
Charles Flahault, BP 14491, 34093 Montpellier cedex 5, C3¢H p-coumaroyl ester 3¢-hydroxylases
France HQT hydroxycinnamoyl-CoA: quinate
V. Mahesh hydroxycinnamoyl transferase
Avesthagen graine, A Plant Genome Biology Laboratory, HCT hydroxycinnamoyl-CoA: shikimate/
9th floor, Discoverer, ITPL, Bangalore, India quinate hydroxycinnamoyl transferase
CODEHOP consensus degenerate hybrid
M. Morant
Department of Plant Biology, Thorvaldsensvej 40, 1871 oligonucleotide primers
Fredericksberg C, Copenhagen, Denmark CYP cytochrome P450 monooxygenases
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146 Plant Mol Biol (2007) 64:145–159
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Plant Mol Biol (2007) 64:145–159 147
Fig. 1 Proposed pathways for the biosynthesis of chlorogenic namoyl CoA shikimate/quinate hydroxycinnamoyltransferase;
acid (5-caffeoyl quinic acid) in plants. The three different routes C3H (and C3¢H), p-coumarate 3¢-hydroxylases; UGCT, UDP
in caffeoyl quinic acid pathway are labeled 1(a and b), 2 and 3. glucose:cinnamate glucosyl transferase; HCGQT, hydroxycinna-
PAL, phenylalanine ammonia-lyase; HQT, hydroxycinnamoyl moyl D-glucose: quinate hydroxycinnamoyl transferase
CoA quinate hydroxycinnamoyltransferase; HCT, hydroxycin-
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148 Plant Mol Biol (2007) 64:145–159
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Plant Mol Biol (2007) 64:145–159 149
Assay conditions for 3¢-hydroxylase activities blocking agent (Biorad, USA) for 2 h). After rinsing
(buffer 1), they were incubated with anti-CYP98A3
Total P450 content was evaluated in each microsome polyclonal serum diluted 1: 1000 in buffer 2 (buffer 1
preparation by spectrophotometric measurement with 1% blocking agent) for 2 h. After 3 washes with
(Omura and Sato, 1964). Assays for p-coumaroyl- buffer 1, the membranes were incubated with 1: 7,500
shikimate/quinate 3¢-hydroxylase (C3¢H) were per- diluted secondary antibody (goat) anti-rabbit IgG
formed in a total volume of 100 ll, as already de- conjugated with alkaline phosphatase (Promega, USA)
scribed by Schoch et al. (2001). The final concentration in buffer 2. After rinsing (buffer 1), protein-antibody
was 0.2 lM for P450 and 30 to 50 lM for the substrate complexes were detected with 5-bromo-4-chloro-3-
(coumaric acid, coumaroylshikimate or coumaroylqui- indolyl phosphate and nitro blue tetrazolium as sub-
nate). The reaction was incubated under shaking in the strates (Sigma, USA). Additional Western-blot analy-
dark at 27C for 30 min and then stopped by addition ses were performed with CYP98A3 antibodies to
of 5 ll of 4 M HCl. After extraction with ethyl acetate, confirm the recognition of CYP98A in a protein extract
the organic phase was evaporated under argon and the from C. canephora leaves and of the recombinant
reaction products dissolved in 150 ll of 10% acetoni- proteins.
trile, 90% water, and 0.2% acetic acid (v/v/v) for re-
verse-phase HPLC analysis (Merck LiChrospher Histochemical detection of caffeoylquinic acids
100RP-18 column, 4 x 15 mm, 5 lm; flow rate of 1 ml/ and lignin
min; 5 min of isocratic 10% acetonitrile, and then a
linear gradient from 10% to 48% acetonitrile in water Coffea canephora seedlings freshly collected samples
containing 0.2% acetic acid). Absorbance was moni- were embedded in 3% (w/v) agarose (type II EEO,
tored at 320 nm with a diode array detector. Substrate Panreac). Cross-sections (40 lm, Leica VT 1000S
amounts and products were calculated according to microtome), were immersed (30 s) in Neu’s reagent
Kühnl et al. (1987). Retention times and UV spectra (1% (w/v) 2-amino-ethyldiphenylborinate (Fluka) in
confirmed the nature of the products. absolute methanol) and mounted in glycerine: water
(10:90, v/v) solution (Neu, 1957). Microscope obser-
Semi-quantitative RT-PCR vations (Nikon Optiphot) under UV light (filter
UV-1A: 365 nm excitation filter, 400 nm barrier filter)
The primers 5¢-CCATCTTCAAGGACTGCACCA- allowed to identify caffeoylquinic acids by a specific
TACC-3¢ and 5¢-TTCTATCGGAGCCAATGACT greenish–white fluorescence (Mondolot-Cosson et al.,
CG-3¢ for CYP98A35 and 5¢-CAGGGCAGAGTCT 1997) while feruloyl derivatives were bright blue. The
ACTAGTGAAG-3¢ and 5¢-AGTCCGTCTCGAT sections were also stained with Mirande’s reagent for
CATAACACGCTC-3¢ for CYP98A36 were designed lignin and cellulose detection (green and red-pink
so as to frame the intron in both sequences. Total RNA coloration respectively) (Mondolot et al., 2001). Pho-
was extracted from about 100 mg of plant tissues using tographs were taken with a digital Nikon coolpix 4500
the RNeasy Plant Mini Kit (Qiagen). RNA was camera.
quantified by spectrophotometry and the cDNA was
synthesized from 2 lg of total RNA using the kit HPLC analysis of hydroxycinnamoyl ester content
Superscript II cDNA synthesis (Invitrogen). The PCR
reactions were conducted in a final volume of 50 ll Hydroxycinnamoyl ester extraction was carried out
under the following conditions: 94C for 5 min, fol- following the method described by Ky et al. (2001).
lowed by 23, 26, 29, 32 or 35 amplification cycles (30 s The different compounds were identified on the basis
at 94C, 50 s at 58C and 60 s at 72C) and a final of their retention time and UV spectra using the HPLC
extension at 72C for 10 min. analytical procedure described by Bertrand et al.
(2003). Retention times were those described by Ky
Tissue print hybridization et al. (1997) for 3-caffeoyl quinic acid (3-CQA,
6.1 min), 3-feruloyl quinic acid (3-FQA, 9.2 min), 4-
Transversal hand cuts of petioles from nodes 1 and 2 of and 5-CQA (10.9 min), 4-FQA (14.3 min), 5-FQA
young shoot tip leaves, and stems, were pressed firmly (15.2 min), 3,4-dicaffeoyl quinic acid (3,4-DiCQA,
for 2 s three times onto a nitrocellulose membrane 18.8 min), 3,5-DiCQA (19.5 min) and 4,5-DiCQA
(0.2 lm) and blotted dry. The membranes were incu- (22.1 min). Coumaroyl quinic, coumaroyl shikimic and
bated 1 h in PBS containing Tween 20 (1% v/v) and caffeoyl shikimic acids were used as control. Quantifi-
then in a blocking buffer (buffer 1 with 5% (w/v) cation was performed by comparison to a 5-CQA
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150 Plant Mol Biol (2007) 64:145–159
standard (Sigma-Aldrich, USA). The hydroxycinna- Alignment with CYP98A3 from A. thaliana (Fig. 2)
moyl quinic acid content of each analyzed sample was showed that both deduced protein sequences contain
expressed as mean percentage of dry weight (% DW) the classically conserved domains of the P450 pro-
from three different extractions. Data were analyzed teins such as the ERR triad, cysteine in the heme-
using Statistica 5.1. Between-organ differences were binding domain and the six probable substrate rec-
tested using a one-way ANOVA (Newman-Keuls). ognition sites specific to CYP98A (SRS). However,
there were significant differences in the SRS1, SRS2,
and SRS6 domains, which may be indicative of dif-
Results ferences in substrate specificity.
Southern blotting, using a C. canephora CYP98As
Isolation of full length C. canephora CYP98A specific probe, showed that each gene was present as
cDNAs a single copy in the C. canephora genome (Fig. 3).
Digestion by EcoRV, whose site is supposed absent
To specifically isolate the CYP98A homologs from from both sequences, gave only one signal of high
the large CYP gene family, PCR primers were con- molecular weight. DraI cuts outside CYP98A36 gene
structed according to the CODEHOP primer design (upper band) and inside CYP98A35 (lower band) and
strategy avoiding the strong consensus regions com- NsiI, which cut differently inside both genes, showed
mon to other P450 families (Fig. 2). Their selectivity a three-band pattern (the third band may be ex-
was increased conducting a touch-down PCR starting plained by a different allelic form of one of the two
at a high annealing temperature (70C). A single genes).
band of the expected size was obtained with each A phylogenetic tree constructed using 16 other
primer pair 98f2-98r1 and 98f3-98r2. After sequenc- CYP98A sequences and the CYP51G2 sequence as
ing and blast analysis, 17 of the fifty clones analyzed outgroup showed that CYP98A35 and CYP98A36
displayed significant homology with genes from the were grouped with other dicot CYP98A sequences
CYP98A family. Even though all of these partial (Fig. 4). Nevertheless, CYP98A36 seemed to be
sequences had 93% similarity with the partial de- more closely related to CYP98A3 from A. thaliana
duced protein sequence of Ocimum basilicum p- than CYP98A35. Genes were clustered in two dis-
coumaryl 3¢-shikimate hydroxylase isoform 1 tinct groups indicating that the encoding genes re-
(AAL99200), it was possible to divide them into two sulted from an early duplication of the ancestral
groups according to their sequence homology. gene.
Among each of the two groups, the sequences were
almost identical with the exception of very few nu- Gene structure
cleotides, which might be due to Taq misamplifica-
tion, and the between group homology didn’t exceed Sequence analysis of C. canephora genomic DNA
70.3%. The full-length cDNA sequences corre- amplified with specific primers designed to match the
sponding to each gene were obtained by PCR respective 5¢ and 3¢-end of CYP98A35 and
amplification using specific primers. CYP98A35 CYP98A36 genes revealed the presence of introns in
(Accession No DQ269126) and CYP98A36 (Acces- both gene sequences (Fig. 5). The two amplified
sion No DQ269127) differed by their 5¢- and 3¢-UTR fragments differed by their length (4 kb and 1.5 kb
(75 and 28 bp for the 5¢-end, and 156 and 194 for the for CYP98A35 and CYP98A36, respectively). Like
3¢-end respectively), but both open reading frames most A-type P450s, both genes shared a phase 0
had the same length and encoded a protein of 508 intron at position 882 on the nucleotide sequence,
amino acids (Fig. 2). Comparison of the coding se- corresponding to the intron labeled M by Paquette
quences showed that they share 72.4% identity at the et al. (2000). However, the two introns differed in
nucleotide level, and 88% similarity at the amino length (106 nucleotides for CYP98A35 and 490 for
acid level. When the deduced sequences were com- CYP98A36) and in sequences. Moreover, the
pared to 13 CYP98As from other plant species, both CYP98A35 genomic sequence showed an additional
showed the best similarity score with Populus 116 bp phase 1 intron at position 484. In this respect
trichocarpa sequences (CYP98A27 and CYP98A23): CYP98A35 differs from the prototype A-clade plant
89% similarity was observed between the CYP98A35 P450s and CYP98As (e.g. CYP98A3 from A. thaliana
and CYP98A27 and 98% between the CYP98A36 or CYP98A36). These features indicate that the
and CYP98A23. For both genes, the lowest score was CYP98A35 additional intron was most likely gained
obtained with CYP98A1 from S. bicolor (82%). after the gene duplication.
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Plant Mol Biol (2007) 64:145–159 151
Enzymatic properties of recombinant CcCYP98A of NADPH (data not shown). This indicates that, as
proteins already observed for A. thaliana CYP98A3, p-coum-
aric acid is not a relevant substrate for CYP98A35
For functional analysis, the two CcCYP98As were and CYP98A36. However, in the presence of p-cou-
co-expressed in yeast with a plant cytochrome P450 maroylshikimate, both recombinant proteins were
reductase. The microsomal fractions, which were ex- able to form caffeoylshikimate (Fig. 6). Despite very
tracted from the recombinant yeasts expressing each similar affinities, CYP98A35 appeared slightly more
of the CcCYP98As, were tested for their enzymatic efficient than CYP98A36 with this substrate, with a
activity and substrate specificity with p-coumaric acid, Kcat/Km of 0.25 instead of 0.15 min–1 lM– 1 (Table 1).
p-coumaroyl shikimic or p-coumaroyl quinic acids as When incubated with p-coumaroylquinate, only
substrates. A negative control consisted in a micro- CYP98A35 was able to form caffeoylquinate. The
somal fraction from yeast transformed with a void enzyme affinity for p-coumaroylquinate was two
plasmid; microsomes from yeast expressing CYP98A3 fold lower than for p-coumaroylshikimate, but its
from A. thaliana were used as positive control. When efficiency was identical with shikimate and quinate
assays were performed in presence of p-coumaric esters. The conversion of p-coumaroylquinate by
acid, no conversion into more oxygenated compound CYP98A36 was too slow to allow reliable determi-
was detected with recombinant CYP98A35, nation of kinetic constants. It is worth mentioning
CYP98A36 and CYP98A3 incubated in the presence that the catalytic turnover of both the CcCYP98As
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152 Plant Mol Biol (2007) 64:145–159
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Plant Mol Biol (2007) 64:145–159 153
Fig. 4 Phylogenetic tree of the CYP98A family. The phylogenetic A. thaliana (CYP98A9 AAM67314), Triticum aestivum
representation of the CYP98 family built by the neighbour-joining (CYP98A10 CAE47489), T. aestivum (CYP98A11, CAE47490),
method and bootstrapped 1,000 times in Mega3.1. CYP51G2 T. aestivum (CYP98A12, CAE47491), Ocimum basilicum (CY-
involved in sterol biosynthesis was used to root the tree. Branch P98A13v1, AAL99200), Solenostemon scutellarioides (CYP98A14,
values under 50% were omitted from the figure. • CYP98s from CAD20576), Pinus taeda (CYP98A19, AAL47685), Sesamum
Coffea h CYP98 from Gymnosperms, D CYP98s from Monocots. indicum (CYP98A20, AAL47545), Ammi majus (CYP98A21,
The sequences used for alignment were those from Sorghum AAT06912), Camptotheca acuminata (CYP98A28, AAS57921),
bicolor (CYP98A1, AAC39316), Glycine max (CYP98A2, C. canephora (CYP98A35, DQ269126), C. canephora (CYP98A36,
AAB94587), Arabidopsis thaliana (CYP98A3, NP850337), Oryza DQ269127). The sterol 14-a-demethylase from A. thaliana
sativa (CYP98A4, AAU44038), Lithospermum erythrorhizon (CYP51G2) was used as outgroup
(CYP98A6, BAC44836), A. thaliana (CYP98A8, AAG52369),
Discussion
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154 Plant Mol Biol (2007) 64:145–159
Table 1 Substrate specificity of the recombinant CYP98As from C. canephora in comparison with the recombinant CYP98A3 from
A. thaliana
Yeast expressed enzyme Substrate Km (lM) Kcat (min–1) Kcat/Km (min–1 M–1)
genes of the A-clade (Paquette et al., 2000), but the CYP98 family described so far (Schoch et al., 2001;
CYP98A35 acquired an additional intron located at Gang et al., 2002; Morant et al., 2007), metabolized
nucleotide 484 from the ATG (between amino acids shikimate esters of p-coumaric acid more efficiently
Pro and Glu). This significant difference in gene than quinate esters. CYP98A35 is the first to catalyze
structure and the only moderate similarity between the with equal efficiency the hydroxylation of quinate and
two paralogs indicate early duplication of the ancestral shikimate esters of p-coumaric acid. It is thus very
gene and, very likely, acquisition of specific functions likely that this enzyme significantly contributes to the
and regulations (Jeong et al., 2006). biosynthesis of chlorogenic acid and other CQAs in the
This hypothesis was supported by the functional coffee plant via the direct route involving hydroxyl-
expression of the two CYP98A35 and CYP98A36 ation of p-coumaroylquinate (Fig. 1, pathway 2).
proteins in yeast. While both recombinant proteins Nevertheless, the low catalytic turnover of both pro-
metabolized p-coumaroyl shikimate at similar rates, teins (compared to CYP98A3 but not to other plant
only CYP98A35 was able to hydroxylate p-coumaroyl P450 enzymes) may also indicate that the shikimate
quinate to form chlorogenic acid. All the members of and quinate esters of p-coumaric acid are not their
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Plant Mol Biol (2007) 64:145–159 155
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156 Plant Mol Biol (2007) 64:145–159
Table 2 Evaluation of the content in the major hydroxycinnamoyl esters present in the different organs of 4-month-old seedlings of C.
canephora
Organ CQA DiCQA 5-FQA HQA
3– 4/5– 3,4– 3,5– 4,5–
e c d b bc cd b
Shoot tip 0.02 4.90 0.13 7.73 0.19 0.10 13.11
ab b c a b cd a
Stem Apical 0.43 5.78 0.22 9.23 0.24 0.10 16.02
bc c b c bc b c
Medium 0.39 4.80 0.30 2.01 0.20 0.19 7.89
d f d de bc de ef
Basal 0.20 1.59 0.15 0.50 0.15 0.05 2.62
e f de de c de f
Hypocotyl 0.04 1.04 0.09 0.35 0.06 0.04 1.61
Petiole
ab
Node 1 Young 0.48 4.16 c 0.15 cd
1.52 cd 0.09 bc
0.18 b
6.59 cd
a
Node 2 Medium 0.55 3.27d 0.22 cd
1.09 cde 0.15 bc
0.13 bc
5.42 d
bc
Node 3 Old 0.37 2.24 ef 0.18 cd
0.51d 0.16 bc
0.08 cde
3.54 def
Leaf
de de d de bc cde de
Node 1 Young 0.12 2.59 0.12 0.97 0.07 0.08 3.94
de de d de bc cde def
Node 2 Medium 0.14 2.53 0.12 0.55 0.07 0.08 3.49
cd de cd de bc cd de
Node 3 Old 0.27 2.92 0.17 0.40 0.14 0.09 3.99
a a a cd a a b
Cotyledon 0.55 7.63 0.70 1.51 1.08 0.47 11.94
e f e d c e f
Root 0.02 1.09 0.03 0.16 0.04 0.02 1.36
Values are expressed in percentage of the dry mass (% DW). For the same class of compounds, values followed by the same letter
indicate no significant between-organ difference at P £ 0.05 according to one-way ANOVA. As shown in Fig. 8, 4-CQA and 5-CQA
cannot be separated in our analytic system
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Plant Mol Biol (2007) 64:145–159 157
Fig. 10 Histolocalization of the caffeoylquinic acids, lignin, and greenish–white fluorescence (arrows) is restricted to the vascular
CYP98A35 and CYP98A36 proteins. Neu’s reagent was used to bundle, in cell layers surrounding the phloem and in the phloem
visualize caffeoylquinic acids under UV light (A1, B1, C2) and cells (ph). The blue fluorescence observed in the cell wall of the
Mirande’s reagent for lignin under visible light (C1) in cross xylem vessels (x) is due to hydroxycinnamic compounds bound
sections of different organs of Coffea canephora 4-month-old to lignin; (B2) In the same way, proteins are abundant in the
plants. Tissue print hybridization was performed on the same vascular bundle, particularly in the phloem compared to xylem
organs (A2, B2, C3) by printing transversal hand cuts onto and cortical parenchyma (cp); (C): Stem 2, situated between the
nitrocellulose membranes. (A): (A1) Developing leaves from the node 2 and 3; (C1) vascular formations are clearly visible using
shoot tip; the greenish–white fluorescence (arrows), characteris- Mirande’s reagent i.e. primary phloem (pph), secondary phloem
tic of caffeoylquinic derivatives, was observed in all the cells, and (sph), xylem (x) and medullar parenchyma (mp); (C2) the level
was particularly abundant in the blade mesophyll (m), and in the of greenish–white fluorescence indicates that caffeoyl esters were
medullar parenchyma (mp); (A2) identical distribution of concentrated around the vascular bundle, principally in the cells
CYP98A35 and CYP98A36 was revealed using polyclonal from the pph and sph, and in the medullar parenchyma; (C3)
antibodies; (B): (B1) Petiole from the leaf of the node 2; the proteins seem to be identically localized. Scale bars = 200 lm
and the proteins were concentrated in the vascular CYP98A3, recognize with the same efficiency both
bundles. Previous studies have shown that caffeoyl CYP98A35 and CYP98A36, we were unable to dif-
quinic acids are localized in some cells forming the leaf ferentiate their respective distributions. Our semi-
bundle sheath, and in the phloem cells and lignifying quantitative PCR experiments indicate that their genes
tissues from petioles and stems (Mondolot et al., 2006). have very similar spatiotemporal expression patterns
As the polyclonal antibodies used, prepared against at the level of the organ (except in old leaves), but
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158 Plant Mol Biol (2007) 64:145–159
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