Differential gene expression analysis of strawberry cultivars that differ in fruit-firmness
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PHYSIOLOGIA PLANTARUM 118: 571–578. 2003 Copyright # Physiologia Plantarum 2003 Printed in Denmark – all rights reserved ISSN 0031-9317 Differential gene expression analysis of strawberry cultivars that differ in fruit-firmness Elma M. J. Salentijn*, Asaph Aharoni, Jan G. Schaart, Marjan J. Boone, Frans A. Krens *Plant Research International, PO Box 16, 6700 AA, Wageningen, The Netherlands *Corresponding author, e-mail: E.M.J.Salentijn@plant.wag-ur.nl Received 17 September 2002; revised 9 January 2003 Firmness is an important selection criterium in the breeding expression, including 10 clones (8 different ones) which of fruit, including strawberry (Fragaria · ananassa Duch.). showed homology to cell wall related genes in the public Clear differences in fruit-firmness are observed between databases. The results from the microarray experiments cultivars. In order to identify candidate genes which might were further confirmed by RNA gel blots, which were also be associated with such textural differences, gene expression used to examine gene expression in a third cultivar, Elsanta, levels were compared for a soft and a firm cultivar showing an intermediate texture phenotype (offspring of a (cv. Gorella and cv. Holiday, respectively). DNA-microarrays cross between Gorella and Holiday). Interestingly, two genes representing 1701 strawberry cDNAs were used for simultan- encoding proteins catalysing successive reactions in lignin eous hybridization of two RNA populations derived from metabolism (cinnamoyl CoA reductase and cinnamyl alcohol red ripe fruit of both cultivars. In total 61 clones (3.6% of dehydrogenase) showed the highest difference in expression the total cDNAs on the arrays) displayed differential level. Introduction Strawberry (Fragaria ananassa Duch.) is an important (i.e. b-galactosidase [EC 3.2.1.23], pectin methylesterase soft fruit in the temperate climate zones of the world. (PME) [pectinesterase; EC 3.1.1.11], endo- and This attractive and tasty fruit, with its high nutritional exo-polygalacturonase [endo-PG, pectinase; EC 3.2.1.15, value, is either sold in the fresh market or as a raw exo-PG, galacturan; EC 3.2.1.67] (Barnes and Patchett material for the food processing industry. Fruit-firmness 1976, Huber 1984, Nogata et al. 1993, Manning 1998b, is an important issue for both markets and is therefore an Llop-Tous et al. 1999, Redondo-Nevado et al. 2001, important target for improvement of strawberry quality Trainotti et al. 2001). Pectate lyase [EC 4.2.2.2] activity (Manning 1998a). Cultivars can differ significantly in is difficult to measure in strawberry. However, two textural aspects like firmness, postharvest shelf life and cDNAs coding for pectate lyase (U63550 and susceptibility to damage. Texture of fruits, including AF339024) are highly expressed in strawberry fruit fruit-firmness, is determined by cell turgor pressure and (Medina-Escobar et al. 1997) and recently, Jiménéz- by characteristics of the cell wall. Modification and turn- Bermudez et al. 2002) reported that transgenic plants over of the primary cell wall is required for both growth with reduced pectate lyase expression (to less than 30% (cell expansion) and for the softening of fruits, whereby of the control plants) showed a higher fruit-firmness. one family of cell wall and middle lamella modifying Another group of cell wall related enzymes such as proteins mediate the activity of other families in a endo-1,4-b-glucanases (cellulases), xyloglucan endo- concerted and synergistic action (Rose and Bennett 1999). transglycolase [EC 2.1.4.207] and expansins may play a In recent years, expression patterns of genes encoding role in the disassembly of the hemicellulose matrix in various different enzymes likely to be involved in the ripening fruits (Abeles and Takeda 1990, Rose and modification of the cell wall and thus in fruit-firmness Bennett 1999). The expression profiles of several have been extensively studied. They mainly include cDNAs coding for endo-1,4-b-glucanases [EC 3.2.1.4] enzymes acting on the pectin fraction of the cell wall have been studied in strawberry (Harpster et al. 1998, Physiol. Plant. 118, 2003 571
Llop Tous et al. 1999, Trainotti et al. 1999a, 1999b). intermediately firm fruits, was selected from the offspring
Transgenic strawberry plants with a considerable of the cross between cv. Holiday and cv. Gorella. Ripe fruits
decrease in cel1 mRNA accumulation, encoding an of the cultivars Holiday and Gorella were used for
endo-1,4-b-glucanase enzyme, did not show a clear poly(A)1 RNA isolation (mRNA purification kit, Amer-
change in firmness (Woolley et al. 2001). Furthermore, sham Pharmacia Biotech, Uppsala, Sweden). These poly(A)
it is evident that expansins are intimately involved in the RNAs were subsequently labelled for use as samples in
fruit-softening process. These proteins help to gain access DNA microarray experiments.
to the cell wall to early hemicellulases and late acting
pectinases in tomato ripening (Brummell et al. 1999,
RNA-isolation and RNA gel blot analysis
Brummell and Harpster 2001). Expansin activity was
also detected in ripening strawberry fruits (Harrison For RNA gel blot analysis, total RNA was isolated from
et al. 2001) and 7 expansins genes (FaExp1–7) were fruit tissue according to Schulz et al. (1994). 6–10 mg of
isolated (Civello et al. 1999, Harrison et al. 2001). glyoxal (1.5 M)-denatured total RNA (6–10 mg) was electro-
DNA microarray technology provides a powerful way phoresed and transferred to a Hybond N1-membrane
to investigate differential gene expression (reviewed by (Amersham). Probes for hybridization were labelled
Aharoni and Vorst 2002). The method is fluorescence- via random prime labelling (Random Prime Labelling
based and allows the simultaneous and quantitative kit; Invitrogen, Carlsbad, CA, USA). For quantifica-
analysis of gene expression for a large set of genes. In tion, RNA gel blots were exposed to a phosphorimager-
the past years, microarrays representing hundreds of screen, and subsequently scanned in a Bioimager device
cDNAs derived from a strawberry fruit cDNA library (Fujix BAS2000). Next, the signals were measured as
have been utilized for monitoring gene expression during photo-simulated-luminescence (OD per mm2) using TINA
fruit development and specifically in achene and recep- software (Raytest, Straubenhardt, Germany). cDNA
tacle tissue (Aharoni and O’Connell 2002). The same sequences of cell wall related genes were used as probes
microarrays have been used for the identification of for RNA gel blot analysis. Strawberry cDNA sequences
auxin dependent and independent genes and oxidative coding for, respectively, a ribosomal protein and a DNA
stress responsive ripening-related genes (Aharoni et al. binding protein were used as internal controls for loading.
2002). Apart from providing a global picture on gene Expression level quantification was determined relative to a
expression during fruit maturation the method facilitated loading control using the same RNA gel blot (after strip-
the identification of individual genes related to fruit ping of the first probe) for both probes.
quality traits (Aharoni et al. 2000). To date, no studies
investigating the phenotypic differences in fruit-
DNA-microarray experiments
phenotype among strawberry cultivars have been
reported. DNA-microarray analysis, development of the fluores-
In this report, we describe the use of DNA microarray cent target probes and statistical analysis of reciprocal
technology to identify genes that may determine cultivar two-colour microarray experiments were performed
differences in fruit-firmness by comparing the expression according to Aharoni et al. (2000) using the same batch
profiles of two strawberry cultivars that differ substan- of microarray slides containing 1701 strawberry cv.
tially for this trait. The expression of candidate genes Elsanta cDNAs spotted in duplicate. Expression ratios
identified in the microarray experiment was further above or below the inverse of the value 2.1 indicated a
analysed in detail using RNA gel blots (Northern blots). significant up- or downregulation.
Remarkably, in a soft fruit like strawberry, our results
indicate that mRNAs coding for the enzymes cinnamyl
Firmness measurements
alcohol dehydrogenase, CAD [EC 1.1.1.195], and
cinnamoyl CoA reductase, CCR [EC 1.2.1.44], which are Mechanical properties of at least 6 freshly harvested ripe
involved in the lignin biosynthesis pathway, show a clear strawberries (cultivars Elsanta, Holiday and Gorella)
differential expression profile among the cultivars. were analysed at two perpendicular sides through
compression and penetration tests by using a universal
testing machine (TA-XT2i Texture Analyser, Stable
Materials and methods Micro System, Godalming, UK).
Plant material
Strawberry (Fragaria ananassa, Duch.) cultivars Results
Elsanta, Holiday and Gorella were used for expression
Comparing a soft and a firm cultivar using strawberry
profiling by microarray and RNA gel blot analysis. The
cDNA microarrays
firm cultivar, Holiday, was selected in 1965 (US) from a
cross between cv. Raritan and breeding line NY844 Among cultivars of strawberry, significant differences in
whereas the soft cultivar Gorella was selected in 1955 in firmness were observed in the ripe stage of fruit development.
the Netherlands from a cross between cv. Juspa and For instance, the difference in firmness in the ripe stage
breeding line US3763. Cultivar Elsanta, that has between cv. Holiday (firm) and cv. Gorella (soft) is
572 Physiol. Plant. 118, 2003obvious and easy to detect by manual inspection. Values Holiday (clones B39 and G61 showing 3.3 and 2.3 higher
of 5.0 and 2.5 (on a scale 1–5) were scored for Holiday expression in cv. Holiday, see Table 1). A gene coding for
and Gorella, respectively, (an average over the years O-methyl transferase (FaOMT, Wein et al. 2002) was not
1991–1997). Mechanical inspection, scored a firmness significantly differentially expressed. This enzyme
of 18.5 and 7.4 (gradient between two points on the (FaOMT) acts in the lignin biosynthesis pathway by
curve force [50 g]/t [s]) for Holiday and Gorrela, respect- conversion of caffeic acid into ferulic acid.
ively. Both cultivars are the parents of cultivar Elsanta, Other differentially expressed genes putatively encod-
which showed an intermediate phenotype for firmness, ing for proteins with a probable influence on the cell wall
when examined by similar criteria (manually: 4.0; were an endo-1,4-b-glucanase, Cel1 (clone C1), acting on
mechanically: 14.7). Apart from the difference in texture, the hemicellulose or cellulose fraction of the cell wall,
the cultivars Holiday and Gorella show obvious differ- and several enzymes that may act on the pectin fraction,
ences in colour, flavour and the shine and fragility of the like sequences with similarity to PME (clones B8 and
skin. Ripe fruits of cv. Holiday are bright red, have a H163) and a PG-like gene (clone B4). Furthermore,
shining skin and a distinct flavour, whereas the fruits of genes putatively encoding structural cell wall proteins,
cv. Gorella are more brownish red, with a more dull like a glycine rich protein (clone H58) and a hybrid
skin which is very fragile and a moderate flavour proline rich protein (HyPRP, clone D65) were differen-
(ir. E.J. Meulenbroek, strawberry breeder at Plant tially expressed.
Research International, personal communication).
To identify genes that might be associated with the
Detailed gene expression analysis using RNA gel blots
phenotypic differences in firmness, we utilized
DNA-microarrays representing 1701 cDNAs isolated The expression level of cell wall related genes, was exam-
from a ripe fruit cDNA library of the cultivar Elsanta ined in the cultivars Elsanta, Holiday and Gorella using
(Aharoni et al. 2000). The microarrays were hybridized quantitative RNA gel blots (Fig. 1). These RNA gel blot
with a mixture of fluorescently labelled targets derived experiments confirmed the data by DNA microarrays for
from the firm cultivar Holiday and the soft cultivar seven out of the eight different genes that were differen-
Gorella. The raw microarray data were further statisti- tial expressed between Holiday and Gorella. The expres-
cally analysed to determine the threshold for a significant sion of the proline rich protein (H58) was below the
change in expression (ratio ¼ 2.1) and a list of differen- detection level of the RNA gel blot analysis and is there-
tially expressed clones between the two samples was fore not presented.
generated. A set of 61 clones out of the 1701 present on In ripe fruits, several genes that may possibly play a
the array (3.6%) showed a significant change in expres- role in cell wall degradation like PME-like genes (H163
sion between the cultivars (Table 1). Based on their and B8) and a PG-like gene (B4) were upregulated in the
homology to genes with a known or putative function soft cultivar. In addition, the cell wall loosening enzyme
present in the public databases, the genes were assigned expansin (FaExp2, B155) and the polygalacturonase
to various functional classes. Ten genes (among them 8 gene (sPG, D15) showed only a slight increase in
different ones) could be associated with cell wall transcript level in the soft cultivar as detected by RNA
metabolism (16.4% of the differentially expressed gel blot (Fig. 1).
genes). Other differentially expressed genes could be The expression levels of CCR and CAD were analysed
associated with pigmentation (3.3%); primary metabo- in six stages of fruit development (from small green to
lism (4.9%); protein degradation (8.2%); amino acid ripe red) of the three cultivars, Holiday (firm), Elsanta
metabolism (11.5%); stress (9.8%) and transport and (intermediate) and Gorella (soft) (Fig. 2). CAD and CCR
signalling (4.9%), whereas 41% of the genes could not gene expression was similarly low between the cultivars
be attributed to a specific function. Gorella and Holiday in the early stages of fruit develop-
ment (small green and green-white). At the big white
stage expression of both genes started to differ between
Differential gene expression associated with cell wall
the two cultivars, being much higher for CCR in cv.
metabolism
Gorella and higher for CAD in cv. Holiday in the
Interestingly, the most dramatic differences in mRNA subsequent stages up to and including the red ripe stage.
levels were observed for a homologue of cinnamoyl
CoA reductase (CCR, homologous to accession no.
Discussion
CAC07424, AJ295838, www.ncbi.nlm.nih.gov/entrez)
encoding an enzyme active in lignin metabolism. The The texture of fruit such as strawberry is by far one of
CCR clones H101 and JB116 showed a more than the most important traits in breeding programmes.
20-fold higher level of expression in the soft cultivar Breeding for firm fruit in strawberry is difficult since it
Gorella compared to the firm cultivar Holiday. Another occasionally seems that texture is negatively linked with
gene, cinnamyl alcohol dehydrogenase (CAD, homolo- good flavour (E.J. Meulenbroek, personal communica-
gous to accession no. AAK28509, AF320110), encoding tion). The trait is complex and more information on the
the enzyme catalysing the subsequent step in the lignin factors that are determining and controlling it are
pathway was expressed higher in the firm cultivar needed. In the present study we identified candidate
Physiol. Plant. 118, 2003 573Table 1. Differentially expressed cDNAs (PRI clone number) among ripe fruits of a firm cultivar (cv. Holiday) and a soft cultivar (cv. Gorella)
as determined by DNA-microarray analysis. Each cDNA was annotated according to the definition and accession of the nucleotide sequence
of the first BlastX homologue from the NCBI database (www.ncbi.nlm.nih.gov). Out of the 61 differetially expressed cDNAs 8 were not
annotated because no hit was found in the database. Positive and negative ratios correspond to cDNAs that are higher expressed in the soft or
firm cultivar, respectively. The number in parentheses indicates the number of the sequence contig in the case when sequences are different but
show similar Blast results
Homologue Definition Ratio
PRI clone (GeneBank accession number) H/G
PIGMENTATION
A5 UDP-GFT, AB047097 12.2
D40 Leucoanthocyanidin dioxygenase, X71360 2.5
PRIMARY METABOLISM
H51 Pyruvate decarboxylase, AF193791 (1) 12.9
H68 Pyruvate decarboxylase, AF193791 (1) 12.4
JB190 Hydroxypyruvate reductase, AB060810 12.6
PROTEIN DEGRADATION
C70 Cysteine proteinase, AF454958 (1) 12.4
E71 Cysteine proteinase, AF454958 (2) 12.6
E93 Cysteine proteinase, AB020961 (3) 12.1
H55 Cysteine proteinase, AF411121 (4) 12.1
H86 Cysteine proteinase, NM_128302 (5) 2.8
AMINO ACID METABOLISM
C151 Glutamine synthetase, K03282 12.2
H154 Asparagine synthetase, AF061740 (1) 13.5
H35 Asparagine synthetase, AB035248 (2) 13.1
JB138 Asparaginase, AF308474 (1) 5.9
JB180 Asparaginase, AY096000 (2) 4.7
C119 Glutamate decarboxylase, AF361836 (1) 12.3
C184 Glutamate decarboxylase, AF506366 (2) 12.3
STRESS
A104 Glutathione S-transferase, NM_111189 (1) 12.3
C88 Glutathione S-transferase, Y07721 (2) 12.1
Jb319 Glutathione S-transferase, AF193439 (3) 2.8
E149 Quinone reductase-like protein, NM_115504 12.6
A125 Ubiquitin-conjugating enzyme, NM_105097 12.3
C137 Superoxide dismutase, AF016893 12.2
TRANSPORT/SIGNALLING
C86 Ns-lipid transfer protein, AJ315844 2.2
JB299 Serine threonine protein kinase, NM_118334 12.4
D156 ADP-ribosylation factor, AP003247.4 17.8
CELL WALL
B4 Endo-PG like protein, NM_116014.1 13.2
C1 Endo-b-1,4-glucanase, cel1, AF074923 2.4
B8 Similarity to pectinesterase, AB019235 12.5
H163 Similarity to pectinesterase, AJ237985 (2) 12.3
H101 Cinnamoyl CoA reductase, AJ295838 (1) 120.3
JB116 Cinnamoyl CoA reductase, AJ295838 (1) 120.4
B39 Cinnamyl alcohol dehydrogenase, AF320110 (1) 3.3
G61 Cinnamyl alcohol dehydrogenase, AF320110 (2) 2.3
D65 Hybrid PRP, AF026382 2.6
H58 Glycin rich protein, AB031227 2.8
OTHERS
B181 Ripening induced protein, AJ001449 (1) 16.9
E177 Ripening induced protein, AJ001449 (1) 17.7
H174 Ripening induced protein, AJ001445 (2) 2.5
H79 Gast-like gene, AF039183 2.8
Jb105 Gibberellin 2 beta-hydroxylase, AF101382 2.3
D105 cold-regulated gene, AF007784 4.9
E187 Cytochrome P450, NM_123002 2.9
F143 3-hydroxyisobutyryl CoA hydrolase, NM_125991 3.0
C3 Histone H2A, M64838 2.4
E103 Putative AAA-type ATPase, AK118992 2.3
C188 FolC-FolP fusion protein, AF227236 2.2
D55 Hypothetical protein, NM_116300 12.2
JB286 Hypothetical protein, NC_003366 2.2
D81 Hypothetical protein, NM_100756 2.8
G89 Unknown protein, NM_121261 2.5
F8 Expressed protein, AY091184 12.2
C91 Expressed protein, NM_111682 12.4
574 Physiol. Plant. 118, 2003OD/mm2 the increment in transcript level was underestimated by
RNA gel blot
DNA microarray analysis compared to the RNA gel blot
Holiday
Holiday
Gorella
Elsanta
Gorella
Elsanta
Ratio
RNA
Ratio
DNA
analysis (Fig. 1).
PRI clone gel blot microarray Genes expected to be involved in cell wall degradation
D15 (sPG) 17.7 15.0 32.1 +2.1 +1.3* such as the genes encoding PME-like and PG-like
proteins were upregulated in the soft cultivar. For two
B155 (FaExp2) 48.8 85.6 103.7 +1.2 +1.0* other important cell wall degrading enzymes, expansin
(FaExp2) and polygalacturonase (sPG), only a slightly
H163 (PME-like) 11.0 2.4 11.1 +4.7 +2.3 higher expression was detected in ripe fruits of the soft
cultivar by RNA gel blot analysis (Fig. 1). Both genes
B8 (PME-like) 153.8 29.0 141.9 +4.9 +2.5
showed a more clear differential expression in the turn-
B4 (PG-like) 0.3 4.5 13.4 +3.0 +3.2 ing stages of fruit-ripening as detected by RNA gel blot
analysis (results not shown), supporting their importance
H101 (CCR) 10.8 1.2 33.7 +27.9 +20.1 in determining differences in fruit-firmness. Further-
more, a cDNA coding for endo-1,4-b-glucanases (Cel1),
B39 (CAD) 44.7 24.1 3.3 –7.4 –3.3 acting on the hemicellulose or hemicellulose fraction of
the cell wall was differentially expressed (with a higher
D65 (HyPRP) 11.6 19.6 11.7 –1.7 –2.6
expression observed in red fruits of the firm cultivar).
C1 (Cel1) 37.9 172.0 110.2 –1.6 –2.4 This may explain why downregulation of Cel1 mRNA
accumulation by antisense inhibition did not result in
DBP increased fruit firmness (Woolley et al. 2001).
Until recently, polygalacturonase (PG) activity in
EtBr strawberry was an issue of debate (Manning 1993). How-
ever, we previously reported on at least two PG encoding
Fig. 1. Confirmation of microarray results using RNA gel blot genes (clones B4 and D15) which are active during fruit
analysis. Probes of candidate genes for fruit texture were used to development (Aharoni and O’Connell 2002). One of
hybridize gel-blots loaded with total RNA derived from the three those genes (D15) was identical to the sPG gene
cultivars, Elsanta (intermediate), Holiday (firm) and Gorella
(soft). The expression levels on the RNA gel-blots were quantified described by Redondo Nevado et al. 2001). The deduced
using a bioimager device (OD/mm2,TINA software, Raytest, sPG protein was classified as a Clade A polygalacturo-
Straubenhardt, Germany) and counted relative to the expression nase, showing up to 45% sequence identity with
levels of an internal standard (gene coding for a DNA binding fruit-ripening endo-polygalacturonases. However, we
protein, DBP) that was expressed at a similar level in red fruits of
the three cultivars analysed. EtBr ¼ ethidium bromide stained RNA observed a higher homology (up to 62% identity)
samples. The ratio of the expression levels between cv. Holiday and with PGs belonging to Clade C (e.g. Salix gilgiana,
cv. Gorella were counted for, respectively, RNA gel blot and DNA- BAA89478). This Clade comprises genes expressed in
micoarray. Positive ratios (1) and negative (–) ratios are reflecting,
respectively, an upregulation and a downregulation of specific gene pollen that are thought to encode exo-PGs (Hadfield
expression in cv. Gorella. Ratios marked with an asterisk indicate a and Bennett 1998). Also, a cystein residue that was
not significant difference in expression as measured by DNA shown to be restricted to pollen specific PGs (Tebbutt
microarray (ration below 1 or 2.1).
et al. 1994) is present at position 273 of the deduced
protein. The second gene with homology to polygalac-
turonases (B4) was also expressed at the early stages of
genes that might be related to differences in texture development (Aharoni and O’Connell 2002). In the
among strawberry varieties. current study it was identified as differentially expressed
For the analysis of differential gene expression DNA between the cultivars at the ripe stage (Fig. 1). The
microarray technology was used which previously has deduced amino acid sequence of the B4 cDNA is hom-
shown to serve as a powerful tool for the identification ologous to Arabidopsis thaliana putative PG sequences
of genes associated with major quality traits in straw- (e.g. NP-190464) and to microbial PGs (e.g. NP-346995).
berry (Aharoni et al. 2000). The cultivars Holiday and The pattern of the PG active-site (pattern PS00502 of the
Gorella, used for the comparisons in the DNA micro- prosite database at http://www.expasy.org/prosite) is
array experiments, are not closely related. Therefore, the present in the sPG protein. However, in the deduced B4
risk existed that the amount of differences was too high protein and homologous PG-like proteins the PG active-
and could mask the specific differences in gene expres- site differs from the one present in known PG proteins.
sion related to the trait of interest. However, this was not Also, the deduced amino acid sequences of the PME-like
the case and only 61 genes showed significant differences sequences (H163, B8) did not contain the consensus
in expression. Expression differences for specific regions that are present in all PMEs that have been
members of gene families are most likely underestimated functionally characterized (Markovic and Jornvall
by cross-hybridization in microarrays and have to be 1992) and their functional role is still unclear.
validated by a secondary procedure, especially those The most pronounced difference between both
with a 2- to 4-fold difference in expression (Rajeevan cultivars was observed for the CCR and CAD genes.
et al. 2001). We found that in most cases (7 out of 9) These genes encode enzymes that are acting successively
Physiol. Plant. 118, 2003 575A
CCR CAD
16 16
H H
14 14
E E
Transcript ratio
12 G 12 G
10 10
8 8
6 6
4 4
2 2
0 0
SG GW BW T O R SG GW BW T O R
B monolignols
CCR CAD
feruloyl CoA coniferaldehyde coniferalcohol guaiacyl lignin
sinapoyl CoA sinapaldehyde sinapylalcohol syringyl lignin
p-coumaroyl CoA p-coumaroyl aldehyde p-coumarylalcohol p-hydroxyphenyl lignin
Flavonoids
Fig. 2. (A) Expression levels of CCR and CAD in fruit developmental stages [small green (SG), green white (GW), white (W), turning (T),
orange (O) and ripe (R)] of three different cultivars: Elsanta (E, intermediate firm), Holiday (H, firm) and Gorella (G, soft), as detected by
RNA gel blots. Each of the blots was checked for RNA amounts by rehybridizing with a strawberry ribosomal RNA probe. Quantification of
the results was performed by measuring the OD/mm2 (TINA software, Raytest, Straubenhardt, Germany) for each band and calculating the
transcript ratio between the gene specific signal and the corresponding band in the ribosomal loading control (transcript ratio). (B) Substrates
and products of CCR and CAD, enzymes that are acting in a branch of the phenylpropanoid pathway leading to lignin biosynthesis (for a
detailed model of the lignin biosynthesis see Humphreys and Chapple 2002).
in the lignin biosynthetic pathway, which is a branch of cultivar and CAD in the firm cultivar. It is not comple-
the phenylpropanoid pathway (CCR acting upstream of tely clear yet how the lignin biosynthetic pathway oper-
CAD). Lignin in a soft fruit like strawberry has been ates. Modifications of lignin by downregulation or
detected in the achenes and in the vascular bundles that overexpression of CCR and CAD showed that severe
connect the achenes to the central pith (Suutarinen et al. changes in CAD activity had a striking effect on the
1998). Jewell et al. (1973) associated the amount of composition of lignin. In contrast, CCR downregulation
vascular tissue with texture and processing quality of effected mainly a decrease of the total lignin content
strawberries. Using a primary anti-strawberry CAD (Chapple and Carpita 1998, Hertzberg et al. 2001,
polyclonal antiserum Aharoni et al. (2002) showed that Abbott et al. 2002, Humphreys and Chapple 2002). In
the corresponding protein was localized specifically to both natural occurring mutants and transgenic plants
immature xylem cells undergoing active lignification. with depleted levels of CAD, increased incorporation of
Also, expression of strawberry CAD in yeast cells and cinnamaldehydes into lignin has been observed. The
enzymatic activity assays demonstrated a cinnamyl cross-linking and physical properties of cinnamaldehyde
alcohol dehydrogenase activity of the recombinant rich lignins differ from that of normal lignins.
enzyme (Blanco-Portales et al. 2002) indicating that it It has been observed that the lignin in CAD deficient
may be involved in lignin biosynthesis. However, it can plants is more susceptible to chemical extraction and
not be ruled out that both enzymes CCR and CAD shows improved pulping characteristics (Halpin et al.
acting in the lignin biosynthesis pathway are associated 1994, Russell et al. 2000, Ruel et al. 2001). Thus, the
with other functions like, for CAD, the biosynthesis of differences in CAD gene expression could be related with
flavour compounds (Mitchell and Jelenkovic 1995). lignin composition, being richer in aldehydes and more
Also, the various branches of the phenylpropanoid susceptible to enzymatic degradation in the soft cultivar.
pathway including those associated with the biosynthesis However, since the lignin content and composition of
of lignin, hydroxycinnamates (ferulic, sinapic, caffeic and both strawberry cultivars has not been determined we
4-coumaric acid) and flavonoids, appear to be closely can not confirm this assumption.
linked. Variations in the flux through the pathway may In this study, DNA-microarray analysis provided a
lead to the biosynthesis of a different pool of hydroxy- method for large-scale identification of differentially
cinnamic acids or –aldehydes with a putative effect on expressed genes in a comparison between a soft and a
flavour or on cell wall bound hydroxycinnamates firm cultivar. The combination of sequence information,
(Kroon and Williamson 1999). information on the biological function of sequence
Expression of the CCR and CAD genes differed homologues and additional gene expression data is
between the cultivars, CCR being higher in the soft needed to select candidate genes for further analysis.
576 Physiol. Plant. 118, 2003The information obtained, although correlative in Harrison EP, McQueen Mason SJ, Manning K (2001) Expression
nature, indicates that enzymes acting in the lignin of six expansin genes in relation to extension activity in devel-
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candidates for fruit-firmness. However, the hypotheses R, Blomqvist K, Bhalerao R, Uhlen M, Teeri TT, Lundeberg J,
concerning gene functions still must be confirmed Sundberg B, Nilsson P, Sandberg G (2001) A transcriptional
roadmap to wood formation. Proc Natl Acad Sci USA 98:
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CCR is underway. polyuronides and hemicelluloses. J Food Sci 49: 1310–1315
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