Pectins, Hemicelluloses and Celluloses Show Specific Dynamics
in the Internal and External Surfaces of Grape Berry Skin
During Ripening
Regular Paper
Marianna Fasoli1,5, Rossana Dell’Anna2,5, Silvia Dal Santo1, Raffaella Balestrini3, Andrea Sanson4,6,
Mario Pezzotti1, Francesca Monti4,* and Sara Zenoni1
1
Department of Biotechnology, University of Verona, 37134 Verona, Italy
Micro Nano Facility, Fondazione Bruno Kessler, 38123 Trento, Italy
3
Institute for Sustainable Plant Protection, CNR, 10125 Torino, Italy
4
Department of Computer Science, University of Verona, 37134 Verona, Italy
5
These authors contributed equally to this work.
6
Present address: Department of Physics and Astronomy, University of Padova, 35131 Padova, Italy.
2
*Corresponding author: E-mail, [email protected]; Fax, +39-045-802-7068.
(Received August 14, 2015; Accepted April 12, 2016)
Grapevine berry skin is a complex structure that contributes
to the final size and shape of the fruit and affects its quality
traits. The organization of cell wall polysaccharides in situ
and their modification during ripening are largely uncharacterized. The polymer structure of Corvina berry skin, its
evolution during ripening and related modifying genes were
determined by combing mid-infrared micro-spectroscopy
and multivariate statistical analysis with transcript profiling
and immunohistochemistry. Spectra were acquired in situ
using a surface-sensitive technique on internal and external
sides of the skin without previous sample pre-treatment,
allowing comparison of the related cell wall polymer dynamics. The external surface featured cuticle-related
bands; the internal surface showed more adsorbed water.
Application of surface-specific normalization revealed the
major molecular changes related to hemicelluloses and pectins in the internal surface and to cellulose and pectins in the
external surface and that they occur between mid-ripening
and full ripening in both sides of the skin. Transcript profiling of cell wall-modifying genes indicated a general suppression of cell wall metabolism during ripening. Genes related
to pectin metabolism—a b-galactosidase, a pectin(methyl)esterase and a pectate lyase—and a xyloglucan endotransglucosylase/hydrolase, involved in hemicellulose
modification, showed enhanced expression. In agreement
with Fourier transform infrared spectroscopy, patterns
due to pectin methyl esterification provided new insights
into the relationship between pectin modifications and
the associated transcript profile during skin ripening.
This study proposes an original description of polymer
dynamics in grape berries during ripening, highlighting
differences between the internal and external sides of the
skin.
Keywords: Berry skin ripening Cell wall Mid-infrared FTIR
micro-spectroscopy Multivariate data analysis Pectin
methyl esterification Transcript profiling.
Abbreviations: ATR, attenuated total reflection; BSA, bovine
serum albumin; FDR, false discovery rate; FTIR, Fourier transform infrared; PBS, phosphate-buffered saline; PC, principal
component; PCA, principal component analysis; PG, polygalacturonase; PME, pectin(methyl)esterase; SAM, significance
analysis of microarray; XET, xyloglucan endotransglucosylase/hydrolase.
Introduction
Grapevine (Vitis vinifera) is an economically important perennial fruit crop used for the production of table grapes, raisins
and wine (Vivier and Pretorius 2002). The final quality traits of
berries reflect a long developmental process that involves a
double-sigmoid growth profile and profound modifications of
physiological, metabolic and physical properties (Coombe and
Hale 1973, Coombe 1976, Conde et al. 2007). At the end of this
process, each berry achieves its final volume and accumulates
sugars and compounds that determine aroma and flavor
(Coombe 1992).
The berry pericarp comprises the mesocarp (pulp), characterized by cells with thin cell walls, and the exocarp (skin),
containing thick-walled epidermal and hypodermal cells with
abundant plastids and polyphenols (Hardie et al. 1996). The
skin cell wall structure and composition determine the extractability of phenolics and other compounds during wine making
(Huang et al. 2005, Ortega-Regules et al. 2006, Schlosser et al.
2008, Bindon et al. 2012). An outer layer of hydrophobic cuticle
waxes rich in triterpenoids is usually considered part of the skin,
and acts as the primary protective barrier against environmental stress (Commenil et al. 1997, Pensec et al. 2014).
Although cuticle waxes affect berry quality and integrity by
minimizing water loss, little is known about the structural integrity and mechanical toughness of the cuticle during fruit
maturation. In contrast, many efforts were devoted to the characterization of structural and compositional changes of the skin
Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080, Advance Access publication on 19 April 2016,
available online at www.pcp.oxfordjournals.org
! The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080
and pulp during ripening, especially changes affecting hemicelluloses, pectins and cellulose microfibrils of the cell wall (Nunan
et al. 1997, Nunan et al. 1998, Vidal et al. 2001, Doco et al. 2003).
During cell expansion, these polysaccharides are rearranged by
the disruption of chemical bonds and the integration of new
synthesized material (Cosgrove 2005), whereas fruit softening
involves extensive polymer degradation and cell wall disassembly (Vicente et al. 2007).
Previous studies have identified differences between the skin
and pulp cell walls, including the total polysaccharide content,
and the composition of pectins and hemicellulose, the latter
also reflecting cultivar-dependent differences (Vidal et al. 2001,
Doco et al. 2003, Ortega-Regules et al. 2008). The dynamic expression profiles of cell wall-modifying enzymes and corresponding changes in cell wall composition reveal that pectin
modification is primarily responsible for the progressive loss
of firmness in ripening and withering fruits (Nunan et al.
1998, Vidal et al. 2001, Doco et al. 2003, Zoccatelli et al.
2013). Furthermore, the xyloglucan content of hemicellulose
decreases significantly during berry growth and ripening,
whereas cellulose levels are only marginally affected (Moore
et al. 2014a).
Over the last two decades, a comprehensive picture of gene
expression dynamics during berry ripening has emerged based
on transcriptome analysis (Fasoli et al. 2012, Tornielli et al. 2012,
Zenoni et al. 2012). Different members of the same cell wallmodifying protein families appear to be expressed in the skin
and pulp (Grimplet et al. 2007, Fasoli et al. 2012), with different
expression profiles during berry ripening (Deluc et al. 2007,
Pilati et al. 2007, Zamboni et al. 2010, Zenoni et al. 2010,
Sweetman et al. 2012, Cookson et al. 2013, Dal Santo et al.
2013). These observations suggest that the synthesis and degradation of cell wall polymers are regulated at the level of transcription, but cell wall changes during berry ripening are not
understood in detail, so we currently lack a complete model of
grape berry maturation.
Fourier transform infrared (FTIR) spectroscopy coupled
to multivariate statistical analysis is an accurate and nondestructive technique to determine with a unique measurement the overall molecular composition of plant cell walls
(Cavagna et al. 2010) and provide information on polysaccharides in situ without the need for extracting or solubilizing, and
therefore altering, the cell wall (Largo-Gosens et al. 2014). As
regards grapevine, thus far FTIR spectroscopy has only been
applied to leaves and roots under biotic or abiotic stress
(Oliveira et al. 2009), and to alcohol-insoluble residue extracts
for the high-throughput analysis of polymer composition in
fully expanded leaves (Moore et al. 2014b) and ripening berries
(Moore et al. 2014a).
Among various FTIR experimental techniques, attenuated
total reflection (ATR) is intrinsically more sensitive to modifications in external cell structures due to the reduced photon
beam penetration depth (Burattini et al. 2008). This allows
measurements to be performed directly on hydrated samples
without pre-treatment, hence preserving the native cell wall
structure and natural biological variability (Cavagna et al.
2010, Monti et al. 2013). Principal component analysis (PCA)
can be successfully applied to the absorption spectra to monitor biochemical changes over time (Cavagna et al. 2010). An
original and statistically reliable method was recently proposed
to enhance spectral differences and facilitate the extraction of
information concerning the molecular processes involved in
cell expansion (Monti et al. 2013).
Here we used our previously proposed method based
on ATR-FTIR micro-spectroscopy, automatic selection of
spectra, PCA and graphical heat map data representation
to investigate the internal and external surfaces of intact
berry skins from the Corvina grapevine variety during three
phenological phases, and to monitor polymer modifications
from veraison, the onset of berry maturation, to ripening.
The FTIR findings were compared with the expression profiles
of genes involved in cell wall polymer and cuticle metabolism
obtained by evaluating transcriptomic changes in the Corvina
berry skin during the same three phenological phases. Pectin
distribution was also evaluated by performing immunohistochemical analyses on the berry skin. Using this multifaceted
approach, we provided a detailed profile of cell wall modifications in grape berry skin during ripening, highlighting
differences between the internal and the external side of the
skin.
Results
FTIR spectroscopy reveals different absorption
bands in the internal and external surfaces of
berry skins during ripening
Corvina berries were collected at veraison, mid-ripening and full
ripening, and we determined the total soluble solid content at
each stage (Fig. 1A). Three skin layers could be distinguished by
optical microscopy: the cuticle (about 5 mm thick, comprising a
soft epicuticular layer of waxes); the epidermis, featuring regular
tiles of small cells known to be characterized by moderately
thick walls; and the hypodermis, composed of six or seven layers
of cells increasing in size in the deeper layers and known to be
characterized by cell walls thicker than the epidermal cells
(Fig. 1B, C; Supplementary Fig. S1). Optical microscopy at
the three ripening stages showed that mesocarp cells were
not present in the internal skin surface of the berry skin and
that the hypodermal cells expanded longitudinally during
ripening while the structure of the cell layers as a whole
became disorganized (Supplementary Fig. S1).
Infrared spectra on the internal and external surfaces of the
berry skins were measured in ATR mode. The ATR objective is
characterized by a short depth of penetration of the photon
beam, ranging from about 0.2 mm at the highest wavenumbers
to about 0.8 mm at the lowest wavenumbers (Burattini et al.
2008), and requires a contact pressure at the sample surface.
Due to the softness of the berry skin, the ATR objective was to
probe cuticle and epidermal cells in measurements made on
the external surface, and hypodermal cells in measurements
made on the internal surface (Fig. 1B).
Typical single-point absorbance spectra on the internal
and external surfaces of the berry skin after baseline correction
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M. Fasoli et al. | Cell wall polymer dynamics in ripening berry skin
Fig. 1 Overview of the sampling strategy. (A) Images showing Corvina berry clusters collected at V (veraison), MR (mid-ripening) and R (full
ripening). Brix values are indicated below each cluster. (B) Diagram of cell layers overlaid with a 100 magnification of the transverse section of a
berry at veraison stained with toluidine blue. (C) A 100 magnification of the transverse section of a berry at V, MR and R (from left to right). The
sections were stained with toluidine blue, and show epidermal and hypodermal skin layers plus mesocarp.
Fig. 2 Typical baseline-corrected and area-normalized spectra on the
external (solid) and internal (dotted line) surfaces of the berry skin at
veraison. The main cuticle-related absorption bands discussed in the
text are reported.
and area normalization are shown in Fig. 2. The main absorption bands are related to the presence of functional chemical
groups assigned to polyesters, water, carbonyl groups in proteins, amides and cell wall polysaccharides (Table 1). The external surface of the berry skin was clearly distinguished by
absorption bands that we assigned to cuticle polyesters and
extracellular matrix structural proteins (Fig. 2), whereas the
internal surface was mainly characterized by a large amount
of adsorbed water. In particular, among the bands related
to the cuticle, we identified two characteristic intense peaks
at 2,928 and 2,840 cm–1, assigned to the asymmetric and
1334
symmetric stretching of CH2 in aliphatic compounds recently
associated with petal cutin (Mazurek et al. 2013), and two
bands at 1,735 and 1,715 cm–1, assigned to carbonyl group
stretching in aliphatic polyesters. Bands assigned to turn structures in amide I (1,685 cm–1) and to carbonyl stretching
(1,390 cm–1) were also specific to the external surface, suggesting that they may correspond either to proteins in the extracellular matrix or to specific proteins present only in cell walls
on the external surface of the skin (Fig. 2; Table 1).
Baseline-corrected and area-normalized average spectra
of the full original data set for all the three phenological
phases are shown in Fig. 3A and B, where major absorption
bands related to cellulose, hemicellulose, pectins, adsorbed
water and proteins are indicated. By applying to the singlepoint absorption spectra a statistically controlled selection
procedure (Monti et al. 2013) based on PCA, as described in
the Materials and Methods, we obtained an enhancement of
the differences already present in the full data set, especially in
the polysaccharide region, without altering the original spectral
information. Subsequent analyses will be reported on the selected spectra. The area-normalized average spectra for
the three phenological phases after selection are shown in
Fig. 3C and D.
The internal and external surfaces of the berry
skin undergo different molecular changes during
ripening that are masked by the water content
and cuticle thinning
PCA results for the area-normalized full range spectra on the
internal and external surfaces of the berry skin are shown in
Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080
Table 1 Main absorption bands and assignments related to the cuticle, adsorbed water, lipids, proteins and cell wall polysaccharides
Absorption bands (cm–1)
Main assignments
Bands related to the cuticle structure
(waxes and structural proteins)
Lipid- and protein-related vibrations
2,928
CH2 asymmetric stretching in lipids
2,840
CH2 symmetric stretching in lipids
1,735
C = O stretching in aliphatic polyesters (aldehydes)
1,715
C = O stretching in aliphatic polyesters (ketones)
1,685
Turn structures in amide I
1,385
C = O of COO– symmetric stretching in proteins
720
CH2 groups rocking in long methylene chains
Bands related to adsorbed water, amides,
proteins and lipids (not waxes)
Adsorbed water-, amide-, protein- and lipid-related vibrations
1,650
H-O-H bending in adsorbed water
1,640/1,600
Amide I
1,540/1,520
Amide II
1,470/1,460
CH2 asymmetric bending in lipids
1,350/1,320
Amide III
Bands mainly related to cellulose
Orientation-sensitive cellulose-related vibrations
1,175/1,165
C-O-C stretching modes of the glycosidic bond in cellulose
1,050
C-O stretching modes mainly of C3-O3H secondary alcohols in cellulose
1,030
C-OH stretching modes mainly of primary alcohols in cellulose
Bands mainly related to hemicellulose
Hemicellulose-related vibrations
1,130/1,135
b1–3 glucans and C-O-C stretching modes of the glycosidic link in xyloglucans
1,065
b1–4 glucans and C-O and C-C stretching modes in xyloglucans
Bands mainly related to pectins
Pectin (mainly homogalacturonans)-related vibrations
1740
C = O stretching in pectin methylesters
1,145/1,150
C-O-C stretching modes of the glycosidic link in pectins
1,100/1,105
C-O and C-C stretching modes in pectins (ring)
1,015
C-O and C-C stretching modes in pectins
990/995
b1–6 glucans in pectins
Assignments are based on the literature (Kačuráková et al. 2000, Wilson et al. 2000, Kačuráková et al. 2002, Cosgrove 2005, Watanabe et al. 2006, Cavagna et al. 2010,
Zenoni et al. 2011).
Fig. 4. The highest variance, captured by principal component 1
(PC1), describes the most significant differences in the score
plot, which can in turn be explained by the increasing or
decreasing intensity of certain absorption bands in the corresponding loading plot. Strong biochemical differences were
observed between the internal and external surfaces of the
skin from veraison to full ripening.
The PC1 scores for the internal surface indicated a gradual
change from veraison to full ripening (Fig. 4A), which can be
mainly ascribed to the increasing intensity of the adsorbed
water band at 1,650 cm–1 and to the behavior of hemicellulose
and pectin absorption bands (Fig. 4B). It is also worth noting
the decrease of the absorption bands at around 1,740 cm–1 that
can be related to the decrease of pectin methylesters
(Kačuráková et al. 2000).
Conversely, for the external surface, although the increase in
water content was also present, the PC1 scores indicate a clear
separation of the veraison stage from the other two stages
(Fig. 4C), which can be ascribed to a decrease in the intensity of
the absorption bands related to aliphatic polyesters (1,735 and
1,715 cm–1) and structural cell wall proteins (1,685 and
1,385 cm–1) (Fig. 4D). This behavior suggests an apparent (not
structural) reduction in the amount of cuticle compounds from
veraison to the subsequent ripening stages, which is most probably
due to the thinning of the berry cuticle after veraison (Commenil
et al. 1997, Pensec et al. 2014). A contribution coming from cellulose and pectin absorption bands (1,050 and 1,015 cm–1, respectively; Fig. 4D) is also present.
To focus on molecular changes related to polysaccharides,
we applied PCA in the restricted range (1,150–800 cm–1). For
the internal surface of the berry skin, the PC1 scores in the
restricted range still indicated a gradual change throughout
ripening, with only a slightly greater separation between
mid-ripening and full ripening than between mid-ripening
and veraison (Fig. 5A). Although adsorbed water might still
affect the real dynamics of polysaccharides, the PC1 score pattern could be attributed to the role of hemicelluloses and
pectins, i.e. a decrease in the intensity of bands assigned to
hemicellulose polymers, in particular xyloglucans (bands at
1,130 and 1,060 cm–1), and an increase in the pectin fraction,
1335
M. Fasoli et al. | Cell wall polymer dynamics in ripening berry skin
Fig. 3 Average baseline-corrected and area-normalized absorbance spectra in the 2,000–700 cm–1 range before (A, B) and after (C, D) the
selection procedure at V (veraison; solid line), MR (mid-ripening; long dashes) and R (full ripening; dashes) acquired on the internal (A, C) and
external (B, D) surfaces of the berry skin. The vertical dotted lines in (A–D) indicate the polysaccharide region.
in particular homogalacturonan polymers (the band at
1,015 cm–1) (Fig. 5B).
In contrast, the PC1 scores in the restricted range for the
external surface spectra behave differently from those obtained
in the full range, with the full ripening stage separated from the
other two stages (Fig. 5C). The PC1 loadings indicated that this
effect can be ascribed to an increase in the pectin fraction
(homogalacturonans, as for the internal surface) and to the
increasing intensity of bands at 1,055 and 1,030 cm–1 which
means a higher degree of orientation of the cellulose microfibrils (Fig. 5D).
To highlight the dynamics of polysaccharides that could be
partially masked by the increase in water content on the internal surface, and by the effect of cuticle thinning on the external surface, we applied a surface-specific normalization. The
spectra acquired on the internal surface of the skin were normalized to the intensity of the adsorbed water band in the
1,654–1,649 cm–1 range (Fig. 6A), and the spectra acquired
on the external surface of the skin were normalized to the
intensity of bands related to waxes and structural proteins in
the 1,787–1,667 cm–1 range (Fig. 6B).
1336
Analysis of internal surface spectra normalized to
water content: major changes occur between
mid-ripening and full ripening, and are due to
hemicellulose and pectins
The most significant results for the internal surface spectra
were obtained after normalization to adsorbed water using
multivariate statistical analysis applied in the polymer-specific
restricted range (1,150–800 cm–1). Analysis in the full range
(2,000–700 cm–1) is shown in Supplementary Fig. S2.
The gradual change previously seen in the PC1 scores from
veraison through mid-ripening to full ripening (Fig. 5A) disappeared after normalization (Fig. 7A, B), allowing the investigation of polysaccharide modifications that were previously
obscured. The heat map shows clustering of the veraison and
mid-ripening samples, whereas the full ripening samples are
separated (Fig. 7A). The average spectra above the heat map
reveal that the differences in the absorbance profile that determine the clustering predominate in the 1,150–950 cm–1 range.
PCA confirmed these findings, showing veraison and midripening samples in the same half-plane of the score plot related
Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080
Fig. 4 Principal component analysis on the area-normalized average spectra in the 2,000–700 cm–1 range of the internal (A, B) and external (C,
D) surfaces of the berry skin at V (veraison), MR (mid-ripening) and R (full ripening) from three collection sites (n = north, ns = middle, s = south).
(A) PC1/PC2 score–score plot and (B) PC1 loading plot for the internal surface of the berry skin; (C) PC1/PC2 score–score plot and (D) PC1
loading plot for the external surface of the berry skin. The letters indicate the relevant wavenumbers by the corresponding cell wall components,
also reported in Table 1: W, water; H, hemicellulose; P, pectins; L, lipids; Pr, proteins; C, cellulose. The vertical dotted lines in (B, D) indicate the
polysaccharide region.
to PC1 (Fig. 7B). Indeed, the most significant first-order effect
(PC1) was the change from mid-ripening to full ripening, due to
a decrease in the intensity of the hemicellulose bands and of the
1,105 cm–1 pectin band (Fig. 7C). The latter can also be related
to the decrease of the around 1,740 cm–1 absorption band in
the full range (Supplementary Fig. S2). As a second-order
effect, indicated by the loadings of PC2 that account for only
7% of the total data variance, there was an increase in the intensity of the two pectin bands at 1,015 and 995 cm–1 (Fig. 7D).
This behavior may be considered an effect parallel to the reduction in the hemicellulose content shown in PC2 during the
transition from veraison to mid-ripening, which is anyway less
intense than the reduction during the transition from midripening to full ripening, as seen in the PC1. The score plot
also indicates that the dynamics of the relationship between
hemicellulose and pectins is slower in the north vineyard sites
(MRn). These characteristics of the process described by PC2
are well represented in the heat map, where they contribute
to the substructures of the veraison plus mid-ripening cluster
(Fig. 7A).
Analysis of external surface spectra normalized to
cuticle thickness: major changes occur between
mid-ripening and full ripening, and are due to
cellulose and pectins
The most significant results for the external surface spectra
were obtained after normalization to the absorption bands
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M. Fasoli et al. | Cell wall polymer dynamics in ripening berry skin
Fig. 5 Principal component analysis of the area-normalized average spectra in the 1,150–800 cm–1 range of the internal (A, B) and external (C, D)
surfaces of the berry skin at V (veraison), MR (mid-ripening) and R (full ripening) from three collection sites (n = north, ns = middle, s = south).
(A) PC1/PC2 score–score plot and (B) PC1 loading plot for the internal surface of the berry skin; (C) PC1/PC2 score–score plot and (D) PC1
loading plot for the external surface of the berry skin. The letters indicate the relevant wavenumbers by the corresponding cell wall components,
as reported in Table 1: H, hemicellulose; P, pectins; C, cellulose.
related to waxes and structural proteins, which account for
cuticle thinning (Fig. 4C, D). In this case, multivariate statistical
analysis was applied both in the full range (2,000–700 cm–1;
Fig. 8) and in the restricted polymer range (1,150–900 cm–1;
Fig. 9). The effect of normalization to the absorption bands
accounting for cuticle thinning was observed by comparing
the PC1 loading plots before and after normalization (Fig. 8C
and Fig. 4D). The contribution of the absorption band at
1,685 cm–1, related to structural cell wall proteins, became negligible, whereas bands related to aliphatic polyesters (1,735 and
1,715 cm–1) clearly lowered in intensity.
Together with the strong reduction in the intensity of
the cuticle thinning signal, Fig. 8D shows a clear enhancement
of the molecular changes related to polysaccharides
(1,150–950 cm–1), in this case predominantly cellulose and to
a lesser extent pectins.
1338
The heat map captures the evolution of the various absorption bands over time (Fig. 8A), which is also represented by the
PCA scores and loadings (Fig. 8B–D). In the 1,700–1,400 cm–1
range, a gradual increase is visible from veraison to full ripening,
whereas the increasing absorption in the polysaccharide range
mainly occurs from mid-ripening to full ripening.
The effect of adsorbed water is still apparent. Interestingly,
the PC1/PC2 score plot (Fig. 8B) shows the same dynamics as
the internal surface of the skin before normalization to adsorbed water (Fig. 4A). Whereas PC1 indicates a clear overall
gradual change in the intensity of the water and polysaccharide
bands from veraison to full ripening, specifically pectin/cellulose on the external surface (Fig. 8C) and pectin/hemicellulose
on the internal surface (Fig. 4B), PC2 shows an initial increase in
the water content relative to the polysaccharides from veraison
to mid-ripening, and a subsequent increase in the
Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080
Fig. 6 (A) Average absorbance spectra for the internal surface of the berry skin at V (veraison; solid line), MR (mid-ripening; long dashes)
and R (full ripening; dashes) in the 2,000–700 cm–1 range after normalization to the intensity of the adsorbed water band in the range
1,654–1,649 cm–1, indicated by the arrow. (B) Average absorbance spectra for the external surface of the berry skin at V (solid), MR (long
dashes) and R (dashes) after normalization to the cuticle-related absorption bands in the 1,787–1,667 cm–1 range, indicated by the vertical
dotted lines.
polysaccharide absorption bands relative to water, which eventually re-establishes in the ripe berries the initial value of the
ratio between the water and polysaccharide content as seen at
veraison (Fig. 8D; Supplementary Fig. S3). No significant
changes were observed by normalizing the external surface
spectra to the intensity of the adsorbed water band
(Supplementary Fig. S4).
In the restricted range (Fig. 9A), only 2% of the total variance was described by PC2, which differentiates among samples
within the ripe berry group more than among the three phenological stages. This indicates that the dominant dynamics of the
present analysis, which are also reproduced in the hierarchical
clustering of the heat map, are in fact described by the PC1
alone. The gradual evolution in the full range throughout ripening (Fig. 8A) disappears once again, as observed for the internal
surface spectra (Fig. 7A). The most significant changes
occurred from veraison plus mid-ripening to fully ripe berries,
as previously seen in Fig. 5C and D, and are mainly due to a
strong increase of the bands related to a higher degree of orientation of the cellulose polymers at the ripening stage, and to an
increase of the pectin-related bands assigned to homogalacturonans (as for the internal surface).
Expression analysis of genes involved in cuticle
and cell wall skin metabolism
The recent Corvina gene expression atlas (Fasoli et al. 2012) was
used to identify genes that are differentially expressed during
berry skin ripening. By applying SAM (significance analysis of
microarray) multiclass comparison among berries collected at
veraison, mid-ripening and ripening, we found 1,399 genes with
significant differential expression in the skin, with a false discovery rate (FDR) of 2% (Supplementary Table S1). On the
basis of the V1 version of the grapevine gene annotation
(Grimplet et al. 2007), we were able to identify genes involved
in cuticle and cell wall metabolism, particularly genes involved
in cellulose, hemicellulose and pectin metabolism (Fig. 10). We
also identified differentially expressed genes encoding expansin
proteins (Cosgrove 2005) which are required for cell expansion
during development (Cosgrove 2001, Zenoni et al. 2011). We
also found that two cuticle proteins and Eceriferum 2 (CER2)
were progressively down-regulated during ripening. One wax
synthase gene (VIT_15s0046g00480) was also down-regulated
during ripening, whereas another was marginally up-regulated
(VIT_12s0028g03480).
Four cellulose synthase genes were gradually down-regulated during ripening whereas two others were marginally upregulated (Fig. 10). Among genes involved in hemicellulose
metabolism, we found two endo-1,3;1,4-b-D-glucanases, two
endo-1,4-b-glucanases, a b-D-glucosidase and two b-D-xylosidases that were down-regulated during ripening (Fig. 10). In
contrast, one gene encoding a xyloglucan endotransglucosylase/hydrolase (XET) was suppressed just after veraison but
was up-regulated between mid-ripening and full ripening, suggesting a role during the last step of ripening. Genes involved
in pectin metabolism showed much more heterogeneous
transcriptional behavior. Many of the genes were downregulated during ripening, whereas one b-galactosidase
(VIT_09s0002g07630), one pectate lyase (VIT_16s0039g00260)
and one pectin(methyl)esterase (PME; VIT_15s0048g00500)
were strongly up-regulated (Fig. 10). Another b-galactosidase
gene (VIT_18s0001g02220) was moderately up-regulated after
veraison and therefore may influence pectin metabolism between veraison and mid-ripening. A polygalacturonase (PG;
VIT_01s0127g00870) was repressed at the mid-ripening stage
but induced later in the ripening process (Fig. 10).
Finally, six expansins representing different subfamilies were
found to be differentially expressed during berry ripening, with
VvEXLB3, VvEXPA2 and VvEXPB4 induced towards the end of
ripening, VvEXPA3 transiently induced during the mid-ripening
1339
M. Fasoli et al. | Cell wall polymer dynamics in ripening berry skin
Fig. 7 Heat map representation and principal component analysis in the restricted 1,150–800 cm–1 range at V (veraison), MR (mid-ripening) and
R (full ripening) from three collection sites (n = north, ns = middle, s = south) for the internal surface average spectra after normalization to the
intensity of the adsorbed water band. (A) The row dendrogram groups the spectra into clusters of hierarchical similarity. The average spectra
shown above are those calculated on the V (solid), MR (long dashes) and R (dashes) phases. (B) PC1/PC2 score–score plot. (C) PC1 loadings.
(D) PC2 loadings. The letters indicate the relevant wavenumbers by the corresponding cell wall components, as reported in Table 1: H,
hemicellulose; P, pectins.
stage and VvEXPA11 and VvEXPA16 only expressed during
veraison (Fig. 10).
Pectin distribution in the internal and external
skin surface during ripening
To examine the pectin dynamics in the two skin surfaces, sections of berry skin at the three ripening stages were treated
with JIM7 and LM19 antibodies. Both antibodies bind
to partially methyl-esterified homogalacturonans, but JIM7
binds preferentially to homogalacturonan with a relatively
high degree of methyl esterification, while LM19 binds preferentially to homogalacturonan with a relatively low degree of methyl esterification (Verhertbruggen et al. 2009)
(Fig. 11).
1340
During the overall maturation process, no differences in the
binding of JIM7 were found between internal and external skin
surfaces (Fig. 11A–C), suggesting a similar degree of methyl
esterification, whereas LM19 showed higher signal in the external (epidermis) than the internal (hypodermis) surface, that
featured a decreasing signal gradient towards the most internal
layers (Fig. 11D–F). This result suggests a different pattern of
homogalacturonans with a low degree of methyl esterification
between the internal and external skin surfaces.
JIM7 signal seemed higher at veraison and mid-ripening
stages compared with the full ripening stage; this decrease in
JIM7 binding may have resulted partly from a reduction in
methyl ester groups on pectin polymers (Moller et al. 2011)
during the process, in particular between the mid-ripening and
full ripening stages. Likewise, LM19 appears slightly lower in the
Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080
Fig. 8 Heat map representation and principal component analysis in the 2,000–700 cm–1 range at V (veraison), MR (mid-ripening) and R (full
ripening) from three collection sites (n = north, ns = middle, s = south) for the external surface average spectra after normalization to the
intensity of the cuticle-related absorption bands. (A) The row dendrogram groups the spectra into clusters of hierarchical similarity. The average
spectra shown above are those calculated on the V (solid), MR (long dashes) and R (dashes) phases. (B) PC1/PC2 score–score plot. (C) PC1
loadings. (D) PC2 loadings. The letters indicate the relevant wavenumbers by the corresponding cell wall components, as reported in Table 1: L,
lipids; Pr, proteins; W, water; C, cellulose; P, pectins.
full ripening stage compared with veraison and mid-ripening
only in the hypodermal cell walls, suggesting that in the epidermis cells homogalacturonans with a relatively low degree of
methyl esterification content did not decrease during the process. No specific signal was observed in the control sections
where primary antibody was omitted (not shown), except for
the cutin layer, which displayed unspecific autofluorescence in
all samples.
Taken together, the differences observed in the labeling
of skin sections during ripening by JIM7 and LM19, even
though not quantitative, seemed consistent with the changes
observed in the infrared absorption bands related to pectin
methyl esters and homogalacturonans during the process and
support the obtained FTIR results regarding major pectin
changes mainly occurring between mid-ripening and full ripening stages.
Discussion
The external and internal surfaces of the berry
skin were clearly distinguished by the presence of
cuticle polyesters and water absorption bands,
respectively
Mid-infrared ATR-FTIR microspectroscopy is intrinsically sensitive to surface features and can therefore be used to study the
organization of cell wall polymers and their functional groups in
1341
M. Fasoli et al. | Cell wall polymer dynamics in ripening berry skin
Fig. 9 Heat map representation and principal component analysis in the restricted 1,150–800 cm–1 range at V (veraison), MR (mid-ripening) and
R (full ripening) from three collection sites (n = north, ns = middle, s = south) for the external surface average spectra after normalization to the
intensity of the cuticle-related absorption bands. (A) The row dendrogram groups the spectra into clusters of hierarchical similarity. The average
spectra shown above are those calculated on the V (solid), MR (long dashes) and R (dashes) phases. (B) PC1/PC2 score–score plot. (C) PC1
loadings. The letters indicate the relevant wavenumbers by the corresponding cell wall components, as reported in Table 1: P, pectins; C,
cellulose.
situ without any prior sample preparation, hence preserving the
natural biological variability of the native cell wall structure
(Cavagna et al. 2010, Monti et al. 2013) and the distinction
between the epidermis and the hypodermis. We combined
ATR-FTIR microspectroscopy with multivariate statistical analysis to investigate the cell wall polymer composition in the
inner and outer sides of skins of V. vinifera cv Corvina berries
and to follow modifications during ripening.
It has been reported that berry skin has a very important role
in regulating the berry growth in terms of expansion and
softening by controlling post-véraison growth through its cell
wall remodeling (Matthews et al. 1987, Huang and Huang 2001,
Huang et al. 2005, Schlosser et al. 2008). Moreover, it was argued
that skin loosening triggers the consequent loosening of pulp
tissue that contributes to berry softening (Huang and Huang
2001, Vicens et al. 2009).
1342
The proposed method is based on the identification of absorption peaks using second derivative calculations and on their
assignment to the specific vibrations reported in the literature
concerning similar biological systems. With no need for quantification, PCA applied on normalized FTIR spectra allows the
study of the evolution over time of the polymer structure and
composition through the relative changes in absorption bands.
These reflect the changing patterns of molecular bonds, which
have a structural and biochemical meaning and are not solely
related to an increase or decrease in the amount of a certain
compound. In contrast, quantitative cell wall compositional analysis can be carried out only on the whole skin extracts, thus
losing the distinction between the inner and outer sides of the
berry skin, which is one of the objectives of the present study.
Measurements were performed on both the internal and the
external surfaces of the skin, whose distinct morphological
Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080
Fig. 10 Cell wall-related gene family members differentially expressed during the ripening of Corvina berry pericarp. Phenological stages V
(veraison), MR (mid-ripening) and R (full ripening) from three collection sites (n = north, ns = middle, s = south) are indicated at the top of the
heat map representation. Genes are grouped according to the corresponding metabolic process in the cell wall. The expression data are scaled by
row and represented by color scale intensity: green and red boxes indicate low and high expression levels, respectively.
features were revealed by optical microscopy also performed on
berry skins to confirm the absence of mesocarp cells in the
internal surface. The external surface spectra showed absorption bands related to the hydrophobic cuticle layer, whereas the
internal surface spectra featured a higher content of water and
cell wall polysaccharides. These differences in polymer composition revealed by FTIR can be associated with distinct physiological roles of the skin during ripening, i.e. the internal
regulation of berry growth and softening by water uptake
and polysaccharide modification, combined with protection
against external abiotic and biotic stress such as water loss
and pathogens.
Optical microscopy revealed that hypodermal cells expand
laterally in the plane of the epidermis during ripening and
the skin cell layers become less organized. This effect has
been previously attributed to the degradation of the middle
lamella (Huang et al. 2005). Furthermore, skin cells began
to accumulate anthocyanins in the vacuole from veraison
onwards, particularly the cells belonging to the hypodermis
(Fig. 1C).
Data analysis was performed in the 2,000–700 cm1 full
range and 1,150–800 cm1 restricted range using an automatic
spectra selection procedure to enhance differences without
altering the original spectral information. The analysis was
first performed on area-normalized spectra for both the internal and external skin surfaces.
The internal surface spectra revealed a gradual change from
veraison to full ripening that could be ascribed to the increasing
intensity of the adsorbed water band and, to a lesser extent, to
the decreasing intensity of the absorption band corresponding
to pectin methyl esters. These are related to the well-known
processes, occurring during ripening, of cell expansion and of
berry growth and softening, respectively (Brummell 2006).
Conversely, the external surface spectra revealed a strong separation between veraison and subsequent ripening stages,
which could be attributed to the decreasing intensity of the
absorption bands assigned to cuticle compounds. Consistently,
the synthesis and deposition of the cuticle occurs before veraison and then gradually declines, so the cuticle becomes thinner as the berry enlarges during ripening (Commenil et al. 1997,
1343
M. Fasoli et al. | Cell wall polymer dynamics in ripening berry skin
Fig. 11 Immunofluorescence labeling of pectins on paraffin sections of Corvina skins during the maturation process (V, veraison; MR, midripening; R, full ripening). Immunofluorescence labeling was performed using the antibody JIM7 for highly esterified pectins (A–C) or LM19 for
de-esterified pectins (D–F). Treatment with JIM7 led to a signal on the cell wall that seems to be higher at V and MR stages (A and B, respectively)
compared with the R stage (C). In (C), a weak red/orange autofluorescence is visible on the cutin layer. Using LM19, the signal appears slightly
higher in V and MR (D and E, respectively) compared with the R stage (F), but only in the hypodermal cell walls, while the green fluorescence
does not seem to decrease in the epidermal layer. e, epidermis; h, hypodermis. Scale bars correspond to 51 mm in (A), 48 mm in (B), 44 mm in (C),
42 mm in (D) and 60 mm in (E) and (F).
Pensec et al. 2014). Because the water content and cuticle thickness could partially screen polysaccharide modifications, the
polysaccharide changes on the internal and external surfaces
during ripening were analyzed in more detail by normalizing
the spectra for water-related and cuticle-related bands,
respectively.
Surface-specific normalization revealed different
polymer dynamics during ripening on the two
sides of the skin
The removal of the effect of gradual water uptake from the
internal surface spectra revealed the underlying decrease in
the intensity of hemicellulose and pectin absorption bands
during the transition from mid- to full ripening (Fig. 7). The
observed decrease of hemicellulose-related bands and the increase of pectin solubilization between mid- and full ripening
may be an indication of the well-described loosening of the cell
1344
wall polymer network that is directly related to skin wall swelling and to an increase in wall porosity that occur later in the
ripening process (Brummell 2006). The changes in berry textural properties related to cell disruption during ripening may
be associated with the release of aroma compounds due to the
interaction among enzymes and substrates previously compartmentalized, as suggested for the lipoxygenase pathway involved
in biosynthesis of fatty acid-derived aromatic compounds
(Buttery 1993, Sanz et al. 1997, El Hadi et al. 2013).
Internal surface spectra are related to the hypodermal cell
wall, and corresponding changes may be attributed to the degradation of the middle lamella during ripening in concert with
the swelling of epidermal and subepidermal cells during the
continuous loosening of skin cell walls (Hardie et al. 1996).
Moreover, our in situ analysis showed that the major loss of
pectin and hemicellulose occurred between mid- and full ripening and not gradually from the onset of ripening as previously
reported (Hardie et al. 1996, Vicens et al. 2009). The precise
Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080
swelling (Redgwell et al. 1997), in agreement also with the
increasing intensity of the homogalacturonan-related band at
1,015 cm–1 (Fig. 9C).
Taken together, the chosen sampling and measurement
strategy accounting for the natural biological variability allowed
a clear overview to be obtained of the modifications of polymer
structure and of their relative composition from the onset of
ripening to complete ripening of the berries, showing important differences between the internal and the external surfaces
of the skin.
Fig. 12 Experimental design for FTIR measurements. Twenty grape
berries were collected at three ripening stages (V, veraison; MR, midripening; R, full ripening) and at three sites in the vineyard (n = north,
ns = middle, s = south), i.e. 180 berries. Three point-by-point absorption spectra were acquired for the internal and for the external surfaces of the skin for each berry, i.e. 1,080 single-point spectra.
profile of polysaccharide changes may have been difficult to
determine in previous studies due to the confounding effect
of water uptake. Pectin demethylesterification may sweep inwards from the homogalacturonan-rich middle lamella
(Brummell 2006, Ortega-Regules et al. 2008, Vicens et al.
2009), whose depolymerization is related to the loss of cell–
cell adhesion. The progressive loss of pectin methylation was
specifically indicated by the decreasing intensity of the around
1,740 cm–1 bands during the transition from mid- to full
ripening.
During cell wall solubilization, hemicellulose modifications
include the depolymerization of xyloglucans, predominantly
starting from the end of the molecules. This is where they are
attached to cellulose or interact with pectins, thus achieving
the gradual weakening of the structure whilst maintaining the
overall integrity of the berry during ripening (Vicens et al. 2009).
The evident decrease in the intensity of hemicellulose-related
bands (Fig. 7C) from mid- to full ripening on the internal surface of the skin highlights the depolymerization of tightly
bound matrix glycans (Brummell 2006). It is therefore possible
that the minor changes in hemicellulose-related bands reported
in Fig. 7D represent an early depolymerization of more loosely
bound hemicelluloses from the onset of ripening to midripening.
The simultaneous effects of water uptake and cuticle thinning make the interpretation of external surface spectra more
challenging. More meaningful results were obtained after normalization to the cuticle thickness. The gradual increase in the
intensity of cellulose and pectin bands corresponds to the swelling of the berry skin epidermis layer (Huang et al. 2005). After
veraison, the walls of the ripening fruits become more hydrophilic and accessible to cell wall-modifying enzymes, hence
contributing to the observed increase in the intensity of cellulose-specific bands (Figs. 8C, 9C, D). A higher degree of orientation of the cellulose microfibrils may increase enzyme
mobility and substrate accessibility, promoting the dismantling
of polymers during fruit maturation (Huang et al. 2005). Pectin
depolymerization and solubilization correlate with skin wall
Identification of putative candidate genes
involved in cell wall polymer skin dynamics
throughout ripening
In order to correlate our FTIR results with the expression profiles of genes involved in cell wall and cuticle metabolism, we
consulted the V. vinifera cv Corvina global expression atlas
based on berry skin at the veraison, mid-ripening and full ripening phenological stages (Fasoli et al. 2012). We observed a general down-regulation during berry ripening of genes related to
cuticle metabolism (Fig. 10), strongly supporting previous findings concerning the termination of cuticle synthesis after veraison (Hardie et al. 1996, Pensec et al. 2014). Notably, we found
a significant down-regulation of CER2, whose direct involvement in the synthesis of 28-carbon cuticle waxes was recently
reported in Arabidopsis (Haslam et al. 2015). This expression
behavior mirrors the decreasing intensity of cuticle-related absorption bands, suggesting the involvement of CER2 in berry
cuticle deposition before veraison. However, the up-regulation
of a wax synthase gene suggests that the production of waxrelated compounds is also required in the ripening berry.
We also observed a general down-regulation during ripening
of genes related to cell wall metabolism, including four members of the cellulose synthase family. This supports transcriptomic data obtained from the berry pericarp (Deluc et al. 2007)
and strongly correlates with the observation that the cellulose
content does not change during ripening (Zenoni et al. 2012).
On the other hand, IRX3 (VIT_11s0037g00530), one of the two
up-regulated cellulose synthase genes, is directly involved in the
synthesis and correct deposition of cellulose in the secondary
wall (Taylor et al. 2003), suggesting that the increasing intensity
of cellulose-related bands in the external surface of the skin may
be related to cellulose re-orientation.
The strong reduction in the intensity of hemicellulose-associated bands on the internal surface at the transition from midto full ripening highlighted the importance of xyloglucan metabolism in cell wall rearrangement during Corvina berry ripening, as recently demonstrated in Cabernet Sauvignon and
Crimson Seedless berries (Moore et al. 2014a). Nevertheless,
the induction of genes involved in the hydrolysis of (1–4)-bD-linked glucan chains during ripening was not detected during
pericarp ripening by Nunan et al. (2001). Only one gene encoding a XET responsible for xyloglucan polymer reassembly was
marginally induced during ripening. XET was previously shown
to promote berry softening by the depolymerization of xyloglucans in Kyoho grapes (Kobayashi et al. 2002), as confirmed for
1345
M. Fasoli et al. | Cell wall polymer dynamics in ripening berry skin
the berry pericarp at the transcriptomic level (Deluc et al. 2007,
Glissant et al. 2008, Sweetman et al. 2012, Dal Santo et al. 2013).
However, the stronger up-regulation of XET on the internal
surface of the skin may have been obscured in the transcriptome data because it was derived from total skin tissue.
Pectin depolymerization by PG and de-esterification by PME
are key processes underlying fruit softening during ripening
(Hyodo et al. 2013). However, a specific PG activity has yet to
be detected during berry ripening, suggesting that pectin solubilization may be promoted by a-galactosidases and b-galactosidases that remove the non-reducing terminal galactosyl
residues from the side chains of pectic polysaccharides
(Nunan et al. 2001, Ortega-Regules et al. 2008). Localization
of pectin polymers by homogalacturonan-specific antibodies
in the skin sections during ripening supported our FTIR results
and interpretation, and allowed us to identify differentially expressed genes possibly involved in polymer changes in the two
skin surfaces that could represent interesting candidates for
further investigations. In particular, the high degree of pectin
esterification recognized by JIM7 antibodies showed that epidermal and hypodermal cells featured the same pattern of
degree of methyl esterification, that appeared slightly lower
at ripening compared with the previous stages. This evidence
can be associated with the decrease in the intensity of the
absorption band related to pectin methyl esters between
mid-ripening and full ripening on the internal skin surface,
and suggests that the PME VIT_15s0048g00500, up-regulated
during ripening, is a good candidate for pectin methyl esterification in all skin cell layers. The signal of LM19 antibodies, that
recognize homogalacturonans with a relatively low degree of
methyl esterification, showed a slight decrease only in hypodermis cell walls and no differences in epidermis during ripening.
This result can be associated with the decrease of homogalacturonan-related infrared absorption bands in the internal
skin surface and with the increase of bands assigned to homogalacturonans in the external skin surface. In this context the
up-regulation of the b-galactosidase VIT_09s0002g07630, that
may increase pectin solubility, and of the pectate lyase
VIT_16s0039g00260, involved in the cleavage of de-esterified
pectins, could be assigned to the internal surface of the skin.
In concert with other cell wall-modifying enzymes, expansins promote fruit softening by the acid-induced disruption of
hydrogen bonds linking cellulose and hemicellulose fibers, thus
stimulating slippage between the polymers (Cosgrove 2000, Dal
Santo et al. 2013). The modulation of expansin genes during berry ripening could promote the hemicellulose rearrangement observed on the internal surface of the skin, and the
pectin solubilization detected on both sides. The transient
up-regulation of VvEXPA3 suggests that it may enhance
pectin solubilization at the mid-ripening stage, correlating
with the second-order dynamics observed on the internal surface of the skin after normalization to adsorbed water.
Interestingly, the significant up-regulation of VvEXLB3, an
EXLB protein that may promote the formation of secondary
cell walls (Dal Santo et al. 2013), indicates a correlation with the
expression of cellulose synthase IRX3, suggesting that these
proteins may co-operate to facilitate cellulose deposition
1346
during the formation of secondary cell walls specifically in the
outermost layer of the ripe berry skin.
In summary, our in situ analysis represents an original approach which allowed us to detect differences in the polymer
structure and composition in the epidermal side and in the
inner skin side of grape berry cell walls, also accounting for
the natural biological variability, and to follow their evolution
from the onset of ripening to complete ripening. During this
period, the obtained profiles of polymer changes showed that
most of the variations take place between mid- and full ripening. The two skin sides feature different cell wall polymer structure and modifications, hence pointing out that attention must
be paid when presenting grape berry skin data as a homogenous matrix.
Materials and Methods
Plant material and sampling
Berry samples were collected from a 7-year-old vineyard of V. vinifera cv.
Corvina located 130 m above sea level at Montorio, Verona Province, Italy
(45 270 1700 N, 11 030 1400 E). The soil composition is 36% sand, 36% clay and
28% silt. The replacement cane Guyot rows were north–south oriented, and
41B was used as the rootstock.
The phenological stages of berry ripening were monitored by recording the
total soluble solids, expressed as degrees of Brix ( B), using a PR-32 bench
refractometer (Atago Co.), and defined according to the modified E-L system
proposed by Coombe (1995).
The berries were collected during the 2006 growing season at the same time
of day (09:30 h) at 71, 98 and 112 d after fruit set, corresponding to veraison,
when berries begin to change color and enlarge (10.4 B; E-L 35), mid-ripening
(15.5 B; E-L 36) and complete ripening (20.0 B; E-L 38). For each phenological
stage, three biological replicates were obtained from vines located at the north,
middle and south sites of the vineyard. Each sample comprised a pool of 20
berries from different bunches representing four plants.
Optical microscopy and immunofluorescence
Flesh Corvina berries and berry skins carefully removed from frozen berries were
cut into small pieces and fixed in 2% formaldehyde and 0.25% glutaraldehyde in
phosphate-buffered saline (PBS; pH 7.5) under vacuum overnight. The fixed
berries and skins were rinsed five times in PBS and then dehydrated in increasing concentrations of ethanol (25, 50, 75 and 100%) before post-fixing overnight
in ethanol containing increasing concentrations of xylene (25, 50, 75 and 100%).
The samples were embedded by progressively substituting the xylene with
Paraplast Plus (Thermo Fisher Scientific). Tissue sections (7 mm thick) were
prepared with a 2035 Leica microtome (Leica Microsystems GmbH), floated
on warm water, and immobilized on slides coated with poly-L-lysine to facilitate
handling. The slides were then air-dried at 37 C. After removing the paraffin by
incubating in 100% xylene for 2 15 min and passing quickly through decreasing concentrations of ethanol (100, 75, 50 and 25%), the sections were stained
with toluidine blue and viewed under a Leica DMRB optical microscope (Leica
Microsystems GmbH).
Skin sections have also been used for immunofluorescence experiments
to localize homogalucturonans. Briefly, sections were blocked in phosphate
buffer containing 1% bovine serum albumin (BSA; w/v) for 30 min, incubated
overnight at 4 C with the LM19 or JIM7 antibody (CarboSource, http://www.
ccrc.uga.edu/carbosource/CSS_mabs7-07.html) diluted 1 : 10 in 1% BSA,
washed three times with phosphate buffer for about 30 min, saturated
for 30 min with 1% (w/v) BSA in phosphate buffer, and incubated at room
temperature in the dark for 3 h with a goat anti-rat IgG conjugated to fluorescein isothiocyanate (FITC) (dilution 1 : 20 in 1% BSA). The sections
were washed as before, mounted, and observed under a Leica TCS SP2 confocal microscope, using a 40 water-immersion objective, at 488 nm. Labeling
specificity was determined by replacing the primary antibody with the buffer.
Plant Cell Physiol. 57(6): 1332–1349 (2016) doi:10.1093/pcp/pcw080
FTIR spectroscopy
The experimental design of FTIR measurement is shown in Fig. 12. Mid-infrared
spectra were acquired in ATR mode in the 4,000–7,00 cm–1 range using a Vertex
70 Bruker spectrometer coupled to a Hyperion 3000 vis/IR microscope
equipped with a photoconductive MCT detector and a 20 germanium
ATR-crystal objective. Before measurement, the skin was removed from the
frozen berries and thawed at room temperature. Measurements were performed on all the samples dried at the same thawing time (10 min at room
temperature). To acquire the infrared signal, a small amount of water released
during the thawing process was also carefully removed manually. By comparing
spectra acquired on a set of fresh and thawed samples, we checked that there
was no significant alteration in the thawed samples with respect to the fresh
samples, especially as regards absorption bands related to cell wall polymers.
For each thawed berry, three point-by-point absorption spectra were collected on the internal and external surfaces of the skin (i.e. 540 absorption
spectra for each side) at 4 cm–1 resolution over a 100 mm diameter area by coadding 64 scans (acquisition time = 27 s). Since in ATR measurements the
penetration depth inside the sample is proportional to the wavelength
(Griffiths and de Haseth 1986), the usual ATR correction was applied by multiplying the absorption value at each spectral channel by the corresponding
wavenumber.
Data processing and analysis
Data treatment was performed in the 2,000–700 cm–1 range where we
found the most interesting spectral information (full range). Single-point absorbance spectra were baseline corrected using the rubber band method, and
area normalized. Average spectra were obtained for each replicate at each
phenological stage. Subsequent data analysis was applied in the full range
and in the polymer-specific restricted range (1,150–800 cm–1) tailored to be
the same for internal and external surface spectra avoiding cutting of the absorption bands. PCA, automatic selection of spectra and heat map analysis were
carried out in the R software environment (Monti et al. 2013, R Core Team
2015).
Principal component analysis
The average spectra calculated for each replicate at each phenological stage
were analyzed though PCA. The first two PCs in our analyses always best
described the spread of the data, and the projection of the absorption spectra
onto this two-dimensional PC plane (score–score plot) allowed us to visualize
the relationships among them as explained by the captured variance. By direct
comparison with the scores, the loading plots allowed us to identify one or
more spectral bands responsible for the grouping and differences highlighted in
the score plots.
Automatic selection of spectra
PCA was applied on all the single-point absorbance spectra. To better discriminate patterns among the scores, a procedure based on an automatic selection
of spectra was applied (Monti et al. 2013). Briefly, the procedure consists of
iteratively discarding the single-point spectra having the lowest Pearson correlation coefficient with the average spectrum. In each cycle, the average spectrum was recalculated until all the correlation coefficients were higher than a
threshold value chosen to preserve biological variability. As a result, the spectral
differences present in the original data set were enhanced and the corresponding score patterns represented a higher percentage of the total variance and
were described by a lower PC.
Heat maps
A spectral data matrix was built containing an average spectrum in each row
and the absorbance values of the same spectral channel across all spectra in
each column. The heat map was then created as a two-dimensional grid, in
which the color of each cell represented the corresponding value of the matrix
(Monti et al. 2013). The lowest value relative to the mean value of each column
was shown as brightest green and the highest value as brightest red. We used
hierarchical clustering (average clustering method and a metric based on the
Pearson correlation coefficient) to order the spectra (the rows of the heat
maps) into a dendrogram based on the similar intensity of common spectral
bands.
Transcriptome analysis
The expression profiles of genes related to cell wall and cuticle metabolism were
analyzed by consulting the V. vinifera cv. Corvina global expression survey of
different organs at various developmental stages (Fasoli et al. 2012). The gene
expression microarray data were obtained by hybridization to a NimbleGen
090818 Vitis exp HX12 microarray (Roche, NimbleGen Inc.) representing 29,549
genes based on the 12 grapevine V1 gene prediction (http://genomes.cribi.
unipd.it/grape/). From this data set, we chose the berry skin tissues collected at
the same phenological stages used for FTIR spectroscopy. Each sample was
analyzed as three biological replicates.
A gene was considered to be expressed if the normalized expression value
for at least two of the three biological replicates was higher than the value
obtained by averaging the fluorescence of the negative control present on the
chip. The genes differentially expressed during ripening were evaluated using
the SAM statistical approach in the TMev software suite v4.3 (http://www.tm4.
org/mev) with an FDR of 2%.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the CARIVERONA Bank
Foundation [within the project ‘Completamento del Centro
di Genomica Funzionale Vegetale’]; the European funded
COST ACTION FA1106 [networking activities within ‘An integrated systems approach to determine the developmental
mechanisms influencing fleshy fruit quality in tomato and
grapevine’]; the Italian Ministry of University and Research
[FIRB RBFR13GHC5 project ‘The Epigenomic Plasticity of
Grapevine in Genotype per Environment Interactions’ to
S.D.S.].
Acknowledgments
We thank Giorgio Pasqua for providing plant material.
Disclosures
The authors have no conflicts of interest to declare.
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