plant physiology preview. published on march 11, 2015, as doi
TRANSCRIPT
1
Research article 1
Running head 2
Metabolic change in grape berry skin by UV 3
4
Corresponding author 5
Katsuhiro Shiratake 6
Laboratory of Horticultural Science, Graduate School of Bioagricultural Sciences, Nagoya 7
University, Chikusa, Nagoya 464-8601, Japan 8
Tel: +81-52-789-4026 9
Fax: +81-52-789-4026 10
e-mail: [email protected] 11
12
Research areas 13
Biochemistry and Metabolism 14
Plant Physiology Preview. Published on March 11, 2015, as DOI:10.1104/pp.114.254375
Copyright 2015 by the American Society of Plant Biologists
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
2
Title 1
Multi omics in grape berry skin revealed specific induction of stilbene synthetic 2
pathway by UV-C irradiation 3
4
Authors 5
Mami Suzukia,b, Ryo Nakabayashic, Yoshiyuki Ogatad, Nozomu Sakuraie, Toshiaki 6
Tokimatsuf,g,Susumu Gotof, Makoto Suzukic, Michal Jasinskih,i, Enrico Martinoiaj, Shungo 7
Otagakia, Shogo Matsumotoa, Kazuki Saitoc,k, Katsuhiro Shiratakea,1 8
9
Author’s affiliations 10
a. Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 11
464-8601, Japan 12
b. National Institute of Vegetables and Tea Science, Taketoyo 470-2351, Japan 13
c. RIKEN Center for Sustainable Resource Science, Tsurumi, Yokohama 230-0045, Japan 14
d. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Naka, 15
Sakai 599-8531, Japan 16
e. Kazusa DNA Research Institute, Kisarazu 292-0818, Japan 17
f. Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji 611-0011, 18
Japan 19
g.Database Center for Life Science, Research Organization of Information and Systems, 20
Kashiwa 277-0871, Japan. 21
h. Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, 22
Dojazd 60-637 Poznań, Poland 23
i. Institute of Bioorganic Chemistry PAS, Noskowskiego 61-704 Poznań, Poland 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
3
j. Institute of Plant Biology, University of Zurich, Zurich 8008, Switzerland 1
k. Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675, 2
Japan 3
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
4
One sentence summary 1
The metabolic pathway map reflecting the transcriptome data and metabolome data clearly 2
showed the specific induction of resveratrol synthetic pathway by UV-C in grape berry skin. 3
4
Footnotes 5
1To whom correspondence should be addressed. E-mail: [email protected]. 6
7
Author contributions: Katsuhiro S., E.M., M.J. and Kazuki S. designed the research; Mami S., 8
Makoto S., R.N., S.O. and S.M. performed the experiments; Mami S., Y.O., N.S., T.T. and 9
S.G. updated the database; and Mami S. and Katsuhiro S. wrote the manuscript. 10
11
Data deposition: The data reported in this paper have been deposited in the Gene 12
Expression Omnibus database, http://www.ncbi.nlm.nih.gov/geo/ (accession no. 13
GSE59436). 14
15
Keywords: Grape berry, Metabolome analysis, Resveratrol, Transcriptome analysis, 16
Ultraviolet irradiation 17
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
5
Abstract 1
Grape (Vitis vinifera L.) accumulates various polyphenolic compounds, which protect 2
against environmental stresses, including ultraviolet-C light (UV-C) and pathogens. In this 3
study, we looked at the transcriptome and metabolome in grape berry skin after UV-C 4
irradiation, which demonstrated the effectiveness of omics approaches to clarify important 5
traits of grape. We performed transcriptome analysis using a genome-wide microarray, 6
which revealed 238 genes up-regulated more than 5-fold by UV-C. Enrichment analysis of 7
Gene Ontology terms showed that genes encoding stilbene synthase, a key enzyme for 8
resveratrol synthesis, were enriched in the up-regulated genes. We performed metabolome 9
analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry, and 10
2,012 metabolite peaks, including unidentified peaks, were detected. Principal component 11
analysis using the peaks showed that only one metabolite peak, identified as resveratrol, 12
was highly induced by UV-C. We updated the metabolic pathway map of grape in the Kyoto 13
Encyclopedia of Genes and Genomes (KEGG) database and in the KaPPA-View 4 KEGG 14
system, then projected the transcriptome and metabolome data on a metabolic pathway 15
map. The map showed specific induction of the resveratrol synthetic pathway by UV-C. Our 16
results showed that multi-omics is a powerful tool to elucidate accumulation mechanisms of 17
secondary metabolites, and updated systems, such as KEGG and KaPPA-View 4 KEGG for 18
grape, can support such studies. 19
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
6
Introduction 1
Grape (Vitis vinifera L.) accumulates many polyphenolic compounds, such as 2
anthocyanin, catechin and resveratrol, in the berry skin. These compounds are important 3
not only for resistance to abiotic and biotic stresses but also for berry qualities such as color, 4
astringency and consumer health benefits. Resveratrol is a polyphenolic compound 5
categorized as a stilbenoid, and it, as well as its dimer, e-viniferin, act as phytoalexins 6
(Lamgcake and Pryce 1977). Resveratrol has been identified as a key compound 7
associated with the French paradox (Renaud and Lorgeril 1992). Resveratrol has also been 8
reported to prevent heart disease and Alzheimer's disease and to have anti-cancer effects 9
(Jang et al. 1997, Marambaud et al. 2005). Recently some clinical trials have been 10
presented for showing the evidence of favorable effects of resveratrol not only on mouse but 11
also on human health (Vang et al. 2011). 12
Accumulation of polyphenolic compounds occurs during berry maturation, but can also 13
be induced by external stimuli, such as light, plant hormones and pathogen infection. The 14
amounts of resveratrol and its analogues are increased by pathogen infection in leaves and 15
berries (Aziz et al. 2003, Pezet et al. 2003). Resveratrol accumulation in mature grape 16
berries is also induced by ultraviolet-C (UV-C) irradiation (Adrian et al. 2000, Versari et al. 17
2001, Takayanagi et al. 2004, Nishikawa et al. 2011). 18
Recent expressed sequence tag (EST) analyses (Quackenbush 2001) and whole 19
genome sequencing of grape (Jaillon et al. 2007, Velasco et al. 2007) have allowed a more 20
comprehensive approach to the study of its secondary metabolites. However, most 21
transcriptome analyses have used EST-based microarrays, and this information is limited, 22
because not all genes are represented on these microarrays. Profiling of metabolites has 23
also been targeted, and only a few selected compounds have been analyzed. Few analyses 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
7
in grape using genome-wide microarrays, and even fewer studies have combined different 1
omes, such as combining transcriptome analysis and metabolome analysis (Zamboni et al. 2
2010). 3
In this study, we performed multi-omics analysis, combining transcriptome and 4
metabolome analyses to study metabolic changes in grape berry skin after UV-C irradiation 5
(Fig. 1). A genome-wide microarray, but not EST-based microarray, was used to determine 6
expressions of the whole grape genes and the obtained data underwent Gene Ontology 7
(GO) enrichment analysis. Non-targeted metabolome analysis in grape berry skin was 8
performed by liquid chromatography-quadrupole time-of-flight mass spectrometry 9
(LC-QTOF-MS), allowing the detection of more than 2,000 metabolite peaks. Principal 10
component analysis (PCA) revealed induction of a single metabolite by UV-C irradiation. 11
Data from the transcriptome and metabolome analyses were projected on a metabolic map 12
using the KaPPA-View 4 KEGG system (http://kpv.kazusa.or.jp/kpv4-kegg-1402/) (Sakurai 13
et al. 2011) after modification to apply the whole grape gene expressions. The map 14
demonstrated the induction of the stilbene synthetic pathway by UV-C irradiation. 15
16
Results 17
Annotation of grape genes 18
The grape genome has been sequenced by two groups; one is at the IASMA Research 19
Center (Velasco et al. 2007) and the other is the French-Italian Public Consortium for 20
Grapevine Genome Characterization (Jaillon et al. 2007). The latter group first released a 21
set of gene predictions of the grape genome called version 0 (V0) (Jaillon et al. 2007), which 22
is available on the GENOSCOPE website (http://www.genoscope.cns.fr/spip/). Later gene 23
predictions of the grape genome, called version 1 (V1), have been made available online 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
8
independently on the CRIBI website (http://genomes.cribi.unipd.it/grape/) by the CRIBI 1
Biotechnology Centre, which includes members of the latter group (Grimplet et al. 2012). 2
The NimbleGen microarray 090818 Vitis exp HX12 (Roche NimbleGen, USA), which was 3
used in this study, was designed based on the V1 gene predictions. To obtain more 4
information about grape gene functions, we searched best-hit homologue of each grape 5
gene in the Arabidopsis gene database (TAIR Ver. 10, http://www.arabidopsis.org) and in 6
the UniprotKB database (http://www.uniprot.org). Out of the 29,549 genes on the grape 7
microarray, 28,801 could be assigned to an Arabidopsis gene, while 28,322 genes had hits 8
in UniprotKB. Some genes did not have a hit to any Arabidopsis genes, but did to genes in 9
UniprotKB. Among the grape genes, 1,847 could be assigned to grape genes in UniprotKB. 10
As a result, 20,302 grape genes were tagged with GO slim terms 11
(http://www.geneontology.org), which could be assigned based on the results of a Blast 12
search against the NCBI non-redundant protein database using the Blast2GO algorithm 13
(Conesa and Götz 2008) (see Methods). In addition, 13,651 grape genes were tagged with 14
network names in VitisNet (Grimplet et al. 2009) 15
(http://www.sdstate.edu/ps/research/vitis/pathways.cfm), which are similar to GO terms but 16
specific for grape (Dataset S1). 17
18
Transcriptome analysis 19
The transcriptome of grape berry skin after UV-C irradiation was analyzed using a 20
microarray covering the whole grape genes. Following UV-C irradiation, 238 genes were 21
more than 5-fold up-regulated, while 24 genes were more than 0.2-fold down-regulated 22
(Dataset S2, S3, Fig. S1). Among the down-regulated genes, “cytochrome P450” 23
(VIT_01s0137g00410, VIT_07s0031g01680, VIT_02s0025g03320), “Wax2” 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
9
(VIT_09s0018g01340, VIT_09s0018g01360) and “pleiotropic drug resistance 4” 1
(VIT_11s0016g04540) were found (Dataset S3). The 238 up-regulated genes (Dataset S2) 2
were investigated for GO functional categories using Blast2GO. Based on a comparison 3
between the rates of occurrence of GO slim terms in all grape genes and those in the genes 4
up-regulated by UV-C irradiation, “response to stress” (GO:0006950) increased from 5.88% 5
to 10.2% and “secondary metabolic process” (GO:0019748) increased from 1.13% to 3.39% 6
in the up-regulated genes (Fig. 2). “Response to stress” included genes related to resistance 7
to pathogens, such as bacterial-induced peroxidase (Chassot et al. 2007), calmodulin 8
binding protein (Wang et al. 2011, Wan et al 2012), and stilbene synthase (Delaunois et al. 9
2009), while “secondary metabolic process” included genes related to secondary metabolite 10
synthesis, such as laccase-14-like (Turlapati et al. 2011) and phenylalanine ammonia lyase 11
(Huang et al. 2010). 12
GO enrichment analysis revealed that two GO terms in Biological Process and 11 GO 13
terms in Molecular Function were enriched in the genes up-regulated by UV-C irradiation 14
(Table 1). “Cell wall modification” (GO:0042545) and “lipid glycosylation” (GO:0030259) 15
were the GO terms in Biological Process. As shown in Figure 3, among the GO terms found 16
in Molecular Function were “pectinesterase activity” (GO:0030599), “chlorophyllase activity” 17
(GO:0047746), “aspartyl esterase activity” (GO:0045330), “trihydroxystilbene synthase 18
activity” (GO:0050350) and “transferase activity, transferring hexosyl groups” (GO:0016758). 19
Also found were “pectinesterase activity” (GO:0030599) and “aspartyl esterase activity” 20
(GO:0045330), which includes pectinesterase 2-like (Louvet et al. 2006) (Table 2). The 21
category “transferase activity, transferring hexosyl groups” (GO:0016758) includes 22
UDP-glycosyltransferase, related to formation of cell wall and cell plate (Eudes et al. 2008), 23
while “trihydroxystilbene synthase activity” includes stilbene synthase (STS) (Table 2). STS 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
10
is a key enzyme of resveratrol synthesis from coumaroyl-CoA and malonyl-CoA (Delaunois 1
et al 2009). As described above, the annotated genes of GO slim term of “response to 2
stress”, including stilbene synthase, were tend to enriched remarkably after UV-C irradiation, 3
and this category includes stilbene synthase. Our transcriptome analysis showed that UV-C 4
irradiation strongly and conspicuously induces STS gene expression. 5
6
Metabolome analysis 7
Next, we analyzed the metabolome, focusing on secondary metabolites, using 8
LC-QTOF-MS. In positive ion mode, 1,197 metabolite peaks were detected, and in negative 9
ion mode, 2,012 were detected, including unidentified peaks. The data from negative ion 10
mode were normalized before PCA. A score scatter plot from PCA showed two groups of 11
data, that is, control (dark) and UV-C irradiation (Fig. 4). Interestingly, a loading scatter plot 12
showed only one metabolite related to UV-C irradiation (Fig. 4), and the metabolite was 13
identified as resveratrol based on chemical reference standards. Its intensity was high, 14
meaning resveratrol accumulation was strongly induced by UV-C irradiation. The same 15
results were obtained when data from positive ion mode were used. 16
We attempted to identify and quantify major phenolic compounds and their intermediates 17
in the phenylpropanoid pathway with reference to 24 standard chemical compounds (Table 18
S1). As a result, 12 metabolite peaks were identified in positive ion mode and 17 in negative 19
ion mode. The concentrations of 10 metabolites identified in negative ion mode were high 20
enough to calculate, while 7 metabolites were not (Table 3). 21
The intensity of catechin was high in both control and UV-C-irradiated samples. Its 22
concentration in control samples was 3,771 µg g-1 FW and 4,043 µg g-1 FW in 23
UV-C-irradiated samples (Table 3). Catechin was the most abundant metabolite among 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
11
those identified, but the difference in catechin concentration between the two sets of 1
samples was only minor. Some reports in the literature have reported high accumulation of 2
proanthocyanidines including catechin in young berries before veraison (Bogs et al. 2005, 3
Fujita et al. 2007). Our results agree with these reports. 4
On the other hand, the intensity of trans-resveratrol was low in control samples, but very 5
high in UV-C-irradiated samples. The trans-resveratrol concentration in UV-C-irradiated 6
samples was 3,492 µg g-1 FW, 355 times higher than in controls (Table 3). In addition, we 7
observed much stronger fluorescence, perhaps due to resveratrol (Nero and Melo 2002), in 8
the grape berry skin of UV-C-irradiated samples than in control samples (Fig. 5). 9
cis-Resveratrol is a minor compound compared with trans-resveratrol in grape berry skin 10
(De Nisco et al. 2013). In our metabolome analysis, measurable cis-resveratrol was 11
detected in UV-C-irradiated samples, although its concentration was 6.3 µg g-1 FW, far lower 12
than the trans-resveratrol concentration (Table 3). Resveratrol analogues viniferin and 13
piceid were induced by UV-C irradiation at detectable levels (Table 3). These minor 14
analogues are thought to be synthesized by modification of trans-resveratrol. 15
Transcriptome and metabolome analyses in this study found clear induction of STS gene 16
expression and accumulation of resveratrol. 17
18
Update of the metabolic pathway map of grape in KEGG and the KaPPA-View 4 KEGG 19
system 20
The transcriptome and metabolome data were integrated and applied to a metabolic 21
pathway map using the KaPPA-View 4 KEGG system (Sakurai et al. 2011). 22
GENESCOPE released the V0 grape genome data in 2007 (Jaillon et al. 2007) and the 23
V0 data were registered as reference sequences in NCBI. Recently, the dataset was 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
12
updated from V0 to V1 by CRIBI (Grimplet et al. 2012). However, the reference sequences 1
of the grape genome in NCBI remain V0. The grape metabolic pathway map in the 2
KaPPA-View 4 KEGG system was constructed based on the grape pathway map in KEGG 3
(http://www.genome.jp/kegg) (Kanehisa 2000). The KEGG system collects gene catalogs 4
from NCBI, as well as another draft genome dataset, DGENES 5
(http://www.genome.jp/kegg/catalog/org_list1.html). 6
On the other hand, the NimbleGen microarray was designed based on the V1 grape 7
genome data (http://genomes.cribi.unipd.it/grape/). Thus, NimbleGen microarray data 8
cannot be applied to the KaPPA-View 4 KEGG system. Therefore, we updated the 9
Kappa-View 4 KEGG system for this study. We added the V1 CDS to the DGENES dataset 10
(T number T10027) with an automatic annotation server, KAAS (Moriya et al. 2007). Then 11
we applied the new pathway map based on the DGENES data of V1 CDS to the 12
KaPPA-View 4 KEGG system. As a result, 5,440 genes of V1 CDS are now reflected on the 13
pathway maps. The updated system is available on the KaPPA-View 4 KEGG web page 14
(http://kpv.kazusa.or.jp/kpv4-kegg-1402/). 15
16
Integration of transcriptome and metabolome data on the metabolic pathway map 17
Our experimental values from transcriptome and metabolome analysis were included at 18
appropriate scales (see Methods) on the metabolic pathway map. Using the updated 19
KaPPA-View 4 KEGG system, we produced an updated map (Fig. 6). It includes levels of 73 20
genes and 17 compounds, and is based on four maps (Fig. S2). 21
Phenolic compounds, such as flavonol, proanthocyanidin, anthocyanin, and resveratrol 22
have a common synthetic pathway from phenylalanine to coumaroyl-CoA, and these 23
pathways branch after coumaroyl-CoA. Coumaroyl-CoA is the substrate of both STS and 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
13
chalcone synthase (CHS). CHS mediates the reaction from coumaroyl-CoA to naringenin 1
chalcone; naringenin chalcone is converted to naringenin by chalcone isomerase (CHI), and 2
this reaction pathway feeds into the proanthocyanidin, flavonol and anthocyanin synthetic 3
pathways. Figure 6 shows that not only STS but also genes involved in the pathway from 4
phenylalanine to coumaroyl-CoA, i.e., phenylalanine ammonia-lyase (PAL), 5
4-coumarate:CoA ligase (4CL) and cinnamate 4-hydroxylase (C4H), were up-regulated by 6
UV-C irradiation. On the other hand, expression of the genes involved in the other pathway 7
branches for phenolic compounds, such as those encoding CHS, CHI and flavonoid 8
3',5'-hydroxylase (F3’5’H), were not changed by UV-C irradiation. Figure 6 also shows that 9
UV-C irradiation induced only accumulation of resveratrol and its analogues, not any other 10
phenolic compounds. This map reveals dynamic and specific metabolic changes in grape 11
berry skin due to UV-C irradiation. 12
13
Discussion 14
To elucidate the effects of UV-C irradiation on grape berry skin, we performed 15
transcriptome analysis using a genome-wide microarray and non-targeted metabolome 16
analysis. Advantages of this study comparing with previous studies are comprehensiveness 17
of ome analyses and data integration of two ome analyses on a metabolic pathway map 18
using the updated KaPPa-View 4 KEGG system. In this study, to apply the transcriptome 19
data of the whole grape genes, we added V1 CDS to the KEGG database and then updated 20
the KaPPa-View 4 KEGG system. By these updates, the KEGG database and KaPPa-View 21
4 KEGG system become more powerful tools for grape research. 22
23
Omics-based studies in grape 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
14
Before the release of the grape genome sequence data by Jaillon et al. (2007) and 1
Velasco et al. (2007), a few omics-based studies had been reported in grape, for example, 2
the transcriptome analyses of pathogen resistance in leaves (Fung et al. 2008, Polesani et 3
al. 2010) and the transcriptome and proteome analyses focused on grape berry 4
development (Deluc et al. 2007, Negri et al. 2008, Lücker et al. 2009). Particularly, the 5
accumulation of secondary metabolites is an active topic in omics-based research in grape 6
(Ali et al. 2011). Although a certain number of publications have reported results of 7
transcriptome analyses in grape, most used EST-based microarrays and their data did not 8
cover the whole grape genes. After the update of the grape genome data by Gimplet et al. 9
(2012), transcriptome analysis using a microarray covering the whole grape genes became 10
popular (Pastore et al., 2011, Fasoli et al., 2012, Gambino et al., 2012, Young et al., 2012, 11
Dal Santo et al., 2013, Pastore et al., 2013, Dai et al., 2014, Carbonell-Bejerano et al., 12
2014a,b, Rienth et al., 2014a,b). Recently, RNA sequencing has also become popular in the 13
transcriptome analysis of grape (Li et al., 2014, Xu et al., 2014, Chitwood et al., 2014, 14
Venturini et al., 2013, Sweetman et al., 2012, Perazzolli et al., 2012., Fasoli et al., 2012, 15
Zenoni et al., 2010, Vitulo et al., 2014). 16
On the other hand, metabolome analyses of various plants have also been reported 17
(Saito and Matsuda 2010). However, only a few studies reporting grape metabolome 18
detected more than 1,000 metabolite peaks (Zamboni et al., 2010,Sternad et al., 2013, 19
Marti et al. 2014). Other studies detected far less number of metabolite peaks and 20
discussed only a small number of metabolites (Dai et al., 2013). 21
With the availability of mass spectrometry and microarray covering the whole grape 22
genes, it has become possible to perform comprehensive multi-omics studies. In this 23
context, there have been a few studies involving integrated multi-omics analyses in grape 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
15
(Zamboni et al., 2010, Agudelo-Romero et al., 2013). We analyzed the expression of 29,549 1
genes and detected 2,012 metabolite peaks corresponding to metabolite. In addition, we 2
combined these data using a metabolic pathway map. Thus, this study is one of the most 3
comprehensive multi-omics studies in grape research. Identification of genes responsible for 4
key enzymes and the compounds produced by them from the detailed pathway map is the 5
first step toward better understanding of the metabolic changes in grape berry skin during 6
the accumulation of polyphenolic components. 7
8
Omics studies in grape berry skin 9
It should be noted that in this study we focused on grape berry skin. Grape berry skin 10
accumulates various secondary metabolites at higher concentrations than other tissues 11
such as leaves and berry flesh (Kader 2002, Steyn 2009). These secondary metabolites 12
determine the important traits of grape berries. Some researchers have used omics 13
approaches to study secondary metabolite accumulation in grape berry skin, for example, 14
changes in the transcriptome before and after ripening (Waters et al. 2005), tissue-specific 15
transcriptome profiles (Grimplet et al. 2007), transcriptome changes after ABA treatment at 16
the veraison stage (Koyama et al. 2010), and comparison of transcriptomes in berry flesh 17
with that in berry skin (Ali et al. 2011). Recently, transcriptome analyses of grape berry with 18
or without the usage of solar UV was reported by Carbonell-Bejerano et al. (2014a). They 19
observed both flavonol and stilbenoid accumulation and induction of their synthetic genes, 20
i.e., phenylpropanoid and stilbenoid synthetic genes with solar UV condition. Although we 21
irradiated grape berries with UV-C light in the present study, interestingly, we observed only 22
specific stilbenoid accumulation and its biosynthetic gene induction and no flavonol 23
accumulation and its synthetic gene induction (Fig. 3 and Table 2). 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
16
1
Transcription factors associated with secondary metabolite accumulation 2
Carbonell-Bejerano et al. (2014a) reported that some transcription factors (TFs) such 3
as MYB and bHLH were up-regulated by solar UV radiation in grape berry skin. In contrast, 4
these TFs were not induced in our experiment (Dataset S1). Pontin et al. (2010) reported 5
that some TFs, including ethylene response factor (ERF), WRKY, and NAC, were 6
up-regulated by UV-B irradiation in grape leaves. Interestingly, ERFs (VIT_07s0005g03220, 7
VIT_15s0021g01630, VIT_05s0049g00490, VIT_07s0005g03260, VIT_07s0005g03230, 8
VIT_17s0000g00200), WRKYs (VIT_04s0069g00970, VIT_14s0068g01770, 9
VIT_04s0008g05750), and NACs (VIT_06s0004g00020) were up-regulated in our 10
experiment (Dataset S2). Carbonell-Bejerano et al. (2014a) applied natural solar UV to 11
berries, whereas in our study and the study by Pontin et al. (2010) applied artificial UV-B or 12
UV-C light. Difference in quality and quantity of UV light may cause differences in TF 13
expressions, and the strong artificial UV in our study and the study by Pontin et al. (2010) 14
may act as stress and also induce stress-related TFs. 15
Höll et al. (2013) identified the R2R3-MYB transcription factors, i.e., MYB14 and MYB15, 16
as TFs implicated in the regulation of stilbene biosynthesis, given that they are 17
co-expressed with STS genes. However, the expression of neither MYB14 18
(VIT_07s0005g03340) nor MYB15 (VIT_05s0049g01020) was induced by UV irradiation in 19
other studies (Pontin et al. 2010, Carbonell-Bejerano et al., 2014a) as well as in our study 20
(Dataset S1). This difference may be due to the use of different cultivars, tissues, and 21
experimental conditions, which suggests that various TFs are responsible for regulation of 22
stilbene biosynthesis by different stimuli. 23
24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
17
Different effects of UV-B and UV-C on accumulation of phenolic compounds 1
Plants accumulate phenolic compounds to prevent UV damage (Caldwell et al. 1983). 2
However, in our experiment, the levels of none of the studied phenolic compounds, including 3
anthocyanin and flavonol, increased after UV-C irradiation (Table 3), although strong 4
resveratrol accumulation was observed (Fig. 4). 5
Different accumulation patterns of phenolic compounds in grapes in response to UV-B 6
and UV-C have been observed. Anthocyanin and flavanol contents in grape berry skin were 7
at the same level during the conditions of the presence and absence of solar UV, although 8
flavonol content was higher during the presence of solar UV (Carbonell-Bejerano et al., 9
2014a). Martínez-Lüscher et al. (2014) showed that UV-B irradiation increased flavonol 10
accumulation in grape berry skin, but did not modify anthocyanin content. Berli et al. (2010) 11
reported that anthocyanin accumulation in grape leaves was not induced by UV-B irradiation 12
alone, but by a combination of UV-B irradiation and ABA treatment. These results show that 13
anthocyanin accumulation is not induced by UV-B alone and that other factors, including 14
ABA, are needed for its induction. ABA is known as a trigger for ripening of grape berry and 15
induces anthocyanin accumulation (Coombe and Hale 1972, Koyama et al. 2010). 16
Therefore, ABA must be an important cofactor for UV-B dependent anthocyanin 17
accumulation in grape. 18
Stilbene biosynthesis is induced not only by UV-C but also by pathogen infection, 19
elicitors, and methyl jasmonate (Adrian et al. 2000, Bru et al. 2006, Versari et al. 2001, Aziz 20
et al. 2003, Pezet et al. 2003, Takayanagi et al. 2004, Nishikawa et al. 2011, Belchı´-Navarro 21
et al. 2012). UV-C induced pathogenesis-related (PR) proteins (Colas et al., 2012), 22
antioxidant defense (Xie et al. 2012), DNA repair mechanism (Molinier et al. 2004), 23
programmed cell death (He et al. 2008), and phenolic compound accumulation, including 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
18
stilbenenoid accumulation (Douillet-Breuil et al. 1999, Adrian et al. 2000, Versari et al. 2001, 1
Pezet et al. 2003, Takayanagi et al. 2004, Nishikawa et al. 2011,). As mentioned above, 2
UV-C may act as stress and induce many stress responses including stilbenoid 3
accumulation. 4
5
Our research provided an overview of grape berry skin metabolism and identified a key 6
factor involved in the response to UV-C irradiation. Based on the transcriptional analysis, 7
genes from the whole genome were tagged with GO terms and used for enrichment analysis. 8
As a result, 238 genes were identified as up-regulated genes (Dataset S2) and we found 9
enrichment of “trihydroxystilbene synthase activity” (GO:0050350), which includes STS, a 10
key enzyme of resveratrol synthesis (Table 2). In the 238 up-regulated genes by UV-C 11
irradiation, 7 STS genes were found (Table 2). On the other hand, using EST-based 12
microarray, Pontin et al. (2010) found 4 STS genes as more than 5-fold up-regulated genes 13
in grape leaves by UV-B irradiation. Only one gene, VIT_10s0042g00870 14
(GSVIVP00031875001) was overlapped between two experiments and 6 STS genes were 15
found newly in this study. Because 33 STS genes, including the 6 genes, were not included 16
in the EST-based microarray, although the genome-wide microarray includes all 39 STS 17
genes of grape. 18
In the non-targeted metabolome analysis, the profiles of metabolite peaks were analyzed 19
by PCA to detect fundamental differences between treatments. Surprisingly, in the 2,012 20
detected metabolite peaks, only resveratrol was identified as a compound strongly 21
responsive to UV-C irradiation (Fig. 4). These data clarified that one of the metabolic 22
changes causing resveratrol to accumulate in berry skin was the uniquely up-regulated 23
expression of the genes for STS. In addition, our updated metabolic map showed the 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
19
UV-C-induced metabolic change in polyphenolic compounds visually (Fig. 6). Because we 1
modified the KaPPA-View 4 KEGG system to include V1 grape genome data, this system 2
has become a more powerful tool in grape omics research. The NimbleGen microarray data 3
directory can be uploaded into the KaPPA-View 4 KEGG system to easily project 4
transcriptome data on a metabolic pathway map. 5
6
Conclusion 7
In this study, we focused on the UV-C-inducible accumulation of phenolic compounds in 8
grape berry skin and showed the specific induction of the resveratrol synthetic pathway by a 9
microarray covering the whole grape genes and LC-QTOF-MS analysis. Development of 10
analytical tools, i.e. the KEGG database and the KaPPA-View 4 KEGG system, and 11
integration of the two thorough comprehensive omics data using the analytical tools could 12
demonstrate metabolic change much more clearly than previous studies. This study is an 13
example demonstrating the effectiveness of omics approaches to analysis of specific traits 14
of grape. 15
16
Materials and Methods 17
Plant materials and treatments 18
Berry clusters of grape (Vitis vinifera L. ‘Pinot Noir’) before veraison stage were 19
harvested at the vineyard of the AZUMI Apple Corporation (Nagano, Japan) in August 2011. 20
Berry clusters were irradiated using a UV-C lamp (253.7 nm, GL-15, TOSHIBA, Japan) from 21
a distance of 50 cm (0.25 μW cm-2) for 1 hour. Dark-treated control samples were covered 22
with a box and placed next to the samples undergoing UV-C irradiation. For transcriptome 23
analysis, berry skins were collected immediately after irradiation. For metabolite extraction, 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
20
after irradiation, berry clusters were kept for 23 hours in the dark at room temperature and 1
then the berry skins were harvested (Fig. 1). All samples were frozen immediately in liquid 2
nitrogen and stored at -80° C. Three biological replicates were performed for each treatment. 3
Each biological replicate comprised a pool of berries from 4 clusters collected from 4 4
different plants randomly. 5
6
RNA extraction and microarray analysis 7
Total RNA was extracted from berry skins according to the hot borate method (Wan and 8
Wilkins 1994). The RNA obtained was further purified using an RNeasy Plant Mini Kit 9
(QIAGEN, Germany). RNA quality and quantity were determined using a spectrophotometer 10
and a Bioanalyzer 2100 instrument (Agilent, USA). The NimbleGen microarray 090818 Vitis 11
exp HX12 (Roche NimbleGen, USA), representing 29,549 predicted genes on the basis of 12
the 12X grape V1 gene prediction (http://genomes.cribi.unipd.it/grape/), was used for 13
microarray analysis according to the manufacturer's specifications. Microarray data were 14
processed using the Subio platform (Subio, Japan). The signal intensities of the four probes 15
for each gene were averaged. Averaged raw data were then analyzed using the Subio 16
platform. After removal of noise signals with intensity less than 3,000, the triplicate data 17
were standardized by a global normalization method with log formation and centering. When 18
the fold-change (UV-C vs. dark) was greater than 5.0 or smaller than 0.2, the gene was 19
considered significantly up-regulated or down-regulated, respectively, by UV-C. All 20
microarray expression data are available in the Gene Expression Omnibus under the series 21
entry GSE59436 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE59436). 22
23
Gene annotation and Gene Ontology (GO) analyses 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
21
The best-hit homologue of each grape gene (V1 coding sequences) in CRIBI 1
(http://genomes.cribi.unipd.it/grape/) was found by searching the Arabidopsis CDS (Tair10, 2
http://arabidopsis.org) or whole organism amino acid sequences in UniprotKB 3
(http://www.uniprot.org) using the basic local alignment search tool (BLAST) within NCBI 4
(http://www.ncbi.nlm.nih.gov). VitisNet ID (VIVID) and the Network name of VitisNet 5
(http://www.sdstate.edu/ps/research/vitis/pathways.cfm) were also searched. GO 6
annotations, classification and enrichment analysis were performed using Blast2GO 7
software (http://www.blast2go.org) based on sequence similarity. For annotation, the default 8
configuration settings were used (blastx against NCBI non-redundant protein database, 9
E-value filter 1.0E-3, 20 BLAST hits per sequence to sequence description tool and 10
annotation cutoff of 55). GO enrichment analysis was performed directly using the default 11
settings. The GOslim ‘‘goslim_plant.obo’’ annotation was added to the dataset to obtain 12
plant-related GO terms. 13
14
Metabolome analysis 15
Fresh samples were extracted with 5 l 80% MeOH containing 2.5 mM lidocaine and 16
10-camphor sulfonic acid per mg fresh weight using a mixer mill with zirconia beads for 7 17
min at 18 Hz and 4C. After centrifugation for 10 min, the supernatant was filtered using an 18
HLB Elution plate (Waters). The extracts (1 l) were analyzed using LC-QTOF-MS (LC, 19
Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). Analytical conditions were: LC 20
column, Acquity bridged ethyl hybrid (BEH) C18 (1.7 m, 2.1 mm 100 mm, Waters); 21
solvent system, solvent A ( 0.1% formic acid in water) and solvent B (acetonitrile including 22
0.1% formic acid); gradient program, 99.5%A/0.5%B at 0 min, 99.5%A/0.5%B at 0.1 min, 23
20%A/80%B at 10 min, 0.5%A/99.5%B at 10.1 min, 0.5%A/99.5%B at 12.0 min, 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
22
99.5%A/0.5%B at 12.1 min and 99.5%A/0.5%B at 15.0 min; flow rate, 0.3 ml/min at 0 min, 1
0.3 ml/min at 10 min, 0.4 ml/min at 10.1 min, 0.4 min/min at 14.4 min and 0.3 ml/min at 14.5 2
min; column temperature, 40C; MS capillary voltage, +3.0 keV, cone voltage, 25.0 V, 3
source temperature, 120C, desolvation temperature, 450C, cone gas flow, 50 l/h; 4
desolvation gas flow, 800 l/h; collision energy, 6 V; mass range, m/z 100‒1500; scan 5
duration, 0.1 s; interscan delay, 0.014 s; data acquisition, centroid mode; polarity, 6
positive/negative; Lockspray (leucine enkephalin) scan duration, 1.0 s; interscan delay, 0.1 s. 7
The data matrix was aligned using MassLynx ver. 4.1 software (Waters). After alignment, 8
de-isotoping, and cutoff of the low-intensity peaks (<500 counts), the intensity values of the 9
remaining peaks were divided by those of 10-camphor sulfonic acid ([M - H]-, m/z 10
231.06910) for normalization. The processed data were used for PCA using the SIMCA-P 11
11.5 program (Umetrics, Sweden). 12
13
Chemicals 14
Standard compounds were purchased from Wako Pure Chemical Industries 15
(http://www.wako-chem.co.jp/), Sigma-Aldrich (http://www.sigmaaldrich.com/), ChromaDex 16
(https://chromadex.com/default.aspx), Cayman Chemical Company 17
(https://www.caymanchem.com/app/template/Home.vm), Enzo Life Sciences 18
(http://www.enzolifesciences.com/), Polyphenols Laboratories AS 19
(http://www.polyphenols.no/), EXTRASYNTHESE S.A. (http://www.extrasynthese.com/), 20
Nacalai Tesque (http://www.nacalai.co.jp/), and Tokyo Chemical Industry 21
(http://www.tcichemicals.com/ja/jp/). A list is shown in Table S1. 22
23
Fluorescence microscopy 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
23
UV-C-treated and control samples were prepared into 100 μm thin sections by a 1
vibrating-type microtome (VT1000 S, Leica, Germany). Sections were placed on a glass 2
slide, covered by a cover slip and observed immediately. The sections were observed under 3
a BZ-9000 fluorescence microscope (KEYENCE, Japan) using a Hg-vapor lamp and filter 4
setting for 4',6-diamidino-2-phenylindole (OP-66834 BZ filter, KEYENCE). 5
6
Gene expression and metabolite profiling 7
The pathway map from phenylalanine to phenolic compounds, such as resveratrol, 8
flavonol, proanthocyanidin and anthocyanin, was originally based on the KEGG pathway 9
database (http://www.genome.jp/kegg) (Kanehisa 2000). The KaPPA View 4 KEGG system 10
(Sakurai et al. 2011) was used for representation of transcriptome and metabolome data on 11
pathway maps. The experimental values were input on a log scale for the gene intensity and 12
on a linear scale for the metabolite intensity in negative ion mode. 13
14
Acknowledgments 15
We thank Mr. Hiroya Saito and Mr. Chiharu Uchikata at AZUMI Apple Corporation for 16
supplying grape berries. We thank Mr. Yutaka Nishikawa and Mr. Kenji Wada of Mie 17
Prefecture Agricultural Research Institute and Dr. Takafumi Tezuka of Nagoya University for 18
helpful advice on UV-C treatment of grape berries. We thank Dr. Mikio Nakazono of Nagoya 19
University for help with preparing grape skin sections. We also thank Mr. Tetsuya Mori at 20
RIKEN CSRS for technical assistance and Dr. Stefan Reuscher for proofreading of the 21
manuscript. 22
This work was technically supported by the Programme for Japan Advanced Plant 23
Science Network and financially supported by the Programme for Promotion of Basic and 24
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
24
Applied Researches for Innovations in Bio-oriented Industry from the Bio-oriented 1
Technology Research Advancement Institution (BRAIN) and by Grants-in-Aid for Scientific 2
Research from The Japan Society for the Promotion of Science (JSPS). 3
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
25
Literature cited 1
Adrian M, Jeandet P, Douillet-Breuil AC, Tesson L, Bessis R (2000) Stilbene content of 2
mature Vitis vinifera berries in response to UV-C elicitation. J Agric Food Chem 48: 3
6103–6105 4
Agudelo-Romero P, Erban A, Sousa L, Pais MS, Kopka J, Fortes AM (2013) Search for 5
transcriptional and metabolic markers of grape pre-ripening and ripening and insights 6
into specific aroma development in three Portuguese cultivars. PLoS One 8: e60422 7
Ali MB, Howard S, Chen S, Wang Y, Yu O, Kovacs LG, Qiu W (2011) Berry skin 8
development in Norton grape: distinct patterns of transcriptional regulation and 9
flavonoid biosynthesis. BMC Plant Biol 11: 7 10
Aziz A, Poinssot B, Daire X, Adrian M, Bézier A, Lambert B, Joubert JM, Pugin A 11
(2003) Laminarin elicits defense responses in grapevine and induces protection 12
against Botrytis cinerea and Plasmopara viticola. Mol Plant Microbe Interact 16: 1118–13
1128 14
Belchí-Navarro S, Almagro L, Lijavetzky D, Bru R, Pedreño MA (2012) Enhanced 15
extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells 16
by using cyclodextrins and methyljasmonate. Plant Cell Rep 31: 81–89 17
Berli FJ, Moreno D, Piccoli P, Hespanhol-Viana L, Silva MF, Bressan-Smith R, 18
Cavagnaro JB, Bottini R (2010) Abscisic acid is involved in the response of grape 19
(Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing 20
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
26
ultraviolet-absorbing compounds, antioxidant enzymes and membrane sterols. Plant 1
Cell Environ 33: 1–10 2
Bogs J, Downey MO, Harvey JS, Ashton AR, Tanner GJ, Robinson SP (2005) 3
Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin 4
reductase and anthocyanidin reductase in developing grape berries and grapevine 5
leaves. Plant Physiol 139: 652–663 6
Bru R, Sellés S, Casado-Vela J, Belchí-Navarro S, Pedreño MA (2006) Modified 7
cyclodextrins are chemically defined glucan inducers of defense responses in 8
grapevine cell cultures. J Agric Food Chem 54: 65–71 9
Caldwell MM, Robberecht R, Flint SD (1983) Internal filters: Prospects for UV-acclimation 10
in higher plants. Physiol Plant 58: 445–450. 11
Carbonell-Bejerano P, Diago MP, Martínez-Abaigar J, Martínez-Zapater JM, Tardáguila 12
J, Núñez-Olivera E. (2014a) Solar ultraviolet radiation is necessary to enhance 13
grapevine fruit ripening transcriptional and phenolic responses. BMC Plant Biol 14: 183 14
Carbonell-Bejerano P, Rodríguez V, Royo C, Hernáiz S, Moro-González LC, 15
Torres-Viñals M, Martínez-Zapater JM (2014b) Circadian oscillatory transcriptional 16
programs in grapevine ripening fruits. BMC Plant Biol 14: 78. 17
Chassot C, Nawrath C, Métraux JP (2007) Cuticular defects lead to full immunity to a 18
major plant pathogen. Plant J 49: 972–980 19
Chitwood DH, Ranjan A, Martinez CC, Headland LR, Thiem T, Kumar R, Covington MF, 20
Hatcher T, Naylor DT, Zimmerman S, et al (2014) A modern ampelography: a 21
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
27
genetic basis for leaf shape and venation patterning in grape. Plant Physiol 164: 259–1
272 2
Colas S, Afoufa-Bastien D, Jacquens L, Clément C, Baillieul F, Mazeyrat-Gourbeyre F, 3
Monti-Dedieu L (2012) Expression and in situ localization of two major PR proteins of 4
grapevine berries during development and after UV-C exposition. PLoS one 7: e43681 5
Conesa A, Götz S (2008) Blast2GO: A comprehensive suite for functional analysis in plant 6
genomics. Int J Plant Genomics 2008: 619832 7
Coombe BG, Hale CR (1973) The hormone content of ripening grape berries and the 8
effects of growth substance treatments. Plant Physiol 51: 629–634 9
Dai ZW, Léon C, Feil R, Lunn JE, Delrot S, Gomès E (2013) Metabolic profiling reveals 10
coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera 11
L.), a non-climacteric fleshy fruit. J Exp Bot 64: 1345–1355 12
Dai ZW, Meddar M, Renaud C, Merlin I, Hilbert G, Delrot S, Gomès E (2014) Long-term 13
in vitro culture of grape berries and its application to assess the effects of sugar supply 14
on anthocyanin accumulation. J Exp Bot 65: 4665–4677 15
Dal Santo S, Tornielli GB, Zenoni S, Fasoli M, Farina L, Anesi A, Guzzo F, Delledonne 16
M, Pezzotti M (2013) The plasticity of the grapevine berry transcriptome. Genome Biol 17
14: r54 18
De Nisco M, Manfra M, Bolognese A, Sofo A, Scopa A, Tenore GC, Pagano F, Milite C, 19
Russo MT (2013) Nutraceutical properties and polyphenolic profile of berry skin and 20
wine of Vitis vinifera L (cv Aglianico). Food Chem 140: 623–629 21
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
28
Delaunois B, Cordelier S, Conreux A, Clément C, Jeandet P (2009) Molecular 1
engineering of resveratrol in plants. Plant Biotechnol J 7: 2–12 2
Deluc LG, Grimplet J, Wheatley MD, Tillett RL, Quilici DR, Osborne C, Schooley DA, 3
Schlauch KA, Cushman JC, Cramer GR (2007) Transcriptomic and metabolite 4
analyses of Cabernet Sauvignon grape berry development. BMC Genomics 8: 429 5
Douillet-Breuil AC, Jeandet P, Adrian M, Bessis R (1999) Changes in the phytoalexin 6
content of various Vitis spp. in response to ultraviolet C elicitation. J Agric Food Chem 7
47: 4456–4461 8
Eudes A, Bozzo GG, Waller JC, Naponelli V, Lim EK, Bowles DJ, Gregory JF 3rd, 9
Hanson AD (2008) Metabolism of the folate precursor p-aminobenzoate in plants: 10
glucose ester formation and vacuolar storage. J Biol Chem 283: 15451–15459 11
Fasoli M, Dal Santo S, Zenoni S, Tornielli GB, Farina L, Zamboni A, Porceddu A, 12
Venturini L, Bicego M, Murino V, et al (2012) The grapevine expression atlas reveals 13
a deep transcriptome shift driving the entire plant into a maturation program. Plant Cell 14
24: 3489-3505 15
Fujita A, Soma N, Goto-Yamamoto N, Mizuno A, Kiso K, Hashizume K (2007) Effect of 16
shading on proanthocyanidin biosynthesis in the grape berry. J Japan Soc Hortic Sci 17
76: 112–119 18
Fung RW, Gonzalo M, Fekete C, Kovacs LG, He Y, Marsh E, McIntyre LM, Schachtman 19
DP, Qiu W (2008) Powdery mildew induces defense-oriented reprogramming of the 20
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
29
transcriptome in a susceptible but not in a resistant grapevine. Plant Physiol 146: 236–1
249 2
Gambino G, Cuozzo D, Fasoli M, Pagliarani C, Vitali M, Boccacci P, Pezzotti M, 3
Mannini F (2012) Co-evolution between Grapevine rupestris stem pitting-associated 4
virus and Vitis vinifera L. leads to decreased defence responses and increased 5
transcription of genes related to photosynthesis. J Exp Bot 63: 5919-5933 6
Grimplet J, Cramer GR, Dickerson JA, Mathiason K, Van Hemert J, Fennell AY (2009) 7
VitisNet: “Omics” integration through grapevine molecular networks. PLoS One 4: 8
e8365 9
Grimplet J, Deluc LG, Tillett RL, Wheatley MD, Schlauch KA, Cramer GR, Cushman JC 10
(2007) Tissue-specific mRNA expression profiling in grape berry tissues. BMC 11
Genomics 8: 187 12
Grimplet J, Van Hemert J, Carbonell-Bejerano P, Díaz-Riquelme J, Dickerson J, 13
Fennell A, Pezzotti M, Martínez-Zapater JM (2012) Comparative analysis of 14
grapevine whole-genome gene predictions, functional annotation, categorization and 15
integration of the predicted gene sequences. BMC Res Notes 5: 213 16
He R, Drury GE, Rotari VI, Gordon A, Willer M, Farzaneh T, Woltering EJ, Gallois P 17
(2008) Metacaspase-8 modulates programmed cell death induced by ultraviolet light 18
and H2O2 in Arabidopsis. J Biol Chem 283: 774–783 19
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
30
Huang J, Gu M, Lai Z, Fan B, Shi K, Zhou YH, Yu JQ, Chen Z (2010) Functional analysis 1
of the Arabidopsis PAL gene family in plant growth, development, and response to 2
environmental stress Plant Physiol 153: 1526–1538 3
Höll J, Vannozzi A, Czemmel S, D'Onofrio C, Walker AR, Rausch T, Lucchin M, Boss 4
PK, Dry IB, Bogs J (2013) The R2R3-MYB transcription factors MYB14 and MYB15 5
regulate stilbene biosynthesis in Vitis vinifera. Plant Cell 25: 4135–4149 6
Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, 7
Vitulo N, Jubin C, et al (2007) The grapevine genome sequence suggests ancestral 8
hexaploidization in major angiosperm phyla. Nature 449: 463–467 9
Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, Fong HH, 10
Farnsworth NR, Kinghorn AD, Mehta RG, et al (1997) Cancer chemopreventive 11
activity of resveratrol, a natural product derived from grapes. Science 275: 218–220 12
Kader AA (2002) Fruits in the global market. In M Knee, eds, Fruit quality and its biological 13
basis, Sheffield Academic Press, Sheffield, pp 1-16 14
Kanehisa M (2000) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 15
28: 27–30 16
Koyama K, Sadamatsu K, Goto-Yamamoto N (2010) Abscisic acid stimulated ripening 17
and gene expression in berry skins of the Cabernet Sauvignon grape. Funct Integr 18
Genomics 10: 367–381 19
Langcake P, Pryce RJ (1977) A new class of phytoalexins from grapevines. Experientia 20
33:151–152 21
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
31
Li Q, He F, Zhu BQ, Liu B, Sun RZ, Duan CQ, Reeves MJ, Wang J (2014) Comparison of 1
distinct transcriptional expression patterns of flavonoid biosynthesis in Cabernet 2
Sauvignon grapes from east and west China. Plant Physiol Biochem 84: 45–56 3
Lijavetzky D, Almagro L, Belchi-Navarro S, Martínez-Zapater JM, Bru R, Pedreño MA 4
(2008) Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis 5
pathway gene expression and resveratrol production in Monastrell grapevine cell 6
cultures. BMC Res Notes 1: 132 7
Lijavetzky D, Carbonell-Bejerano P, Grimplet J, Bravo G, Flores P, Fenoll J, Hellín P, 8
Oliveros JC, Martínez-Zapater JM (2012) Berry flesh and skin ripening features in 9
Vitis vinifera as assessed by transcriptional profiling. PLoS One 7: e39547 10
Louvet R, Cavel E, Gutierrez L, Guénin S, Roger D, Gillet F, Guerineau F, Pelloux J 11
(2006) Comprehensive expression profiling of the pectin methylesterase gene family 12
during silique development in Arabidopsis thaliana. Planta 224: 782–791 13
Lücker J, Laszczak M, Smith D, Lund ST (2009) Generation of a predicted protein 14
database from EST data and application to iTRAQ analyses in grape (Vitis vinifera cv 15
Cabernet Sauvignon) berries at ripening initiation. BMC Genomics 10: 50 16
Marambaud P, Zhao H, Davies P (2005) Resveratrol promotes clearance of Alzheimer’s 17
disease amyloid-beta peptides. J Biol Chem 280: 37377–37382 18
Marti G, Schnee S, Andrey Y, Simoes-Pires C, Carrupt PA, Wolfender JL, Gindro K 19
(2014) Study of leaf metabolome modifications induced by UV-C radiations in 20
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
32
representative Vitis, Cissus and Cannabis species by LC-MS based metabolomics and 1
antioxidant assays. Molecules 19: 14004–14021 2
Martínez-Lüscher J, Torres N, Hilbert G, Richard T, Sánchez-Díaz M, Delrot S, 3
Aguirreolea J, Pascual I, Gomès E (2014) Ultraviolet-B radiation modifies the 4
quantitative and qualitative profile of flavonoids and amino acids in grape berries. 5
Phytochemistry 102: 106–114 6
Molinier J, Ramos C, Fritsch O, Hohn B (2004) CENTRIN2 modulates homologous 7
recombination and nucleotide excision repair in Arabidopsis. Plant Cell 16: 1633–1643 8
Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M (2007) KAAS: an automatic 9
genome annotation and pathway reconstruction server. Nucleic Acids Res 35: 10
W182-W185 11
Negri AS, Prinsi B, Rossoni M, Failla O, Scienza A, Cocucci M, Espen L (2008) 12
Proteome changes in the skin of the grape cultivar Barbera among different stages of 13
ripening. BMC Genomics 9: 378 14
Nishikawa Y. Tominori S, Wada K, Kondo H (2011) Effect of cultivation practices on 15
resveratrol content in grape berry skins. Hortic Res (in Japanese) 10: 249-253 16
Pastore C, Zenoni S, Fasoli M, Pezzotti M, Tornielli GB, Filippetti I (2013) Selective 17
defoliation affects plant growth, fruit transcriptional ripening program and flavonoid 18
metabolism in grapevine. BMC Plant Biol 13: 30 19
Pastore C, Zenoni S, Tornielli GB, Allegro G, Dal Santo S, Valentini G, Intrieri C, 20
Pezzotti M, Filippetti I (2011) Increasing the source/sink ratio in Vitis vinifera (cv 21
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
33
Sangiovese) induces extensive transcriptome reprogramming and modifies berry 1
ripening. BMC Genomics 12: 631 2
Perazzolli M, Moretto M, Fontana P, Ferrarini A, Velasco R, Moser C, Delledonne M, 3
Pertot I (2012) Downy mildew resistance induced by Trichoderma harzianum T39 in 4
susceptible grapevines partially mimics transcriptional changes of resistant genotypes. 5
BMC Genomics 13: 660 6
Pezet R, Perret C, Jean-Denis JB, Tabacchi R, Gindro K, Viret O (2003) Delta-viniferin, a 7
resveratrol dehydrodimer: one of the major stilbenes synthesized by stressed 8
grapevine leaves. J Agric Food Chem 51: 5488–5492 9
Polesani M, Bortesi L, Ferrarini A, Zamboni A, Fasoli M, Zadra C, Lovato A, Pezzotti M, 10
Delledonne M, Polverari A (2010) General and species-specific transcriptional 11
responses to downy mildew infection in a susceptible (Vitis vinifera) and a resistant (V. 12
riparia) grapevine species. BMC Genomics 11: 117 13
Pontin MA, Piccoli PN, Francisco R, Bottini R, Martinez-Zapater JM, Lijavetzky D 14
(2010) Transcriptome changes in grapevine (Vitis vinifera L) cv Malbec leaves induced 15
by ultraviolet-B radiation. BMC Plant Biol 10: 224 16
Quackenbush J (2001) The TIGR gene indices: analysis of gene transcript sequences in 17
highly sampled eukaryotic species. Nucleic Acids Res 29: 159–164 18
Renaud S, De Lorgeril M (1992) Wine, alcohol, platelets, and the French paradox for 19
coronary heart disease. Lancet 339:1523–1526 20
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
34
Rienth M, Torregrosa L, Kelly MT, Luchaire N, Pellegrino A, Grimplet J, Romieu C 1
(2014a) Is transcriptomic regulation of berry development more important at night than 2
during the day? PLoS one 9: e88844 3
Rienth M, Torregrosa L, Luchaire N, Chatbanyong R, Lecourieux D, Kelly MT, 4
Romieu, C (2014b) Day and night heat stress trigger different transcriptomic 5
responses in green and ripening grapevine (Vitis vinifera) fruit. BMC Plant Biol 14: 108 6
Saito K, Matsuda F (2010) Metabolomics for functional genomics, systems biology, and 7
biotechnology. Annu Rev Plant Biol 61: 463–489 8
Sakurai N, Ara T, Ogata Y, Sano R, Ohno T, Sugiyama K, Hiruta A, Yamazaki K, Yano K, 9
Aoki K, et al (2011) KaPPA-View4: a metabolic pathway database for representation 10
and analysis of correlation networks of gene co-expression and metabolite 11
co-accumulation and omics data. Nucleic Acids Res 39: D677–D684 12
Sternad Lemut M, Sivilotti P, Franceschi P, Wehrens R, Vrhovsek U (2013) Use of 13
metabolic profiling to study grape skin polyphenol behavior as a result of canopy 14
microclimate manipulation in a “Pinot noir” vineyard. J Agric Food Chem 61: 8976–15
8986 16
Steyn WJ (2009) Prevalence and functions of anthocyanins in Fruits. In C Winefield, K 17
Davies, K Gould, eds, Anthocyanins, Springer, New York, pp 86-105 18
Sweetman C, Wong DC, Ford CM, Drew DP (2012) Transcriptome analysis at four 19
developmental stages of grape berry (Vitis vinifera cv. Shiraz) provides insights into 20
regulated and coordinated gene expression. BMC Genomics 13: 691 21
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
35
Takayanagi T, Okuda T, Mine Y, Yokotsuka K (2004) Induction of resveratrol biosynthesis 1
in skins of three grape cultivars by ultraviolet irradiation. J Japan Soc Hortic Sci 73: 2
193-199 3
Turlapati PV, Kim KW, Davin LB, Lewis NG (2011) The laccase multigene family in 4
Arabidopsis thaliana: towards addressing the mystery of their gene function(s). Planta 5
233: 439–470 6
Velasco R, Zharkikh A, Troggio M, Cartwright DA, Cestaro A, Pruss D, Pindo M, 7
Fitzgerald LM, Vezzulli S, Reid J, et al (2007) A high quality draft consensus 8
sequence of the genome of a heterozygous grapevine variety. PLoS One 2: e1326 9
Venturini L, Ferrarini A, Zenoni S, Tornielli GB, Fasoli M, Dal Santo S, Minio A, Buson 10
G, Tononi P, Zago ED, et al (2013) De novo transcriptome characterization of Vitis 11
vinifera cv. Corvina unveils varietal diversity. BMC Genomics 14: 41 12
Versari A, Parpinello GP, Tornielli GB, Ferrarini R, Giulivo C (2001) Stilbene compounds 13
and stilbene synthase expression during ripening, wilting, and UV treatment in grape 14
cv. Corvina. J Agric Food Chem 49: 5531-5536 15
Vitulo N, Forcato C, Carpinelli EC, Telatin A, Campagna D, D'Angelo M, Zimbello R, 16
Corso M, Vannozzi A, Bonghi C, et al (2014) A deep survey of alternative splicing in 17
grape reveals changes in the splicing machinery related to tissue, stress condition and 18
genotype. BMC Plant Biol 14: 99 19
Wan CY, Wilkins TA (1994) A modified hot borate method significantly enhances the yield 20
of high-quality RNA from cotton (Gossypium hirsutum L). Anal Biochem 223: 7–12 21
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
36
Wan D, Li R, Zou B, Zhang X, Cong J, Wang R, Xia Y, Li G (2012) Calmodulin-binding 1
protein CBP60g is a positive regulator of both disease resistance and drought 2
tolerance in Arabidopsis. Plant Cell Rep 31: 1269–1281 3
Wang L, Tsuda K, Truman W, Sato M, Nguyen le V, Katagiri F, Glazebrook J (2011) 4
CBP60g and SARD1 play partially redundant critical roles in salicylic acid signaling. 5
Plant J 67: 1029–1041 6
Waters DLE, Holton TA, Ablett EM, Lee LS, Henry RJ (2005) cDNA microarray analysis 7
of developing grape (Vitis vinifera cv Shiraz) berry skin. Funct Integr Genomics 5: 40–8
58 9
Xie Y, Xu D, Cui W, Shen W (2012) Mutation of Arabidopsis HY1 causes UV-C 10
hypersensitivity by impairing carotenoid and flavonoid biosynthesis and the 11
down-regulation of antioxidant defence. J Exp Bot 63, 3869–3883 12
Xu W, Li R, Zhang N, Ma F, Jiao Y, Wang, Z (2014) Transcriptome profiling of Vitis 13
amurensis, an extremely cold-tolerant Chinese wild Vitis species, reveals candidate 14
genes and events that potentially connected to cold stress. Plant Mol Biol 86: 527–541 15
Young PR, Lashbrooke JG, Alexandersson E, Jacobson D, Moser C, Velasco R, Vivier 16
MA (2012) The genes and enzymes of the carotenoid metabolic pathway in Vitis 17
vinifera L. BMC Genomics 13: 243 18
Zamboni A, Di Carli M, Guzzo F, Stocchero M, Zenoni S, Ferrarini A, Tononi P, Toffali 19
K, Desiderio A, Lilley KS, et al (2010) Identification of putative stage-specific 20
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
37
grapevine berry biomarkers and omics data integration into networks. Plant Physiol 1
154: 1439–1459 2
Zenoni S, Ferrarini A, Giacomelli E, Xumerle L, Fasoli M, Malerba G, Bellin D, Pezzotti 3
M, Delledonne M (2010) Characterization of transcriptional complexity during berry 4
development in Vitis vinifera using RNA-Seq. Plant Physiol 152: 1787–1795 5
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
38
Figure legends 1
2 Figure 1. 3
Overview of experimental design in this study. 4
5
Figure 2. 6
Distribution of grape genes based on GO slim classification. A, Functional classifications 7
for all microarray genes (n=29,549 genes). B, Functional classifications for up-regulated 8
genes (n=238 genes) by UV-C irradiation. For illustration purposes only, biological 9
process level 3 ontology terms were selected. Values indicate the percentage of each 10
GO category in total. 11
12
Figure 3. 13
Gene Ontology hierarchy and enrichment statistics for the molecular function ontology. 14
Some GO terms within the molecular function ontology were significantly enriched by 15
UV-C irradiation. Different shades of pink indicate activity level according to GO term 16
analysis. Significance was calculated by False Discovery Rate (FDR). 17
18
Figure 4. 19
Principal component analysis of grape berry skin metabolome. A, Score scatter plot of a 20
PCA model on the berry skin metabolome in darkness () and after UV-C treatment () 21
(n=6 for each treatment). B, Loading scatter plot from the PCA analysis of berry skin in 22
darkness and after UV-C treatment (n=2012 metabolite peaks). Each annotation showed 23
the retention time and m/z. 24
25
Figure 5. 26
Accumulation of fluorescent substances at berry skin. The sections of grape berry of 27
UV-C-irradiated (A, B) and control (C, D). A, C: Bright-field images. B, D: Fluorescence 28
images. 29
30
Figure 6. 31
Modulation of secondary metabolism after UV-C irradiation. The schematic 32
representation was determined by transcriptome and metabolome data integration using 33
updated KaPPA-View 4 KEGG. Fold change of each gene expression after log10 34
transformation is divided into 10 levels and depicted using the following color: red, 35
up-regulated by UV-C; blue, down-regulated by UV-C; gray, not detected. Fold change of 36
contents of each metabolite is depicted using the following colors: red, up-regulated by 37
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
39
UV-C; white, not identified. PAL; phenylalanine ammonia-lyase, 4CL; 4-coumarate:CoA 1
ligase, C4H; cinnamate 4-hydroxylase, CHS; chalcone synthase, STS; stilbene synthase, 2
ROMT; resveratrol O-methyltransferase, CHI; chalcone isomerase, F3H; flavanone 3
3-hydroxylase, F3’H; flavonoid 3'-hydroxylase, F3’5’H; flavonoid 3',5'-hydroxylase, FLS; 4
flavonol synthase, DFR; dihydroflavonol-4-reductase, LDOX; leucoanthocyanidin 5
dioxygenase, LAR; leucoanthocyanidin reductase, ANR; anthocyanidin reductase, BZ1; 6
anthocyanidin 3-O-glucosyltransferase, 3MaT1; anthocyanin 7
3-O-glucoside-6''-O-malonyltransferase, GT1; anthocyanidin 5,3-O-glucosyltransferase, 8
UGAT; cyanidin-3-O-glucoside 2''-O-glucuronosyltransferase. 9
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
40
Table
Table 1. List of enrichment GO terms
GO-ID Term Category FDR P-Value
GO:0052689 carboxylic ester hydrolase activity F 1.E-05 2.13E-09
GO:0050350 trihydroxystilbene synthase activity F 7.E-05 2.10E-08
GO:0030599 pectinesterase activity F 1.E-03 5.25E-07
GO:0045330 aspartyl esterase activity F 2.E-03 8.89E-07
GO:0016747 transferase activity, transferring acyl groups other than amino-acyl groups F 3.E-03 2.35E-06
GO:0016758 transferase activity, transferring hexosyl groups F 3.E-03 2.90E-06
GO:0016746 transferase activity, transferring acyl groups F 5.E-03 4.71E-06
GO:0016757 transferase activity, transferring glycosyl groups F 6.E-03 6.62E-06
GO:0042545 cell wall modification P 6.E-03 8.19E-06
GO:0016740 transferase activity F 7.E-03 9.57E-06
GO:0030259 lipid glycosylation P 2.E-02 2.45E-05
GO:0047746 chlorophyllase activity F 2.E-02 2.62E-05
GO:0003824 catalytic activity F 2.E-02 4.38E-05
Thirteen GO terms were picked out at enrichment analysis graph by Blast2GO. Category F; Molecular Function,
P; Biological process.
w
ww
.plantphysiol.orgon M
arch 25, 2018 - Published by
Dow
nloaded from
Copyright ©
2015 Am
erican Society of P
lant Biologists. A
ll rights reserved.
41
Table 2. List of enrichment GO terms and gene annotations for the molecular function
ontology.
GO Gene ID
Fold Change Annotation of Blast2GO (NCBI,
Blastx) (UV/Dark)
GO
:00
305
99
pe
ctin
este
rase
act
ivity
VIT_06s0009g02630 14.9 pectinesterase 2-like
VIT_06s0009g02590 39.5 pectinesterase 2-like
VIT_03s0038g03570 5.6 l-ascorbate oxidase homolog
VIT_02s0154g00600 8.6 probable pectinesterase 68-like
VIT_06s0009g02570 9.8 pectinesterase 2-like
VIT_06s0009g02600 18.6 pectinesterase 2-like
VIT_06s0009g02560 6.0 pectinesterase 2-like
VIT_07s0005g00720 7.5 probable pectinesterase
pectinesterase inhibitor 41-like
GO
:00
477
46
chlo
rop
hylla
se
activ
ity
VIT_07s0151g00210 12.9 chlorophyllase 1
VIT_07s0151g00130 10.2 chlorophyllase 1
VIT_07s0151g00270 10.2 chlorophyllase 2
GO
:00
4533
0 a
spar
tyl e
ster
ase
act
ivity
VIT_06s0009g02630 14.9 pectinesterase 2-like
VIT_06s0009g02590 39.5 pectinesterase 2-like
VIT_02s0154g00600 8.6 probable pectinesterase 68-like
VIT_06s0009g02570 9.8 pectinesterase 2-like
VIT_06s0009g02600 18.6 pectinesterase 2-like
VIT_06s0009g02560 6.0 pectinesterase 2-like
VIT_07s0005g00720 7.5 probable pectinesterase
pectinesterase inhibitor 41-like
GO
:005
035
0
trih
ydro
xyst
ilben
e
syn
tha
se a
ctiv
ity VIT_16s0100g01000 6.5 stilbene synthase 4-like
VIT_16s0100g01140 6.2 stilbene synthase 1
VIT_16s0100g01130 6.0 resveratrol synthase
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
42
VIT_10s0042g00890 5.4 stilbene synthase 1
VIT_10s0042g00870 6.1 stilbene synthase
VIT_16s0100g00770 9.9 stilbene synthase
VIT_16s0100g01190 9.3 resveratrol synthase G
O:0
01
675
8 t
rans
fera
se a
ctiv
ity, t
ran
sfe
rrin
g h
exo
syl g
rou
ps
VIT_05s0062g00310 5.6 udp-glycosyltransferase 75d1-like
VIT_05s0062g00270 8.0 udp-glycosyltransferase 75d1-like
VIT_05s0062g00700 8.0 udp-glycosyltransferase 75d1-like
VIT_05s0062g00660 5.6 udp-glycosyltransferase 75d1-like
VIT_05s0062g00300 7.8 udp-glycosyltransferase 75d1-like
VIT_03s0017g01990 7.6 udp-glucose flavonoid
3-o-glucosyltransferase 6-like
VIT_17s0000g08100 5.7 udp-glycosyltransferase-like protein
VIT_06s0004g07250 6.0 udp-glycosyltransferase 87a1-like
VIT_06s0004g07240 6.1 udp-glycosyltransferase 87a1-like
VIT_12s0034g00030 5.2 udp-glucose flavonoid
3-o-glucosyltransferase 6-like
VIT_04s0023g01120 6.1 cazy family gt8
VIT_18s0041g00740 5.3 udp-glycosyltransferase 88a1-like
VIT_15s0021g02060 17.8 hydroquinone glucosyltransferase
VIT_03s0017g02000 5.8 udp-glucose flavonoid
3-o-glucosyltransferase 6
VIT_01s0011g03850 6.3 protein
VIT_06s0004g01670 8.2 udp-glycosyltransferase 92a1-like
VIT_05s0062g00340 6.2 udp-glycosyltransferase 75d1-like
VIT_17s0000g04760 6.5 udp-glycosyltransferase 89b1-like
VIT_02s0025g01860 5.1 cellulose synthase-like protein g3
Five GO terms were picked out as terms of the lowest level of the hierarchy in
enrichment graph (Fig. 3).
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
43
Table 3. List of metabolites identified by one-point calibration in darkness and after
treatment with UV.
Category KEGG
ID Compound
Intensity UV/Dark Conc.(μg g-1FW)
Dark UV Ratio Dark UV
C00079 L-Phenylalanine 0.152 0.103 0.678 61.8 41.9
Resveratrol
C03582 trans-resveratrol, 3,4',5-Trihydroxystilbene 0.091 32.43 355.176 9.8 3492.3
- cis-resveratrol *0.051 0.088 1.724 - 6.3
C10275 Piceid *0.051 0.171 3.35 - 45.3
C10289 (-)-epsilon-Viniferin, epsilon-Viniferin 0.389 2.273 5.839 79.2 462.3
Anthocyanin
C12138 Delphinidin 3-glucoside (Dp3G),(Mirtillin) *0.051 *0.052 1.008 - -
C12140 Malvidin3-glucoside(Mv3G), (Oenin) *0.051 *0.052 1.008 - -
C12139 Petunidin3-glucoside (Pt3G) *0.051 *0.052 1.008 - -
C08604 Cyanidin3-glucoside(Cy3G) *0.051 *0.052 1.008 - -
C12141 Peonidin 3-O-glucoside *0.051 *0.052 1.008 - -
Proanthocyanidin
C06562 (+)-Catechin 21.313 22.85 1.072 3771.2 4043.3
C09727 (-)-Epicatechin 0.635 0.598 0.942 114.6 108
C12136 L-Epigallocatechin 0.061 0.062 1.005 17.9 18
- (-)-Epicatechin 3-O-gallate *0.051 *0.052 1.008 - -
Flavonol
C00389 Quercetin *0.051 0.073 1.42 - 8.2
C10107 Myricetin *0.051 *0.052 1.008 - -
C05623 quercetin-3-O-glucoside(Hirsutrin) 0.946 1.162 1.228 227.6 279.4
*Low intensities (≦0.052) were detected at background noise levels.
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
44
Short legends for Supplemental Data
Dataset S1. List of transcripts modulated by UV-C trradiation, with detailed annotations.
Dataset S2. List of 238 up-regulated genes by UV-C irradiation with annotations.
Dataset S3. List of 24 down-regulated genes by UV-C irradiation with annotations.
Table S1. Chemical compounds used to identify metabolites.
Figure S1.
Volcano plot of microarray analysis. Shown is the fold-change of each gene plotted
against its p-value. Each data point represents one gene (n=23,096 genes; after removal
of low signal as a noise). Fold-change was calculated as the ratio of UV-C treatment vs.
darkness. Significance was calculated by Student’s t-test corrected for multiple
comparisons. The red lines denote a 5-fold threshold. For each set of conditions, data
from three replicate samples were averaged.
Figure S2. The grape metabolic pathways of KaPPA View 4 KEGG, uploaded with
microarray and metabolome datasets. A: 00940, phenylpropanoid biosynthesis, B: 00945,
stilbenoid, diarylheptanoid and gingerol biosynthesis, C: 00941, flavonoid biosynthesis,
D: 00942, anthocyanin biosynthesis.
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Figure 1
Sample (grape berries)
UV 1 hour
Dark 23 hoursImmediately
Dark 1 hour
Berry skins Berry skins
Microarray analysis
LC-QTOF-MS analysis
mRNA extraction Metabolite extraction
Observation of fluorescence
image
Figure 1. Overview of experimental design in this study.
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Figure 2
Figure 2. Distribution of grape genes based on GO slim classification. A, FunctionalFigure 2. Distribution of grape genes based on GO slim classification. A, Functionalclassifications for all microarray genes (n=29,549 genes). B, Functional classifications forupregulated genes (n=238 genes) by UV-C irradiation. For illustration purposes only,biological process level 3 ontology terms were selected. Values indicate the percentage ofeach GO category in total.
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Figure 3
molecular_function
catalytic activity GO:0003824
FDR: 2.3E-2, p-Value: 4.4E-5
hydrolase activity
GO:0016787
transferase activity GO: 0016740
FDR: 6.7E-3, p-Value: 9.6E-6
molecular_function
GO:0003674
is
isis
GO:0016787 FDR: 6.7E-3, p-Value: 9.6E-6
hydrolase activity, acting on
ester bonds GO:0016788
transferase activity,
transferring acyl groups
GO:0016746
FDR: 4.7E-3, p-Value: 4.7E-6
transferase activity transferring
glycosyl groups GO:0016757
FDR: 5.8E-3, p-Value: 6.6E-6
carboxylic ester aspartyl esterase activity transferase activity, transferring transferase activity,
is is is
is isis is
carboxylic ester
hydrolase activity
GO:0052689
aspartyl esterase activity
GO:0045330
FDR: 1.5E-3, p-Value:
8.9E-7
transferase activity, transferring
acyl groups other than amino-
acyl groups GO:0016747
FDR: 3.3E-3, p-Value: 2.4E-6
transferase activity,
transferring hexosyl groups
GO:0016758
FDR: 3.4E-3, pValue: 2.9E-6
pectinesterase activity
GO:0030599
FDR: 1.2E-3, p-Value:
5.3E-7
chlorophyllase activity
GO:0047746
FDR: 1.5E-2, p-Value: 2.6E-5
trihydroxystilbene synthase activity
GO:0050350
FDR: 7.3E-5, p-Value: 2.1E-8
isis is
Figure 3. Gene Ontology hierarchy and enrichmentstatistics for the molecular function ontology. Some GOterms within the molecular function ontology weresignificantly enriched by UV-C irradiation. Different shadesof pink indicate activity level according to GO term analysis.Significance was calculated by False Discovery Rate (FDR).
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
-12-10-8-6-4-202468
1012
-20 -10 0 10 20
PC1 51.4%
PC
2 16
.3% Dark
UV
Score Scatter PlotA
Figure 4
-0.2
-0.1
-0.0
0.1
0.2
0.0 0.1 0.2 0.3 0.4 0.5 0.6
1.0016_209.06581.0023_1161.2671.0034_351.93131.0037_129.01791.0045_491.11871.005_59.01231.0053_195.04921.0053_227.93521.0062_683.22791.0063_259.02151.0068_732.2091.0069_544.03421.007_141.97171.0077_103.00271.0089_1123.315
1.0091_439.0794
1.0102_87.00791.0117_133.01331.0117_781.1983
1.0125_149.00831.0126_191.0191.0138_377.08551.0145_130.99791.015_341.10811.0186_475.12861.0228_528.04251.027_185.96141.0288_819.1481.0362_320.95131.0384_319.94831.0396_173.00831.0418_323.02841.0432_203.0188
1.0435_111.00781.0435_154.99781.0449_535.03711.0473_606.07421.0477_565.04721.053_683.2221.0538_87.00811.0549_528.04191.0559_149.00831.0569_781.19761.0584_439.07831.0595_341.10851.0613_103.0021
1.0615_96.95931.0617_491.98621.0647_191.01841.0702_304.99061.0856_260.93751.0915_154.9993
1.0925_133.0133
1.093_115.0027
1.1018_341.10721.1019_71.01291.1026_185.96121.1038_141.97151.1107_203.0191.1125_173.00841.1127_103.00211.1127_323.02791.113_565.04821.1131_319.94811.1145_528.04061.115_111.00771.1161_191.0189
1.1169_96.95931.1181_149.00821.1182_320.95481.1183_229.95111.1188_275.95581.124_87.00791.1252_260.93281.1255_89.02371.1544_185.96191.1564_87.00821.1584_320.95231.1758_439.07941.1837_203.01851.1884_149.00811.1885_503.1591.1896_179.05531.1916_341.10851.2095_96.96021.2266_203.01851.2367_133.01291.2371_115.00261.2506_341.1081.2658_323.09821.2754_115.00281.3165_289.11431.3172_253.09291.3175_271.10471.3195_341.10891.3259_179.05551.3293_149.0073
1.33_297.11811.3309_133.01331.3326_145.01381.3343_115.00261.3373_101.0238
1.3398_142.04981.3406_605.19311.3429_337.07761.3442_188.05541.3739_111.00771.3788_605.19281.4001_337.07691.4132_111.00761.4218_341.1084
1.486_155.9511.4863_111.00791.4864_191.01891.4891_257.97981.4919_87.0081.4919_199.94321.4923_129.01941.4928_85.02881.4935_203.01941.4965_435.9591.5292_435.9574
1.531_191.0191.5323_111.00781.5326_199.94291.5338_257.98221.535_87.00781.5358_203.01911.546_173.00831.5483_155.95071.554_129.01851.5562_85.02861.5973_447.96031.6015_111.00781.6018_191.01871.6046_257.98251.6049_435.95751.6056_87.0081.6056_199.9406
1.6094_203.01871.6099_85.02861.6103_170.00391.6136_155.95061.6141_173.00721.6147_129.01831.7417_191.01851.7454_111.00761.747_204.0871.7481_128.03431.7626_133.04841.7998_180.06551.8029_163.03971.8036_763.21451.8107_765.22961.8144_111.00751.8388_241.88381.841_147.0273
1.8414_117.01851.842_73.02841.8433_331.06531.8651_130.0867
1.8755_147.02871.8767_763.21391.8949_331.06651.8968_645.18711.8994_322.11381.9104_647.2034
1.9214_387.09231.9313_130.08661.9566_99.04461.9568_156.06631.958_200.05571.9607_161.04481.9675_331.06721.9731_645.18861.9778_387.09041.9829_129.01821.9983_609.12342.0282_205.07082.0356_779.24782.0589_265.09222.0627_197.80732.0638_881.19112.0665_283.06772.0674_181.05022.0679_151.01362.068_169.01212.0686_331.06612.1193_345.08112.1197_211.06242.1197_331.06642.1197_881.19162.1235_181.052.1242_139.0392.1244_197.80812.1263_121.02792.1272_301.09212.1306_195.80852.1346_153.01822.1809_197.80922.181_359.0612.182_284.12722.182_419.1283
2.1857_383.15442.1857_865.1972.1858_139.03852.1929_301.0918
2.2303_865.1977
2.2377_863.18192.253_395.1552.2563_315.07262.2872_359.11392.2875_411.02332.2924_863.1816
2.2976_865.197
2.3087_331.066
2.3125_425.0864
2.3126_155.0357
2.3126_593.12912.3127_407.0756
2.3147_164.07052.3151_591.11322.3171_479.11892.3175_439.06512.3179_147.04432.3202_289.07052.3221_169.01312.3226_465.09212.3268_641.17112.3282_323.13362.3414_397.17292.3875_331.06642.3933_144.04452.3943_179.05312.3945_457.10052.3966_315.072.3974_397.17082.3975_421.13352.3976_368.09792.4005_353.14442.4028_167.03412.4036_129.01832.404_329.08582.4041_253.88312.4072_85.02922.4078_897.1861
2.4083_452.13292.4089_416.1551
2.4123_443.19122.4172_383.20662.4271_163.03812.4276_345.11752.4284_331.10352.4305_152.01112.4369_457.0989
2.4431_593.12962.4445_423.06352.4455_323.13292.4462_315.07112.4478_161.03922.4485_153.01862.4497_621.14572.4521_165.01822.4556_125.02352.4559_219.0652.456_179.03412.456_303.05012.4567_259.05992.457_261.07562.4572_137.02342.4574_139.03882.4583_611.13912.4595_149.0112
2.4602_305.0656
2.4615_275.05532.4618_235.06032.4622_285.03922.4622_429.93152.463_619.13142.4635_221.04462.4642_279.10762.4645_380.15552.4656_167.03422.4677_218.1012.4679_217.04932.4701_241.04822.4738_319.0452.4776_591.11162.478_193.0135
2.478_593.1291
2.4789_477.12392.4791_183.02762.5058_131.03422.5183_305.06532.5223_315.10472.5246_467.1162.5456_425.09142.5526_279.10792.5583_897.18792.564_305.06542.5641_593.12922.5722_182.02042.5724_359.09652.5731_197.04422.5735_895.17712.5738_675.21462.5741_439.9716
2.5744_153.0547
2.5753_123.0442.5755_351.0841
2.5761_315.1064
2.5792_481.09622.5805_137.02472.5817_479.08372.5818_463.08722.582_239.05522.5839_627.16122.5841_179.03432.5852_233.10152.5858_299.07622.5885_197.80522.6042_359.09672.6042_787.19042.606_153.0549
2.606_397.17052.6072_197.04472.626_479.08552.6299_593.13162.631_1185.25262.6325_897.18772.6346_591.1152.6366_125.02352.6373_179.03472.6373_355.06632.6376_289.06772.6379_447.15142.6457_481.0972.6461_301.03712.6502_463.08752.6711_293.12292.6716_139.03862.6731_641.13762.6896_411.18612.69_119.052.6916_289.06972.6928_329.08782.6962_163.03922.7009_391.1235
2.7065_881.19152.7149_631.16492.7152_879.1757
2.7229_206.04532.723_463.08612.7248_1191.230
2.7254_146.0242.7263_144.04442.7283_118.0296
2.7288_177.01832.7306_133.0285
2.7313_105.03392.7334_311.0398
2.7342_329.08712.7346_87.00822.7346_309.02412.7371_149.0084
2.7379_103.00262.7438_122.0362.7451_195.02882.7534_881.19332.7535_879.17592.7542_289.06982.7796_671.96532.7803_194.02022.7811_933.12182.7817_72.99112.7817_121.02812.7824_619.05782.7826_637.06442.7827_673.98582.7829_639.08452.7834_674.99092.7837_935.13482.785_128.98192.7851_267.05012.7875_112.98722.7879_661.04412.7885_621.07252.7903_675.9994
2.7905_177.01832.7912_133.0286
2.7912_577.13382.7916_105.03372.7919_623.08782.7922_313.0452
2.793_195.0288
2.7934_309.0242
2.7937_130.99782.7941_136.04722.7944_181.0395
2.7946_425.08692.7947_327.03482.7949_87.0078
2.7952_149.0082
2.7954_407.07542.7955_103.00262.799_193.01212.7992_1465.3042.8003_203.0806
2.802_311.0396
2.8022_677.00652.8025_887.16632.8056_905.17782.8056_1466.314
2.8067_889.1815
2.8075_1467.3222.8089_759.142.8096_885.15122.8128_289.06992.8147_209.04492.816_122.03642.8187_575.11682.8211_125.02442.8213_1153.2582.8217_299.05792.8225_1155.275
2.8249_559.1252.8252_194.02232.8253_451.10212.8258_102.99982.8272_423.07182.832_495.11122.8332_577.1341
2.8334_407.07652.8349_777.1548
2.835_425.0868
2.8431_359.0977
2.8434_179.034
2.8455_465.10262.8513_285.03952.8518_447.09162.8542_327.03422.8549_195.02982.8549_305.06562.8553_477.09962.8569_887.16852.8577_1169.257
2.8601_889.18182.8609_759.14492.8629_309.02452.8632_177.0184
2.864_881.19212.8641_593.1329
2.8649_149.0083
2.8651_87.00822.8651_133.02852.8657_311.03972.867_623.13092.8677_355.068
2.8724_193.0492.8753_461.12852.8765_451.10842.8783_728.15022.8793_121.02772.8813_487.14892.89_879.17712.8944_575.23112.8944_591.11312.8977_595.16632.8982_1153.260
2.8999_235.1177
2.9001_122.0358
2.9001_367.16
2.9007_443.19122.9008_881.19442.9009_403.13642.9013_611.16142.9013_1169.2522.9018_193.04472.9021_495.11312.9026_413.16282.9031_315.05052.9033_149.0083
2.9034_448.09582.9035_311.03982.9036_179.0341
2.9036_493.09652.9037_309.02562.906_477.10242.9062_449.10712.907_161.08072.9076_625.14092.9086_880.18332.9095_177.01852.9102_491.13822.9124_133.02882.9239_447.09162.9396_1457.3172.9398_615.17182.9466_889.185
2.9497_577.13412.95_425.0867
2.9502_728.65472.9506_451.10132.9506_465.10222.9508_285.03982.9508_303.05
2.9514_509.13192.9515_1155.2742.9521_728.15572.9524_443.19092.9524_477.10312.9527_367.162.9528_311.04042.953_407.0768
2.9532_625.13752.954_495.11342.9548_655.15392.9549_177.01872.9557_423.0722.9563_575.11912.9566_289.0705
2.9569_327.10962.9569_591.11142.9577_447.0932.9596_133.02852.9607_125.02352.9619_149.0082.9624_287.0552.9627_865.20272.9683_880.68562.9699_880.18262.9753_881.19312.9787_245.0512.981_549.25232.9815_872.18762.9855_872.68322.9899_179.03462.9899_863.18132.9933_315.0643.0057_341.08823.0064_879.17813.0095_477.10213.0125_153.0179
3.0132_865.1969
3.0165_713.14873.0181_652.13143.0193_880.18443.0205_728.65683.0205_757.17553.0224_577.13273.024_711.13683.0244_133.02743.0274_137.02343.0319_407.078
3.0321_615.1728
3.0348_299.06493.0351_289.07073.0354_720.15873.0367_863.18173.0372_413.08863.0378_245.06153.0382_739.16933.0389_177.01823.0405_125.02353.0413_479.11833.0432_491.0983.0434_287.0543.0437_406.06763.0445_500.10123.0452_355.06613.0458_575.11873.0461_451.10213.0463_317.06663.0463_461.10753.0464_347.07563.0465_161.0229
3.0471_1153.260
3.0472_1151.2443.0476_728.15513.0477_367.16
3.0478_576.12583.0497_431.15533.0497_864.18853.0522_149.0081
3.0542_181.0496
3.0546_872.68743.0553_285.04213.0563_728.6597
3.0582_865.19783.0593_881.19323.0596_303.0501
3.061_465.10143.061_872.18663.0627_629.14943.0664_343.10183.0684_1441.3253.0703_720.65773.0726_1016.2243.073_1016.7161
3.0769_864.18993.078_720.15963.0792_470.11763.0817_123.04493.0823_311.043.0831_415.93093.0845_133.02633.0859_633.0683.0872_217.0749
3.0878_289.0708
3.0885_227.07043.0887_1001.2573.0893_179.0343
3.0895_231.033.0897_593.12983.0901_329.06623.0901_983.24293.0905_863.20523.0907_188.04613.0907_796.65873.091_579.15013.0918_165.01833.0919_347.0763.0924_617.10723.0927_187.03943.0929_491.11753.0934_125.0234
3.0935_796.16473.0936_109.02853.0937_271.063.0939_1019.2683.094_509.12883.0941_469.11293.0945_307.0814
3.0953_205.0497
3.0954_325.0485
3.0955_245.0811
3.0958_137.02343.0958_203.0704
3.0958_201.05483.0959_341.08963.0962_632.06253.097_161.05373.0987_287.05533.0989_243.06633.099_193.01343.0995_175.03963.1013_177.01893.1013_259.05993.1019_413.93593.1032_465.10173.1037_461.10763.1037_515.09743.1055_299.05553.1059_527.09683.107_864.68893.1075_317.06583.1075_923.22343.1077_479.11813.1087_303.05023.1098_989.25413.11_865.1983.1103_355.0663.1103_971.24423.1107_941.23393.1111_1024.2123.1114_953.23473.1116_149.0093.1145_872.18883.1166_880.18673.117_561.14223.117_1016.21333.1181_881.19193.1192_959.24063.1239_327.03853.1243_529.11873.1247_1016.7223.1264_545.10383.1284_164.01893.1291_728.15653.1324_872.6863.1376_323.1333.1384_720.6581
3.1395_864.18943.1413_983.24613.1415_491.11863.142_1019.26623.1427_347.07623.1427_425.08723.1428_1153.2593.1429_509.13023.143_153.05523.1431_357.11813.1432_293.1233.1435_1001.2563.1448_863.18123.1451_329.06633.1456_593.12963.1457_575.1193
3.1461_119.04853.1461_1017.213.1474_720.15833.15_1441.32583.151_971.24593.1515_161.02713.1515_317.06613.1516_303.05143.1518_923.22293.1519_205.04973.1519_461.10773.1521_515.09633.1522_137.02363.1524_953.2353.1524_941.23293.1525_245.0815
3.1525_644.13653.1527_299.05533.1527_872.18623.1533_325.0922
3.1533_355.06563.1537_125.02253.1538_287.05483.1538_635.1313.1541_289.07023.1542_145.02793.1542_989.25593.1547_865.19693.156_527.09783.1563_635.63263.1574_193.01283.1576_479.11873.1648_889.18373.1821_775.17113.1852_59.01313.1877_72.99233.1891_591.0994
3.1897_887.1509
3.1913_149.0082
3.192_757.16023.1932_645.01553.1934_87.0079
3.1935_805.18323.1936_1083.2263.1941_629.05513.1947_644.00983.1959_103.0028
3.196_295.045
3.1963_130.9977
3.1968_435.1273
3.1971_163.0392
3.1978_112.987
3.1986_379.1615
3.1988_119.0493
3.2002_787.1733.2047_1019.2713.2082_479.11763.2088_873.18443.2095_1001.2533.2103_347.07663.2142_355.0663.2144_509.12923.2151_731.18373.2184_193.01233.2211_461.10853.2233_983.24493.2251_527.09423.2284_545.10853.2294_927.24583.2339_379.16013.2341_293.12183.2349_137.02283.2372_435.12863.2412_491.11893.2419_329.06743.2452_167.03443.2461_1008.7203.2468_864.18483.2478_401.14423.2503_607.14453.2505_983.24343.2518_787.17483.2519_881.19353.2521_455.09153.2526_351.20063.2541_864.6917
3.2547_87.00813.2549_289.07083.255_119.0493
3.2551_149.00823.2552_103.00293.2557_481.098
3.2569_295.0451
3.2573_112.9873.2573_131.03453.2578_269.102
3.2586_163.0391
3.2603_461.10693.2605_447.1487
3.2609_1017.2233.2612_303.04963.2619_728.14953.2631_177.01763.2649_285.03963.2652_1008.222
3.2667_1016.7203.272_1016.21973.2751_1008.7213.2798_1152.7503.2836_865.19683.284_125.02283.2861_161.06763.2902_287.05463.2941_401.14483.2941_741.17983.2943_863.1819
3.2962_449.10853.2972_1152.2493.3001_261.13373.3003_423.18523.3005_285.04023.3008_864.1883.3009_451.08783.3014_1153.2603.3021_306.07813.3033_447.14623.3055_301.06993.3099_163.0391
3.3103_119.04933.3109_295.04513.3117_379.15913.3134_289.07133.3134_575.11913.3144_577.13323.3157_217.10723.3185_149.00843.3225_1151.2523.3252_125.02373.3258_863.18233.326_277.12193.3283_1152.7513.329_193.0493.3314_720.1533.3403_872.17733.3426_1008.2193.3471_331.08113.3483_355.10253.349_175.03933.3503_217.04993.3509_509.12983.3514_865.1989
3.3514_1008.7203.3523_1152.2533.3524_349.18513.3554_261.13553.3559_173.08063.3575_1016.213.3592_306.07513.36_461.10773.3629_521.20343.3653_1017.2103.3717_729.1463.3722_491.11683.3733_864.18683.3818_295.04483.3821_1296.2823.3836_112.9873.3846_1152.7553.3848_293.08543.3856_796.1618
3.3865_447.1472
3.3875_269.10233.3875_437.12083.3881_401.14443.3881_477.1035
3.3906_149.00853.3928_119.04963.3956_161.03493.3985_431.1914
3.3987_325.05573.399_134.03623.3993_193.04963.4024_575.1183.4028_179.03573.4031_1008.226
3.4032_1153.2593.4033_163.0393.4036_863.1811
3.4043_865.1974
3.4048_387.16243.4063_125.02383.4067_577.13383.4075_1296.7893.4117_1152.2553.413_245.081
3.4151_289.0708
3.4153_203.07073.4185_205.04793.4206_509.12713.4208_491.11873.4223_425.08693.4242_287.05483.441_720.66083.4421_517.22833.4447_295.04413.4466_864.18853.4484_261.1339
3.4487_453.11783.4499_423.18623.4517_429.21243.4518_451.1043.4526_381.17643.4532_325.05573.4537_1441.325
3.4538_613.15573.4542_577.13323.455_193.053.4553_1153.258
3.4571_865.1973.4572_203.08143.4573_289.07093.4575_197.0473.4577_720.15943.458_323.1227
3.4589_189.06643.4603_245.06743.462_319.08323.4633_125.02363.4654_863.18583.4664_389.12543.4685_269.07693.4725_163.0393.4914_463.21823.4941_259.06013.4967_287.05523.4991_235.1183.4998_449.10773.4999_341.12333.5017_179.07023.5034_625.143.5043_415.163.5052_779.25313.5062_385.18563.507_431.18893.5083_613.1557
3.5122_363.16523.5132_333.02763.5159_285.0383.5162_245.0443.5165_465.10243.5251_639.11933.5259_1008.2233.5272_1008.7183.5383_269.0812
3.5383_389.12363.5385_163.03953.5411_377.18173.5471_577.13363.5492_423.18583.5498_405.11853.5501_241.053.5501_243.10113.5503_289.06333.5503_404.10713.5517_363.16533.5517_720.15693.5533_465.1033.5536_613.15023.5549_217.10723.5572_864.18543.5576_739.16683.5583_403.10093.5591_449.10443.5664_281.13833.5688_237.14973.5722_895.20613.5732_451.10433.5741_287.05523.5744_125.02373.5752_575.11633.5753_1153.2653.5762_407.07513.5819_577.13443.5824_425.08723.5861_479.21473.5867_865.19623.5891_864.6915
3.5895_227.07053.5969_729.14553.5973_355.06553.6002_809.21633.6007_317.06623.6007_479.11883.604_193.01333.6052_641.14023.6061_465.10083.6074_299.05483.6095_1008.2183.6099_231.06893.6102_711.19753.6119_739.16533.6159_417.1532
3.6171_381.1759
3.6175_463.08683.6176_249.13353.6179_495.11563.6269_655.11773.6365_741.20353.6424_329.06563.6424_1019.2643.6427_509.12933.6491_355.06683.6515_433.1133
3.6517_347.076
3.6524_463.2179
3.6531_989.2243.6554_611.25453.6595_479.08243.6617_493.063.6639_315.0143.6673_231.06833.6909_509.12923.6922_113.063.6955_157.05053.6991_337.15063.7004_379.16023.7007_209.04353.7013_329.0867
3.7019_167.0343.7023_191.0343.7026_463.21743.7034_479.08153.7044_303.14393.7049_529.11083.7067_493.13443.7093_331.0823.7095_347.07633.7119_313.07153.7122_221.04523.714_739.16653.7175_133.06443.7182_241.10563.7186_403.1623.7194_633.1951
3.7234_597.1609
3.724_210.07733.7278_539.21173.7353_507.11213.7409_289.04183.7418_333.02393.7433_779.2533.7442_283.05783.7466_319.0453.7474_259.06063.7474_501.07893.7478_447.09223.7482_287.05533.7485_178.99733.7485_299.01873.7485_463.08723.7487_273.03963.7487_565.02733.7488_339.07113.7491_481.09843.7494_931.21323.7496_193.01343.7496_465.10263.7497_301.03443.7504_151.00283.7507_929.21773.7508_739.16633.7527_331.0829
3.7528_435.10013.7529_329.08943.7532_479.08063.7545_463.22123.7559_479.2105
3.7563_597.1607
3.7564_317.06173.7568_365.18013.7577_901.20453.7583_347.11473.7596_499.09893.7628_415.15963.7629_503.07163.7644_285.043.7671_551.17763.7674_303.04963.772_832.23083.7801_463.11623.7802_517.1482
3.7811_261.1343.7821_366.11793.7852_349.1077
3.7854_943.2966
3.7857_121.02883.786_377.10153.7861_507.11953.7879_255.0651
3.7881_471.1437
3.7884_627.15643.7984_436.0673.8019_521.19953.802_597.15983.8031_435.08523.8246_371.09843.8321_487.1441
3.8401_401.14443.8403_443.1548
3.8415_383.13373.8466_415.16053.8485_723.17043.8518_465.10253.8519_449.1082
3.8535_471.1441
3.8535_609.1454
3.8622_301.03543.8639_181.04883.8954_391.1372
3.8974_433.11493.913_307.13913.9174_405.17583.9179_609.14633.9203_243.12263.9226_393.1762
3.931_387.10963.9692_751.19263.9696_781.19783.9723_985.26113.9775_433.11263.9986_633.1973.9999_779.25544.0002_425.09994.0003_435.12914.0004_389.12324.0008_227.0706
4.0066_463.08714.0111_493.09784.0158_317.03154.0167_475.0503
4.0169_941.16014.0173_927.1844
4.0173_955.1404
4.0175_491.08224.0175_953.12484.0176_509.22064.0182_299.019
4.0183_301.0343
4.0188_477.0661
4.0195_939.14564.0202_461.07134.0203_449.2014.0244_287.14864.0306_453.23684.033_507.07824.0339_206.08014.0379_531.1273
4.0569_441.08164.0578_243.06644.0587_405.11794.0605_493.09834.0619_389.12344.0625_403.10234.0653_299.0193
4.0683_301.0346
4.0686_227.071
4.0696_463.087
4.0704_447.09414.0821_449.20074.0834_161.04484.0862_269.1028
4.087_477.0665
4.0892_311.11234.0926_723.16994.0936_287.05514.0979_827.25574.0998_463.2177
4.101_471.14394.102_121.02854.1023_507.12094.1024_517.14994.1025_349.1077
4.1037_469.1284.1048_943.29944.1055_347.116
4.1064_301.03454.1115_227.06984.1122_463.08774.1153_225.05494.1199_419.09694.1221_389.12864.1291_547.24144.1428_593.14884.1564_463.21664.1583_349.10674.1594_227.98864.1596_433.07534.1596_477.06644.1598_471.14584.1606_118.02964.1614_151.0037
4.1618_485.08364.162_125.02384.1622_177.0189
4.163_303.04994.1631_285.03964.1633_899.2241
4.1635_449.10774.1657_323.07684.1731_431.0894.1738_487.07134.1779_549.0643
4.1821_505.098
4.1955_523.2164.1959_405.11474.1964_435.09274.1997_419.13454.2001_447.09334.2004_479.11864.2009_125.02444.2011_257.08114.202_485.07834.2021_899.22434.2033_151.00254.2064_303.05034.2089_285.03964.2114_449.10734.2491_263.12744.2504_153.09214.2534_397.14994.2542_303.0527
4.2553_415.16084.2562_457.16964.2566_271.06134.259_285.0391
4.2612_561.25354.2693_447.09574.2974_315.08624.301_751.18894.3022_463.2175
4.3022_781.19994.3109_509.22264.313_183.02834.3132_447.0922
4.3133_363.07174.3174_165.01884.3184_239.03364.3195_477.1034.3196_207.1014.3221_285.03754.3244_461.07144.325_499.19334.3267_195.06514.3278_485.1212
4.3481_535.18074.3519_507.11364.353_417.15314.3598_271.15334.3672_447.09274.3672_463.21584.3745_503.254.3767_521.16694.3776_452.10974.3818_197.0594.3818_477.103
4.3825_243.0653
4.3828_213.05514.3844_199.03914.3874_201.05614.3886_373.1872
4.3892_241.0497
4.3898_285.03954.3907_303.04994.3911_451.10274.3913_341.06524.3926_449.10734.393_395.19144.4043_447.08864.4088_507.11314.4138_345.06024.4203_491.08574.4209_315.04984.4229_289.07034.4338_451.10244.4347_341.0657
4.4355_299.05184.5088_243.0655
4.5094_271.06034.5139_287.05694.5153_505.22954.5156_269.04564.5157_433.11324.5227_477.13994.526_501.15554.5356_471.1441
4.5408_225.05454.5451_387.1084.5598_433.11464.5719_463.12374.579_287.0874.5875_417.21664.5918_449.10774.5937_817.20914.5938_227.07034.5939_425.09914.5941_779.2542
4.5945_389.12284.5948_477.2336
4.5981_471.14364.6001_317.19564.6045_479.0834.6277_163.99344.6301_458.05764.6305_429.10424.631_280.04624.6351_326.05174.6502_257.08084.6513_419.13444.6543_389.1227
4.6548_359.09184.6556_227.0703
4.6557_453.133
4.6588_907.27214.6615_499.13924.6667_459.12754.6774_317.0294.6811_341.0664.683_274.07624.6893_723.17034.696_451.1026
4.7034_471.14354.7094_359.09294.7125_453.13384.7134_435.1281
4.7176_273.0757
4.7193_227.07034.7205_255.06294.7422_511.21644.7701_273.07644.7747_297.07614.7911_289.07034.7911_409.20754.7977_483.14314.7999_387.10664.7999_423.08494.8002_775.22154.8008_225.0545
4.8032_297.07514.8038_273.1695
4.8212_435.10754.8223_341.0659
4.8314_289.07124.8364_271.06074.8381_485.1555
4.8405_371.1335
4.8411_209.081
4.8502_469.1277
4.8503_433.11214.8517_273.17054.8637_609.27264.8809_271.06064.8913_871.2265
4.8943_435.1077
4.8945_681.2118
4.8954_341.0659
4.9141_469.12554.9175_485.1272
4.9205_273.1694.946_681.2126
4.947_269.17544.9601_435.1052
4.9658_485.12364.9699_907.2748
4.9714_453.1336
4.9714_723.17274.9722_359.0918
4.9736_447.22244.9737_315.18064.9737_493.22714.986_263.04894.9887_415.1235
4.9909_227.0705
4.9929_159.08034.9929_183.08074.9937_143.04874.9938_185.05994.9938_580.01174.9941_157.06524.9942_471.14454.9948_683.22584.9952_121.02824.997_241.0584.9972_225.0549
4.9984_243.06555.0009_681.20965.0014_447.22465.0021_359.09095.0023_403.14145.0023_493.2263
5.0028_453.13325.0617_325.09375.0639_511.21695.0651_263.12815.0767_163.03855.0843_399.1295.0896_483.199
5.0917_227.0704
5.0923_493.2272
5.0939_447.22255.1044_435.1072
5.151_417.24915.1518_463.2522
5.1527_435.10775.1538_447.22355.1564_497.23845.1573_478.10235.1587_316.04995.159_318.05025.1937_242.0571
5.1966_257.0812
5.1974_423.22235.1989_359.0918
5.1992_453.13355.2208_681.2129
5.2255_166.00825.2259_282.06055.226_149.00845.2267_458.05725.2268_326.0525.2682_345.227
5.2832_453.13335.294_511.2165.2995_349.16415.3051_227.07145.3138_497.23895.3191_287.09125.3236_905.26425.33_515.13245.3315_939.2644
5.3317_379.08065.3326_505.1028
5.3341_469.1282
5.3344_467.11125.356_581.18115.3568_387.99255.3572_109.05535.3572_152.08315.3572_219.1385.3572_527.26365.3574_847.32555.3575_153.0915.3575_353.02155.3578_263.12755.358_138.06735.3583_382.02285.3585_136.05215.3588_111.04425.3589_363.05265.3593_201.12725.3594_205.1175.3596_204.11445.3603_423.96875.3605_580.1745.3612_137.06025.3621_425.96555.3622_161.09595.3636_151.07485.364_565.22065.3741_301.0326
5.3936_241.0865.3949_403.13515.4081_905.2575
5.4093_489.2386
5.4115_469.128
5.4172_423.22185.4564_329.23285.4566_469.1267
5.4627_153.0915.4631_527.26285.4942_343.04525.5036_493.2285.5055_459.2579
5.5062_447.22245.5072_399.23795.5082_329.23285.51_417.24835.5851_227.07065.5886_531.28165.5907_459.2372
5.5944_447.2225.6211_469.12745.6338_459.22225.6516_315.08665.6526_125.02345.7177_485.1227
5.7195_239.03215.7564_469.12535.7578_489.11085.7584_363.08715.7588_907.2774
5.7598_453.1334
5.8341_453.13345.839_679.19736.0019_453.1335
6.0078_363.08396.0377_327.2186.1762_531.2813
6.1779_577.28336.1892_679.1964
6.192_467.10776.1957_489.11066.197_907.2695
6.1974_453.1329
6.1985_499.13896.1988_347.09116.2181_242.17186.239_497.25546.2421_451.2536.3036_483.14096.3878_329.2326
7.5667_561.28457.9954_471.3478.3412_265.14688.4124_265.14798.5117_265.14698.5598_711.33918.5605_721.3646
8.5624_675.35938.8168_291.19629.1517_473.36519.4069_513.30779.4082_277.21559.4095_559.31299.4107_453.33629.4122_487.34199.5012_476.27759.5379_504.3088
9.5385_564.32919.585_471.34589.7086_295.22539.7467_469.33189.7591_295.22689.9156_293.2111
9.9966_116.9273
PC1 51.4%
PC
2 16
.3%
Loading Scatter PlotB
Figure 4. Principal component analysis of grape berry skin metabolome. A,Score scatter plot of a PCA model on the berry skin metabolome in darkness(�) and after UV-C treatment (�) (n=6 for each treatment). B, Loadingscatter plot from the PCA analysis of berry skin in darkness and after UV-Ctreatment (n=2012 metabolite peaks). Each annotation showed the retentiontime and m/z.
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Figure 5
Figure 5. Accumulation of fluorescent substances at
berry skin. The sections of grape berry of UV-C-irradiated
(A, B) and control (C, D). A, C: Bright-field images. B, D:
Fluorescence images.
Dark
UV
Bright-field Fluorescence
A B
C D
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Figure 6
Figure 6. Modulation of secondary metabolism after UV-C irradiation. The schematic representation was determined by
transcriptome and metabolome data integration using updated KaPPA-View 4 KEGG. Fold-change of each gene
expression after log10 transformation is divided into 10 levels and visualized by colors: red, upregulated by UV-C; blue,
downregulated by UV-C; gray, not detected. Fold-change of each metabolite content is visualized by 4 colors: red,
upregulated by UV-C; white, not identified.
www.plantphysiol.orgon March 25, 2018 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.