plant physiology preview. published on march 11, 2015, as doi

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Research article 1

Running head 2

Metabolic change in grape berry skin by UV 3

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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

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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

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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



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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



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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

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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

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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

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Keywords: Grape berry, Metabolome analysis, Resveratrol, Transcriptome analysis, 16

Ultraviolet irradiation 17



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



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



25

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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



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



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

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nloaded from

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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_03s0038g03570 5.6 l-ascorbate oxidase homolog

VIT_02s0154g00600 8.6 probable pectinesterase 68-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



GO

:00

4533

0 a

spar

tyl e

ster

ase

act

ivity



VIT_02s0154g00600 8.6 probable pectinesterase 68-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



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_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



3-o-glucosyltransferase 6-like

VIT_04s0023g01120 6.1 cazy family gt8


VIT_15s0021g02060 17.8 hydroquinone glucosyltransferase


3-o-glucosyltransferase 6

VIT_01s0011g03850 6.3 protein



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).



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.



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.



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.



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.



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).



-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

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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

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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

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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

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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

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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

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4.7034_471.14354.7094_359.09294.7125_453.13384.7134_435.1281

4.7176_273.0757

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4.8032_297.07514.8038_273.1695

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4.8943_435.1077

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4.9658_485.12364.9699_907.2748

4.9714_453.1336

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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.



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



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.



plant physiology preview. published on march 11, 2015, as doi

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