ORIGINAL ARTICLE
Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions
Akifumi Azuma •Hiroshi Yakushiji •
Yoshiko Koshita •Shozo Kobayashi
Received:20March 2012/Accepted:6April 2012/Published online:9May 2012ÓSpringer-Verlag 2012
Abstract Temperature and light are important environ-mental factors that affect flavonoid biosynthesis in grape berry skin.However,the interrelationships between tem-perature and light effects on flavonoid biosynthesis have not been fully elucidated at the molecular level.Here,we investigated the effects of temperature and light conditions on the biosynthesis of flavonoids (anthocyanins and flavo-nols)and the expression levels of related genes in an in vitro environmental experiment using detached grape berries.Sufficient anthocyanin accumulation in the grape skin was obrved under a low temperature (15°C)plus light treat-ment,whereas high temperature (35°C)or dark treatment verely suppresd anthocyanin accumulation.This indi-cates that the accumulation of anthocyanins is dependent on both l
ow temperature and light.qRT-PCR analysis showed that the respons of three MYB -related genes (VlMYBA1-3,VlMYBA1-2,and VlMYBA2)to temperature and light dif-fered greatly even though the products of all three genes had the ability to regulate anthocyanin biosynthesis pathway genes.Furthermore,the expression levels of other MYB -related genes and many flavonoid biosynthesis pathway genes were regulated independently by temperature and light.We also found that temperature and light conditions affected the anthocyanin composition in the skin through
the regulation of flavonoid biosynthesis pathway genes.Our results suggest that low temperature and light have a syn-ergistic effect on the expression of genes in the flavonoid biosynthesis pathway.The findings provide new infor-mation about the relationships between environmental factors and flavonoid accumulation in grape berry skin.Keywords Anthocyanins ÁFlavonols ÁGrape ÁSkin color ÁLight ÁTemperature Abbreviations ABA
Abscisic acid
喜迎新年手抄报
antho-MATE Anthocyanin multidrug and toxic extrusion CHI Chalcone isomera CHS Chalcone syntha
DFR Dihydroflavonol 4-reducta F3H Flavanone 3-hydroxyla F30H Flavonoid 30-hydroxyla F3
050H Flavonoid 3050-hydroxyla FLS4Flavonol syntha
GST Glutathione-S-transfera
LDOX Leucoanthocyanin dioxygena
西藏路线NCED19-cis-epoxycarotenoid dioxygena 1OMT O-methyltransfera
Pn3G Peonidin-3-monoglucoside
Pn3pG5G Peonidin-3-(p -coumarylglucoside)-5-glucoside
UFGT
UDP-gluco:flavonoid 3-O-glucosyltrans-fera
Introduction
Flavonoids such as anthocyanins and flavonols are c-ondary plant metabolites that accumulate in fruits,flowers,
A.Azuma (&)ÁH.Yakushiji ÁS.Kobayashi Grape and Persimmon Rearch Station,
National Institute of Fruit Tree Science,National Agriculture and Food Rearch Organization (NARO),Akitsu 301-2,Higashi Hiroshima,Hiroshima 739-2494,Japan e-mail:jp
Y.Koshita
National Institute of Fruit Tree Science,National Agriculture and Food Rearch Organization (NARO),Tsukuba,Ibaraki 305-8605,Japan
Planta (2012)236:1067–1080DOI 10.1007/s00425-012-1650-x
and leaves.Flavonoids play important roles in resistance to UV light and pathogens,and in attraction of animal poll-inators(Harbone and Williams2000;Koes et al.2005). Furthermore,many rearchers have studied the beneficial effects offlavonoids in grapes and wines on human health. The benefits include antioxidant activity,prevention of coronary heart dia,anticancer activity,and others (Harbone and Williams2000).For the reasons,it is valuable to understand howflavonoid biosynthesis is affected by environmental conditions such as temperature and light,as this knowledge may contribute to the devel-opment and production of high-flavonoid grapes.
The skin color of grapes is mainly determined by the content and composition of anthocyanins,andfla
vonols contribute to wine color through co-pigmentation with anthocyanins(Baranac et al.1997).The genes encoding the enzymes of theflavonoid biosynthesis pathway in grape have been isolated(Sparvoli et al.1994).Boss et al. (1996a,b)showed that the expression of the gene for UDP-gluco:flavonoid3-O-glucosyltransfera(UFGT)is critical for anthocyanin biosynthesis in grapes.Koes et al. (2005)suggested that anthocyanin biosynthesis was con-trolled by a transcription complex compod of three reg-ulatory proteins belonging to the MYB,bHLH,and WD40 class,which activate the structural genes in theflavonoid pathway.In grape,some genes for MYB-related transcrip-tion factors have been isolated and shown to regulate anthocyanin biosynthesis.Two MYB-related transcription factor genes that regulate anthocyanin biosynthesis, VvMYBA1and VvMYBA2,have been isolated from Vitis vinifera grapes(Kobayashi et al.2004;Walker et al.2007), whereas three functional MYB-related genes,(VlMYBA1-2, VlMYBA1-3,and VlMYBA2)are prent in the Vitis9labruscana genome(Azuma et al.2008,2011; Kobayashi et al.2002).Forflavonol biosynthesis,the activity offlavonol syntha(FLS)is required(Holton et al.1993).Downey et al.(2003)suggested that expres-sion of FLS increas during ripening coincident with the accumulation offlavonols in the grape berry skin. Recently,Czemmel et al.(2009)isolated the MYB-related transcription factor MYBF1and confirmed that it was a transcriptional regulator of FLS.Furthermore,veral reports have indicated that MYB5a,MYB5b,MYBPA1, and MYBPA2appear
to regulate veral genes in the common steps of theflavonoid pathway(Bogs et al.2007; Deluc et al.2006,2008;Terrier et al.2009).
Temperature and light are important environmental factors that affectflavonoid biosynthesis in many plants (Bowler and Chua1994;Christie et al.1994;Christie and Jenkins1996;Dela et al.2003;Frohnmeyer et al.1997; Leyva et al.1995;Lo Piero et al.2005;Shvarts et al.1997; Ubi et al.2006).In grape,the accumulation of anthocya-nins in berry skin begins after the ont of ripening (veraison)and is affected by the environmental conditions in the vineyard(Kliewer and Torres1972).Kliewer and Torres(1972)reported that‘Tokay’grapes developed the strongest fruit coloration under day temperatures between 15and25°C and night temperatures between10and 20°C,and that temperatures of35°C in the day or30°C at night inhibited the development of coloration.Tomana et al.(1979a,b)reported that a lower temperature(15°C) around the berry clusters affected color development more strongly than higher cluster temperatures in‘Delaware’and ‘Kyoho’grapes.Recent studies have demonstrated that low ambient temperature during the maturation stages of grape berries increas anthocyanin accumulation,whereas high temperature decreas the expression of genes related to flavonoid biosynthesis in grape skins and increas antho-cyanin degradation(Mori et al.2005,2007;Yamane et al. 2006).In veral studies,exposure of g
rape bunches to light significantly incread the accumulation offlavonoids and the expression of their biosynthesis genes,whereas shading reduced them(Cortell and Kennedy2006;Downey et al.2004;Fujita et al.2006;Jeong et al.2004;Matus et al.2009).
Although many studies have been performed to deter-mine the impact of environmental factors onflavonoid accumulation in grape berries,our knowledge is still incomplete becau such studies are complex underfield conditions and the results are highly variable.For example, it is difficult to parate the environmental effects of temperature and light in afield tting.Furthermore,it is difficult to control the nutrient state of thefield ud for the experiments.For the reasons,there are few reports con-cerning the interrelationship of temperature and light effects onflavonoid accumulation in grape skin.Further-more,it has not yet been elucidated howflavonoid bio-synthesis pathway genes and MYB-related genes respond to various combinations of temperature and light.In this study,we performed in vitro environmental experiments using detached grape berries to clarify the interrelationship of temperature and light effects onflavonoid accumulation and on the expression offlavonoid biosynthesis-related genes.
Materials and methods中国园林建筑
In vitro environmental experiments using detached grape berries
窝窝团购美食
营销方式都有哪些
Grape berries beginning to color(early stages of veraison) were collected from mature vines of‘Pione’(V.9labrus-cana)growing in a vineyard at the Grape and Persimmon Rearch Station,National Institute of Fruit Tree Science, Hiroshima,Japan.Berry weight and total soluble solids of
the berries ud in this study were10.6–11.9g and 9.2–10.8°Brix,respectively(data not shown).The surface of the grape berries was sterilized in a1%sodium hypo-chlorite(v/v)with0.02%Tween20(v/v)for10min and then washed three times in sterile distilled water.Sterilized berries were placed on0.7%agar medium,and three ts of 15berries per treatment were incubated in a multi incubator (TG-180CCFL-5LD,Nippon Medical and Chemical Instruments,Osaka,Japan)for10days under one of four conditions:15°C/light(white light(cold cathodefluores-cent lamp)?UV light(FL10BLB,Toshiba,Japan), 80l mol m-2s-1,continuous irradiation),15°C/dark, 35°C/light,and35°C/dark.After incubation,the berry skins were peeled off,immediately frozen in liquid nitro-gen,and kept at-80°C until required.This experiment was performed twice:once in2008and again in2009. Analysis of anthocyanin content,composition,
andflavonol content
The anthocyanin analysis was performed as described by Shiraishi et al.(2007).Total anthocyanin content was expresd as milligrams of cyanidin-3-monoglucoside (Extrasynthe`,Genay,France)equivalent per gram of fresh berry skin.The anthocyanin compositions were ana-lyzed by high-performance liquid chromatography(HPLC; LC-10A,Shimadzu Co.Ltd.,Kyoto,Japan).Solvent A was 1.5%phosphoric acid(v/v),and solvent B consisted of 1.5%phosphoric acid(v/v)combined with20%acetic acid(v/v)and25%acetonitrile(v/v).The proportion of solvent B was linearly incread from25to85%over 40min at aflow rate of0.8mL/min.Anthocyanin com-positions were determined as the percentage ratio of the area of the individual peaks to the total area of all peaks. Totalflavonol contents were analyzed as described by Mazza et al.(1999)with slight modifications.Samples (*1g each)were diluted with10mL of70%methanol (v/v)combined with2%formic acid(v/v).A0.25-mL sample was measured into a test tube,and4.75mL of 0.2M sodium acetate/hydrochloric acid solution(pH1.0) was added.The solution was thoroughly mixed and allowed to stand for approximately15min and the absor-bance at360nm was read with a spectrophotometer (ND-1000,Thermo Fisher Scientific,Waltham,MA,USA). Analysis of abscisic acid(ABA)content
The ABA contents in the berry skins in the2008experi-ment were analyzed three times under each of the four environmental conditions by the method of Kojima et al. (1999)with slight modifications.Each
sample(*1g)was added to2H6-ABA as an internal standard and the mixture was homogenized in cold(approximately4°C)80%acetone containing100mg L-1of butylhydroxytoluene using a Polytron(PT10/35,Kinematica GmbH,Kriens-Luzern,Switzerland).The resulting extract wasfiltered and acetone was removed by rotary evaporation.The pH of the solution was adjusted to 2.5and the acidified aqueous pha was partitioned three times against diethyl ether.The organic layer was evaporated to dryness.Further purifica-tion was done by HPLC using an ODS column(PEGASIL ODS,6i.d.9150mm,Senshu Scientific,Tokyo,Japan). The zone of elution corresponding to that of the ABA standard was collected and vacuum-concentrated to dry-ness.The residue was methylated with1mL diethyl ether in10%methanol(v/v)and400l L diazomethane.The reagents were eliminated by evaporation and the residues dissolved in200l l acetonitrile.The quantity of methylated ABA was then determined by gas chromatography/mass spectroscopy-lective ion monitoring(GC/MS-SIM; GCMS-QP5000,Shimadzu Co.Ltd.).
Expression analysis of MYB-related genes
andflavonoid biosynthesis-related genes by qRT-PCR Total RNA was extracted as described by Reid et al.(2006) from three samples for each of the four experimental conditions,and cDNAs were synthesized using a Prime-Script II cDNA synthesis kit(Takara,Shiga,Japan)as described in the man
ufacturers’manuals.We determined the expression levels of MYB-related genes(VlMYBA1-2, VlMYBA1-3,VlMYBA2,MYB4,MYBPA1,MYB5a,MYB5b, and MYBF1),flavonoid biosynthesis-related genes(chal-cone syntha[CHS]2and CHS3,chalcone isomera [CHI]1and CHI2,flavanone3-hydroxyla[F3H]1and F3H2,flavonoid30-hydroxyla[F30H],flavonoid3050-hydroxyla[F3050H],dihydroflavonol4-reducta[DFR], leucoanthocyanin dioxygena[LDOX],UDP-gluco:flavonoid3-O-glucosyltransfera[UFGT],O-methyl-transfera[OMT],glutathione-S-transfera[GST], anthocyanin multidrug and toxic extrusion[antho-MATE],flavonol syntha[FLS4];Fig.1),and9-cis-epoxycarote-noid dioxygena1(NCED1),a gene involved in ABA biosynthesis in berry skins by quantitative real-time PCR (qRT-PCR)using a7300Real-Time PCR system(Applied Biosystems,Foster City,CA,USA)and a QuantiTect SYBR Green PCR kit(Qiagen,Hilden,Germany)as described in the manufacturers’manuals.Primer information for ampli-fication of the genes analyzed in the prent study is given in Table1.The relative expression level of each gene was determined by a standard curve-bad method.The standard DNA for each calibration curve was prepared by making a ries offive dilutions of plasmid DNA of each gene to cover the range of template concentrations.Data were calculated using calibration curves and normalized against Ubiquitin1 (Bogs et al.2006).qRT-PCR was carried out with three
replicates per sample,and the average expression level of each gene was calculated as the molar ratio relative to the copy number of Ubiquitin1.Statistical significance was evaluated using the Tukey–Kramer test.
Results
Total anthocyanin content and composition in the in vitro environmental experiment
Color development of berries in the15°C/light treatment started after4–5days of incubation and sufficient colora-tion was obrved after10days of incubation,whereas high temperature(35°C)or dark treatment suppresd anthocyanin accumulation verely(Fig.2a).The total anthocyanin contents of berry skin in the2008experiment were0.76,0.08,0.06,and0.03mg g-1fresh weight in the 15°C/light,15°C/dark,35°C/light,and35°C/dark treatments,respectively(Fig.2b).The total anthocyanin content in the15°C/light treatment was significantly higher than in the other three treatments.The total antho-cyanin content for each treatment in the2009experiment was similar to that in the2008experiment(Fig.2b).The results suggest that light irradiation at low temperature induces anthocyanin accumulation,but that dark or high-temperature treatment verely reduces it.
The anthocyanin compositions of the treatments are shown in Fig.3.Although the anthocyanin comp
osition of the berry skin in the35°C/dark treatment could not be determined becau the total anthocyanin content of the treated berry skins was too low,anthocyanin compositions varied among the three other treatments.In15°C/light, peonidin and malvidin derivatives were dominant(40.2and 28.1%of the total anthocyanin content,respectively)but the levels of malvidin derivatives decread in15°C/dark (17.6%),and both peonidin and malvidin derivatives decread in35°C/light(30.7and7.3%,respectively). Interestingly,peonidin-3-monoglucoside(Pn3G)was the dominant anthocyanin form in15°C/light(17.8%),but the percentage of Pn3G decread in15°C/dark(5.1%) and35°C/light(4.8%).On the other hand,the relative abundance of peonidin-3-(p-coumarylglucoside)-5-gluco-side(Pn3pG5G)incread in15°C/dark(24.0%)and 35°C/light(13.8%)compared to15°C/light.In the experiments,both temperature and light conditions affected the anthocyanin compositions in grape berry skin. Expression levels of MYB-related transcription factor genes
qRT-PCR analysis was carried out to compare the expression levels of MYB-related transcription factor genes in the berry skins among the four treatments.We detected
Table1Primers ud for the quantification of gene expression levels by qRT-PCR
Gene name Accession number Primer Sequence(50?30)Reference
唱歌游戏Ubiquitin1BN000705F GTGGTATTATTGAGCCATCCTT Bogs et al.(2006)
R AACCTCCAATCCAGTCATCTAC
VlMYBA1-2AB427164F TGGTCACCACTTGAAAAAGA Azuma et al.(2011)
R GAATGTGTTAGGGGTTTATT
VlMYBA1-3AB427165F GGCTTCTGGAGAGGTGCTTA Azuma et al.(2011)
R CTGTGTTGGGAAAATCCCA
VlMYBA2AB073013F GCTGAGCATGCTCAAATGGAT Azuma et al.(2011)
R TCCCACCATATGATGTCACCC
MYB4EF113078F ACCGGACGTTACAACCATATC Matus et al.(2008)
R TCCGTAACTGGGTTTTTCTCA
MYBPA1AM259485F CATGCACGTGCTCACCTT This study
R CCGCACGTATCGCTATTATAAG
MYB5a AY555190F GTGCAGCAGCCATCTAATGTG Matus et al.(2009)
R GCAGCAGGTTCCCAGACAGT
MYB5b AY899404F GGTGTTCTTTAATTTGGCTTCA Deluc et al.(2008)
R CACAACAACACAACCACATACA
MYBF1GQ423422F GGAGGTTGAGGGGTTGTG Czemmel et al.(2009)
R AAGTTGGGGAAGAGCAGGAG
CHS2AB066275F GAAGATGGGAATGGCTGCTG Jeong et al.(2004)
R AAGGCACAGGGACACAAAAG
CHS3AB066274F TCGGCTGAGGAAGGGCTGAA Jeong et al.(2004)
R GGCAAGTAAAGTGGAAACAG
CHI1X75963F CAGGCAACTCCATTCTTTTC Jeong et al.(2004)
R TTCTCTATCACTGCATTCCC
CHI2TC51784F TCCAGATCAAGTTCACAGCA Jeong et al.(2008)
R GAAATAAGAGCCTCAAAGAA
F3H1X75965F CCAATCATAGCAGACTGTCC Jeong et al.(2004)
R TCAGAGGATACACGGTTGCC
F3H2TC45490F CTGTGGTGAACTCCGACTGC Jeong et al.(2004)
R CAAATGTTATGGGCTCCTCC
F30H z F GCCTCCGTTGCTGCTCAGTT Jeong et al.(2006)
R GAGAAGAGGTGGACGGAGCAAATC
F3050H AB213606F AAACCGCTCAGACCAAAACC Jeong et al.(2006)
R ACTAAGCCACAGGAAACTAA
DFR X75964F GAAACCTGTAGATGGCAGGA Jeong et al.(2004)
R GGCCAAATCAAACTACCAGA
LDOX X75966F AGGGAAGGGAAAACAAGTAG Jeong et al.(2004)
R ACTCTTTGGGGATTGACTGG
UFGT AF000372F GGGATGGTAATGGCTGTGG Jeong et al.(2004)
R ACATGGGTGGAGAGTGAGTT
OMT FJ460168F GTTCAACTTCATGAGATGGA Azuma et al.(2009)
R GGAGAACTACCTCAACTACCA
GST RT043D09F ACTTGGTGAAGGAAGCAGGA Terrier et al.(2005)
R CAGCGAGCTCCATGACTTTT
电影行业antho-MATE FJ264202F GCAAACAACAGAGAGGATGC Cutanda-Perez et al.(2009)穿山甲有毒吗
R AGACCTCGACAATGATCTTAC
FLS4AB092591F AAACCACCTACTTACAGAGC Fujita et al.(2006)
R ACCTAACCCCAGTGACAGAC
