Identification of Yellow Pigmentation Genes in Brassica rapa ssp. pekinensis Using Br300 Microarray

Research ArticleIdentification of Yellow Pigmentation Genes in Brassica rapassp pekinensis Using Br300 Microarray

Hee-Jeong Jung1 Ranjith Kumar Manoharan1 Jong-In Park1 Mi-Young Chung2

Jeongyeo Lee3 Yong-Pyo Lim4 Yoonkang Hur5 and Ill-Sup Nou1

1Department of Horticulture Sunchon National University Suncheon Jeonnam 540-950 Republic of Korea2Department of Agricultural Education Sunchon National University Suncheon Jeonnam 540-950 Republic of Korea3Plant Systems Engineering Research Center KRIBB Daejeon 305-806 Republic of Korea4Department of Horticulture Chungnam National University Daejeon 305-764 Republic of Korea5Department of Biological Sciences Chungnam National University Daejeon 305-764 Republic of Korea

Correspondence should be addressed to Yoonkang Hur ykhurcnuackr and Ill-Sup Nou nissunchonackr

Received 14 October 2014 Accepted 14 December 2014 Published 31 December 2014

Academic Editor Mohamed Salem

Copyright copy 2014 Hee-Jeong Jung et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The yellow color of inner leaves in Chinese cabbage depends on its lutein and carotene content To identify responsible genes foryellow pigmentation in leaves the transcriptome profiles of white (Kenshin) and yellow leaves (Wheessen) were examined usingthe Br300K oligomeric chip in Chinese cabbage In yellow leaves genes involved in carotene synthesis (BrPSY BrPDS BrCRTISOand BrLCYE) lutein and zeaxanthin synthesis (BrCYP97A3 and BrHYDB) were upregulated while those associated with carotenedegradation (BrNCED3 BrNCED4 and BrNCED6) were downregulatedThese expression patterns might support that the contentof both lutein and total carotenoid was much higher in the yellow leaves than that in the white leaves These results indicate thatthe yellow leaves accumulate high levels of both lutein and 120573-carotene due to stimulation of synthesis and that the degradation rateis inhibited A large number of responsible genes as novel genes were specifically expressed in yellow inner leaves suggesting thepossible involvement in pigment synthesis Finally we identified three transcription factors (BrA20AN1-like BrBIM1 and BrZFP8)that are specifically expressed and confirmed their relatedness in carotenoid synthesis from Arabidopsis plants

1 Introduction

Chinese cabbage (Brassica rapa ssp pekinensis) which iswidely used vegetable in Asia particularly in China Japanand Korea is rich in vitamin C and minerals such aspotassium calcium magnesium and zinc This subspeciesis typical of the Brassica A genome and has a large numberof genomic resources including a mapping population andBAC libraries due to its small genome (529Mb) relative toother Brassica species The inner leaves can be white yellowor orange Earlier reports have revealed that the color ofthe inner leaves is primarily associated with their carotenoidcomposition Yellow inner leaves of Chinese cabbage havehigher nutritional values than white inner leaves [1] Inrecent years Asian countries have preferred yellow inner leaf

cultivars due to its high nutritional values and significanteconomic benefits

Carotenoids represent a class of red orange and yellowpigments widely distributed in nature that are mainly C

40

isoprenoids and play a critical role in human nutrition andhealth Because humans are unable to synthesize vitaminA denovo from endogenous isoprenoids precursors it is primarilyobtained through dietary plant carotenoids Apart from theirnutritional significance carotenoids have been implicatedin reducing the risk of cancer and cardiovascular diseasesvia their antioxidant activity [2 3] Thus development ofcarotenoid enriched food crops provides the most effectiveand sustainable approach to maximizing the nutritionaland health benefits of carotenoids to a large number ofpopulations worldwide

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2014 Article ID 204969 11 pageshttpdxdoiorg1011552014204969

2 International Journal of Genomics

The carotenoid pigment composition has been investi-gated inmanyBrassica vegetables including broccoli Brusselssprouts white cabbage red cabbage kale and cauliflower [4]and these investigations have indicated that lutein and 120573-carotene were the dominant carotenoids Chinese cabbagealso contains lutein and 120573-carotene as the major carotenoidpigment [5] with levels of 001 to 003mg100 g fresh weightgenerally being observed [6] The orange-yellow pigmenta-tion of Chinese cabbage expressed in inner leaves and petalsis governed by a single recessive gene The locus of the pig-mentation has beenmapped from threeRFLPmarkers closelylinked to each other [7] Microarray technology enablesgenome wide analyses that can be used to identify plantgene expression under various conditions including plantorganogenesis and interactions with microorganisms [8 9]Microarray experiments have been employed to analyze geneexpression changes in a number of crop species includingChinese cabbage [10 11]

The objective of this study was to identify the genesresponsible for yellow color pigmentation in B rapa innerleaves using a 300K Brassica rapa microarray with 47548unigenes

2 Materials and Methods

21 Plant Materials Two lines of Chinese cabbage (Brassicarapa ssp pekinensis) were used in this study hybrid cul-tivar Wheessen (Woori Seed Co Korea) for yellow innerleaves and the inbred line Kenshin for white inner leaves(Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014204969) Seedlings were trans-planted to cabbage patch at Chungnam National UniversityDaejeon Korea on September 1 After completion of theheading process (November 1) inner leaves were harvestedfrom 10 plants The blade portion of the leaves (excluding themidrib) was separated frozen in liquid nitrogen and storedat minus70∘C until use

The wild-type strain and T-DNA insertional mutants(SALK 045674 SALK 008677C SALK 098442C andSALK 012835) of Arabidopsis thaliana (L) Heynh varColumbia (Col-0) were collected from ABRC (httpwwwarabidopsisorg) Seeds were sterilized with 50 bleachsolution with 01 triton X-100 (Sigma USA) followedby germination on 60mm times 60mm pots in potting soilPlants were grown in growth chamber at 22 plusmn 05∘C with aphoton flux density of 140 plusmn 2 120583molm2sec and for a 16 hlight8 h dark photoperiod Mutants were confirmed by PCRusing primers that were specific for the gene sequence andthe T-DNA left border (Table S1) After 3 weeks all leavesfrom 20 plants were collected and subjected to measure thecarotenoid content

22 Measurement of Lutein and Carotene Content fromChinese Cabbage Leaf samples from each plant were groundin liquid nitrogen using a mortar and pestle after which1mL DMSO and 10mL methanol were added to 1 g ofsample and mixed well The sample was then incubated indarkness for 30min after which 5mL of hexane and 10mLof 20NaCl were added Next samples were centrifuged at

2100timesg for 10mins after which the supernatant was collectedand amended with 05 g of sodium sulfate The sampleswere then vortex and centrifuged at 2100timesg for 10minFinally the supernatant was filtered through a 045120583m PTFEand analyzed by high performance liquid chromatography(HPLC)

The lutein and carotene content were analyzed using anHPLC (Waters Corp Milford Mass USA) equipped with a2707 autosampler a 1525 binary pump and a C18 reversed-phase symmetry analytical column (26120583m times 100mm times46mm) with a guard column containing the same packingmaterial as the analytical column (PhenomenexKinetex)Thefollowing binary mobile phases were used during analysissolvent A 75 methanol in HPLC grade water solvent Bethyl acetateThe gradient for the HPLC analysis was linearlyapplied as follows 0 B at zero min 75 B at 10min and100 B at 14min Flow rate was set to 10mLmin at constantroom temperature the Waters UVvisible detector was setat 450 nm and carotenoid standards [lutein xanthophyll(alfalfa sigma) and 120573-carotene (wako)] were used to generatecharacteristic UV visible spectra and calibration curves

Individual carotenoids in the samples were tentativelyidentified based on comparison of their individual UV-visiblespectrum and retention time Lutein and 120573-carotene contentwere analyzed for each sample on the day of analysis Thepercentage of lutein and 120573-carotene was calculated per 1 g ofraw sample

23 Measurement of Total Carotenoid Content fromArabidop-sis Plants The content of total carotenoids from Arabidopsisplants was assayed by a simple and efficient method by Porraet al [12] Leaves were ground in liquid nitrogen with mortarand pestle OnemL of DMF (dimethylformamide) was addedto ca 100mg of powder in the dark The suspension wasmicrofuged by 12000 rpm at 4∘C for 10min and supernatantwas diluted by 10 times with DMF The absorbance of theresulting solution was measured at 461 and 664 nm Thecarotenoid content (120583gmL) was calculated by an equation[A461minus (0046 times A

664)] times 4 Finally its value was transformed

into 120583gg tissue

24 Construction of the Br300KOligomeric Chip andMicroar-rayAnalysis A300kmicroarray chip (Br50K version 20) forB rapa designed from 47548 unigenes (Table S2) was man-ufactured by NimbleGen Inc (httpwwwnimblegencom)as recently described [13] To assess the reproducibility ofthe microarray analysis we repeated the experiment twotimes using independently prepared total RNAThedatawerethen normalized and processed with cubic spline normal-ization using quantile to adjust signal variations betweenchips and robust multichip analysis (RMA) using a medianpolish algorithm implemented in NimbleScan [14 15] RNApreparation GeneChip hybridization and data analyses werealso conducted following previously described methods [16]

25 RT-PCR Analysis Total RNA (5 120583g) from each samplewas added with random hexamer primers in a superscriptfirst-strand cDNA synthesis system according to the man-ufacturerrsquos instructions (Invitrogen USA) Complementary

International Journal of Genomics 3

Table 1 Content of lutein and 120573-carotene in two contrastingChinese cabbage cultivars

B rapa cultivar Lutein(120583gg dry weight)

120573-carotene(120583gg dry weight)

Kenshin (whiteinner leaves) 019 plusmn 008 069 plusmn 008

Wheessen (yellowinner leaves) 4749 plusmn 159 1200 plusmn 061

DNA was diluted 10-fold and 1120583L of the diluted cDNAwas used in a 20120583L PCR mixture RT-PCR primers arelisted in Table S1 and the primers for BrACT1 which wasused as a control were 51015840-AGATCGTCCCCGGCTTCAAA-31015840 (forward) and 51015840-CAAGGCGTAAACGACGCAGG-31015840(reversed) Standard PCR was performed by subjecting thesamples to 5min initial denaturation at 94∘C followed by 25cycles of 94∘C for 30 s 55∘C for 30 s and 72∘C for 90 s PCRproducts were electrophoresed on a 1 agarose gel

3 Results

31 Carotenoid Analysis We compared the carotenoid com-position including lutein and120573-carotene in the inner leaves ofWheessen (yellow inner leaves) with those of Kenshin (whiteinner leaves) using HPLC analysis The content of lutein and120573-carotene is listed in Table 1 A lutein-representing peak waspresent in both Wheessen and Kenshin although Wheessenshowed a peak area higher than that found in Kenshin(data not shown) The content of lutein and 120573-carotene inWheessen inner leaves was higher than that of Kenshin innerleaves by 250- and 17-fold respectively These data wereconsistent with those observed for standard lutein and 120573-carotene The results supported that carotenoid becomes themain pigment in inner leaves of Chinese cabbage and isresponsible for their yellow color

32 Microarray Analysis To evaluate the pigmentation ofyellow inner leaves in Chinese cabbage we conductedmicroarray analyses using the version 2 Br300K chip (TableS2) Among 47548 genes on the Br300K chip 11543 genesshowed probe intensity (PI) values of less than 500 from twotested leaf samples To reduce the false positives we ignoredthese 11543 genes in subsequent analyses while the remain-ing 36005 genes were subjected to significance analysis ofmicroarray (SAM) [17] Thus genes with adjPValue or false(FDR) discovery rate below 005 were collected and furtherselected for those genes with expression greater than 2-foldor less than minus2-folds A total of 10199 were differentiallyexpressed 4807 that were upregulated and 5392 that weredownregulated in yellow inner leaves (Table S3) Highlyexpressed genes in yellow leaves like the top 20 upregulatedgenes in yellow inner leaves (Tables S3 and S4) appeared to benew genes whose functions have not been identified in plantsup to now Some of these novel genes might be directly orindirectly involved in the determination of yellow color inChinese cabbage

Interestingly Br300K microarray included 8542 uni-genes classified as no hit found upon initial analysis withArabidopsis thaliana annotation Among these 180 unigeneswere specifically expressed in yellow leaves (Table S5) Whenthese sequences were subjected to BlastN analysis some ofthe unigenes showed low similarity to Brassica sp and otherplant unigenes without known domain up to now (data notshown) Identification of function of these genes will beindispensable for further researches if the efficient and fastmethod of B rapa transformation is available

33 Functional Categorization of Genes Gene functions wereanalyzed based on gene ontology (GO) to simplify the anno-tation process using controlled vocabularies and hierarchiesincluding three major categories cellular component (CC)biological process (BP) and molecular function (MF) [18]All EST sequences that showed greater than 2-fold increaseswere classified into 2312 2334 and 2546 ESTs of CC BPandMF respectively while there were 2499 2546 and 2662downregulated genes of CC BP and MF respectively Themajority of the differently expressed genes were shown tobe involved in metabolic and cellular process and somewere components of the plasma membrane and plastids(chloroplast) The unique sequences were grouped under thefirst category of CC (Figure 1) which contained cell wallchloroplast cytosol ER extracellular region Golgi appara-tus mitochondria nucleus other cellular components othercytoplasmic components other intracellular componentsother membranes plasma membranes plastid ribosomeand unknown cellular components Previous studies havereported that carotenoids are also synthesized and accumu-lated in lipid bodies inside the chromoplasts and plastidswhich accumulate pigments in flowers fruits and storageroots Carotenoids are more stable in chromoplasts than inplastids because they are protected from light [19]The secondcategory included BP (Figure 2) with subcategories suchas cell organization and biogenesis development processesDNA or RNAmetabolism electron transport or energy path-ways other biological processes cellular processes metabolicprocesses protein metabolism response to abiotic or bioticstimulus stress signal transduction transcription trans-port and unknown biological processes Although enzymesappear to be of particular importance during biological pro-cess responses some carotenogenic enzymes such as geranyl-geranyl diphosphate (GGDP) synthase (GGPS) and phytoenesynthase (PSY) from the isoprenoid pathway are part of asoluble large protein complex that catalyzes the formationof phytoene in plastid stroma [20] A large protein complexPDS catalyzes the synthesis of 120572- and 120573-carotene fromphytoene in the plant plastid membrane [21] This phytoeneenzyme plays a key role in the carotenoid biosynthetic path-way The large numbers of unique sequences were groupedunder the third category of MF (Figure 3) with DNA orRNA binding hydrolase activity kinase activity nucleic acidbinding nucleotide binding other binding other enzymebinding othermolecular functions protein binding receptorbinding or activity structural molecule activity transcriptionfactor activity transferase activity transporter activity andunknownmolecular functionsThe expression ofmany genes


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Figure 1 Gene ontology characterization by loci for cellularcomponents

targeted to plastids is regulated through a signal between thenucleus and the plastidsThese signals whether environmen-tal or developmental are supposed to include reactive oxygenspecies (ROS) carotenoids and hormones Thus plastidsseem to be important to carotenoid biosynthesis [22]

34 Expression of Lutein and 120573-Carotene Synthesis-RelatedGenes To understand the relatedness of known genes thatregulate the carotenoid level expression of several categoriesof B rapa (Br) orthologous genes was analyzed (Table 2)These genes include 120572- and 120573-carotene synthesis-relatedgenes (phytoene synthase (PSY) PDS carotene isomerase(CRTISO) and lycopene epsilon cylase (LUT2)) luteinand zeaxanthin synthesis-related genes (carotene epsilon-monooxygenase (RUT1) lutein deficient 5 (LUT5) carotene120573-hydroxylase 1 (HYDB1) and HYDB2) and carotenoiddegradation genes (9-cis-epoxycarotenoid dioxygenase 3(NCED3) NCED4 and NCED6) Three known color-relatedgenes (pale CRESS1 (PAC1) cauliflower orange (OR) andimmutans (IM)) were used for references As shown inTable 2 the expression of genes associated with carotenoidsynthesis was higher in yellow inner leaves compared to thatin white inner leaves whereas the expression of degradationrelated genes was lower The level of Or gene that controls 120573-carotene content [23] was similar in both inner leaves whilePAC1 level that controls early chloroplast development [24]was high in white leaves suggesting these genes may notbe related to the determination of yellow color Howeverthe level of IM which determines source-sink interactions


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Table 2 Carotene and lutein synthesis-related genes

Classification Gene name Br Chip ID At locus PI value RT-PCRWhite Yellow W Y

Alpha- beta-carotenesynthesis

BrPSY Brapa ESTC045764 At5g17230 2591 8978

BrPDS Brapa ESTC002927 At4g14210 198 644

BrCRTISO Brapa ESTC016346 At1g06820 199 565

BrRUT2 Brapa ESTC013530 At5g57030 172 522

Lutein and zeaxanthinsynthesis



BrHYDB1 Brapa ESTC013493 At4g25700 1995 2768


Degradation

BrNCED3 Brapa ESTC007729 At3g14440 1848 1622



Color

BrPAC1 Brapa ESTC007240 At2g48120 4656 1719

BrOR Brapa ESTC013943 At5g61670 901 702

BrIM Brapa ESTC010363 At4g22260 1285 1523

BrACT1

which appeared to be little higher in yellow leaves ratherthan white inner leaves suggesting somewhat relatednessof chloroplast development with carotenoid biosynthesisMicroarray analysis was conducted in duplicate using bio-logical repeated samples and correlation coefficient betweenreplicated microarray analyses was found to be greater morethan 096 (Figure S2)TheRT-PCR expression ratio was com-pared with our microarray results and correlation coefficientvalues were found to be greater than 08 (Figure S3) Unfor-tunately some RT-PCR results were not consistent with PIvalues suggesting requirement of real-time PCR experiment

35 Identification of Yellow-Specific Responsive Genes Inaddition to carotenoid biosynthetic genes some of genesthat are predominantly expressed in yellow inner leaveswill be either directly or indirectly related to the yellowcolor determination in Chinese cabbage To identify leaf-color specific genes we selected genes as follows Yellow-specific genes were defined as those that had PI values ofover 1000 in yellow leaves but less than 500 in white leaves(Table S6) while white specific genes were those that hadPI values of over 1000 in white leaves but less than 500 inyellow leaves (Table S7) The total numbers of yellow and


white specific genes were 761 and 647 respectively implyingthat the expression of a large number of genes is essentialfor determination of pigmentation and other traits in eachline These inner color-specific genes also include manyunidentified genes up to now We confirmed expression ofsome of color-specific genes by RT-PCR (Table 3) As shownin Table 3 many genes were unidentified genes or B rapaspecific genes In addition some genes like chitinase andtaumatin protein gene might not be related to leaf colordetermination These results indicate that many of color-specifically expressed genes could be due to cultivar traitswhile some of them might be related to color determination

36 Identification of Transcription Factor Genes InducedSpecifically in Yellow Leaves Transcription factors controlthe expression of a genome and play vital roles in theplant life cycle Therefore we examined the transcriptionfactors associated with yellow pigmentation [25] Among2336 transcription factor genes 536 clones were foundto be expressed in low levels with a PI value lt 500 forboth yellow and white leaves while 312 clones were con-stitutively expressed in both leaves (Table S8) Twenty-onetranscription factors responsive to yellow color in B rapawere identified by comparison with Arabidopsis information(Table 4) Although these TFs have not been reported fortheir function with respect to carotenoid biosynthesis yetthere is a possibility of involvement of these genes in colordetermination in B rapa directly or indirectly Particularlythree genes showing high levels of expression in yellow innerleaves will be good candidates for yellow color regulationThese include Brapa ESTC013161 (A20AN1-like zinc fingerprotein) Brapa ESTC025847 (BIM1 (BES1-interacting MYC-like protein 1)) and Brapa ESTC006452 (ZFP8 (Zinc fingerprotein8))These genes may be responsible for the carotenoidsynthesis or related to pigmentmetabolismduring carotenoidproduction

Four T-DNA knock-out Arabidopsis mutants corre-sponding to three B rapa genes (Brapa ESTC013161Brapa ESTC025847 and Brapa ESTC006452) were analyzedfor carotenoid content by comparison with Arabidopsis wildtype (Table S9) The content of total carotenoid was greatlyreduced in T-DNA knock-out mutants implying theirpossible function in the carotenoid biosynthesis Particularlyknock-out of A20AN1-like zinc finger protein gene greatlyaffected the carotenoid content

4 Discussion

Transcriptome studies using microarray analyses are increas-ingly being used to identify key genes involved in plantgrowth development and responses to environmentalchanges Nevertheless microarray technology has not beenwidely applied to the study of Brassica species owing tothe absence of a high density Brassica microarray withsufficient coverage of the entire genome Instead Arabidopsismicroarray has been widely employed for analysis of Brassicaspecies because both species belong to the same family(Brassicaceae) and have an evolutionary close relationship

The Brassica genome has generally diverged fromArabidopsis[26 27] and consists of approximately 46000 genes [28]Although the Arabidopsismicroarray chip may provide someinformation regarding gene regulation of Brassica plants itis not sufficient to provide information regarding Brassicabiology [29] Therefore to accurately conduct transcriptomestudies a Brassica specific microarray Br300K was used inthe present study This microarray will also be useful ininvestigations of other Brassica species including B napusand B oleracea

Carotene and lutein contents are known key factorsassociated with yellow pigmentation in Chinese cabbageHowever no reports of specific genes involved in yellowcolor determination in Chinese cabbage have been publishedto date except the involvement of BrCRTISO in orangecolor determination [30] Researches to identify genes reg-ulating yellow inner leaves and to confirm inheritance ofits characteristics in Chinese cabbage are essential for theimprovement of Chinese cabbage qualityWe identifiedmanygenes that were specifically expressed in yellow inner leavesusing microarray experiment and confirmed some of themby RT-PCR Furthermore a high correlation was observedbetween microarray data of the two samples In addition ahigh correlation between RT-PCR analysis and microarraydata indicated that our data from theBr300Kmicroarraywerereliable and reproducible

Wheessen and Kenshin were used for HPLC analysis tocompare carotenoid composition In a previous study luteinwas found to be predominantly accumulated in yellow innerleaves [5 31] Carotenoid composition especially lutein inthe inner leaves of yellow cultivar was higher than thatin the inner leaves of orange cultivar upon HPLC analysis[30] Similar results were obtained from yellow inner leavescompared to white inner leaves (Table 1) Collectively thesedata confirmed that carotenoid profiles of the yellow innerleaf cultivars of Chinese cabbage were obviously higher thanand different from those of common white leaf cultivars [1]However there is no report on the involvement of relatedgenes and its expression We found out that expressionof lutein and 120573-carotene biosynthesis-related genes (BrPSYBrPDS BrRUT BrHYDB and BrCRTISO) is high in yellowinner leaves whereas expression of its degradation-relatedgenes is low (Table 2) In the carotenoid biosynthetic pathwayphytoene synthase (PSY) catalyzes the first committed reac-tion of the head to head condensation of two geranylgeranyldiphosphate (GGPP) molecules [20] BrHYDB acts as acatalyst for the production of zeaxanthin hydrolyzed from120573-carotene [32ndash34] BrPSY and BrHYDB are believed to beresponsible for120573-carotene and zeaxanthin synthesis [20]Theprimary role of BrCRTISO is as an enzyme that convertsprolycopene into lycopene in B rapa plants In this studycomparative analysis with Arabidopsis counterparts revealedthat BrCRTISO is likely responsible for carotenoid biosynthe-sisThese results are similar to those reported forArabidopsiswhich encodes a functional carotenoid isomerase and causesprolycopene accumulation when mutated [35] Lee et al[30] reported that BrCRTISO1 was not normally expressedin orange cultivars and this result indicates that a lack ofBrCRTISO1 transcript was the cause of sequence variation in


Table 3 Genes expressed specifically in either yellow or white inner leaves

Expression pattern Gene name Br SEQ ID At locus PI value RT-PCRWhite Yellow W Y

Yellow specific

Unknownprotein Brapa ESTC005061 At4g30660 37 11124

Chitinase Brapa ESTC005522 At3g47540 34 13990

CA18 Brapa ESTC013802 At5g14740 38 8063

Dynein lightchain Brapa ESTC010078 At4g27360 124 20072


Reductase Brapa ESTC013704 At5g52100 462 18255

Calciumbinding Brapa ESTC035004 At1g15860 497 11041

APX4 Brapa ESTC013724 At4g09010 437 12439

SHM2 Brapa ESTC017490 At5g26780 466 4033

SBPase Brapa ESTC047526 At3g55800 440 7571

Thylakoidprotein Brapa ESTC011620 At5g52970 456 6459


Brapa ESTC048334 no hits 13 21498








Table 3 Continued

Expression pattern Gene name Br SEQ ID At locusPI value RT-PCR

White Yellow W Y




White specific

Thaumatinprotein Brapa ESTC024359 At4g36010 7666 125




BrACT1

the orange cultivars when compared to the counterpart genein the yellow leaves cultivars This could be one of the causesresponsible for the yellow inner leaves phenotype

We identified 1 unknown gene and 9 putative genesresponsible for yellow pigmentation (Table 3) These genesshowed high expression in yellow leaves when comparedwithwhite leavesMoreover some stress-related genes-like such aschitinase dynein light chain and reductase genes [36] alsoshowed high expression in yellow leaves (Table 3) Althoughthese genes are not involved in carotenoid biosynthesis thismight be one of the causes of pigmentation In additiona large number of no hit found genes (novel genes in Brapa) (180 genes) were specifically expressed in yellow leaves(Table S5) It will be essential for identifying function of thesegenes in relation to leaf color determination Based on thesefindings functional studies should be conducted to clarify thenature of these unknown genes in Chinese cabbage

We identified 21 putative transcription factor genes asso-ciated with yellow color in B rapa Basic helix-loop-helixproteins and MYB proteins also function together to controlflower pigmentation in snapdragon [37] and petunia [38]MYB transcription factors act as the primary componentof color differences between plant varieties such as potatotomato pepper [16] and grape [39] and in some species ofAntirrhinum [40] Zinc binding proteins are triggered viaspecific mechanisms related to transcription factors involvedin important biological processes [41] These facts maysupport that these TFs will participate in the regulation ofcarotenoid content in B rapa

Regarding the three transcription factors that were highlyexpressed in yellow inner leaves (Table 4 Table S9) none ofthem has been reported as gene as determining color for-mation Arabidopsis orthologous (A20AN1-like zinc fingerprotein) of Brapa ESTC013161 shows induced expression bystress with redox-dependent regulation [42] and Arabidop-sis BIM1 controls embryonic patterning through brassinos-teroid signaling [43] There is no report on the functionof Brapa ESTC006452 orthologous (ZFP8) yet All theseresults suggest that transcription factors showing yellow-specific expression might be related to control the carotenoidbiosynthesis in B rapa

In conclusion we identified three genes as functionallynovel genes that are involved in yellow color pigmentationand also a large number of unknown genes exhibit func-tionally diverse transcription factor activity using newlydeveloped 300K Brassica rapa microarrays These unknowngenes may be responsible for yellow pigmentation in Chinesecabbage and the results showed that biological functionsof particular transcription factors may be activated in thepigmentation pathways Despite identification of some stressrelated genes not related to carotenoid biosynthesis-likechitinase reductase proteins might be one of the causes forthe yellow color trait Overall the results presented hereinwill provide a tool for future transcriptome studies andinvestigation of genes involved in carotenoid biosynthesis ofChinese cabbage as well as valuable insight into the responseto yellow pigmentation production


Table 4 List of transcription factors expressed specifically in yellow inner leaves

B rapa SEQ ID TAIR7 cds ID A thaliana homologue Description PI value Fold Change (YW)White leaves Yellow leaves

Brapa ESTC013161 AT1G51200 A20AN1-like zinc finger protein 161 2674 166Brapa ESTC025847 AT5G08130 BIM1 (BES1-interacting Myc-like protein 1) 280 3517 125Brapa ESTC006452 AT2G41940 ZFP8 (ZINC FINGER PROTEIN 8) 269 3323 124Brapa ESTC024811 AT5G64810 WRKY51 (WRKY DNA-binding protein 51) 155 1002 65Brapa ESTC017689 AT1G51600 ZML2 (ZIM-LIKE 2) 472 3009 64Brapa ESTC017762 AT2G18328 DNA binding 401 2336 58Brapa ESTC004343 AT4G16420 ADA2B (PROPORZ1) 286 1593 56

Brapa ESTC051029 AT1G49950 ATTRB1TRB1 (TELOMERE REPEATBINDING FACTOR 1) 396 1918 48


Brapa ESTC032962 AT4G31060 AP2 domain-containing transcriptionfactor putative 382 1582 41

Brapa ESTC006124 AT5G64810 WRKY51 (WRKY DNA-binding protein 51) 312 1166 37

Brapa ESTC001675 AT4G17810 Nucleic acid bindingtranscriptionfactorzinc ion binding 442 1601 36

Brapa ESTC011816 AT3G51960 bZIP family transcription factor 309 1079 35Brapa ESTC037364 AT4G12020 WRKY19 (WRKY DNA-binding protein 19) 412 1394 34Brapa ESTC014402 AT2G40750 WRKY54 (WRKY DNA-binding protein 54) 322 1021 32


Brapa ESTC011257 AT4G34680 GATA transcription factor 3 putative(GATA-3) 371 1095 30

Brapa ESTC025927 AT3G17609 HYH (HY5-HOMOLOG) 392 1152 29Brapa ESTC035196 AT4G30410 Transcription factor 480 1248 26

Brapa ESTC025999 AT3G50890 ATHB28 (ARABIDOPSIS THALIANAHOMEOBOX PROTEIN 28) 464 1189 26

Brapa ESTC030153 AT1G79180 AtMYB63 (myb domain protein 63) 470 1023 22

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by Golden Seed Project (Cen-ter for Horticultural Seed Development nos 213003-04-2-CG100 and 213003-04-2-SB230) Ministry of AgricultureFood and Rural Affairs (MAFRA) Ministry of Oceans andFisheries (MOF) RuralDevelopmentAdministration (RDA)and Korea Forest Service (KFS)

References

[1] P F ChenThe Study of Identify and Accumulation Mechanismsand Regulation Control of Carotenoid on Chinese CabbageYangzhou University 2008

[2] E Giovannucci ldquoTomatoes tomato-based products lycopeneand cancer review of the epidemiologic literaturerdquo Journal ofthe National Cancer Institute vol 91 no 4 pp 317ndash331 1999

[3] C W Hadley E C Miller S J Schwartz and S K Clin-ton ldquoTomatoes lycopene and prostate cancer progress andpromiserdquo Experimental Biology and Medicine vol 227 no 10pp 869ndash880 2002

[4] A Podsedek ldquoNatural antioxidants and antioxidant capacity ofBrassica vegetables a reviewrdquo LWTmdashFood Science and Technol-ogy vol 40 no 1 pp 1ndash11 2007

[5] R B H Wills and A Rangga ldquoDetermination of carotenoids inChinese vegetablesrdquo Food Chemistry vol 56 no 4 pp 451ndash4551996

[6] J Singh A K Upadhyay K Prasad A Bahadur and M RaildquoVariability of carotenes vitamin C E and phenolics in Brassicavegetablesrdquo Journal of Food Composition and Analysis vol 20no 2 pp 106ndash112 2007

[7] E Matsumoto C Yasui M Ohi and M Tsukada ldquoLinkageanalysis of RFLP markers for clubroot resistance and pig-mentation in Chinese cabbage (Brassica rapa ssp pekinensis)rdquoEuphytica vol 104 no 2 pp 79ndash86 1998

[8] H Qin T Feng S A Harding C-J Tsai and S Zhang ldquoAnefficient method to identify differentially expressed genes inmicroarray experimentsrdquo Bioinformatics vol 24 no 14 pp1583ndash1589 2008

[9] Z Zhang X Kou K Fugal and J McLaughlin ldquoComparisonof HPLC methods for determination of anthocyanins and


anthocyanidins in bilberry extractsrdquo Journal of Agricultural andFood Chemistry vol 52 no 4 pp 688ndash691 2004

[10] C Kim S Kikuchi K Satoh et al ldquoGenetic analysis of seedspecific gene expression for pigmentation in colored ricerdquoBiochip Journal vol 3 no 2 pp 125ndash129 2009

[11] K A Yang C J Lim J K Hong et al ldquoIdentification ofChinese cabbage genes up-regulated by prolonged cold by usingmicroarray analysisrdquo Plant Science vol 168 no 4 pp 959ndash9662005

[12] R J Porra W A Thompson and P E Kriedemann ldquoDeter-mination of accurate extinction coefficients and simultaneousequations for assaying chlorophyll a and b extracted with fourdifferent solvents verification of the concentration of chloro-phyll standards by atomic absorption spectroscopyrdquo Biochimicaet Biophysica vol 975 pp 384ndash394 1989

[13] X Dong H Feng M Xu et al ldquoComprehensive analysis ofgenic male sterility-related genes in Brassica rapa using a newlydeveloped Br300K oligomeric chiprdquo PLoS ONE vol 8 no 9Article ID e72178 2013

[14] R A Irizarry BM Bolstad F Collin LMCope BHobbs andT P Speed ldquoSummaries of Affymetrix GeneChip probe leveldatardquo Nucleic acids research vol 31 no 4 article e15 2003

[15] C Workman L J Jensen H Jarmer et al ldquoA new non-linear normalization method for reducing variability in DNAmicroarray experimentsrdquo Genome Biology vol 3 no 9 ArticleID research00481 2002

[16] W S De Jong N T Eannetta D M de Jong and M BodisldquoCandidate gene analysis of anthocyanin pigmentation loci inthe Solanaceaerdquo Theoretical and Applied Genetics vol 108 no3 pp 423ndash432 2004

[17] V G Tusher R Tibshirani and G Chu ldquoSignificance analysisof microarrays applied to the ionizing radiation responserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 98 no 9 pp 5116ndash5121 2001

[18] The Gene Ontology Consortium ldquoThe gene ontology in 2010extensions and refinementsrdquo Nucleic Acids Research vol 38supplement 1 pp D331ndashD335 2009

[19] M Vishnevetsky M Ovadis and A Vainstein ldquoCarotenoidsequestration in plants the role of carotenoid-associated pro-teinsrdquo Trends in Plant Science vol 4 no 6 pp 232ndash235 1999

[20] S Lu and L Li ldquoCarotenoid metabolism biosynthesis regula-tion and beyondrdquo Journal of Integrative Plant Biology vol 50no 7 pp 778ndash785 2008

[21] A B Lopez Y Yang T W Thannhauser and L Li ldquoPhytoenedesaturase is present in a large protein complex in the plastidmembranerdquo Physiologia Plantarum vol 133 no 2 pp 190ndash1982008

[22] I Egea C Barsan W Bian et al ldquoChromoplast differentiationcurrent status and perspectivesrdquo Plant and Cell Physiology vol51 no 10 pp 1601ndash1611 2010

[23] S Lu J van Eck X Zhou et al ldquoThe cauliflower Or geneencodes a DnaJ cysteine-rich domain-containing protein thatmediates high levels of 120573-carotene accumulationrdquo The PlantCell vol 18 no 12 pp 3594ndash3605 2006

[24] R S Reiter S A Coomber T M Bourett G E Bartley and PA Scolnik ldquoControl of leaf and chloroplast development by theArabidopsis gene pale cressrdquo Plant Cell vol 6 no 9 pp 1253ndash1264 1994

[25] C K Kim S H Park S Kikuchi et al ldquoGenetic analysis ofgene expression for pigmentation in Chinese cabbage (Brassicarapa)rdquo Biochip Journal vol 4 no 2 pp 123ndash128 2010

[26] C M OrsquoNeill and I Bancroft ldquoComparative physical mappingof segments of the genome of Brassica oleracea var alboglabrathat are homoeologous to sequenced regions of chromosomes4 and 5 of Arabidopsis thalianardquo The Plant Journal vol 23 no2 pp 233ndash243 2000

[27] DRana T van denBoogaart CMOrsquoNeill et al ldquoConservationof themicrostructure of genome segments inBrassica napus andits diploid relativesrdquo Plant Journal vol 40 no 5 pp 725ndash7332004

[28] T-J Yang J S Kim S-J Kwon et al ldquoSequence-level analysisof the diploidization process in the triplicated FLOWERINGLOCUS C region of Brassica rapardquo Plant Cell vol 18 no 6 pp1339ndash1347 2006

[29] S C Lee M H Lim J A Kim et al ldquoTranscriptome analysisin Brassica rapa under the abiotic stresses using Brassica 24KOligo microarrayrdquo Molecules and Cells vol 26 no 6 pp 595ndash605 2008

[30] S Lee S-C Lee D H Byun et al ldquoAssociation of molecularmarkers derived from the BrCRISTO1 gene with prolycopene-enriched orange-colored leaves in Brassica rapardquo Theoreticaland Applied Genetics vol 127 no 1 pp 179ndash191 2014

[31] MWatanabe KMusumi and J Ayugase ldquoCarotenoid pigmentcomposition polyphenol content and antioxidant activitiesof extracts from orange-colored Chinese cabbagerdquo LWT-FoodScience and Technology vol 44 no 9 pp 1971ndash1975 2011

[32] L Tian V Musetti J Kim M Magallanes-Lundback and DDellaPenna ldquoThe Arabidopsis LUT1 locus encodes a memberof the cytochrome P450 family that is required for carotenoid120576-ring hydroxylation activityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no1 pp 402ndash407 2004

[33] N Galpaz G Ronen Z Khalfa D Zamir and J HirschbergldquoA chromoplast-specific carotenoid biosynthesis pathway isrevealed by cloning of the tomato white-flower locusrdquo PlantCell vol 18 no 8 pp 1947ndash1960 2006

[34] J Kim and D DellaPenna ldquoDefining the primary route forlutein synthesis in plants the role of Arabidopsis carotenoid120573-ring hydroxylase CYP97A3rdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no9 pp 3474ndash3479 2006

[35] H Park S S Kreunen A J Cuttriss D DellaPenna and BJ Pogson ldquoIdentification of the carotenoid isomerase providesinsight into carotenoid biosynthesis prolamellar body forma-tion and photomorphogenesisrdquo Plant Cell vol 14 no 2 pp321ndash332 2002

[36] N U Ahmed J-I Park H-J Jung et al ldquoMolecular charac-terization of stress resistance-related chitinase genes of Brassicarapardquo Plant Physiology and Biochemistry vol 58 pp 106ndash1152012

[37] J Goodrich R Carpenter and E S Coen ldquoA common generegulates pigmentation pattern in diverse plant speciesrdquo Cellvol 68 no 5 pp 955ndash964 1992

[38] F Quattrocchio J F Wing K van derWoude J N MMol andR Koes ldquoAnalysis of bHLH andMYB domain proteins species-specific regulatory differences are caused by divergent evolutionof target anthocyanin genesrdquo Plant Journal vol 13 no 4 pp475ndash488 1998

[39] S Kobayashi M Ishimaru K Hiraoka and C Honda ldquoMyb-related genes of the Kyoho grape (Vitis labruscana) regulateanthocyanin biosynthesisrdquo Planta vol 215 no 6 pp 924ndash9332002


[40] K Schwinn J Venail Y Shang et al ldquoA small family of MYB-regulatory genes controls floral pigmentation intensity andpatterning in the Genus antirrhinumrdquo Plant Cell vol 18 no 4pp 831ndash851 2006

[41] H Takatsuji ldquoZinc-finger transcription factors in plantsrdquoCellu-lar andMolecular Life Sciences vol 54 no 6 pp 582ndash596 1998

[42] E Stroher X J Wang N Roloff P Klein A Husemann andK J Dietz ldquoRedox-dependent regulation of the stress-inducedzinc-finger protein SAP12 in arabidopsis thalianardquo MolecularPlant vol 2 no 2 pp 357ndash367 2009

[43] J W Chandler M Cole A Flier and W Werr ldquoBIM1 abHLH protein involved in brassinosteroid signalling con-trols Arabidopsis embryonic patterning via interaction withDORNROSCHEN and DORNROSCHEN-LIKErdquo Plant Molec-ular Biology vol 69 no 1-2 pp 57ndash68 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


The carotenoid pigment composition has been investi-gated inmanyBrassica vegetables including broccoli Brusselssprouts white cabbage red cabbage kale and cauliflower [4]and these investigations have indicated that lutein and 120573-carotene were the dominant carotenoids Chinese cabbagealso contains lutein and 120573-carotene as the major carotenoidpigment [5] with levels of 001 to 003mg100 g fresh weightgenerally being observed [6] The orange-yellow pigmenta-tion of Chinese cabbage expressed in inner leaves and petalsis governed by a single recessive gene The locus of the pig-mentation has beenmapped from threeRFLPmarkers closelylinked to each other [7] Microarray technology enablesgenome wide analyses that can be used to identify plantgene expression under various conditions including plantorganogenesis and interactions with microorganisms [8 9]Microarray experiments have been employed to analyze geneexpression changes in a number of crop species includingChinese cabbage [10 11]

The objective of this study was to identify the genesresponsible for yellow color pigmentation in B rapa innerleaves using a 300K Brassica rapa microarray with 47548unigenes

2 Materials and Methods

21 Plant Materials Two lines of Chinese cabbage (Brassicarapa ssp pekinensis) were used in this study hybrid cul-tivar Wheessen (Woori Seed Co Korea) for yellow innerleaves and the inbred line Kenshin for white inner leaves(Figure S1 in Supplementary Material available online athttpdxdoiorg1011552014204969) Seedlings were trans-planted to cabbage patch at Chungnam National UniversityDaejeon Korea on September 1 After completion of theheading process (November 1) inner leaves were harvestedfrom 10 plants The blade portion of the leaves (excluding themidrib) was separated frozen in liquid nitrogen and storedat minus70∘C until use

The wild-type strain and T-DNA insertional mutants(SALK 045674 SALK 008677C SALK 098442C andSALK 012835) of Arabidopsis thaliana (L) Heynh varColumbia (Col-0) were collected from ABRC (httpwwwarabidopsisorg) Seeds were sterilized with 50 bleachsolution with 01 triton X-100 (Sigma USA) followedby germination on 60mm times 60mm pots in potting soilPlants were grown in growth chamber at 22 plusmn 05∘C with aphoton flux density of 140 plusmn 2 120583molm2sec and for a 16 hlight8 h dark photoperiod Mutants were confirmed by PCRusing primers that were specific for the gene sequence andthe T-DNA left border (Table S1) After 3 weeks all leavesfrom 20 plants were collected and subjected to measure thecarotenoid content

22 Measurement of Lutein and Carotene Content fromChinese Cabbage Leaf samples from each plant were groundin liquid nitrogen using a mortar and pestle after which1mL DMSO and 10mL methanol were added to 1 g ofsample and mixed well The sample was then incubated indarkness for 30min after which 5mL of hexane and 10mLof 20NaCl were added Next samples were centrifuged at

2100timesg for 10mins after which the supernatant was collectedand amended with 05 g of sodium sulfate The sampleswere then vortex and centrifuged at 2100timesg for 10minFinally the supernatant was filtered through a 045120583m PTFEand analyzed by high performance liquid chromatography(HPLC)

The lutein and carotene content were analyzed using anHPLC (Waters Corp Milford Mass USA) equipped with a2707 autosampler a 1525 binary pump and a C18 reversed-phase symmetry analytical column (26120583m times 100mm times46mm) with a guard column containing the same packingmaterial as the analytical column (PhenomenexKinetex)Thefollowing binary mobile phases were used during analysissolvent A 75 methanol in HPLC grade water solvent Bethyl acetateThe gradient for the HPLC analysis was linearlyapplied as follows 0 B at zero min 75 B at 10min and100 B at 14min Flow rate was set to 10mLmin at constantroom temperature the Waters UVvisible detector was setat 450 nm and carotenoid standards [lutein xanthophyll(alfalfa sigma) and 120573-carotene (wako)] were used to generatecharacteristic UV visible spectra and calibration curves

Individual carotenoids in the samples were tentativelyidentified based on comparison of their individual UV-visiblespectrum and retention time Lutein and 120573-carotene contentwere analyzed for each sample on the day of analysis Thepercentage of lutein and 120573-carotene was calculated per 1 g ofraw sample

23 Measurement of Total Carotenoid Content fromArabidop-sis Plants The content of total carotenoids from Arabidopsisplants was assayed by a simple and efficient method by Porraet al [12] Leaves were ground in liquid nitrogen with mortarand pestle OnemL of DMF (dimethylformamide) was addedto ca 100mg of powder in the dark The suspension wasmicrofuged by 12000 rpm at 4∘C for 10min and supernatantwas diluted by 10 times with DMF The absorbance of theresulting solution was measured at 461 and 664 nm Thecarotenoid content (120583gmL) was calculated by an equation[A461minus (0046 times A

664)] times 4 Finally its value was transformed

into 120583gg tissue

24 Construction of the Br300KOligomeric Chip andMicroar-rayAnalysis A300kmicroarray chip (Br50K version 20) forB rapa designed from 47548 unigenes (Table S2) was man-ufactured by NimbleGen Inc (httpwwwnimblegencom)as recently described [13] To assess the reproducibility ofthe microarray analysis we repeated the experiment twotimes using independently prepared total RNAThedatawerethen normalized and processed with cubic spline normal-ization using quantile to adjust signal variations betweenchips and robust multichip analysis (RMA) using a medianpolish algorithm implemented in NimbleScan [14 15] RNApreparation GeneChip hybridization and data analyses werealso conducted following previously described methods [16]

25 RT-PCR Analysis Total RNA (5 120583g) from each samplewas added with random hexamer primers in a superscriptfirst-strand cDNA synthesis system according to the man-ufacturerrsquos instructions (Invitrogen USA) Complementary








3 Results






700

600

500

400

300

200

100

0

Num

ber o

f gen

es

Cel

l wal

lCh

loro

plas

tCy

toso

lER

Extr

acel

lula

rG

olgi

appa

ratu

sM

itoch

ondr

iaN

ucle

usO

ther

cellu

lar c

ompo

nent

sO

ther

cyto

plas

mic

com

pone

nts

Oth

er in

trac

ellu

lar c

ompo

nent

sO

ther

mem

bran

esPl

asm

a mem

bran

esPl

astid

Ribo

som

eU

nkno

wn

cellu

lar c

ompo

nent

s







1400

1200

1000

800

600

400

200

0

Num

ber o

f gen

es


Cell

org

aniz

atio

n an

d bi

ogen

esis

Dev

elopm

ent p

roce

ss

DN

A o

r RN

A m

etab

olism

Elec

tron

tran

spor

t or e

nerg

y pa

thw

ays

Oth

er b

iolo

gica

l pro

cess

es

Oth

er ce

llula

r pro

cess

es

Oth

er m

etab

olic

pro

cess

es

Prot

ein

met

abol

ism

Resp

onse

to ab

iotic

or b

iotic

stim

ulus

Resp

onse

to st

ress

Sign

al tr

ansd

uctio

n

Tran

scrip

tion

Tran

spor

t

Unk

now

n bi

olog

ical

pro

cess


800

700

600

500

400

300

200

100

0

Num

ber o

f gen

es

DN

A o

r RN

A b

indi

ngH

ydro

lase

activ

ityKi

nase

activ

ityN

ucle

ic ac

id b

indi

ngN

ucle

otid

e bin

ding

Oth

er b

indi

ngs

Oth

er en

zym

e bin

ding

sO

ther

mol

ecul

ar fu

nctio

nsPr

otei

n bi

ndin

gRe

cept

or b

indi

ng o

r act

ivity

Stru

ctur

al m

olec

ule a

ctiv

ityTr

ansc

riptio

n fa

ctor

activ

ityTr

ansfe

rase

activ

ityTr

ansp

orte

r act

ivity

Unk

now

n m

olec

ular

func

tions

















Degradation




Color




BrACT1







4 Discussion








Yellow specific



CA18 Brapa ESTC013802 At5g14740 38 8063


















Table 3 Continued


White Yellow W Y




White specific





BrACT1























Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








3 Results






700

600

500

400

300

200

100

0

Num

ber o

f gen

es

Cel

l wal

lCh

loro

plas

tCy

toso

lER

Extr

acel

lula

rG

olgi

appa

ratu

sM

itoch

ondr

iaN

ucle

usO

ther

cellu

lar c

ompo

nent

sO

ther

cyto

plas

mic

com

pone

nts

Oth

er in

trac

ellu

lar c

ompo

nent

sO

ther

mem

bran

esPl

asm

a mem

bran

esPl

astid

Ribo

som

eU

nkno

wn

cellu

lar c

ompo

nent

s







1400

1200

1000

800

600

400

200

0

Num

ber o

f gen

es


Cell

org

aniz

atio

n an

d bi

ogen

esis

Dev

elopm

ent p

roce

ss

DN

A o

r RN

A m

etab

olism

Elec

tron

tran

spor

t or e

nerg

y pa

thw

ays

Oth

er b

iolo

gica

l pro

cess

es

Oth

er ce

llula

r pro

cess

es

Oth

er m

etab

olic

pro

cess

es

Prot

ein

met

abol

ism

Resp

onse

to ab

iotic

or b

iotic

stim

ulus

Resp

onse

to st

ress

Sign

al tr

ansd

uctio

n

Tran

scrip

tion

Tran

spor

t

Unk

now

n bi

olog

ical

pro

cess


800

700

600

500

400

300

200

100

0

Num

ber o

f gen

es

DN

A o

r RN

A b

indi

ngH

ydro

lase

activ

ityKi

nase

activ

ityN

ucle

ic ac

id b

indi

ngN

ucle

otid

e bin

ding

Oth

er b

indi

ngs

Oth

er en

zym

e bin

ding

sO

ther

mol

ecul

ar fu

nctio

nsPr

otei

n bi

ndin

gRe

cept

or b

indi

ng o

r act

ivity

Stru

ctur

al m

olec

ule a

ctiv

ityTr

ansc

riptio

n fa

ctor

activ

ityTr

ansfe

rase

activ

ityTr

ansp

orte

r act

ivity

Unk

now

n m

olec

ular

func

tions

















Degradation




Color




BrACT1







4 Discussion








Yellow specific



CA18 Brapa ESTC013802 At5g14740 38 8063


















Table 3 Continued


White Yellow W Y




White specific





BrACT1























Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


700

600

500

400

300

200

100

0

Num

ber o

f gen

es

Cel

l wal

lCh

loro

plas

tCy

toso

lER

Extr

acel

lula

rG

olgi

appa

ratu

sM

itoch

ondr

iaN

ucle

usO

ther

cellu

lar c

ompo

nent

sO

ther

cyto

plas

mic

com

pone

nts

Oth

er in

trac

ellu

lar c

ompo

nent

sO

ther

mem

bran

esPl

asm

a mem

bran

esPl

astid

Ribo

som

eU

nkno

wn

cellu

lar c

ompo

nent

s







1400

1200

1000

800

600

400

200

0

Num

ber o

f gen

es


Cell

org

aniz

atio

n an

d bi

ogen

esis

Dev

elopm

ent p

roce

ss

DN

A o

r RN

A m

etab

olism

Elec

tron

tran

spor

t or e

nerg

y pa

thw

ays

Oth

er b

iolo

gica

l pro

cess

es

Oth

er ce

llula

r pro

cess

es

Oth

er m

etab

olic

pro

cess

es

Prot

ein

met

abol

ism

Resp

onse

to ab

iotic

or b

iotic

stim

ulus

Resp

onse

to st

ress

Sign

al tr

ansd

uctio

n

Tran

scrip

tion

Tran

spor

t

Unk

now

n bi

olog

ical

pro

cess


800

700

600

500

400

300

200

100

0

Num

ber o

f gen

es

DN

A o

r RN

A b

indi

ngH

ydro

lase

activ

ityKi

nase

activ

ityN

ucle

ic ac

id b

indi

ngN

ucle

otid

e bin

ding

Oth

er b

indi

ngs

Oth

er en

zym

e bin

ding

sO

ther

mol

ecul

ar fu

nctio

nsPr

otei

n bi

ndin

gRe

cept

or b

indi

ng o

r act

ivity

Stru

ctur

al m

olec

ule a

ctiv

ityTr

ansc

riptio

n fa

ctor

activ

ityTr

ansfe

rase

activ

ityTr

ansp

orte

r act

ivity

Unk

now

n m

olec

ular

func

tions

















Degradation




Color




BrACT1







4 Discussion








Yellow specific



CA18 Brapa ESTC013802 At5g14740 38 8063


















Table 3 Continued


White Yellow W Y




White specific





BrACT1























Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Degradation




Color




BrACT1







4 Discussion








Yellow specific



CA18 Brapa ESTC013802 At5g14740 38 8063


















Table 3 Continued


White Yellow W Y




White specific





BrACT1























Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4 Discussion








Yellow specific



CA18 Brapa ESTC013802 At5g14740 38 8063


















Table 3 Continued


White Yellow W Y




White specific





BrACT1























Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Yellow specific



CA18 Brapa ESTC013802 At5g14740 38 8063


















Table 3 Continued


White Yellow W Y




White specific





BrACT1























Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 3 Continued


White Yellow W Y




White specific





BrACT1























Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Identification of Yellow Pigmentation Genes in Brassica rapa ssp. pekinensis Using Br300 Microarray

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Transcript of Identification of Yellow Pigmentation Genes in Brassica rapa ssp. pekinensis Using Br300 Microarray