Ageorges, A.

Fernandez, L.

Torregrosa, L.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 45 (1), 51–52 (2006)Research NoteA putative NAP homolog specificallyexpressed during grapevine flowerand berry developmentL. FERNANDEZ, A. AGEORGES and L. TORREGROSAIntroduction: In flowering plants, fruit morphogenesisis a distinct and unique process following fertilization. Mostfleshy fruits as grapevine develop from ovary tissues (EAMESand MACDANIELS 1947) and exhibit convergent characteris-tics such as pericarp cell proliferation and enlargement. Theearly molecular events that control the dynamics of fruitmorphogenesis remain obscure. From preliminary analysisof the fleshless berry mutant (Vitis vinifera L. cv. Ugni Blanc)which is unable to specify ovary cell fate later in fleshypericarp development, we have identified a gene homologof PISTILLATA up-regulated in the mutant ovary. InArabidopsis, NAP (AJ222713) appeared as an immediate tar-get of the floral homeotic genes PISTILLATA/APETALA3(SLABLOWSKI and MEYEROWITZ 1998). NAP genes belong tothe NAC domain gene family encoding transcriptional regu-lators required for meristem establishment and other plantdevelopmental processes. The original genes NO APICALMERISTEM (NAM) from petunia, ATAF1, 2 and CUP-SHAPED COTYLEDON (CUC2) from Arabidopsis definedthe highly conserved NAC protein domain (AIDA et al. 1997).We report, here, quantitative expression patterns of the clos-est NAP grapevine homolog (TC38700 from the TIGR data-base), VvNAP, during grapevine development of the flbmutant and the wild type genotypes.Material and Methods: The flb mutation was identifiedin 1996 in a vineyard located at Prades-le-Lez (France) on aVitis vinifera cv. Ugni-Blanc anticlinal chimeric plant bear-ing some shoots with berries dramatically reduced in size.For flb mutant and wild-type Ugni Blanc, samples corre-sponded to inflorescences from stage 17 (mature inflores-cence with all flowers formed and separated) and 19 (firstflower caps loosening) of the E-L system (COOMBE 1995) andberries from 0 to 20 d after anthesis collected in the green-house (ENSA.M-INRA, Montpellier). For V. vinifera cv.Cabernet-Sauvignon, samples corresponded to vegetativeorgans (young roots, shoot tips including 2 to 3 expandedleaves and young leaves), inflorescences from stages 7 (im-mature inflorescence emerging from buds) and 17, flowers atflowering time (50 % caps off) and seedless berries(2-16 weeks post flowering), harvested in the field (ClareValley, Australia).Total RNA was extracted using RNeasy Plant Mini kit(Qiagen) for Ugni Blanc and according to REZAIAN and KRAKE(1987) with slight modifications as described in BOSS et al.(2001) for Cabernet-Sauvignon.After DNase treatment (RNase-Free DNase Set, Qiagen),reverse transcription of 1 µg total RNA was carried out us-ing Superscript III reverse transcriptase (Invitrogen) with a20mer-dT primer. Gene specific primer pairs were designedto amplify 150-250 bp in the 3’end of the VvNAP (TC38700)(F_ CTTAGCTCGGTTGCGGTTAC, R_ CTAGCGCTCTT-CCCTGACAA), VvUBI (CF406001) (F_AGTAGATGA-CTGGATTGGAGGT, R_GAGTATCAAAACAAAA-GCATCG) and Vvef1alpha (BQ799343) (F_GAACTGGG-TGCTTGATAGGC, R_AACCAAAATATCCGGAG-TAAAAGA) genes.PCR amplifications were performed from 1 µl of 1/10 di-luted ss cDNA, using the SYBR Green PCR Master Mix(Perkin -Elmer, Applied Biosystems) with a Rotor-Gene 2000(Corbett Research, NSW, Australia) or a 7700 Sequence De-tection System (Applied Biosystems, Warrington, UK) ap-paratus. Quantification was obtained by plotting the cyclethreshold value against the linear calibration curve obtainedwith serially diluted cDNA of the target gene. Sample valueswere corrected using the corresponding expression level ofthe control genes (VvUBI and Vvef1alpha) and expressedas the relative abundance ± SD.Results and Discussion: Soon after fertilization thefleshless berry (flb) mutation inhibits the development ofthe mesocarp resulting in a pericarp reduced in size by ap-proximately 20-fold at maturity. The flb mutant and its wild-type counterpart were previously used to screen genes po-tentially involved in early fruit morphogenesis. Among genesup-regulated in the mutant, a transcript coding for aPISTILLATA homolog was identified through suppressionsubtractive hybridizations (data not shown). This factor wasshown to activate the expression of NAP that was suggestedto function in the transition between growth by cell divisionand cell expansion in Arabidopsis floral tissues (SLABLOWSKIand MEYEROWITZ 1998). Search for NAP (AJ222713) similari-ties through tblastn programs in the grapevine TIGR data-base identified the TC38700 contig (VvNAP) showing a highdegree of similarity in the NAC protein domain (Fig. 1). Inthe flb mutant, VvNAP expression exhibited a constant over-expression compared to the wild-type, in both flower andyoung fruit organs (Fig. 2 A), which was compatible withPISTILLATA over-expression observed in this genotype (datanot shown). We supposed that PISTILLATA activated VvNAPexpression in grapevine, as observed for NAP in Arabidopsis.No expression of VvNAP was found in grapevine vegetativeorgans such as leaves, shoots or roots (Fig. 2 B), also simi-lar to NAP in Arabidopsis (SLABLOWSKI and MEYEROWITZ1998). Significant expression was detected in mature inflo-rescence, flower and in fruit at any stage of development,following a bimodal profile with a maximum of expressionlevel at stage of complete ripening (Fig. 2 B). The presentresults appeared somewhat paradoxical since up-regulationin the mutant suggested a negative effect of VvNAP on ovarydevelopment, while its specific expression in reproductiveorgans suggested an ongoing role on flower and fruit devel-opment. Over-expression of VvNAP could be a distant con-sequence of the flb mutation and, in this case, is possiblynot directly correlated to the mutant phenotype. Alterna-tively, a defined level of VvNAP may be necessary for properCorrespondance to: Dr. L. TORREGROSA, UMR BEPC Campus Agro-M/INRA, 2 place Viala, 34060, Montpellier Cedex 01, France.Fax: +33-4-9961-2616. E-mail laurent.torregrosa@ensam.inra.fr52 L. FERNANDEZ et al.Tissue typeI17 I19 0 3 6 10 15 20Transcript relative abundancy (%)020406080100 Wild-typeflb mutantTissue typeR S L I7 I17 F 2 4 6 8 10 12 16 Transcript relative abundancy (%)020406080100A BB (DAA) B (WPF)growth, as was considered for NAP since in its absence,cells cannot shift to the elongation mode, and with sus-tained expression, the transition between cell division andexpansion can not be finished (SLABLOWSKI and MEYEROWITZ1998). No expression of NAP was reported in the Arabidopsissilique and the role of NAC domain genes in fruit develop-ment remains poorly documented. In grapevine berries, ex-pression of some members of the NAC gene family havebeen reported (TERRIER et al. 2005). Although initial studiesfocused on the SAM function of NAC genes, it is increas-ingly clear that the NAC domain genes in the Arabidopsisgenome represent a broad class of regulatory genes uniqueto plants that functions in controlling diverse plant func-tions and probably inducing non-meristematic functionssuch as hormonal control and defense (DUVAL et al. 2002).We report here expression data about VvNAP, the putativeArabidopsis NAP ortholog, possibly important for grape-vine flower and fruit development. Additional experimentssuch as in situ localization and specific gene silencing stud-ies would be necessary to determine the precise function ofthis gene in grapevine.The authors thanks Drs M. R. THOMAS and C. DAVIES (CSIROPlant Industry, Adelaide) for Cabernet-Sauvignon total RNA,N. TERRIER and C. ROMIEU for technical assistance.AtNAP:     6  QSTLPPGFRFHPTDEELIVYYLRNQTMSKPCPVSIIPEVDIYKFDPWQLPEKTEFGENEW  Consensus:    Q  LPPGFRFHPTDEEL V YL     S P PV II EVD YKFDPW LP K  FGE EW VvNAP:     12 QPQLPPGFRFHPTDEELVVHYLKKKASSAPLPVAIIAEVDLYKFDPWELPAKASFGEQEW   AtNAP:        YFFSPRERKYPNGVRPNRAAVSGYWKATGTDKAI--HSGSSNVGVKKALVFYKGRPPKGI  Consensus:    YFFSPR RKYPNG RPNRAA SGYWKATGTDK      G   VGVKKALVFY G PPKGI VvNAP:        YFFSPRDRKYPNGARPNRAATSGYWKATGTDKPVLTSGGTQKVGVKKALVFYGGKPPKGI   AtNAP:        KTDWIMHEYRLHDSRKASTKRNG--------SMRLDEWVLCRIYKK 161 Consensus:    KT WIMHEYRL D  K  TK  G        S RLD WVLCRIYKK  VvNAP:        KTNWIMHEYRLADN-KVNTKPPGCDMGNKKNSLRLDDWVLCRIYKK 176 AIDA, M.; ISHIDA, T.; FUKAKI, H.; FUJISAWA, H.; TASAKA, M.; 1997: Genesinvolved in organ separation in Arabidopsis: An analysis of thecup-shaped cotyledon mutant. Plant Cell 9, 841-857.BOSS, P. K.; VIVIER, M.; MATSUMOTO, S.; DRY, I. B.; THOMAS, M. R.; 2001:A cDNA from grapevine (Vitis vinifera L.), which shows homol-ogy to AGAMOUS and SHATTERPROOF, is not only expressedin flowers but also throughout berry development. Plant Mol.Biol. 45, 541-553.COOMBE, B. G.; 1995: Adoption of a system for identifying grapevinegrowth stages. Aust. J. Grape Wine Res. 1, 104-110.DUVAL M.; HSIEH T. F.; KIM S. Y.; THOMAS T. L.; 2002: Molecularcharacterization of AtNAM: A member of the Arabidopsis NACdomain superfamily. Plant Mol. Biol. 50, 237-48.EAMES, A. J.; MACDANIELS, L. H.; 1947: An Introduction to PlantAnatomy. MacGraw-Hill, New York.REZAIAN, M. A.; KRAKE, L. R.; 1987: Nucleic acid extraction and virusdetection in grapevine. J. Virol. Methods 17, 277-285.SLABOWSKI, R. W. M.; MEYEROWITZ, E. M.; 1998: A homolog of NoApical Meristem is an immediate target of the floral homeoticgenes APETAL3/PISTILLATA. Cell 92, 93-103.TERRIER, N.; GLISSANT, D.; GRIMPLET, J.; BARRIEU, F.; ABBAL, P.; COUTURE, C.;AGEORGES, A.; ATANASSOVA, R.; LÉON, C.; RENAUDIN, J. P.; DÉDALDÉCHAMP,F.; ROMIEU, C.; DELROT, S.; HAMDI, S.; 2005: Isogene specific oligoarrays reveal multifaceted changes in gene expression duringgrape berry (Vitis vinifera L.) development. Planta (in press).Received September 5, 2005Fig. 1: Alignment of the NAC proteic domain, an N-terminal module of ~160 amino acids, of VvNAP (from TC38700, 353 amino acids)and AtNAP (CAA10955, 268 amino acids). Numbers corresponded to the NAC domain position in the protein.Fig. 2: Real time PCR expression profiling of VvNAP. A: Expression in the flb mutant and the wild-type: inflorescences from stage 17 (I17)and 19 (I19) of the E-L system (COOMBE 1995) and berries (B) at 0, 3, 6, 10, 15 and 20 d after anthesis (DAA). B: Expression in CabernetSauvignon grapevine organs and at various developmental stages: roots (R), shoots (S), leaves (L), inflorescences at stages 7 (I7) and stage17 (I17), seedless berries at 2, 4, 6, 8, 10, 12, 16 weeks post flowering (WPF). Expression values have been normalized with Vvef1á andVvUBI in A and B respectively (bars = SD of PCR technical triplicates).Transcript relative abundancy (%)Tissue type Tissue typeTranscript relative abundance (%)

A putative NAP homolog specifically expressed during grapevine flower and berry development

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A putative NAP homolog specifically expressed during grapevine flower and berry development

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