Research NoteEmbryogenic and non-embryogenic cell lines were obtained from the rootstock 41B (Vitis vinifera cv. Chasselas x V. berlandieri). A few extracellular proteins from culture media of embryogenic and non-embryogenic grapevine cell lines during multiplication showed a positive reaction with anti-chitinase antibodies (embryogenic cell lines) and anti-β-1,3-glucanase antibodies (non-embryogenic cell lines)

Bierne, J.

Bonnet, E.

Deloire, A.

Kraeva, E.

Mauro, M. C.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 36 (1), 51-52 (1997) Research Note Immunodetection of chitinase-like and ß-1,3-glucanase-like proteins secreted in vitro by embryogenic and non-embryogenic cells of grapevines A. DELOIRE1), E. KRAEvA1), M. C. MAuRo2), E. BoNNET3) and J. BIERNE3) S u m m a r y : Embryogenie and non-embryogenic cell lines were obtained from the rootstock 41B (Vitis vinifera cv. Chasselas x V. berlandieri). A few extracellular proteins from culture media of embryogenic and non-embryogenic grapevine cell lines during multiplication showed a positive reaction with anti-chitinase antibodies (embryogenic celllines) and anti-ß-1,3-glucanase antibodies (non-embryogenic cell lines). K e y w o r d s : grapevine somatic embryogenesis, chitinase, ß-1 ,3-glucanase, Western blotting. Introduction: The genetic transformation of grape-vine by Agrobacterium tumefaciens is one way to protect grapevines agairrst diseases. This technology requires an efficient regeneration system after cell transformation to obtain genetically improved plants (MARTINELLI et al. 1993; LE GALL et al. 1994; MAURO et al. 1995). The study of intra- and extracellular proteins of cells by in vitro culture would enlarge our understanding of the ability of plants for cell multiplication, development and regeneration (DE V RIESet al. 1988; Coums-THEVENOT et al. 1992; DoMoN et al. 1994 ). Recent results suggest both, a structural role and a developmental function of proteins which are involved in defense mechanisms of plants ('pathogenesis-related pro-teins', PRs), as described by VAN LooN et al. 1994). We have compared pattems of secreted proteins from grape-vine cell suspension culture media (containing NOA) of embryogenic and non-embryogenic celllines at the multi-plication stage. We focused on PRs-like proteins detected by antibodies raised agairrst two families of tobacco PR-proteins: ß-1 ,3-glucanase (PRs-2) and chitinase (PRs-3). Materials and methods: Different sources of explants of the rootstock cultivar 41B (Vitis vinifera cv. Chasselas x V. berlandieri) were used to produce initial calli. Embryogenie cell lines (E-cells) were established from diploid tissue deriving from anther culture whereas non-embryogenic cell lines (NE-cells) originated from leaves 1) ENSA-INRA, U.F.R. de Viticu1ture, 2 place Viala, F-34060 Montpellier cedex 1, France. Corresponding authors. 2) Moet & Chandon, BP 140, F-51333 Epemay, France. 3) Laboratoire de Biologie cellulaire, Universite de Reims Champagne-Ardenne, BP 1039, F-51687 Reims, France. of in vitro plantlets (DELOIRE, unpubl. data). For multipli-cation of E-cell and NE-cell lines, subcultures were car-ried out in liquid media, containing 5 mM 2-naphthoxy-acetic acid (NOA), at 22 °C in the dark, according to MAURO et al. (1995). Proteins secreted in liquid media were ex-tracted following the method described by CouTos-THEVENOT et al. (1992). SOS-PAGE electrophoresis was performed using the minislab gel apparatus mini Proteans II (Bio-Rad, Rieb-mond, CA) and the method of LAEMMLI (1970). Immuno-logical detection was performed according to TRIRIET and ALBERT (1995). Antisera of rabbits were used (provided by B. FRITIG, CNRS, Strasbourg), which were raised agairrst the tobacco PRs-2 (ß-1,3-glucanase) and PRs-3 (chitinase) families (KAUFFMANN et a/. 1987; LEGRAND et al. 1987). The immuno precipitates were detected using a peroxidase conjugated anti-rabbit IgG revealed by diaminobenzidine. Results: Differentkinds of cell growth and subsequent development according to the origin of the cultures were observed. Only E-cells regenerated to embryos when the cells were transferred to the medium (CouTos-THEVENOT et al. 1992 b) without phytohormone, unlike NE-cells which grew unorganized comparable to the growth of undifferentiated calli cells. A comparison of Western blottings of extracellular pro-teins showed a clear difference between lines of E- and NE-cells at the multiplication stage. Anti-chitinase (anti-Q) antiserum reacted only with proteins secreted into the E-cell line culture media during multiplication and developmen-tal stages (bands around 34 kDa) from 10-d-old cultures. No reaction was detected with the secreted proteins from NE-cells (Fig. 1 ). In contrast, anti-ß-1 ,3-glucanase antisera (anti-2 and anti-gluc b) showed reactions only with pro-teins secreted from NE-celllines (bands around 35 kDa), from 4-, 7- and 10-d-old cultures. No reaction was detected with proteins secreted from the E-celllines (Fig. 2). a b c d Fig. 1: Western blot of secreted proteins from embryogenic cells at the multiplication stage (a) and at the developmental stage (b). The antiserum used is raised against tobacco PR-3 chitinase (Anti-Q). A 34 kDa chitinase-like proteinband is detected among extracellular proteins in both stages (a, b). No band was detected among extracellular proteins of non-embryogenic cells at the multiplication (c) and at the developmental stages (d). 52 A. DELOIRE, E . KRAEVA, M . C. MAURO, E . BaNNET and J. BIERNE NE10 NE7 NE4 E10 E7 E4 NE10 NE7 NE4 E10 E7 E4 Fig. 2: Western blots of secreted proteins from embryogenic cells (E; 4, 7 and 10 days) and non-embryogenic cells (NE; 4, 7 and 10 days). The antisera used are raised against tobacco PR-2, ß-1 ,3-glucanase (anti-2 and anti-gluc b ). The 35 kDa ß-1,3-glucanase-like protein bands were detected among extracellular proteins of non-embryogenic cells with both, anti-2 (A) and anti-gluc b (B) antisera. No band was detected among secreted pro-teins from embryogenic cells (E; 4, 7 and 10 days). Discussion: The embryogenic and non-embryogenic grapevine celllines incubated under the same culture con-ditions differed in their total pattern of secreted proteins (KRAEVA, unpubl. data). Among the released proteins, chitinase and ß-1,3-glucanase activities were detected in different culture systems, both in embryogenic and non-embryogenic cultures (ESAKA et al. 1990; GAVISH et al. 1991). For the first time, we have shown with immuno-logical techniques a) the presence of chitinase-like pro-teins among extracellular proteins of embryogenic celllines and b) the presence ofß-1,3-glucanase-like proteins among extracellular proteins in non-embryogenic cell lines in grapevines. In the absence of a known chitinase substrat (i.e. chitin) in plant cells, we may raise the question whether chitinase plays a role in plants multiplication or development. RöHRIG et al. (1995) have considered that lipochitooligosacchar-ides (LCOs), which may be present in plant cell walls (or homologous structures), are hydrolysed by chitinase. In tobacco protoplast cultures, the absence of phytohormones was compensated by the addition of synthetic LCOs. The authors conclude that LCOs, which are hydrolysed by chitinase, can be considered as plant growth regulators. We have shown a correlation between extracellular chitinase-like proteins and embryogenic cells in a peren-nial plant. We pursue this study with grapevine homolo-gaus chitinase and glucanase probes and/or antibodies, comparing intra- and extracellular cell proteins in vitro. The authors are grateful to Prof. B. FRITIG (CNRS Stras-bourg, France) for antisera; Dr. M. BouLAY (Moet Hennessy Louis Vuitton Recherche, France) and Prof. J. ScHELL (Max Planck Institut, Germany) for kind advices. Part of the financial support of this work was provided by the Moet & Chandon Company. Couros-THEVENOT P.; GoEBEL-TouRANT 1.; MAURO M.C.; JouANNEAU J.P.; BouLAY M.; DELOIRE A.; GuERN J.; 1992 b: Somatie embryogenesis from grapevine cells. I. lmprovement of embryo deve1opment by ehanges in eulture eonditions. Plant Cell Tiss. Org. Cult. 29, 125-133. - -; MAEs, 0.; JouENNE, T.; MAURO, M. C.; BouLAY, M.; DELOIRE, A.; GuERN, J. ; 1992: Extraeellular protein pattems of grapevine cell suspensions in embryogenic and non embryogenie situations. Plant Sei . 86, 137-145. DE V RIES, S. C.; Boou, H.; JANSSENS, R.; VOGELS, R.; SARIS, L.; LoscHJAVO, F.; TERZ!, M.; V AN KAMMEN, A.; 1988: Carrot somatie embryogenesis depends on the phytohormone controlled presenee of eorreetly glyeosylated extracellular proteins. Genes Develop. 2, 462-476. DoMoN, J. M.; MEYER, Y. ; FAYE,L.; DAVIo,A.; DAVID, H.; 1994: Extracellular (glyeo)proteins in embryogenie and non-embryogenic celllines of Caribbean pine. Camparisan between phenotypes of stage one so-matic embryos. Plant Physiol. Biochem. 32, 137-147. EsAKA, M.; ENoKI, K.; KoucHI, B.; SASAKI, T.; 1990: Purifieation and char-aeterization of abundant seereted protein in suspension-eultured pumpkin eells. Plant Physiol. 93, 1037-1041. GAVISH, H.; VARDI, A.; FLUHR, R.; 1991 : Extraeellular proteins and early embryo development in nuclear cell eultures. Physiol. Plant. 82, 606-616. KAUFFMANN, S.; LEGRAND, M.; ÜEOFFROY, P.; FRITIG, B.; 1987: Biological funetion of "pathogenesis-related" proteins: four PR proteins of to-baeeo have 1,3-ß-g1ucanase activity. Embo J. 6, 3209-3212. LAEMMLI, U.; 1970: Cleavage of struetural proteins during the assembly of head of baeteriophage T4. Nature 277, 680-685. LE GALL, 0. ; ToRREGROSA, L.; DANGLOT, Y.; CANDRESSE, T. ; BouQuET, A.; 1994: Agrobaeterium-mediated genetic Iransformation of grapevine somatic embryos and regeneration of transgenie p1ants expressing the eoat protein of grapevine ehrome mosaic nepovirus (GCMV). Plant Sei. 102, 161-170. LEGRAND M.; KAuFFMANN S.; GEOFFROY P. ; FRITIG B.; 1987: Biological fune-tion of pathogenesis-re1ated proteins: Four tobaeeo pathogenesis-re-lated proteins are ehitinases. Proe. Nat. Aead. Sei. 84, 6750-6754. MARTINELLI L.; BRAGAGNA P. ; PaLETTI V.; SciENZA A.; 1993: Somatie embryogenesis from leaf- and petio1e-derived eallus of Vitis rupestris. Plant Cell Rep. 12, 207-210. MAURO, M. C.; TouTAIN, S.; WALTER, B.; PINCK, L.; 0TTEN, L.; Couros-THEVENOT, P.; DELOIRE, A.; BARBIER, P.; 1995: High effieiency regen-eration of grapevine plants transformed with the GFLV eoat protein gene. Plant Sei. 112, 97-106. RöHRIG, H.; ScHMIDT, J.; W ALOEN, R.; CzAJA, 1.; MIKLASEVICS, E.; WIENEKE, U .; ScHELL, J .; MicHAEL, J.; 1995: Growth of tobacco protoplasts stimu-lated by synthetic lipo-ehitooligosaeeharides. Scienee 269, 841-843. THIRIET, C.; ALBEIIT, P.; 1995: Rapid and effective Western blotting ofhistones from AUT and SDS polyacrylamide gels: two different approaehes depending on the subsequent qualitative or quantitative analysis. Eleetrophoresis 16, 357-361. VAN LooN, L. C.; PIERPOINT, W. S.; BoLLER, T.; CoNEJERO, V. ; 1994: Recom-mendations for naming plant Pathogenesis-Related proteins. Plant Mol. Bio!. Rep. 3, 245-264. 

Immunodetection of chitinase-like and beta-1,3-glucanase-like proteins secreted in vitro by embryogenic and non-embryogenic cells of grapevines

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Immunodetection of chitinase-like and beta-1,3-glucanase-like proteins secreted in vitro by embryogenic and non-embryogenic cells of grapevines

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