Southern analysis of genomic DNA indicated the presence of a multigene osmotin-like family in the grapevine genome. The expression of one of these genes that corresponds to the previously isolated pVVOSM1 cDNA is developmentally regulated showing high transcript levels in root, stem and leaf tissues of in vitro-grown plants, intermediate in calluses and berries and low in cell suspensions. Treatment of cell suspension cultures with abscisic acid (ABA), ethylene or NaCl resulted in high levels of expression of the pVVOSM1 gene suggesting regulation of the osmotin-like gene by the abiotic signals mentioned above

Loulakakis, K. A.

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Vitis 36(3), 157-158 (1997) Research Note Genomic organization and expression of an osmotin-like gene in Vitis vinifera L. K. A. LOULAKAKIS S u m m a r y : Southern analysis of genomic DNA indi-cated the presence of a multigene osmotin-like family in the grape-vine gei:tome. The expression of one of these genes that corre-sponds to the previously isolated pVVOSMl cDNA is developmentally regulated showing high transcript Ievels in root, stem and leaf tissues of in vitro-grown plants, intermediate in calluses and berries and low in cell suspensions. Treatment of cell suspension cu!tures with abscisic acid (ABA), ethylene or NaCI resulted in high Ievels of expression of the pVVOSMl gene sug-gesting regulation of the osmotin-like gene by the abiotic signals mentioned above. K e y w o r d s : Vitis vinifera, abscisic acid, ethylene, gene expression, osmotic stress, osmotin-like proteins. Introduction: Osmotin was originally isolated from to-bacco cell cultures adapted to an environment of Iow os-motic potential and numerous studies have implicated a putative function for osmotin and the related osmotin-like proteins in osmotic stress (SINGH et al. 1989; LARosA et al. 1992; ZHu et al. 1995). Basedon similarities in amino acid sequences andin expression pattems, osmotin and osmotin-like proteins have been classified as members of the fam-ily 5 of pathogenesis-related (PR) proteins (SINGH et al. 1989; BoL et al. 1990). Osmotin and osmotin-like pro-teins share significant sequence identity with the bifunc-tional a-amylase/trypsin inhibitor from maize, the sweet protein thaumatin and PR proteins (SINGH et al. 1989; CAsAs et al. 1992). The expression of osmotin and osmotin-like genes has been linked to development and is induced by various stress conditions provoked by biotic or abiotic Stimuli (LAROSA et al. 1992; NELSON et al. 1992; ZHu et al. 1995). Although osmotin and osmotin-like proteins are now relatively well-known in annual species they have not been extensively studied in perennial plants . Recently, the nucleotide sequence of the full-length pVVOSMl cDNA clone from grapevine was described (LouLAKAKIS 1997). This clone shows significant sequence similarities to annual plant osmotin and osmotin-like proteins. The purpose of this study was to investigate the gene organization and the expression pattems of the gene, corresponding to pVVOSMl, in vari-ous grapevine tissues and in response to abscisic add (ABA), ethylene and osmotic stress. Materialsand methods: Grapevine (Vitis vinifera L. cv. Sultanina) callus and cell suspension cultures were de-veloped and maintained as described by LouLAKAKIS and Department of Biology, University of Crete, P.O. Box 2208, GR-71409 Heraklion, Crete, Greece. Fax: +33-81-239155 or-233669. E-mail: loulakakis@nefeli.imbb.forth.gr RouBELAKIS-ANGELAKIS (1996). For induction experiments, filter-sterilized concentrated solutions of ABA, NaCI, su-crose and ethephon were added to culture medium 3 d after inoculation to final concentrations of 75 J.LM, 100 mM, 170 mM and 1 mM, respectively. In addition, Ieaf, stem and root tissues from in vitro-grown grapevine plants and berry tissue from greenhouse-grown plants were used in this work. High molecular weight genornic DNA was extracted from leaves of in vitro-grown grapevine plants by a modified cetyltrimethyl ammonium bromide procedure (LooHI et al. 1994). Genomic DNA was digested with the appropriate re-striction enzymes, fractionated on 0.7 .% agarose gels, blot-ted and hybridized at high stringency conditions as de-scribed by LouLAKAKIS and RouBELAKIS-ANGELAKIS (1996). Total RNA extraction and northem blot analysis were performed as described by LouLAKAKIS and RouBELAKIS-ANGELAKIS ( 1996). Results and Discussion: The genomic organization of the grapevine osmotin"Iike gene family was determined by Southem blot hybridization using the pVVOSMl insert as a probe. As illustrated in Fig. 1, multiple hybridizing bands were revealed; 9 EcoRI, 5 EcoRV and 4 Kpnl genomic frag-ments hybridized. It must be noticed that the pVVOSM1 nucleotide sequence contains one intemal EcoRI site but no EcoRV or Kpnl sites. These restriction data indicate that in grapevine there is a multigene osmotin-like family con-sisting of approximately 4 or 5 members. Thus, the grape-vine osmotin-Iike gene family seems tobe similar in com-plexity to the well characterized osmotin-like gene families of other plants (CAsAs et al. 1992; ZHu et al. 1995). The insert of pVVOSM1 hybridized to a transcript of approximately 1.0 kb which showed varying abundance in the grapevine tissues tested (Fig. 2 A). Loading of RNA from different tissues was normalized with regard to ribosomal RNA (Fig. 2 B). High Ievels of expression ofthe pVVOSM1 gene were observed in root, stem and leaf tissues, interme-diate in callus and berry tissues and low in cell suspensions at the stationary phase (12 d after inoculation). These re-sults are in accordance with previous reports on the differ-ential tissue specificity of osmotin and osmotin-like pro-teins from other plants. For example, tobacco osmotins are developmentally regulated in intact plants with higher Iev-els of expression in roots (SINGH et al. 1989; LAROSA et al. 1992; NELSON et al. 1992). The high transcript accumulation observed in grapevine leaves (Fig. 2) could be a characteris-tic of a particular stage of development of in vitro-grown plants. The expression ofthe grapevine osmotin-like genewas further characterized by northem blot analysis of total RNA extracted from cell cultures treated with ABA, 2-chloro-ethylphosphonic acid (ethephon, an ethylene-releasing agent), NaCI and sucrose for 2 d. Loading of RNA was nor-malized with regard to ribosomal RNA (data not shown). As shown in Fig. 3, in the control cells transcript Ievels remained rather constant during this period. Culture of cells in the presence of ABA or ethephon resulted to increase steady-state mRNA Ievels that progressively accumulated for up to 48 h. In addition, NaCI- (Fig. 3) and sucrose-treated 158 K. A. LOULAKAKIS A -21.2 B - 5.1 -4.3 A -3.5 -2.0 B - 1.6 - 1.4 c D =~····· 0 6 12 24 48 Time (h) 1.0 kb @ Fig. I (left): Southem blot analysis of grapevine genomic DNA. Blot of genomic DNA (10 ~-tg per lane) digested with EcoRI, EcoRV or Kpnl was probed with the pVVOSMI insert. Arrows denote the position ofthe more weakly hybridizing bands. The position ofthe size standards are indicated on the right in kb. Fig. 2 (right, top): RNA gel blot analysis of osmotin-like transcript in different grapevine tissues. A. Blot of total RNA (20 11g per lane) isolated from roots, stems and leaves of in vitro-grown plants, cal-luses, cell suspensions at the stationary phase and immature berries. The blot was hybridized to 32P-labeled pVVOSMl cDNA. B. Ethidium bromide stained native agarose gel of the respective total RNAs. R, roots; S, stems; L, leaves; Ca, calluses; Ce, cells; ' B, berries. Fig. 3 (right, bottom): Induction of osmotin-like mRNA Ievel in grape-vine cells by ABA, ethylene and NaCI. Cell cultures were treated with ABA (B) or ethephon (C) or NaCI (D) or an equal volume of water (A, control) for the indicated times. Blots oftotal RNA (20 )lg per lane) were hybridized to 32P-labeled pVVOSMI cDNA. cells (data not shown) accumulated the osmotin-like tran-script 12 h after the low water potential treatment and reached high Ievels after 48 h. lt must be noticed that the rate of accumulation of osmotin-like mRNA was similar between the 4 treatments tested. The above mentioned re-sults indicate that expression of the grapevine osmotin-like gene is upregulated by all stress inducing factors that were tested. Regulation of osmotin and osmotin-like gene expres-sion appears to be very complex in annual plants (LARosA et al. 1992; ZHu et al. 1995). These genes are develop-mentally regulated and also controlled by multiple signals including ABA, salicylic acid, virus infection, low water potentials, cold temperature, wounding and ethylene (SINGH et al. 1989; KoNONOWICZ et al. 1992; LARosA et al. 1992; ZHu et al. 1995). In addition, products of these genes have been shown to exhibit in vitro antifungal activity (WoLOSHUK et al. 1991 ). Thus, it was proposed that induc-tion of osmotins by osmotic stress may merely reflect the activation of a general stress response of plants which evolved fo defend pathogens (KoNONOWICZ et al. 1992; LARosA et al. 1992). The expression of grapevine osmotin-like gene is clearly induced by ABA and ethylene, the two plant hormones related to both, osmotic- and pathogen-induced stresses. However, further experiments are needed to determine the properties of this gene and the biological function of its product. The author thanks Prof. K.A. RouBELAKIS-ANGELAKIS for her support, for providing Iabaratory space and reading the manu-script, Prof. N. PANOPOULOS for his constructive comments on the manuscript and Mr. N. PRIMIKIRlOS for he1pful discussions. This work was supported in part by a grant from Peripheral Operational Programme of Crete to K.A. RouBELAKIS-ANGELAKIS. BoL, J. F.; LINTHORST, H. J. M.; CORNELISSEN, B. J. C.; 1990: Plant pathogenesis-related proteins induced by virus infection. Ann. Rev. Phytopathol. 28, 113-138. CASAS, A. M.; NELSON, D. E.; RAGHOTHAMA, K. G.; D'URZO, M. P.; S!NGH, N. K.; BRESSAN, R .A.; HASEGAWA, P. M.; 1992: Expression of osmotin-like genes in the halophyte Atriplex numrnularia L. Plant Physiol. 99, 329-337. KoNoNowrcz, A. K.; NELSON, D. E.; SrNGH, N. K.; HASEGAWA, P. M.; BRESSAN, R. A.; 1992: Regulation of the osmotin gene promoter. Plant Cell 4, 513-524. LAROSA, P. C.; CHEN, Z.; NELSON, D. E.; S!NGH, N. K.; HASEGAWA, P. M.; BRESSAN, R. A.; 1992: Osmotin gene expression is posttranscriptionally regulated. Plant Physiol. 79, 138-142. LoDHI, M. A.; YE, G. N.; WEEDEN, N. F.; REISCH, B. I.; 1994: A simple and efficient method for DNA extraction from g~apevine cultivars and Vitis species. Plant Mol. Biol. Reptr. 12, 6-13. LouLAKAKIS, K. A.; 1997: Nucleotide sequence of a Vitis vinifera L. cDNA (Accession No. Yl0992) encoding for osmotin-like pro-tein. (PGR97-064), Plant Physiol. 113, 1464. - -; RouBELAKIS-ANGELAKIS, K. A.; 1996: Characterization of Vitis vinifera L. glutamine synthetase and molecular cloning of cDNAs for the cytosolic enzyme. Plant Mol. Bio!. 31, 983-992. NELSON, D. E.; RAGHOTHAMA, K. G.; SINGH, N. K.; HASEGAWA, P, M.; BRESSAN, R. A.; 1992: Analysis of structure and transcriptional activation of an osmotin gene. Plant Mol. Biol. 19, 577-588. S!NGH, N. K.; NELSON D. E.; KUHN D.; HASEGAWA P. M.; BRESSAN R. A.; 1989: Molecular cloning of osmotin and regulation of its ex-pression by ABA and adaptation to low water potential. Plant Physiol. 90, 1096-1101. WoLOSHUK, C. P.; MEULENHOF, J. S.; SELA-BUURLAGE, M.; VAN DEN ELZEN, P. J. M.; CoRNELISSEN, B.J.C.; 1991: Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. Plant Cell3, 619-628. ZHu, B.; CHEN, T. H. H.; LI, P. H.; 1995: Activation of two osmotin-like protein genes by abiotic stimuli and fungal pathogen in transgenic potato plants. Plant Physiol. 108, 929-937. 

Genomic organization and expression of an osmotin-like gene in Vitis vinifera L.

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Genomic organization and expression of an osmotin-like gene in Vitis vinifera L.

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