In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5–6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.

[1]  C. Clément,et al.  Bioproduction of resveratrol and viniferins by an elicited grapevine cell culture in a 2 L stirred bioreactor , 2011 .

[2]  Weidong Huang,et al.  Distribution of resveratrol and stilbene synthase in young grape plants (Vitis vinifera L. cv. Cabernet Sauvignon) and the effect of UV-C on its accumulation. , 2010, Plant physiology and biochemistry : PPB.

[3]  Markus Riederer,et al.  Biology of the plant cuticle , 2006 .

[4]  G. Hoos,et al.  Influence of resveratrol on germination of conidia and mycelial growth of Botrytis cinerea and Phomopsis viticola. , 1990 .

[5]  Ismael Moya,et al.  Ultraviolet-induced fluorescence for plant monitoring: present state and prospects , 1999 .

[6]  W. Dercks,et al.  The significance of stilbene phytoalexins in the Plasmopara viticola-grapevine interaction , 1989 .

[7]  S. Citterio,et al.  Localization of stilbene synthase in Vitis vinifera L. during berry development , 2008, Protoplasma.

[8]  Robert C. Weast,et al.  Handbook of chemistry and physics : a readyreference book of chemical and physical data , 1972 .

[9]  F. García-Carmona,et al.  Aggregation state and pKa values of (E)-resveratrol as determined by fluorescence spectroscopy and UV-visible absorption. , 2008, Journal of agricultural and food chemistry.

[10]  J. Harborne.  The comparative biochemistry of phytoalexin induction in plants , 1999 .

[11]  J. Poulain,et al.  The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla , 2007, Nature.

[12]  A. Kortekamp,et al.  Investigation of the interaction of Plasmopara viticola with susceptible and resistant grapevine cultivars. , 1998 .

[13]  M. Sbaghi,et al.  Production of the Phytoalexin Resveratrol by Grapes as a Response to Botrytis Attack Under Natural Conditions , 1995 .

[14]  R. Pezet,et al.  Method to determine resveratrol and pterostilbene in grape berries and wines using high-performance liquid chromatography and highly sensitive fluorimetric detection , 1994 .

[15]  H. Felle,et al.  The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors. , 2002, Journal of experimental botany.

[16]  Z. Cerovic,et al.  QUANTIFICATION OF STILBENE IN GRAPEVINE LEAVES BY DIRECT FLUOROMETRY AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY: SPATIAL LOCALISATION AND TIME COURSE OF SYNTHESIS , 2010 .

[17]  P. Langcake,et al.  A new class of phytoalexins from grapevines , 1977, Experientia.

[18]  Z. Cerovic,et al.  Optical Properties of Plant Surfaces , 2007 .

[19]  B. M. Lange,et al.  Elicitor-induced formation of free and cell-wall-bound stilbenes in cell-suspension cultures of Scots pine (Pinus sylvestris L.) , 1994, Planta.

[20]  D. Merdinoglu,et al.  Determination and imaging of metabolites from Vitis vinifera leaves by laser desorption/ionisation time-of-flight mass spectrometry. , 2010, Rapid communications in mass spectrometry : RCM.

[21]  R. Hammerschmidt.  PHYTOALEXINS: What Have We Learned After 60 Years? , 1999, Annual review of phytopathology.

[22]  W. Kaiser,et al.  pH regulation in acid-stressed leaves of pea plants grown in the presence of nitrate or ammonium salts: studies involving 31P-NMR spectroscopy and chlorophyll fluorescence , 1997 .

[23]  P. Langcake.  Disease resistance of Vitis spp. and the production of the stress metabolites resveratrol, ε-viniferin, α-viniferin and pterostilbene , 1981 .

[24]  Z. Cerovic,et al.  Fast and local assessment of stilbene content in grapevine leaf by in vivo fluorometry. , 2007, Journal of agricultural and food chemistry.

[25]  C. Andary,et al.  Histochemical responses of leaves of in vitro plantlets of Vitis spp. to infection with Plasmopara viticola , 1995 .

[26]  L. Frémont.  Biological effects of resveratrol. , 2000, Life sciences.

[27]  T. K. Kirk,et al.  Lignification as a Mechanism of Disease Resistance , 1980 .

[28]  W. Hillis,et al.  The chromatographic and spectral properties of stilbene derivatives. , 1968, Journal of chromatography.

[29]  Y. Chupeau,et al.  Phytoalexin production in grapevine protoplasts during isolation and culture , 2003 .

[30]  C. Andary,et al.  Histochemical studies on the interaction between three species of grapevine, Vitis vinifera, V. rupestris and V. rotundifolia and the downy mildew fungus, Plasmopara viticola , 1995 .

[31]  K. Gindro,et al.  The effectiveness of stilbenes in resistant Vitaceae: ultrastructural and biochemical events during Plasmopara viticola infection process. , 2011, Plant physiology and biochemistry : PPB.

[32]  J. Siegel,et al.  Time-resolved fluorescence anisotropy imaging applied to live cells. , 2004, Optics letters.

[33]  K. Gindro,et al.  Glycosylation and oxidative dimerization of resveratrol are respectively associated to sensitivity and resistance of grapevine cultivars to downy mildew , 2004 .

[34]  Z. Cerovic,et al.  UV-induced blue-green and far-red fluorescence along wheat leaves: a potential signature of leaf ageing. , 2003, Journal of experimental botany.

[35]  S. Reuber,et al.  Tissue localization of phenolic compounds in plants by confocal laser scanning microscopy , 1998 .

[36]  Z. Cerovic,et al.  Quantitative study of fluorescence excitation and emission spectra of bean leaves. , 2006, Journal of photochemistry and photobiology. B, Biology.

[37]  P. Hugueney,et al.  Metabolism and roles of stilbenes in plants , 2009 .

[38]  R. J. Pryce,et al.  Identification of pterostilbene as a phytoalexin from Vitis vinifera leaves , 1979 .

[39]  K. Gindro,et al.  Role of stilbenes in the resistance of grapevine to powdery mildew , 2008 .

[40]  J. Kuc.  Phytoalexins, stress metabolism, and disease resistance in plants. , 1995, Annual review of phytopathology.

[41]  K. Gindro,et al.  Delta-viniferin, a resveratrol dehydrodimer: one of the major stilbenes synthesized by stressed grapevine leaves. , 2003, Journal of agricultural and food chemistry.

[42]  V. Dumas,et al.  GENETIC ANALYSIS OF DOWNY MILDEW RESISTANCE DERIVED FROM MUSCADINIA ROTUNDIFOLIA , 2003 .

[43]  D. Merdinoglu,et al.  Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues. , 2007, Micron.

[44]  P. Langcake,et al.  The production of resveratrol by Vitis vinifera and other members of the Vitaceae as a response to infection or injury , 1976 .

[45]  E. Lesniewska,et al.  Cell wall modification in grapevine cells in response to UV stress investigated by atomic force microscopy. , 2004, Ultramicroscopy.

[46]  D. Merdinoglu,et al.  Nonhost versus host resistance to the grapevine downy mildew, Plasmopara viticola, studied at the tissue level. , 2008, Phytopathology.

[47]  Histological and biochemical criteria for objective and early selection of grapevine cultivars resistant to Plasmopara viticola , 2006 .

[48]  C. Clément,et al.  Bioproduction of resveratrol and stilbene derivatives by plant cells and microorganisms. , 2009, Trends in biotechnology.