Multicolor Fluorescence Imaging of Leaves—A Useful Tool for Visualizing Systemic Viral Infections in Plants †

Multicolor fluorescence induced by UV light is a sensitive and specific tool that may be used to provide information about the primary and secondary metabolism of plants by monitoring signals of the chlorophyll fluorescence (Chl‐F) and blue‐green fluorescence (BGF), respectively. We have followed the systemic infection of Nicotiana benthamiana plants with the Pepper mild mottle virus (PMMoV) by means of a multicolor fluorescence‐imaging system, to detect differences between two strains of PMMoV during the infection process and to establish a correlation between the virulence and changes induced in the host plant. Changes in both BGF and Chl‐F were monitored. BGF increased mainly in the abaxial side of the leaf during pathogenesis and the corresponding images showed a clear vein‐associated pattern in leaves of infected plants. HPLC analysis of leaf extracts was carried out to identify compounds emitting BGF, and determined that chlorogenic acid was one of the main contributors. BGF imaging was able to detect viral‐induced changes in asymptomatic (AS) leaves before detection of the virus itself. Chl‐F images confirmed our previous results of alterations in the photosynthetic apparatus of AS leaves from infected plants that were detected with other imaging techniques. Fluorescence ratios F440/F690 and F440/F740, which increase during pathogenesis, were excellent indicators of biotic stress.

[1]  Sándor Lenk,et al.  Multicolor fluorescence imaging for early detection of the hypersensitive reaction to tobacco mosaic virus. , 2007, Journal of plant physiology.

[2]  Ladislav Nedbal,et al.  Visualization of dynamics of plant-pathogen interaction by novel combination of chlorophyll fluorescence imaging and statistical analysis: differential effects of virulent and avirulent strains of P. syringae and of oxylipins on A. thaliana. , 2007, Journal of experimental botany.

[3]  M. Vandeven,et al.  Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus , 2007, Photosynthesis Research.

[4]  Sándor Lenk,et al.  Distribution of UV-shielding of the epidermis of sun and shade leaves of the beech (Fagus sylvatica L.) as monitored by multi-colour fluorescence imaging. , 2006, Journal of plant physiology.

[5]  D. Van Der Straeten,et al.  Robotized thermal and chlorophyll fluorescence imaging of pepper mild mottle virus infection in Nicotiana benthamiana. , 2006, Plant & cell physiology.

[6]  C. Nautiyal,et al.  Induction of Plant Defense Enzymes and Phenolics by Treatment With Plant Growth–Promoting Rhizobacteria Serratia marcescens NBRI1213 , 2006, Current Microbiology.

[7]  Z. Cerovic,et al.  Time-resolved spectral studies of blue-green fluorescence of artichoke (Cynara cardunculus L. Var. Scolymus) leaves: identification of chlorogenic acid as one of the major fluorophores and age-mediated changes. , 2005, Journal of agricultural and food chemistry.

[8]  E. Weis,et al.  Photosynthesis and carbohydrate metabolism in tobacco leaves during an incompatible interaction with Phytophthora nicotianae , 2005 .

[9]  H. Lichtenthaler,et al.  Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system , 2005, Photosynthetica.

[10]  T. Teraoka,et al.  Time-course analysis of the accumulation of phenols in tomato seedlings infected with Potato Virus X and Tobacco mosaic virus , 2005 .

[11]  Z. Cerovic,et al.  Optically assessed contents of leaf polyphenolics and chlorophyll as indicators of nitrogen deficiency in wheat (Triticum aestivum L.) , 2005 .

[12]  T. Roitsch,et al.  Complex regulation of gene expression, photosynthesis and sugar levels by pathogen infection in tomato , 2004 .

[13]  T. Roitsch,et al.  Complex regulation of gene expression, photosynthesis and sugar levels by pathogen infection , 2004 .

[14]  D. Hagenbeek,et al.  Thermal and chlorophyll-fluorescence imaging distinguish plant-pathogen interactions at an early stage. , 2004, Plant & cell physiology.

[15]  Cathie Martin,et al.  Engineering plants with increased levels of the antioxidant chlorogenic acid , 2004, Nature Biotechnology.

[16]  M. Barón,et al.  Proteomic analysis of the oxygen‐evolving complex of photosystem II under biotec stress: Studies on Nicotiana benthamiana infected with tobamoviruses , 2004, Proteomics.

[17]  Shiv O. Prasher,et al.  Laser-induced fluorescence signatures as a tool for remote monitoring of water and nitrogen stresses in plants , 2003 .

[18]  Ismael Moya,et al.  The use of chlorophyll fluorescence excitation spectra for the non‐destructive in situ assessment of UV‐absorbing compounds in leaves , 2002 .

[19]  É. Hideg,et al.  The distribution and possible origin of blue—green fluorescence in control and stressed barley leaves , 2002, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[20]  D. Straeten,et al.  Seeing is believing: imaging techniques to monitor plant health. , 2001, Biochimica et biophysica acta.

[21]  Ismael Moya,et al.  Dual-excitation FLIDAR for the estimation of epidermal UV absorption in leaves and canopies , 2001 .

[22]  M. Sowinska,et al.  Multicolour Fluorescence Imaging of Sugar Beet Leaves with Different Nitrogen Status by Flash Lamp UV-Excitation , 2000, Photosynthetica.

[23]  H. Lichtenthaler,et al.  Imaging of the Blue, Green, and Red Fluorescence Emission of Plants: An Overview , 2000, Photosynthetica.

[24]  Hartmut K. Lichtenthaler,et al.  Detection of photosynthetic activity and water stressby imaging the red chlorophyll fluorescence , 2000 .

[25]  B. Logan,et al.  Energy dissipation and radical scavenging by the plant phenylpropanoid pathway. , 2000, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[26]  M. Barón,et al.  Inhibition of photosynthesis by viral infection : Effect on PSII structure and function , 2000 .

[27]  R. S. Nelson,et al.  Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana. , 2000, The Plant journal : for cell and molecular biology.

[28]  K. Oparka,et al.  THE GREAT ESCAPE: Phloem Transport and Unloading of Macromolecules1. , 2000, Annual review of plant physiology and plant molecular biology.

[29]  S. Rolfe,et al.  Infection of Arabidopsis thaliana leaves with Albugo candida (white blister rust) causes a reprogramming of host metabolism. , 2000, Molecular plant pathology.

[30]  C. Osmond,et al.  Infection with Phloem Limited Abutilon Mosaic Virus Causes Localized Carbohydrate Accumulation in Leaves of Abutilon striatum: Relationships to Symptom Development and Effects on Chlorophyll Fluorescence Quenching During Photosynthetic Induction , 2000 .

[31]  M. Barón,et al.  Effect of Tobamovirus Infection on Thermoluminescence Characteristics of Chloroplasts from Infected Plants , 1999 .

[32]  Hartmut K. Lichtenthaler,et al.  The Chlorophyll Fluorescence Ratio F735/F700 as an Accurate Measure of the Chlorophyll Content in Plants , 1999 .

[33]  Ulrich Lüttge,et al.  Chlorophyll Fluorescence Quenching During Photosynthetic Induction in Leaves of Abutilon striatum Dicks. Infected with Abutilon Mosaic Virus, Observed with a Field-Portable Imaging System , 1998 .

[34]  A. Roberts,et al.  Phloem Unloading in Sink Leaves of Nicotiana benthamiana: Comparison of a Fluorescent Solute with a Fluorescent Virus. , 1997, The Plant cell.

[35]  Hartmut K. Lichtenthaler,et al.  Fluorescence imaging as a diagnostic tool for plant stress , 1997 .

[36]  Sharon A. Robinson,et al.  Concepts of plant biotic stress. Some insights into the stress physiology of virus‐infected plants, from the perspective of photosynthesis , 1997 .

[37]  S. Rolfe,et al.  Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata): quantitative imaging of chlorophyll fluorescence , 1996, Planta.

[38]  Z. Cerovic,et al.  Time-resolved blue-green fluorescence of sugar beet (Beta vulgaris L.) leaves. Spectroscopic evidence for the presence of ferulic acid as the main fluorophore of the epidermis , 1996 .

[39]  A. Nassuth,et al.  Influence of wheat streak mosaic virus infection on phenylpropanoid metabolism and the accumulation of phenolics and lignin in wheat , 1995 .

[40]  S. Rolfe,et al.  Quantitative imaging of chlorophyll fluorescence. , 1995, The New phytologist.

[41]  R. Dixon,et al.  Stress-Induced Phenylpropanoid Metabolism. , 1995, The Plant cell.

[42]  Z. Cerovic,et al.  Characterization of Blue-Green Fluorescence in the Mesophyll of Sugar Beet (Beta vulgaris L.) Leaves Affected by Iron Deficiency , 1994, Plant physiology.

[43]  A. Maule,et al.  Complex, localized changes in CO2 assimilation and starch content associated with the susceptible interaction between cucumber mosaic virus and a cucurbit host , 1994 .

[44]  C. Osmond,et al.  Susceptibility of Tobacco Leaves to Photoinhibition following Infection with Two Strains of Tobacco Mosaic Virus under Different Light and Nitrogen Nutrition Regimes , 1994, Plant physiology.

[45]  C. Osmond,et al.  Diagnosis of the Earliest Strain-Specific Interactions between Tobacco Mosaic Virus and Chloroplasts of Tobacco Leaves in Vivo by Means of Chlorophyll Fluorescence Imaging , 1994, Plant physiology.

[46]  W. Niessen,et al.  Free and cell wall-bound phenolics and other constituents from healthy and fungus-infected carnation (Dianthus caryophyllus L.) stems , 1991 .

[47]  O. Kooten,et al.  Photosynthetic electron transport in tobacco leaves infected with tobacco mosaic virus , 1990 .

[48]  R. Hartley,et al.  Detection of bound ferulic acid in cell walls of the Gramineae by ultraviolet fluorescence microscopy , 1976, Nature.

[49]  G. Samuel.  The Movement of Tobacco Mosaic Virus Within the Plant , 1934 .

[50]  Ladislav Nedbal,et al.  Chlorophyll Fluorescence Imaging of Leaves and Fruits , 2004 .

[51]  Z. Szigeti,et al.  Changes in the photosynthetic functions in leaves of Chinese cabbage infected with turnip yellow mosaic virus , 2002 .

[52]  Y. R. Chen,et al.  Steady-state multispectral fluorescence imaging system for plant leaves. , 2001, Applied optics.

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

[54]  Hartmut K. Lichtenthaler,et al.  Principles and characteristics of multi-colour fluorescence imaging of plants , 1998 .

[55]  Ismael Moya,et al.  Time-resolved blue-green fluorescence of sugar beet leaves. Temperature-induced changes and consequences for the potential use of blue-green fluorescence as a signature for remote sensing of plants , 1998 .

[56]  M. Barón,et al.  Effect of Tobamovirus Infection on PSII Complex of Infected Plants , 1998 .

[57]  Hartmut K. Lichtenthaler,et al.  Cell wall bound ferulic acid, the major substance of the blue-green fluorescence emission of plants. , 1998 .

[58]  Hartmut K. Lichtenthaler,et al.  Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements , 1998 .

[59]  W. Lüdeker,et al.  Detection of Fungal Infection of Plants by Laser-induced Fluorescence: An Attempt to Use Remote Sensing , 1996 .

[60]  Hartmut K. Lichtenthaler,et al.  Differences in Fluorescence Excitation Spectra of Leaves between Stressed and Non-Stressed Plants , 1996 .

[61]  Francine Heisel,et al.  Detection of Nutrient Deficiencies of Maize by Laser Induced Fluorescence Imaging , 1996 .

[62]  Francine Heisel,et al.  Fluorescence Imaging of Water and Temperature Stress in Plant Leaves , 1996 .

[63]  Francine Heisel,et al.  Detection of vegetation stress via a new high resolution fluorescence imaging system , 1996 .

[64]  M. Barón,et al.  PSII Response to Biotic and Abiotic Stress , 1995 .

[65]  H. Nilsson.  Remote sensing and image analysis in plant pathology. , 1995, Annual review of phytopathology.

[66]  R. Hammerschmidt,et al.  Phenolic Compounds and Their Role in Disease Resistance , 1992 .

[67]  H. Lichtenthaler,et al.  Fluorescence emission spectra of plant leaves and plant constituents , 1991, Radiation and environmental biophysics.

[68]  M. Serra,et al.  A Tobamovirus Causing Heavy Losses in Protected Pepper Crops in Spain , 1989 .

[69]  J. McMurtrey,et al.  Laser-induced fluorescence of green plants. 1: A technique for the remote detection of plant stress and species differentiation. , 1984, Applied optics.

[70]  D. Altschuh,et al.  Pepper mild mottle virus, a tobamovirus infecting pepper cultivars in Sicily , 1984 .