Photosynthesis Research

, Volume 90, Issue 2, pp 161–172

A method for quantitative analysis of spatially variable physiological processes across leaf surfaces

Original paper


Many physiological processes are spatially variable across leaf surfaces. While maps of photosynthesis, stomatal conductance, gene expression, water transport, and the production of reactive oxygen species (ROS) for individual leaves are readily obtained, analytical methods for quantifying spatial heterogeneity and combining information gathered from the same leaf but with different instruments are not widely used. We present a novel application of tools from the field of geographical imaging to the multivariate analysis of physiological images. Procedures for registration and resampling, cluster analysis, and classification provide a general framework for the analysis of spatially resolved physiological data. Two experiments were conducted to illustrate the utility of this approach. Quantitative analysis of images of chlorophyll fluorescence and the production of ROS following simultaneous exposure of soybean leaves to atmospheric O3 and soybean mosaic virus revealed that areas of the leaf where the operating quantum efficiency of PSII was depressed also experienced an accumulation of ROS. This correlation suggests a causal relationship between oxidative stress and inhibition of photosynthesis. Overlaying maps of leaf surface temperature and chlorophyll fluorescence following a photoinhibition treatment indicated that areas with low operating quantum efficiency of PSII also experienced reduced stomatal conductance (high temperature). While each of these experiments explored the covariance of two processes by overlaying independent images gathered with different instruments, the same procedures can be used to analyze the covariance of information from multiple images. The application of tools from geographic image analysis to physiological processes occurring over small spatial scales will help reveal the mechanisms generating spatial variation across leaves.


Chlorophyll fluorescence imaging Quantitative image analysis Reactive oxygen species Spatial heterogeneity Thermal imaging 


  1. Aldea M, Hamilton JG, Resti JP et al (2005) Indirect effects of insect herbivory on leaf gas exchange in soybean. Plant Cell Environ 28:402–411CrossRefGoogle Scholar
  2. Aldea M, Hamilton JG, Resti JP et al (2006) Comparison of photosynthetic damage from arthropod herbivory and pathogen infection in understory hardwood saplings. Oecologia 149:221–232PubMedCrossRefGoogle Scholar
  3. Bi JL, Felton GW (1995) Foliar oxidative stress and insect herbivory: primary compounds, secondary metabolites, and reactive oxygen species as components of induced resistance. J Chem Ecol 21:1511–1529CrossRefGoogle Scholar
  4. Bown AW, Hall DE, MacGregor KB (2002) Insect footsteps on leaves stimulate the accumulation of 4-aminobutyrate and can be visualized through increased chlorophyll fluorescence and superoxide production. Plant Physiol 129:1430–1434PubMedCrossRefGoogle Scholar
  5. Buckley TN, Mott KA, Farquhar GD (2003) A hydromechanical and biochemical model of stomatal conductance. Plant Cell Environ 26:1767–1785CrossRefGoogle Scholar
  6. Canny MJ (1990) Tansley review No. 22—what becomes of the transpiration stream? New Phytol 114:341–368CrossRefGoogle Scholar
  7. Chaerle L, Hagenbeek D, De Bruyne E et al (2004) Thermal and chlorophyll-fluorescence imaging distinguish plant–pathogen interactions at an early stage. Plant Cell Physiol 45:887–896PubMedCrossRefGoogle Scholar
  8. Chalfie M, Tu Y, Euskirchen G et al (1994) Green fluorescent protein as a marker for gene-expression. Science 263:802–805PubMedCrossRefGoogle Scholar
  9. Chen P, Buss GR, Tolin SA (2004) Reaction of soybean to single and double inoculation with different soybean mosaic virus strains. Crop Prot 23:965–971CrossRefGoogle Scholar
  10. Clearwater MJ, Clark CJ (2003) In vivo magnetic resonance imaging of xylem vessel contents in woody lianas. Plant Cell Environ 26:1205–1214CrossRefGoogle Scholar
  11. Dixit R, Cyr R, Gilroy S (2006) Using intrinsically fluorescent proteins for plant cell imaging. Plant J 45:599–615PubMedCrossRefGoogle Scholar
  12. Eckstein J, Beyschlag W, Mott KA et al (1996) Changes in photon flux can induce stomatal patchiness. Plant Cell Environ 19:1066–1074CrossRefGoogle Scholar
  13. Ehrlich D, Estes JE, Singh A (1994) Applications of NOAA-AVHRR 1 km data for environmental monitoring. Int J Remote Sens 15:145–161CrossRefGoogle Scholar
  14. Fiscus EL, Booker FL, Burkey KO (2005) Crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning. Plant Cell Environ 28:997–1011CrossRefGoogle Scholar
  15. Frank TD (1988) Mapping dominant vegetation communities in the Colorado Rocky Mountain front range with Landsat Thematic Mapper and digital terrain data. Photogramm Eng Remote Sens 54:1727–1734Google Scholar
  16. Fryer MJ, Ball L, Oxborough K et al (2003) Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves. Plant J 33:691–705PubMedCrossRefGoogle Scholar
  17. Fryer MJ, Oxborough K, Mullineaux PM et al (2002) Imaging of photo-oxidative stress responses in leaves. J Exp Bot 53:1249–1254PubMedCrossRefGoogle Scholar
  18. Gaff DF, O-Ogola O (1971) The use of nonpermeating pigments for testing the survival of cells. J Exp Bot 22:756–758CrossRefGoogle Scholar
  19. Gamon JA, Surfus JS (1999) Assessing leaf pigment content and activity with a reflectometer. New Phytol 143:105–117CrossRefGoogle Scholar
  20. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92Google Scholar
  21. Gog L, Berenbaum MR, DeLucia EH et al (2005) Autotoxic effects of essential oils on photosynthesis in parsley, parsnip and rough lemon. Chemoecol 15:115–119CrossRefGoogle Scholar
  22. Grassi G, Magnani F (2005) Stomatal, mesophyll conductance and biochemical limitations to photosynthesis as affected by drought and leaf ontogeny in ash and oak trees. Plant Cell Environ 28:834–849CrossRefGoogle Scholar
  23. Gray GR, Hope BJ, Qin X et al (2003) The characterization of photoinhibition and recovery during cold acclimation in Arabidopsis thaliana using chlorophyll fluorescence imaging. Physiol Plantarum 119:365–375CrossRefGoogle Scholar
  24. Gussoni M, Greco F, Vezzoli A et al (2001) Magnetic resonance imaging of molecular transport in living morning glory stems. Magn Reson Imaging 19:1311–1322PubMedCrossRefGoogle Scholar
  25. Haefner JW, Buckley TN, Mott KA (1997) A spatially explicit model of patchy stomatal responses to humidity. Plant Cell Environ 20:1087–1097CrossRefGoogle Scholar
  26. Hall DE, MacGregor KB, Nijsse J et al (2004) Footsteps from insect larvae damage leaf surfaces and initiate rapid responses. Eur J Plant Pathol 110:441–447CrossRefGoogle Scholar
  27. Haseloff J, Amos B (1995) GFP in plants. Trends Genet 11:328–329PubMedCrossRefGoogle Scholar
  28. Heiser I, Osswald W, Elstner EF (1998) The formation of reactive oxygen species by fungal and bacterial phytotoxins. Plant Physiol Biochem 36:703–713CrossRefGoogle Scholar
  29. Hernandez JA, Rubio M, Olmos E et al (2004) Oxidative stress induced by long-term plum pox virus infection in peach (Prunus persica). Physiol Plant 122:486–495CrossRefGoogle Scholar
  30. Jensen JR (2005) Introductory digital image processing: a remote sensing perspective, 3rd edn. Upper Saddle River, NJ, Prentice HallGoogle Scholar
  31. Jones H (1999) Use of thermography for quantitative studies of spatial and temporal variation of stomatal conductance over leaf surfaces. Plant Cell Environ 22:1043–1055CrossRefGoogle Scholar
  32. Leon J, Rojo E, Sanchez-Serrano JJ (2001) Wound signaling in plants. J Exp Bot 52:1–9PubMedCrossRefGoogle Scholar
  33. McGarigal K, Cushman S, Stafford S (2000) Multivariate statistics for wildlife and ecology research. Springer-Verlag, New York, NYGoogle Scholar
  34. McVicar TR, Jupp DLB (1998) The current and potential operational uses of remote sensing to aid decisions on drought exceptional circumstances in Australia: a review. Agric Syst 57:399–468CrossRefGoogle Scholar
  35. Morgan PB, Bernacchi CJ, Ort DR et al (2004) An in vivo analysis of the effect of season-long open-air elevation of ozone to anticipated 2050 levels on photosynthesis in soybean. Plant Physiol 135:2348–2357PubMedCrossRefGoogle Scholar
  36. Mott KA, Buckley TN (1998) Stomatal heterogeneity. J Exp Bot 49:407–417CrossRefGoogle Scholar
  37. Mott KA, Buckley TN (2000) Patchy stomatal conductance: emergent collective behaviour of stomata. Trends Plant Sci 5:258–262PubMedCrossRefGoogle Scholar
  38. Mott KA, Franks PJ (2001) The role of epidermal turgor in stomatal interactions following a local perturbation in humidity. Plant Cell Environ 24:657–662CrossRefGoogle Scholar
  39. Nilsson HE (1980) Remote sensing and image processing for disease assessment. Protection Ecol 2:271–274Google Scholar
  40. Nilsson HE (1995) Remote-sensing and image-analysis in plant pathology. Annu Rev Phytopathol 33:489–527CrossRefPubMedGoogle Scholar
  41. Omasa K, Takayama K (2003) Simultaneous measurement of stomatal conductance, non-photochemical quenching, and photochemical yield of Photosystem II in intact leaves by thermal and chlorophyll fluorescence imaging. Plant Cell Physiol 44:1290–1300PubMedCrossRefGoogle Scholar
  42. Ort DR, Baker NR (2002) A photoprotective role for O2 as an alternative electron sink in photosynthesis. Curr Opin Plant Biol 5:193–198PubMedCrossRefGoogle Scholar
  43. Oxborough K (2004) Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance. J Exp Bot 55:1195–1205PubMedCrossRefGoogle Scholar
  44. Oxborough K (2005) Using chlorophyll a fluorescence imaging to monitor photosynthetic performance. In: Govindjee, Papageorgiou GC (eds) Chlorophyll fluorescence: a signature of photosynthesis. Kluwer Academic Press, Dordrecht, pp 409–428Google Scholar
  45. Oxborough K, Hanlon ARM, Underwood GJC et al (2000) In vivo estimation of the Photosystem II photochemical efficiency of individual microphytobenthic cells using high-resolution imaging of chlorophyll a fluorescence. Limnol Oceanogr 45:1420–1425CrossRefGoogle Scholar
  46. Peak D, West JD, Messinger SM et al (2004) Evidence for complex, collective dynamics and emergent, distributed computation in plants. Proc Natl Acad Sci USA 101:918–922PubMedCrossRefGoogle Scholar
  47. Prytz G, Futsaether CM, Johnsson A (2003) Thermography studies of the spatial and temporal variability in stomatal conductance of Avena leaves during stable and oscillatory transpiration. New Phytol 158:249–258CrossRefGoogle Scholar
  48. Repka V (2002) Chlorophyll-deficient mutant in oak (Quercus petraea L.) displays an accelerated hypersensitive-like cell death and an enhanced resistance to powdery mildew disease. Photosynthetica 40:183–193CrossRefGoogle Scholar
  49. Rolfe SA, Scholes JD (1995) Quantitative imaging of chlorophyll fluorescence. New Phytol 131:69–79CrossRefGoogle Scholar
  50. Smith WK, Brodersen CR, Hancock TE et al (2004) Integrated plant temperature measurement using heat-sensitive paint and colour image analysis. Funct Ecol 18:148–153CrossRefGoogle Scholar
  51. Swain PH, Davis SM (eds) (1978) Remote sensing: the quantitative approach. McGraw-Hill International Book Co., New York, NYGoogle Scholar
  52. Tang JY, Zielinski RE, Zangerl AR et al (2006) The differential effects of herbivory by first and fourth instars of Trichoplusia ni (Lepidoptera: Noctuidae) on photosynthesis in Arabidopsis thaliana. J Exp Bot 57:527–536PubMedCrossRefGoogle Scholar
  53. Thordal-Christensen H, Zhang Z, Wei Y et al (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11:1187–1194CrossRefGoogle Scholar
  54. West JD, Peak D, Peterson JQ et al (2005) Dynamics of stomatal patches for a single surface of Xanthium strumarium L. leaves observed with fluorescence and thermal images. Plant Cell Environ 28:633–641CrossRefGoogle Scholar
  55. Wright KM, Duncan GH, Pradel KS et al (2000) Analysis of the NGene hypersensitive response induced by a fluorescently tagged tobacco mosaic virus. Plant Physiol 123:1375–1385PubMedCrossRefGoogle Scholar
  56. Zangerl AR, Hamilton JG, Miller TJ et al (2002) Impact of folivory on photosynthesis is greater than the sum of its holes. Proc Natl Acad Sci USA 99:1088–1091PubMedCrossRefGoogle Scholar
  57. Zou JJ, Rodriguez-Zas S, Aldea M et al (2005) Expression profiling soybean response to Pseudomonas syringae reveals new defense-related genes and rapid HR-specific downregulation of photosynthesis. Mol Plant–Microbe Interact 18:1161–1174PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Mihai Aldea
    • 1
  • Thomas D. Frank
    • 2
  • Evan H. DeLucia
    • 3
  1. 1.Program in Ecology and Evolutionary BiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Department of GeographyUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  3. 3.Department of Plant BiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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