Viewpoint: Avoiding common pitfalls of chlorophyll fluorescence analysis under field conditions.

The determination of chlorophyll fluorescence emission is a powerful tool for assessing the status of PSII and the allocation of absorbed light to photosynthesis v. photoprotective energy dissipation. The development of field-portable fluorometers has enabled growing numbers of scientists to measure fluorescence emission from plants in diverse field settings. However, the ease of operation of contemporary fluorometers masks the many challenges associated with collecting meaningful and interpretable fluorescence signals from leaves exposed to relevant environmental conditions. Here, we offer methodological advice aimed at, but not limited to, the non-specialist for the proper measurement of fluorescence parameters, with an emphasis on avoiding common errors in the use of fluorescence under field conditions. Chief among our suggestions is (1) to delay use of automatically calculated fluorescence parameters, presented by the instrument software, until raw data 'traces' have been carefully inspected to ensure the integrity of findings, and (2) to combine chlorophyll fluorescence analysis, as a rapid, preliminary method of assessing plant responses to stress, with additional methods of characterising the system of interest (e.g. gas exchange, foliar pigment composition, thylakoid protein composition).

[1]  D. Barker,et al.  The xanthophyll cycle and energy dissipation in differently oriented faces of the cactus Opuntia macrorhiza , 1997, Oecologia.

[2]  Klaus Winter,et al.  The ratio of variable to maximum chlorophyll fluorescence from photosystem II, measured in leaves at ambient temperature and at 77K, as an indicator of the photon yield of photosynthesis , 2004, Planta.

[3]  A. Verhoeven,et al.  'Photoinhibition' During Winter Stress: Involvement of Sustained Xanthophyll Cycle-Dependent Energy Dissipation , 1995 .

[4]  E. Weis Chlorophyll fluorescence at 77 K in intact leaves: Characterization of a technique to eliminate artifacts related to self-absorption , 1985, Photosynthesis Research.

[5]  W. W. Adams,et al.  Photosynthesis and Chlorophyll Fluorescence Characteristics in Relationship to Changes in Pigment and Element Composition of Leaves of Platanus occidentalis L. during Autumnal Leaf Senescence. , 1990, Plant physiology.

[6]  K Maxwell,et al.  Chlorophyll fluorescence--a practical guide. , 2000, Journal of experimental botany.

[7]  B. Demmig‐Adams,et al.  Xanthophyll cycle and light stress in nature: uniform response to excess direct sunlight among higher plant species , 1996, Planta.

[8]  David Baker,et al.  Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipa , 2008 .

[9]  A. Melis,et al.  Photosystem-II damage and repair cycle in chloroplasts: what modulates the rate of photodamage ? , 1999, Trends in plant science.

[10]  W. W. Adams,et al.  Up‐regulation of a photosystem II core protein phosphatase inhibitor and sustained D1 phosphorylation in zeaxanthin‐retaining, photoinhibited needles of overwintering Douglas fir , 2005 .

[11]  C. Osmond,et al.  Rapid changes in xanthophyll cycle‐dependent energy dissipation and photosystem II efficiency in two vines, Stephania japonica and Smilax australis, growing in the understory of an open Eucalyptus forest , 1999 .

[12]  W. Bilger,et al.  Relationships among violaxanthin deepoxidation, thylakoid membrane conformation, and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.) , 1994, Planta.

[13]  W. Adams,et al.  Capacity for Energy Dissipation in the Pigment Bed in Leaves With Different Xanthophyll Cycle Pools , 1994 .

[14]  Ulrich Schreiber,et al.  Determination of the quantum efficiency of photosystem II and of non-photochemical quenching of chlorophyll fluorescence in the field , 1995, Oecologia.

[15]  W. Bilger,et al.  Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer , 2004, Photosynthesis Research.

[16]  W. Adams,et al.  Carotenoids and Photosystem II Characteristics of Upper and Lower Halves of Leaves Acclimated to High Light , 1996 .

[17]  A. Mehler Studies on reactions of illuminated chloroplasts. I. Mechanism of the reduction of oxygen and other Hill reagents. , 1951, Archives of biochemistry and biophysics.

[18]  W. W. Adams,et al.  Characteristics and Species-Dependent Employment of Flexible Versus Sustained Thermal Dissipation and Photoinhibition , 2008 .

[19]  W. Bilger,et al.  Temperature dependence of violaxanthin de-epoxidation and non-photochemical fluorescence quenching in intact leaves of Gossypium hirsutum L. and Malva parviflora L. , 1991, Planta.

[20]  W. W. Adams,et al.  Photoprotection in an ecological context: the remarkable complexity of thermal energy dissipation. , 2006, The New phytologist.

[21]  O. Björkman,et al.  Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution in leaves of higher plants , 1987, Planta.

[22]  W. W. Adams,et al.  Energy Dissipation and Photoinhibition: A Continuum of Photoprotection , 2008 .

[23]  Wolfgang Bilger,et al.  Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis , 1990, Photosynthesis Research.

[24]  W. W. Adams,et al.  Photoprotective Strategies of Overwintering Evergreens , 2004 .

[25]  J. Briantais,et al.  The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence , 1989 .

[26]  S. Robinson,et al.  Internal and external photoprotection in developing leaves of the CAM plant Cotyledon orbiculata , 1997 .

[27]  Ulrich Schreiber,et al.  Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview , 2004 .

[28]  C. Warren Estimating the internal conductance to CO2 movement. , 2006, Functional plant biology : FPB.

[29]  W. W. Adams,et al.  Modulation of PsbS and flexible vs sustained energy dissipation by light environment in different species , 2006 .

[30]  W. W. Adams,et al.  Photosynthesis and Photoprotection in Overwintering Plants , 2002 .

[31]  O. Björkman,et al.  Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins , 1987, Planta.

[32]  W. Bilger,et al.  Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis , 1994 .

[33]  S. Robinson,et al.  Internal Gradients of Chlorophyll and Carotenoid Pigments in Relation to Photoprotection in Thick Leaves of Plants With Crassulacean Acid Metabolism , 1994 .

[34]  N. Huner,et al.  Photosynthesis of overwintering evergreen plants. , 2001, Annual review of plant biology.