Eriola Zhuri, University of Durresi ’’Aleksander Moisiu‘‘, Albania Veledin Çako, Dep.Physics, University of Vlora ’’Ismail Qemali‘‘, Albania Fatbardha Babani, Biotechnology Department, University of Tirana, Albania Liri Dinga, Botanical Garden, University of Tirana, Albania Theodhor Karaja, Physics Department, University of Tirana, Albania 1st Winter Summit at the Anatolian Summit (WISAS) February 23-26, 2012 Erzurum/Turkey

Chlorophyll (Chl) fluorescence signatures of leaves have been widely applied as non-invasive techniques for the in vivo analysis of plant stress. Chlorophyll (Chl) fluorescence signatures of leaves have been widely applied as non-invasive techniques for the in vivo analysis of plant stress. The Chl fluorescence provides ample information on the photosynthetic apparatus. The Chl fluorescence provides ample information on the photosynthetic apparatus. The high resolution multi-colour Chl fluorescence imaging techniques for whole leaves offer the new possibility to study the distribution and patchiness of fluorescence signatures over the whole leaf area. The high resolution multi-colour Chl fluorescence imaging techniques for whole leaves offer the new possibility to study the distribution and patchiness of fluorescence signatures over the whole leaf area. Various ratios of the Chl fluorescence determined from the induction kinetics can be used as indicators of the stress effect to the photosynthetic apparatus. Various ratios of the Chl fluorescence determined from the induction kinetics can be used as indicators of the stress effect to the photosynthetic apparatus. Introduction Introduction

To evaluate To evaluate Efficiency of photosynthetic apparatus of analyzed endemic plants grown in different environmental stress conditions via chlorophyll fluorescence imaging during induction kinetics and chlorophyll fluorescence imaging during induction kinetics and various fluorescence ratios various fluorescence ratios which describe the photosynthetic light processes and quantum conversion of light. Objective Objective

Characterize the effect of environmental factors on photosynthetic performance Characterize the effect of environmental factors on photosynthetic performance as well as Estimate the variations between endemic plants in stress conditions Estimate the variations between endemic plants in stress conditionsby differences on imaging of chlorophyll fluorescence signature and differences on imaging of chlorophyll fluorescence signature and photosynthetic pigment metabolism of leaves photosynthetic pigment metabolism of leaves Aim Aim

MATERIALS AND METHODS  Chlorophyll (Chl) fluorescence induction kinetics of pre- darkened leaves (30 min) was measured using the FluorCam 700MF kinetics imaging system - Photon Systems Instrument. FluorCam kinetic fluorescence camera 1.Control Panel 2.Sample Chamber FluorCam 1 2  Chlorophyll fluorescence induction kinetics

 FluorCam is using a rapidly modulated excitation and synchronously gated CCD camera to capture kinetics and 2-dimensional imaging of key fluorescence parameters. Control panel with LCD display (3) control keys (4) sample chamber with CCD camera (5) and sample area (6) FluorCam

 Images of Chl fluorescence intensity were obtained on false colour, whereby black is the lowest (zero) and white the highest fluorescence.  FluorCam 700MF can monitor photosynthesis in objects with a maximal dimension around 10 cm. FluorCam kinetic fluorescence camera Control Panel Sample Chamber

Images of chlorophyll fluorescence during induction kinetics were measured on certain state.  Images of chlorophyll fluorescence These image fluorescence parameters are : F 0 - minimum fluorescence in dark-adapted state Fm - maximum fluorescence in dark-adapted state F 0 ‘ - minimum fluorescence in light Fm‘- maximum fluorescence in light F P - peak fluorescence during the initial phase of the Kautsky effect F S - steady-state fluorescence in light

The images of various Chl fluorescence ratios were obtained by pixel to pixel arithmetic operations performed by FluorCam software : The images of various Chl fluorescence ratios were obtained by pixel to pixel arithmetic operations performed by FluorCam software : maximum quantum yields of Photosystem II. Fv/Fm= (Fm-Fo)/Fm and Fm/Fo effective quantum yields of Photosystem II Fv'/Fm‘) = (Fm’-Fs)/Fm’ fluorescence decline ratio in steady-state (assess plant vitality) Rfd=(F P – F s )/F s where Fv=Fm-Fo and Fv’=Fm’-Fo’  Images of chlorophyll fluorescence ratios

non photochemical quenching during light adaptation NPQ = (Fm - Fm’ )/ Fm non photochemical quenching qN = (Fv - Fv’ )/ Fv  Pigment determination The leaf pigments were extracted with 100% acetone using a mortar. Chlorophylls (Chla and Chlb) and total carotenoids (x+c) were determined spectrophotometrically (SQ-4802 Double Beam Scanning UV/Visible Spectrophotometer) and calculated using the re-evaluated equations of Lichtenthaler. The values represent the mean of 6 separate extracts.

Endemic plants Endemic plants Cercius siliquastrum Cercius siliquastrum Study area Study area optimal physiological conditions – Dajti, shadow area optimal physiological conditions – Dajti, shadow area Stress conditions - Krrabe Stress conditions - Krrabe Stress and pollution - Elbasan Stress and pollution - Elbasan Plant material

RESULTS AND DISCUSSION Fluorscence images and fluorescence image ratios of leaves of Cercius siliquastrum Fluorscence images and fluorescence image ratios of leaves of Cercius siliquastrum in three different area characterize by different conditions: in three different area characterize by different conditions: Dajti area - optimal physiological conditions Dajti area - optimal physiological conditions Krrabe area - Stress conditions (drought stress, high temperature and high light) Krrabe area - Stress conditions (drought stress, high temperature and high light) Elbasan area - Stress and pollution (particularly drought, high light - high temperature) Elbasan area - Stress and pollution (particularly drought, high light - high temperature)

Cercius siliquastrum optimal physiological conditions – Dajti area Image of the maximum fluorescence in the dark Fm maximum fluorescence in light Fm’ maximum fluorescence in light Fm’ Difference of images Fm-Fm' Difference of images Fm-Fm' and R FD ratio image (pseudoscale 0-4) (pseudoscale 0-4)

Histogram of fluorescence during induction kinetics of leaves in Cercis siliquastrum in optimal conditions (Dajti area)

Induced fluorescence kinetics of leaves of Cercius siliquastrum - Dajti area Induced fluorescence kinetics of leaves of Cercius siliquastrum - Dajti area

Induced fluorescence image parameters of some leaves of Cercius siliquastrum - Dajti area Image Fluorescence parameters FoFmFvFo'Fm'Fv' Leaf Leaf Leaf Leaf No significant differences between leaves

Image Fluorescence ratios Quenching coefficients Fm/FoFv/FmFm'/Fo'Fv'/Fm'RfdqNNPQ Leaf Leaf Leaf Leaf Fluorescence ratios of some leaves Cercius siliquastrum in optimal conditions, Dajti - area Four leaves of Cercius siliquastrum were analyzed - new fully green leaves, belong to different branch at same positions - characterized by the high photosynthetic activity, as reflect by the values of fluorescence ratios - almost the same between leaves analyzed

The fluorescence decline ratio image Rfd (pseudoscale 0-3): Cercius siliquastrum Stress conditions – Krrabe area Sun leaves: (A) green leaf and (B) stress leaf Rfd images presented at the same pseudoscale clearly show changes of the values ​​ of this indicator between two leaves and their distributions over leaves area

Image Fluorescence ratios Fm/FoFv/FmFm'/Fo'Fv'/Fm'Rfd Sun green leaf Stress leaf Quenching coefficients qNNPQ Sun green leaf Stress leaf Image Fluorescence ratios of two leaves of Cercius siliquastrum in stress conditions (Krrabe – area) Sun green leaves - characterized by higher photosynthetic activity Stress sun leaves - characterized by lower photosynthetic activity

Cercius siliquastrum Stress and pollution – Elbasan area Image Fluorescence ratio: Rfd ratio (pseudoscale 0-3) A - green leaf with small damaged parts B - damaged leaf C - new green leaf Rfd values of damaged parts of the leaf ​​ (B) are very low compared to other parts.

Histogram of fluorescence during induction kinetics of some leaves of Cercis siliquastrum in stress and pollution conditions (Elbasan area) A.B. A - green leaf with small damaged parts B - damaged leaf Different distributions of fluorescence signatures over leaf area related to Fm, Fm’ and their differences Fm-Fm’

Induced fluorescence kinetics of leaves of Cercius siliquastrum in stress and pollution conditions (Elbasan area) Green leaf (C)

Image fluorescence parameters FoFmFm' Leaf (A) Leaf (B) Leaf (C) Image fluorescence parameters and image fluorescence ratios of some leaves of Cercius siliquastrum - in Elbasan area (Stress and pollution) Image fluorescence ratios Quenching coefficients Fm/FoRfdqNNPQ Leaf (A) Leaf (B) Leaf (C) Leaf (B), a damaged leaf – is characterized by lower photosynthetic activity -as is reflected by the values of the fluorescence ratios (Rfd, qN) Leaf (C), new green leaf - is characterized by higher photosynthetic activity

Cercis siliquastrum Photosynthetic pigments Chl(a+b) (mg/g) x+c (mg/g) Opt. conditions Stress conditions Stress - pollution Photosynthetic pigments The total Chl (a+b) content and total carotenoids (x+c) content were significantly higher in leaves of both endemic plants grown in optimal conditions – Dajti area than of plants grown in stress conditions. The total Chl (a+b) content and total carotenoids (x+c) content were significantly higher in leaves of both endemic plants grown in optimal conditions – Dajti area than of plants grown in stress conditions. The decrease of chlorophylls was faster than that of carotenoids. The decrease of chlorophylls was faster than that of carotenoids.

CONCLUSIONS  Fluorescence images measured at different states during induction kinetics, induced kinetics of Chl fluorescence and histograms of fluorescence distributions in the plants grown in optimal conditions ( Dajti area ) show a high photosynthetic activity as is demonstrated by the values of fluorescence ratios which evaluate the plant vitality and quantum yield of photosynthetic apparatus. Cercius siliquastrum: Rfd = 1.63, Fm/Fo = 3.12)

 Activity of photosynthetic apparatus of leaves of analyzed endemic plants grown in stress conditions ( drought, high light and high temperature - Krrabe area ) was generally lower than activity of plants grown in optimal conditions ( Dajti area ). Cercius siliquastrum: Rfd = 1.52, Fm/Fo = 3.37)

 Activity of photosynthetic apparatus of leaves of analyzed plants grown in stress and pollution conditions ( particularly drought, high light-high temperature; dust and chemical contamination - Elbasan area ) demonstrated reduction compared to other areas as is expressed by -the lowest values of fluorescence decline ratio (Rfd); -increased of non-uniformity distribution and heterogeneity of signal of fluorescence images; -shape of induction kinetics and fluorescence histograms. Cercius siliquastrum: Rfd=1.3)

 The photosynthetic pigments, chlorophylls and carotenoids, could be considered functionally organized in plants grown in optimal conditions ( Dajti area ).  The reduce of pigment content observed in both endemic plants grown in stress conditions ( Krrabe area ) as well as in stress-pollution conditions ( Elbasan area ) compared to optimal conditions indicated a possible modifications in pigment composition during stress events.

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