SISTEMI NERVOR DHE S

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1st Winter Summit at the Anatolian Summit (WISAS)
Differences on stress response of endemic plants
via screening of photosynthetic activity by
chlorophyll fluorescence imaging
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
February 23-26, 2012 Erzurum/Turkey
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• Introduction

• 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 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.

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• Objective

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

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• Aim

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

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• MATERIALS AND METHODS

• Chlorophyll fluorescence induction kinetics

• 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

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1. Control Panel
2. Sample Chamber

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• 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
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• 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
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• Images of chlorophyll fluorescence

Images of chlorophyll fluorescence during
induction kinetics were measured on certain state.
These image fluorescence parameters are F0 -
minimum fluorescence in dark-adapted state Fm -
maximum fluorescence in dark-adapted state F0 -
minimum fluorescence in light Fm- maximum
fluorescence in light FP - peak fluorescence
during the initial phase of the Kautsky
effect FS - steady-state fluorescence in light
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• Images of chlorophyll fluorescence ratios

• 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(FP Fs)/Fs
• where FvFm-Fo and FvFm-Fo

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• 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 (xc) 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.

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Plant material

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

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RESULTS AND DISCUSSION

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

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Cercius siliquastrum optimal physiological
conditions Dajti area
Image of the maximum  fluorescence
in the dark Fm maximum fluorescence
in light  Fm  Difference  of images
Fm-Fm'  and  RFD ratio  image (pseudoscale 
0-4) 
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Histogram of fluorescence during
induction kinetics of leaves in Cercis siliquastru
m in optimal conditions (Dajti area)
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Induced fluorescence kinetics of  leaves of Cerci
us siliquastrum  - Dajti area   
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Induced fluorescence image parameters of some
 leaves of Cercius siliquastrum - Dajti  area
Image Fluorescence parameters Image Fluorescence parameters Image Fluorescence parameters Image Fluorescence parameters Image Fluorescence parameters Image Fluorescence parameters Image Fluorescence parameters
Fo Fm Fv Fo' Fm' Fv'
Leaf 1 129.73 410.4 280.67 143.3 186 42.7
Leaf 2 135.77 404.21 268.44 146.39 185.12 38.73
Leaf 3 128.36 405.86 277.5 137.96 176.68 38.72
Leaf 4 129.36 406.86 278.5 138.96 177.68 39.72
No significant differences between leaves
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Fluorescence ratios of some leaves Cercius siliqua
strum in optimal conditions, Dajti - area
Image Fluorescence ratios Image Fluorescence ratios Image Fluorescence ratios Image Fluorescence ratios Image Fluorescence ratios Image Fluorescence ratios Quenching coefficients Quenching coefficients
Fm/Fo Fv/Fm Fm'/Fo' Fv'/Fm' Rfd qN NPQ
Leaf 1 3.164 0.684 1.298 0.23 1.678 0.848 1.206
Leaf 2 2.977 0.664 1.265 0.209 1.644 0.856 1.184
Leaf 3 3.162 0.684 1.281 0.219 1.638 0.86 1.297
Leaf 4 3.162 0.684 1.255 0.203 1.583 0.868 1.273
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
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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

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Image Fluorescence ratios of two leaves of
Cercius siliquastrum in stress conditions (Krrabe
area)
Image Fluorescence ratios Image Fluorescence ratios Image Fluorescence ratios Image Fluorescence ratios Image Fluorescence ratios Image Fluorescence ratios
Fm/Fo Fv/Fm Fm'/Fo' Fv'/Fm' Rfd
Sun green leaf 3.489 0.713 1.288 0.224 1.62
Stress leaf 3.257 0.693 1.343 0.256 1.42
Sun green leaves - characterized by higher
photosynthetic activity Stress sun leaves -
characterized by lower photosynthetic activity
Quenching coefficients Quenching coefficients Quenching coefficients
qN NPQ
Sun green leaf 0.864 1.311
Stress leaf 0.85 1.456
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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.
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Histogram of fluorescence during
induction kinetics of some leaves of Cercis siliqu
astrum  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
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Induced fluorescence kinetics of leaves of Cerciu
s siliquastrum in stress and pollution conditions
(Elbasan area)
Green leaf (C)
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Image fluorescence parameters and image
fluorescence ratios of some leaves of
Cercius siliquastrum - in Elbasan area (Stress
and pollution)
Image fluorescence parameters Image fluorescence parameters Image fluorescence parameters Image fluorescence parameters
Fo Fm Fm'
Leaf (A) 115.86 315.67 111.52
Leaf (B) 115.44 415.92 131.64
Leaf (C) 124.69 457.28 167.37

• 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

Image fluorescence ratios Image fluorescence ratios Image fluorescence ratios Quenching  coefficients Quenching  coefficients
Fm/Fo Rfd qN NPQ
Leaf (A) 2.725 1.43 0.454 1.831
Leaf (B) 3.603 1.17 0.57 2.16
Leaf (C) 3.667 1.87 0.876 1.732
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Photosynthetic pigments
Cercis siliquastrum Cercis siliquastrum Cercis siliquastrum
Photosynthetic pigments Chl(ab) (mg/g) xc (mg/g)
Opt. conditions 2.125 0.637
Stress conditions 1.796 0.468
Stress - pollution 1.468 0.377

• The total Chl (ab) content and total carotenoids
  (xc) 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.

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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)

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• 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)

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• 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 Rfd1.3)

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• 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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