Transcript Leaf - esruc

1st Winter Summit at the Anatolian Summit (WISAS)
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





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.


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.



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
MATERIALS AND METHODS
 Chlorophyll fluorescence induction kinetics
 Chlorophyll (Chl) fluorescence induction kinetics of predarkened leaves (30 min) was measured using the
FluorCam 700MF kinetics imaging system - Photon
Systems Instrument.
FluorCam kinetic
fluorescence
camera
1
2
1.Control Panel
2.Sample Chamber
FluorCam
 FluorCam is using a rapidly modulated excitation and
synchronously gated CCD camera to capture kinetics
and 2-dimensional imaging of key fluorescence
parameters.
3
4
5
6
FluorCam
Control panel with LCD
display (3)
control keys (4)
sample chamber with
CCD camera (5) and
sample area (6).
 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
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
 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 Fv=Fm-Fo and Fv’=Fm’-Fo’



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.
Plant material

Endemic plants


Cercius siliquastrum
Study area



optimal physiological conditions – Dajti,
shadow area
Stress conditions - Krrabe
Stress and pollution - Elbasan
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)
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)
Histogram of fluorescence during induction kinetics
of leaves in Cercis siliquastrum in optimal
conditions (Dajti area)
5000
Histogram
4500
Cercis siliquastrum
Nr. pixsel
4000
3500
Fm
3000
Fm'
Fm-Fm'
2500
2000
1500
1000
500
0
0
200
400
Fluorescence [rel. units]
600
800
Induced fluorescence kinetics of leaves
of Cercius siliquastrum - Dajti area
500
Kinetics
Fluorescence [rel. unit]
450
Cercis siliquastrum
400
350
300
250
200
150
100
50
0
0
20
40
60
Time [s]
80
100
120
Induced fluorescence image parameters of
some leaves of Cercius siliquastrum - Dajti area
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
Fluorescence ratios of some leaves Cercius siliquastrum
in optimal conditions, Dajti - area
Quenching
coefficients
Image Fluorescence ratios
Rfd
qN
NPQ
0.23
1.678
0.848
1.206
1.265
0.209
1.644
0.856
1.184
0.684
1.281
0.219
1.638
0.86
1.297
0.684
1.255
0.203
1.583
0.868
1.273
Fm/Fo
Fv/Fm Fm'/Fo' Fv'/Fm'
Leaf 1
3.164
0.684
1.298
Leaf 2
2.977
0.664
Leaf 3
3.162
Leaf 4
3.162
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
Cercius siliquastrum
Stress conditions – Krrabe area
The fluorescence decline ratio image Rfd
(pseudoscale 0-3):
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 of two leaves
of Cercius siliquastrum in stress conditions
(Krrabe – area)
Image Fluorescence ratios
Sun green leaf
Stress leaf
Fm/Fo
3.489
3.257
Fv/Fm
0.713
0.693
Quenching coefficients
qN
NPQ
Sun green leaf 0.864 1.311
Stress leaf
0.85
1.456
Fm'/Fo'
1.288
1.343
Fv'/Fm'
0.224
0.256
Rfd
1.62
1.42
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.
A - green leaf with small
damaged parts
B.
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 and image fluorescence
ratios of some leaves of Cercius siliquastrum - in Elbasan area
(Stress and pollution)
Image fluorescence parameters
Leaf (A)
Leaf (B)
Leaf (C)
Fo
115.86
115.44
124.69
Fm
315.67
415.92
457.28
Fm'
111.52
131.64
167.37
Image fluorescence
Quenching
ratios
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
1.87 0.876 1.732
Leaf (C) 3.667
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
Photosynthetic pigments
Cercis siliquastrum
Photosynthetic Chl(a+b) x+c
(mg/g)
(mg/g)
pigments
Opt. conditions
2.125 0.637
Stress conditions
1.796 0.468
Stress - pollution
1.468 0.377
•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.
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.
REFERENCES
• Babani F. and Lichtenthaler H.K. (1996) Light-induced and age-dependent development of
chloroplasts in etiolated barley leaves as visualized by determination of photosynthetic
pigments, CO2 assimilation rates and different kinds of chlorophyll fluorescence ratios. J Plant
Physiol 148: 555-566
• Buschmann C. and Lichtenthaler H.K. (1998). Principles and characteristics of multi-colour
fluorescence imaging of plants. - J. Plant Physiol. 152, 297-314
• Krause G.H. and Weis E. (1991). Chlorophyll fluorescence and photosynthesis: the basics. Ann
Rev Plant Physiol Plant Mo. Biol 42: 313-349
• Langsdorf G., Buschmann C., Sowinska M., Babani F., Mokry M., Timmermann F., Lichtenthaler
H. K., (2000) Measurement of differences in red chlorophyll fluorescence and photosynthetic
activity between sun and shade leaves by fluorescence imaging. Photosynthetica 38: 539-551.
• Lichtenthaler H.K. (1987). Chlorophylls and carotenoids, the pigments of photosynthetic
biomembranes. In: Douce R, Packer L (eds) Methods Enzymol 148, pp. 350-382. Academic
Press Inc, New York
• Lichtenthaler H.K. (1996). Vegetation stress: an introduction to the stress concept in plants. J
Plant Physiol 148: 4-14
• Lichtenthaler H.K. and Babani F. (2000) Detection of photosynthetic activity and water stress
by imaging the red chlorophyll fluorescence. Plant Physiology Biochemistry 38: 889-895
• Lichtenthaler H.K. and Buschmann C. (2001) Chlorophylls and carotenoids–Measurement and
characterisation by UV-VIS. Current Protocols in Food Analytical Chemistry (CPFA),
(Supplement 1), pp. F4.3.1 - F 4.3.8. John Wiley, New York
• Lichtenthaler H.K., Babani F., Langsdorf G., Buschmann C. (2000). Measurement of differences
in red chlorophyll fluorescence and photosynthetic activity between sun and shade leaves by
fluorescence imaging. Photosynthetica38: 521-529.
• Lichtenthaler H.K. and Miehe J.A. (1997) Fluorescence imaging as a diagnostic tool for plant
stress. Trends Plant Sci 2: 316-320.
THANK YOU