Experimental in vivo measurements of light emission in plants
E. Çaçi
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Paper presented in 1-st International Scientific Conference
on Professional Sciences, “Alexander Moisiu” University, Durres
November 2016
EXPERIMENTAL IN VIVO MEASUREMENTS OF LIGHT EMISSION IN
PLANTS
EMILIA ÇAÇI
Abstract
Light emission from plants is of several kinds: prompt fluorescence (PF), delayed fluorescence
(DF), thermo luminescence, and phosphorescence. Chlorophyll (Chl) fluorescence, though
corresponding to a very small fraction of the dissipated energy from the photosynthetic
apparatus, is widely accepted to provide an access to the understanding of its structure and
function. Many experimental techniques are available today for the investigation of the
energetic behaviour of a photosynthetic system. m-PEA (multifunctional Plant Efficiency
Analyser) developed by Hansatech Instruments Ltd (Kings Lynn, UK)allowing for fast and
very informative analysis (in vivo and in situ) of the functional status of the photosynthetic
apparatus in plants. It is based on a simultaneous registration of the kinetic characteristics of
prompt chlorophyll fluorescence emission (PF), delayed chlorophyll fluorescence (DF) and
modulated light scattering and reflection of the actinic incident light at 820 nm (MR). Leaves
from olive plants at different physiological states have been analysed. The prompt fluorescence
signal provides information about electron transport fluxes through Photosystem II and
Photosystem I. The modulated reflection signal at 820 nm provides information about the
activity of the donor and acceptor side of Photosystem I. The delayed fluorescence signals
provide information about the oxygen evolving complex and the acceptor side of Photosystem
II presenting structural information as rate constants, related to the whole photosynthetic
apparatus.
Key words: photosynthesis, chlorophyll fluorescence, prompt fluorescence, delayed
fluorescence, JIP test.
Introduction
All
chlorophyll
based
photosynthetic
organisms contain light-gathering antenna
systems (Green and Parson, 2002). These
systems function to absorb light and transfer
the energy in the light to a trap, which
quenches or deactivates the excited state. In
most cases, trap is the reaction centre itself,
and the excited state is quenched by
photochemistry with energy storage. In some
cases, however, the quenching is by some
other process, such as fluorescence or internal
conversion. Fluorescence is a member of the
luminescence family of processes in which
chromospheres (pigment bearing) molecules
emit light from electronically excited singlet
states produced either by a physical,
mechanical, or a chemical mechanism.
Generation of luminescence through excitation
of a molecule by UV or visible light is a
phenomenon
termed
photoluminescence,
which is formally divided into two categories,
fluorescence and phosphorescence, depending
upon the electronic configuration of the
excited state and the emission pathway.
Fluorescence is the property of some atoms
and molecules that absorb light at a particular
wavelength and subsequently emit light,
usually at longer wavelengths, after a brief
interval. Lifetime of this fluorescence gives
information on the rate constant of this
process.
Measurement and analysis of fluorescence is
one of the most powerful ways to probe
photosynthetic systems. This fluorescence is
from excited states that were lost before
photochemistry took place (except delayed
fluorescence, which arises from reversal of
photochemistry). It usually represents a small
fraction of the excited state decay in a
functional
photosynthetic
complex.
Nevertheless, the fluorescence is an extremely
informative quantity, because it reports on the
energy transfer and trapping. A simple
208 Interdisplinary Journal of Research and Development
“Aleksandër Moisiu“ University, Durrës, Albania
Vol (IV), No.2, 2017
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quantitative relation between the observed
fluorescence yield and the fraction of closed
reaction centres gives insight into the pigment
organization.
In
most
photosynthetic
organisms, the fluorescence yield is increased
when the trap is closed, whether by
photochemistry or by chemical treatment. This
is because one of the decay pathways of the
excited state has been removed, so other
pathways, such as fluorescence, assume a
larger role in excited state decay.
Fluorescence excitation spectra are direct
evidence for energy transfer. The basic
concept of photosynthetic antennas is that light
absorbed by one pigment may subsequently be
transferred to other pigments. A convenient
way to monitor this energy transfer process is
to irradiate a sample with light that is
selectively absorbed by one set of pigments
and then monitor fluorescence that originates
from a different set of pigments. A plot of the
intensity of fluorescence emission at a fixed
wavelength versus the wavelength of
excitation is called a fluorescence excitation
spectrum. If light is absorbed by one set of
pigments and emitted by another set, energy
transfer must have taken place between the
two groups of pigments. This type of
fluorescence excitation experiment can also be
used to measure quantitatively the efficiency
of energy transfer from one set of pigments to
another.
For the sake of illustration, we will consider an
idealized case in which pigment A transfers
energy to pigment B, which then fluoresces
(Fig. 1). Pigment B will also fluoresce if it is
directly excited. We monitor the fluorescence
emission of pigment B at wavelength λB. The
intensity of emission at λB is measured as we
scan the excitation wavelength, as shown in
Fig. 1. The fluorescence excitation spectrum is
thereby recorded. The second part of the
experiment involves simply measuring the
absorption spectrum of the sample. Actually,
the proper quantity to use in this comparison is
not the absorption spectrum, but a related
quantity, the 1 - T spectrum, where T is the
intensity of light that is transmitted by the
sample is called the transmission (T): T = I0/I.
The 1 – T spectrum is the intensity of light that
is absorbed as a function of wavelength. For
absorbance values of 0.1 or less, the absorption
and 1 - T spectra have essentially the same
shape.
Figure 1: Energy transfer efficiency from
fluorescence excitation measurements. A schematic
picture of absorbing (A) and emitting (B)
molecules, with energy transfer between them, is
shown at the top.
Fluorescence excitation spectrum compared
with absorption spectrum, normalized at the
maxima of each spectrum for pigment B. The
curves expected when energy transfer
efficiencies from A to B are 10%, 50% and
90% are shown.
The study of chlorophyll (Chl) a fluorescence
signals and the description how fluorescence
transients, known commonly as Kautsky
curves, exhibited by photosynthetic organisms
under different conditions can be analysed to
provide detailed information about the
structure, conformation and function of the
photosynthetic apparatus and especially of
photo system (PS)II. The analytical
formulation of the biophysics of the
photosynthetic apparatus, according to models
of different complexity regarding both the
architecture of the photosynthetic unit and the
modes of energetic communication among the
pigment assemblies, as well as their links to
the experimental fluorescence signals are
derived, based on the so-called ‘Theory of
Energy Fluxes in Bio membranes’ (Strasser,
1978, 1981). The theory of energy fluxes,
leads to simple algebraic equations expressing
the equilibration of the total energy influx with
the total energy out flux for each pigment
209 Experimental in vivo measurements of light emission in plants
E. Çaçi
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system under consideration, derive analytical
solutions, valid for any model, for the energy
fluxes in units with open and closed RCs; in
this framework, the definition and derivation
of the ‘overall grouping probability’, which
takes into account all possible pathways of
communication.
Many experimental techniques are available
today for the investigation of the energetic
behaviour of a photosynthetic system. There is
a general agreement that at room temperature,
Chl a fluorescence of plants, algae and cyan
bacteria, in the 680–740 nm spectral region, is
emitted mainly by photosystem (PS)II and it
can therefore serve as an intrinsic probe of the
fate of its excitation energy. Both the spectra
and the kinetics of Chl fluorescence have
proven to be powerful, non-invasive tools for
such investigations. Concerning the kinetics,
the fluorimeters used are of two basic types,
one recording fluorescence signals induced by
continuous, and the other by modulated
excitation. Really important is to make the
links between the fluorescence signal and the
biophysics of the system, for which
appropriate theory/methodology is needed.
Depending on the origin of the excited state
and the time between the moment of
absorption of the light quant and the moment
of radiation, fluorescence can be prompt or
delayed.
When the photosynthesizing samples, adapted
in dark (10-30mins), get radiated by a
powerful photosynthesizing active (actinic)
light, the chlorophyll molecules begin to emit
fluorescence, the intensity of which varies with
time and describes a specific pathway known
as induction curve (IC. The phenomenon is
known also like the Kautsky effect (Kautsky
and Hirsch 1931). As it can be observed during
the illumination period, fluorescence is
labelled as prompt (PF). For the plants and
algae IC of PF, measured during illumination
consist of a rapid phase of growth (within a
second) and slow downs (within minutes) and
have several characteristic points / phases (Fig.
2). Each phase of the induction kinetics reflect
different state of PHSA in transition by dark to
light adaptation.
The second type of luminescence emitted by
plants, delayed fluorescence, was discovered
by Strehler and Arnold in 1951.The light
quanta of DF is broadcast from chlorophyll
molecules of PS II, secondary excited as a
result of the radiating recombination of the
charges in the reaction centre( Lavorel, 1975;
Malkin, 1978; Radenovich et al., 1994;
Goltsev et al., 2009).They carry information
for both direct and inverse reactions for the
electronic transfer in the donor and acceptor
side of PHS II. Due to the low probability of
the reverse reactions the intensity of the DF is
extremely low (several orders lower than that
of PF). The emission spectra a PF and DF are
identical (Arnold and Thompson 1956;
Clayton 1969; Sonneveld et al. 1980), which
shows that DF also emanates from S1 level of
antenna Chl a (Krause and Weis 1991; Lang
and Lichtenthaler 1991). Like PF, DF is
emitted mainly, but not only, by the PHS II
(Jursinic 1986).
Hansatech Instruments Ltd (Kings Lynn, UK)
developed a new tool - m PEA
(multifunctional Plant Efficiency Analyzer)
(Fig. 3) allowing for fast and very informative
sub-millisecond time resolution analysis (in
vivo and in situ) of the functional status of the
photosynthetic apparatus in plants. It is based
on a simultaneous signal 16-bit resolution
registration of the kinetic characteristics of
prompt chlorophyll fluorescence emission,
delayed
chlorophyll
fluorescence
and
modulated light scattering and reflection of the
actinic incident light at 820 nm (Fig.4).
Figure 2: The M-PEA system allows simultaneous
measurement of prompt and delayed chlorophyll
fluorescence and 820 nm reflection signals as well
as relative chlorophyll content in living plant
tissues (http://www.hansatech-instruments.com).
210 Interdisplinary Journal of Research and Development
“Aleksandër Moisiu“ University, Durrës, Albania
Vol (IV), No.2, 2017
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Figure 3: A typical Chl a fluorescence transient
The analysis of strong actinic light-induced
(Kautsky curve) exhibited upon illumination of a
fluorescence rise kinetics O-J-I-P by, what we
dark-adapted pea leaf by saturating red light (peak
call the ‘JIP-test, can be applied at any
at 650 nm), plotted on a logarithmic time scale.
physiological state and for the study of any
state transition, including the analytical
derivation of formulae for the translation of
selected data stored in the O-J-I-P transient to
structural
and
functional
parameters
quantifying PS II behaviour. The calculated
parameters are: the so-called ‘specific’ (per
RC) and ‘phenomenological’ (per excited
cross section, CS, of a photosynthetic sample)
energy fluxes of absorption, trapping and
electron transport; the quantum yields of
primary photochemistry and of electron
transport, as well as the efficiency by which a
trapped exaction moves an electron into the
electron transport chain further than QA; the
density of QA-reducing RCs which, by
comparison of samples under different
conditions, leads to the calculation of the
difference in the fraction of non-QA-reducing
RCs (heat sinks or ‘silent’ centres); the socalled ‘performance indexes’ and ‘driving
forces’; the turnover number expressing the
number of QA reduction events until all QA
molecules are reduced; the average excitation
energy of open RCs from time 0 (when all RCs
are open) until the time that all become closed;
and the overall grouping probability.
Fluorescence values are expressed as F/F0,
where F0 is the initial fluorescence (at50 µs).
The actinic light was focused on the sample
surface to provide a homogeneous irradiation
of the exposed area (4 mm diameter); the
maximal intensity of the light source (600 W
m–2, or 3200 µmol photons m–2 s–1), was
used. The O-J-I-P fluorescence rise is followed
by a decline to a steady-state S. Inserts show
the same transient on different linear time
scales: (a; centre) upto 120 s, (b; top left) up to
200 ms and (c; top right) up to 20 ms.
Figure 4: Induction kinetics of simultaneously
measured prompt and delayed chlorophyll
fluorescence and 820nm light reflection signals in
intact bean leaves, illuminated with 2000 µmol
photons· m-2· s-1 light intensity.
DF emitted at different dark time intervals are
integrated and presented as values of DF
induction curves.
211 Experimental in vivo measurements of light emission in plants
E. Çaçi
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Table 1. Definition of terms and formulae for
calculation of the JIP-test parameters from the Chl
a fluorescence transient OJIP emitted by darkadapted leaves (modified from Strasser et al. 2010).
Fluorescence
parameter
ϕ PO = (1 − FO ) FM
ϕ EO = (1 − FJ FM )(1 − VJ )
ϕ RO = (1 − FI FM )(1 − VI )
Description
Maximum quantum yield of
primary photochemistry (at t = 0)
Efficiency/probability that an
electron moves further than QA
Quantum yield for reduction of
end electron acceptors at the PSI
acceptor side (RE)
ψ E = 1 − VI
Probability (at t = 0) that a trapped
exciton moves an electron into the
electron transport chain beyond
Q A-
δR O = (1 − VI ) (1 − VJ )
Efficiency/probability with which
an electron from the intersystem
electron carriers moves to reduce
end electron acceptors at the PSI
acceptor side (RE)
γ RC = Chl RC / Chltotal
Probability that a PSII Chl
molecule functions as RC
O
M0
Approximated initial slope (in ms–
1
) of the fluorescence transient V =
f(t)
ABS / RC = 1 − γ RC / γ RC
Absorption flux (of antenna Chls)
per RC
TRo / RC = M 0 (1 / VJ )
PI ABS =
γ RC ϕ Po ψ Eo
. .
1 − γ RC 1 − ϕ Po 1 −ψ Eo
PI total = PI ABS
δ Ro
1 − δ Ro
Trapping flux (leading to QA
reduction) per RC
Performance index (potential) for
energy conservation from exciton
to the reduction of intersystem
electron acceptors
Performance index (potential) for
energy conservation from exciton
to the reduction of PSI end
acceptors
212 Conclusions
Simultaneous measurement of prompt and
delayed chlorophyll a fluorescence and 820
nm light reflection in intact leaves by the
MPEA allows a precise detailed evaluation of
the plant physiological state to be made,
obtaining wide range of parameters that give
information about the state of the
photosynthetic machinery structures such as:
• Relative antennae size – ABS/RC (Table
1)
• Active PSII reaction centres - γRC (Table 1)
• PSII donor side activity (OEC) – K-band
• Grouping of the photosynthetic units – L –
band
• PSII acceptor side activity – dark drops
• Electron capacity of the electron transport
chain – Sm/Ss
• Efficiency of the electron transfer in
different sites – φPo, φEo, φRo, ψEo, δRo – JIP
test (Table 1)
• Overall efficiency of the photosynthetic
machinery – PIABS and PItotal – JIP test
(Table 1)
• Rate constants of the electron transfer
reactions within PSII – DF decay kinetics
(Fig.4)
• Rates of the photo induced PSII
oxidation/re-reduction (activity of PSI and
PSI + PSII) – MR820
• Cyclic electron flow around PSI - MR820
References
1. Goltsev, V. et al (2013) Analysis of
Dark Drops, Dark-Induced Changes in
Chlorophyll Fluorescence during the
Recording of the OJIP Transient. In:
Kuang T, Lu C, Zhang L (eds)
Photosynthesis Research for Food,
Fuel and the Future. Springer-Verlag,
Berlin Heidelberg, pp 179-183
2. Kalaji, M.H.,
Łoboda, T. (2009)
Chlorophyll fluorescence to in plants’
physiological
state
researches.
Publisher: Warsaw University of Life
Sciences -SGGW, Warsaw 2009
3. Kalaji, M.H. et al (2014) The use of
chlorophyll fluorescence kinetics
analysis to study the performance of
Interdisplinary Journal of Research and Development
“Aleksandër Moisiu“ University, Durrës, Albania
Vol (IV), No.2, 2017
__________________________________________________________________________________________
4.
5.
6.
7.
8.
photosynthetic machinery in plants. In:
P.
Ahmad
(Ed):
Emerging
Technologies and Management of
Crop Stress Tolerance. Elsevier, Vol.
II.
DOI:
http://dx.doi.org/10.1016/B978-0-12800875-1.00015-6
Reto J.S et al: Analysis of the
Chlorophyll a Fluorescence Transient.
Rubin A.B. Teoricheskaya (2004)
Biofozoka. Tom1, 2 Hardcover.
Maxwell, PC., Biggins, J (1976) Role
of cyclic electron transport in
photosynthesis as measured by the
photoinduced turnover of P700 in
vivo. Biochemistry 15: 18:3975-3981.
Strasser, R. J. et al (2010)
Simultaneous in vivo recording of
prompt and delayed fluorescence and
820 nm reflection changes during
drying and after rehydration of the
resurrection
plant
Haberlea
rhodopensis. BBA, 1797, 1313–1326
Гольцев В. Н. et al (2014).
Переменная
и
замедленная
флуоресценция хлорофилла a –
теоретические
основы
и
практическое
приложение
в
исследовании растений. – М.–
Ижевск: Институт компьютерных
исследований, – 220 с.
213