Remote sensing of vegetation fluorescence can yield estimates of sunlight absorbed inside a
complex canopy and sense the on/off status of the photosynthetic machinery.
Ladislav Nedbal1,2, Michal Marek1 and Martin Trtílek3
1
Institute of Landscape Ecology CAS and 2Institute of Physical Biology JU, Zámek 136, 37333 Nové
Hrady, Czech Republic / nedbal@greentech.cz
3
Photon Systems Instruments, spol. s r.o., Kolackova 39, 621 00 Brno, Czech Republic / www.psi.cz
Abstract
Plant physiology and, in particular, research of plant fluorescence signals can help in identifying the
information content of the data potentially acquired by a space-based imaging fluorometer. From this
perspective, we propose that it is feasible to retrieve reliable information on how much of the incident
irradiance is absorbed by photosynthetic apparatus within a vegetation of high complexity. We suggest
that this is new and highly relevant information that cannot be obtained on a global scale by any existing
alternative reflectance technique as all the reflectance data inform only about the top, visible layer of the
canopy. Second, we propose that it is possible to use the fluorescence data to identify the on/off status of
the photosynthetic machinery. The status change can be deduced from the day-to-day variability of the
fluorescence emission during the transition periods that occur with seasonal changes and with severe
stress induction or relaxation.
We propose that the space-acquired information must be supplemented for validation by information
from a network of ground-based calibration stations spread over relevant vegetation types that will
provide an extensive set of measured features, e.g., F656, F760, FS/FM’ as well as REI, PRI, and other. For
this purpose, we constructed a pen-size, low-cost measuring instrument. The data-to-information
conversion must be supported by extending the present modeling/validation effort towards synergistic
effects of irradiance and temperature on steady-state Chl-fluorescence. For this we propose to
interconnect the FluoMOD project with our open web portal www.e-photosynthesis.org that will be
available in early 2005.
Introduction
Planning of a space-based chlorophyll fluorometer requires identification of challenges both in
instrument design and in conversion of data into useful information. Here, we neglect the first type of
the challenge and assume that it is possible to build a perfect instrument that can deliver noise-free
information on fluorescence emitted by terrestrial vegetation. We focus only on the spectral windows of
chlorophyll fluorescence in the 656nm H-Frauenhofer line and in the 760nm O2-absorption line. We
assume that the fluorescence measurement will be complemented by parallel hyperspectral reflectance
imaging in VIS/NIR region. We assume that it will be possible to obtain the fluorescence and reflectance
signals on daily basis from the same ground location with a resolution of ca. 500 x 500 meters.
Variability of chlorophyll fluorescence emission in a dynamic light
Chlorophyll fluorescence emission of a healthy higher plant can change several fold within seconds (Dau
1994, Govindjee 1995, Falkowski and Kolber 1995, Kramer and Crofts 1996, Strasser et al. 1998, Lazár
1999, Krause and Jahns 2002, Nedbal and Whitmarsh 2004, Nedbal and Koblížek 2005). The change
from a dark-adapted level (F0) to flash-saturated level (FM) constitutes the largest fraction of this
variability range (typically 5-fold increase). This change of chlorophyll fluorescence emission is due to
reduction of the plastoquinone pool and, consequently, of the primary quinone acceptor of Photosystem
II in a bright flash of light (Duysens and Sweers 1963). Among the additional factors that can modulate
the chlorophyll fluorescence yield are the quantum efficiency of Photosystem II (PSII), the transitions
between the S-states of the oxygen evolving complex, the rate of steady state and cyclic electron
transport, biotic and abiotic stress, and much more. The dynamics of the chlorophyll fluorescence is
widely studied and used in photosynthesis research with emerging applications in ecology,
biotechnology and in precision farming (e.g., Bolhar-Nordenkampf et al. 1989, Mohammed et al. 1995,
Lichtenthaler and Miehé 1997, Jalink et al. 1998, DeEll et al. 1999, Nedbal et al. 2000).
Interpretation of the dynamic changes in chlorophyll fluorescence emission of higher plants typically
relies on application of elaborate light protocols with periods of dark adaptation and periods of constant
actinic light that are supplemented by saturating bright flashes (reviewed in Nedbal and Koblížek 2005).
This approach cannot be used in remote sensing. The gap between the active fluorescence techniques
developed for a leaf level and remote sensing is partially bridged by a proposal of Z.Kolber (to our
knowledge published only in 2000 during the 12th International Congress on Photosynthesis, Brisbane,
Australia; see also Kolber 2002). Kolber suggested using a grid of laser diodes at the bottom of a lowaltitude-flying aircraft to generate a periodic wave-like pattern of eye-safe actinic light. With the aircraft
flying, the pattern would move over the landscape generating a harmonically modulated irradiance
transient in individual plants. We confirm by our own independent experiments that the harmonically
modulated light generates information rich transients (Nedbal and Brezina 2002, Nedbal et al. 2003 and
Nedbal et al. 2004). Currently, we experiment with frequencies of harmonic light modulation that
approach those anticipated from a flying aircraft. We propose that the method originally proposed by
Kolber has an enormous potential for extending the active fluorescence techniques from the leaf level to
a large canopy level monitored from an aircraft. However, we do not anticipate that a similar approach
could be used in a satellite-based chlorophyll fluorometer in a near future.
Stability of chlorophyll fluorescence emission in a stationary or slowly changing light.
Resigning on our capability to generate, from space, dynamic light patterns, we can turn to the seemingly
opposite experimental strategy. Evolution of photosynthesis is, among other constraints, driven by
selection of genes for photochemical energy conversion that is robust and yet, effective, in a fluctuating
light environment. Variable cloud cover, moving canopy and ocean waves are among the dynamic
factors that have been shaping the photosynthetic regulation since the first ancestors of modern plants
emerged. It is sensible to assume that the regulatory networks concerting the activity of the two
photosystems and of the coupled dark reactions are tuned to light that fluctuates around a mean level
with characteristic periods extending from seconds to minutes (Pearcy 1990, Pearcy et al. 1994, Külheim
et al. 2002, Zhu 2004). We showed recently (Nedbal et al. 2004) that, in a slowly changing light, the
chlorophyll fluorescence emission yield is amazingly stable within a wide range of irradiance levels.
This is due to regulatory feedback mechanisms that sustain plant homeostasis (the contrasting dynamic
features reported above occur only with very rapid irradiance changes). Similar stability of fluorescence
signals was stressed, during the present workshop, by J.Harbinson.
Assuming that the chlorophyll fluorescence yield of a healthy, photosynthetically active, light-adapted
higher plant depends only slightly on the instantaneous incident irradiance, one can propose to use the
emission as a reporter on the solar energy actually absorbed by photosynthetic pigments within plant
canopy. This quantity necessarily differs from the information obtained from the red-edge index of the
reflectance measured at the canopy top layer. The difference is due to the absorption of the light that
penetrates lower, inside the canopy. The chlorophyll fluorescence emission detected at 760nm easily
penetrates from the lower levels of the canopy back to the sky, as it is not absorbed by chlorophyll.
Also, the long-wavelength emission is less variable than the short-wavelength fluorescence because it
consists of relatively larger contribution of Photosystem I compared to Photosystem II (Genty et al.
1990, Roelofs et al. 1992). This makes it a more reliable signal to sense the absorbed irradiance.
We propose that the space-sensed chlorophyll fluorescence signal measured at 760nm can yield estimate
of the sunlight absorbed by photosynthetic pigments inside a complex plant canopy; information that
cannot be accessed by measuring the red-edge index of the top canopy layer. The chlorophyll
fluorescence emission measured at 656nm can supplement the information by reflecting more sensitively
the variability imposed by environmental factors (light, temperature, stress).
Long-term trends in stationary chlorophyll fluorescence yields.
The relative stability of the chlorophyll fluorescence yield in average daylight conditions is broken once
the internal plant homeostasis is affected by a major stress. Long-lasting drought or frost induce strong
changes both in the primary photosynthetic reactions as well as in regulatory feedbacks. As a result, the
steady-state chlorophyll fluorescence largely fluctuates during onset as well as during relaxation of the
environmental stresses. We propose to detect these fluctuations as markers of on/off transitions in plant
canopy monitored from space.
Experimental strategy.
The two objectives outlined above, i.e., the estimate of sunlight captured within a complex canopy and
sensing of the on/off transitions of the photosynthetic machinery, are humble compared to the more
ambitious goals discussed in the workshop. Yet, we consider these objectives not only feasible to reach
but also highly attractive, experimentally challenging and, last-not-least, including acceptable risk. The
risk comes from the present lack of ground data to support our assumptions for various types of canopies
and under various environmental conditions. In order to minimize the risk:
1) it is necessary to measure the steady-state fluorescence emission at 760nm and at 656nm on
ground in various ecosystems, in changing irradiance and temperature; reference measurements
of gas exchange or, at least, of complex fluorescence transients in modulated actinic light are
needed;
2) it is necessary to prove that the 760nm fluorescence emission indeed radiates through in various
types of canopy architecture;
3) it is necessary to prove by more extensive data sets that the onset and relaxation of various
stresses can be detected by fluctuations of the steady-state chlorophyll fluorescence yield in
light-adapted plants.
A strong proposal for a space-based chlorophyll fluorometer requires risk minimization by a sufficiently
broad and rapid collection of experimental data on ground. Here, we propose that this goal can be most
effectively achieved by a large number of portable, low-cost fluorometers that would be collecting
specific signals that are accessible from space.
Experimental tool
We have constructed for this purpose a low-cost, pen-size instrument that measures incident irradiance
and fluorescence emission yield actively excited by modulated beam. The sensitivity range of the PIN-
diode detection can be, as an option, limited to the 656nm H-Frauenhofer line or to the 760nm O2absorption line. Equally so, blue and green fluorescence can be detected providing the excitation in the
UV-A band.
In order to enhance the interpretation capacity of our technique, we initiated construction of open web
portal for modeling of the chlorophyll fluorescence transients (www.e-photosynthesis.org). Asymptotic
solutions that are a necessary product of the modeling effort will be used to assess the steady-state levels
that are among the anticipated outputs of the space-based measurements
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