Journal of Experimental Botany, Vol. 52, No. 356,
Compartmentation Special Issue, pp. 615±621, April 2001
High resolution imaging of photosynthetic activities of
tissues, cells and chloroplasts in leaves
Neil R. Baker1, Kevin Oxborough, Tracy Lawson and James I.L. Morison
Department of Biological Sciences, University of Essex, Colchester, Essex CO4 3SQ, UK
Received 31 March 2000; Accepted 19 October 2000
Abstract
Introduction
Through imaging of chlorophyll fluorescence, it is
possible to produce parameterized fluorescence
images that estimate the operating quantum efficiency of photosystem II (PSII) photochemistry and
which can be used to reveal heterogeneous patterns
of photosynthetic performance within leaves. The
operating quantum efficiency of PSII photochemistry
is dependent upon the effective absorption crosssection of the light-harvesting system of PSII and
the photochemical capacity of PSII. The effective
absorption cross-section is decreased by the process of down-regulation, which is widely thought to
operate within the pigment matrices of PSII and
which results in non-photochemical quenching of
chlorophyll fluorescence. The photochemical capacity is non-linearly related to the proportion of PSII
centres in the `open' state and results in photochemical quenching of chlorophyll fluorescence.
Examples of heterogeneity of the operating quantum
efficiency of PSII photochemistry during the induction
of photosynthesis in maize leaves and in the chloroplast populations of stomatal guard cells of a leaf of
Tradescantia albifora are presented, together with
analyses of the factors determining this heterogeneity. A comparison of the operating quantum efficiency
of PSII photochemistry within guard cells and
adjacent mesophyll cells of Commelina communis is
also made, before and after stomatal closure through
a change in ambient humidity.
Chlorophyll ¯uorescence has long been an important tool
for the estimation of a range of photosynthetic parameters in leaves. The development and commercial
availability of modulated ¯uorometers, together with an
increased understanding of the factors that determine the
yield of chlorophyll ¯uorescence, has led to the widespread use of chlorophyll ¯uorometry as a non-invasive
method for assessing photosynthetic performance in
leaves. Perhaps the single most useful ¯uorescence parameter is (F 9m F 9)uF 9m , which is theoretically proportional to the operating quantum ef®ciency of PSII
photochemistry and which frequently exhibits a strong,
quantitative relationship with the quantum yield of CO2
assimilation (F CO2 ) (Genty et al., 1989, 1990; Di Marco
et al., 1990; Krall and Edwards, 1990; Edwards and
Baker, 1993; Cornic, 1994). Since the light-driven oxidation of water by PSII ultimately provides the reducing
power for CO2 assimilation, it is not too surprising that
such a relationship exists in many situations.
It is well established that changes in photosynthetic
activity within leaves can be heterogeneous. Imaging of
chlorophyll ¯uorescence from leaves has revealed heterogeneous responses to changing CO2 concentration (Genty
and Meyer, 1995; Siebke and Weis, 1995a, b; Bro et al.,
1996), changing photon ¯ux density (Eckstein et al.,
1996), low growth temperature (Oxborough and Baker,
1997a), treatment with abscisic acid (Daley et al., 1989),
in regions of fungal infection (Scholes and Rolfe, 1996)
and during induction of photosynthesis (Oxborough and
Baker, 1997b). It is now possible to generate images of
chlorophyll ¯uorescence, under very low light and at
the cellular and subcellular levels of organization. This
allows for a detailed assessment of the differences in
Key words: Chlorophyll fluorescence, chloroplasts, downregulation, imaging, leaves, photosynthesis, photosystem II
photochemistry, stomata.
1
To whom correspondence should be addressed. Fax: q44 1206 873416. E-mail: [email protected]
Abbreviations: cI, intercellular CO2 concentration; F9, fluorescence level at any point between F9o and F9m; Fm, maximal fluorescence level from darkadapted leaves; F9m , maximal fluorescence level from leaves in light; Fo , minimal fluorescence level from dark-adapted leaves; F9o , minimal fluorescence
F); Fv, variable fluorescence level from dark-adapted leaves
level of leaves in light; F9q , difference in fluorescence between F9m and F (F9
q ˆF9
m
(F v ˆFm Fo ); F9v , variable fluorescence level of leaves in light (F9v ˆF9m F9o ).
ß Society for Experimental Biology 2001
616
Baker et al.
photosynthetic activities between cells to be made and
has the potential to provide an understanding of the
reasons for the heterogeneity in photosynthetic metabolism across leaves. In this article, the ¯uorescence
parameters that are required to carry out such analyses
are reviewed and examples are provided of how the
technique has been used, with intact leaves, to estimate
and analyse the photosynthetic performances of different
cell types and individual chloroplasts within cells.
Materials and methods
Hardware
The instrument used in these experiments is essentially the
same as that described previously (Oxborough and Baker,
1997a). One important change to the instrument has been
modi®cation of the lower light source (which is used to
illuminate the opposite side of the leaf to the one being imaged)
so that a much larger area of leaf (a circle of 2.5 cm diameter)
is illuminated. This was required in studies of stomatal regulation because only a small area is illuminated by the upper light
source (through the lens) and this can result in the internal CO2
concentration (ci) being determined by diffusion within the
leaf, from the surrounding (non-illuminated) tissue, rather than
by changes in stomatal aperture. With most leaves, very little
of the light from the lower light source is transmitted through to
the upper surface. However, the system has been designed in
such a way that the upper and lower light sources are under
independent computer control. This allows the change in light
output from the upper and lower light sources during a
saturating pulse to be matched or for the lower light source to
be shuttered out completely while imaging is taking place.
Fluorescence terminology
The calculation of useful ¯uorescence parameters, irrespective
of whether ¯uorescence measurements are made with conventional modulated ¯uorometers or imaging systems, requires that
the ¯uorescence signal is recorded while the photosynthetic
system is in known states. With dark-adapted material, the Fo
level of ¯uorescence is recorded at very low PPFD (generally less
than 1 mmol m 2 s 1), which leaves virtually all PSII centres in
the `open' state (capable of photochemistry). The Fm level of
¯uorescence is recorded during a short pulse at very high PPFD
(typically less than 1 s at several thousand mmol m 2 s 1), which
transiently drives a very high proportion of PSII centres into the
`closed' state (making the capacity for photochemistry close
to zero). With light-adapted material, the equivalent terms are
F o9 and F m
9 : At any point between F o9 and F m
9 (where a variable
proportion of PSII centres are in the `open' state), the
¯uorescence signal is termed F9. The difference between Fm
and Fo is termed Fv and the difference between F m
9 and F o9 is
termed F v9: While Fv and F v9 have been widely used for a number
of years, there is no equivalent speci®c term, in general usage, to
quantify the difference between F m
9 and F9 (although DF has
been used in this context, this is actually a general term denoting
the difference in the ¯uorescence signal measured at two points
that has been used in many other contexts). The speci®c term
F q9 has recently been introduced to denote the difference
between F m
9 and F 9 measured immediately before application
of the saturating pulse used to measure F m
9 (Oxborough and
Baker, 2000; Oxborough et al., 2000). The diagram in Fig. 1
illustrates this terminology and shows the sequence of events
leading to the acquisition of the ¯uorescence images required for
construction of parameterized images.
As noted above, F q9 uF 9m is theoretically proportional to the
operating quantum ef®ciency of PSII photochemistry (hereafter
referred to as PSII operating ef®ciency). On the same theoretical
basis, F q9 uF 9m is actually the product of two other useful
parameters, F v9uF m
9 and F q9 uF 9v as shown by Equation 1.
F q9 F 9v F9q
ˆ
F 9m F9m F 9v
(1)
9 provides an estimate of the maximum quantum ef®ciency
F v9uF m
of PSII photochemistry (hereafter referred to as PSII maximum
ef®ciency), i.e. the PSII operating ef®ciency if all PSII centres
were in the `open' state at the point of measurement. In most
situations, its value is largely determined by down-regulation,
which appears to involve the operation of one or more processes
that increase the rate constant for non-radiative decay of
excitation energy within the pigment matrix associated with
PSII. The remaining parameter, F q9 uF 9v ; is a factor (hereafter
referred to as the PSII ef®ciency factor) that relates the PSII
maximum ef®ciency to the PSII operating ef®ciency. Its value is
Fig. 1. Fluorescence trace illustrating the terminology used and the
sequence of events leading to the acquisition of the raw ¯uorescence
images that are required for the construction of parameterized images.
The exposure time of the Fo or F image could be anywhere from a few
tens of ms to several min, depending upon the material and incident
9 image is generally within the
PPFD. The exposure time of the Fm or F m
range of 20±100 ms.
Fig. 2. Theoretical data showing the relationship between F 9q uF v9 and the
fraction of PSII centres in the open state (given by wQAx). The dashed line
represents a PSII population with zero connectivity. The solid lines
represent a PSII population with perfect connectivity, at different levels
of down-regulation (the arrow indicates the effect of increasing downregulation). The model used to generate these data is described in
Oxborough and Baker (Oxborough and Baker, 2000).
Imaging photosynthesis of leaf cells 617
Fig. 3. Images of F 9v uF 9m (A, D, G), F 9q uF 9v (B, E, H) and F 9q uF 9m (C, F, J) from a healthy maize leaf during induction at a PPFD of 815 mmol m 2 s 1.
(A), (B) and (C) were taken at 2 min, (D), (E) and (F) after 8 min, and (G), (H) and (I) after 1 h. For each image the left histogram shows the range of
values within the image on a scale of possible values, while the right histogram shows how these values are mapped to the colour palette. Data have
been mapped to the palette in such a way as to emphasize the heterogeneity within each image.
non-linearly related to the proportion of PSII centres in the
open state.
The term WPSII has often been used to denote F q9 uF9m in the
literature and, in this context, has generally been used as a proxy
for the quantum yield of electron transfer from water, through
PSII and into the plastoquinone pool. One reason for not using
WPSII is that the symbol W is already employed in the widely
used term, WCO2, which denotes the quantum yield of CO2
assimilation. In the case of WCO2, W refers to the quantum yield
of photosynthetically-active photons that are absorbed by a
leaf, while the W in WPSII refers to the quantum yield of
photosynthetically-active photons that are absorbed only by
618
Baker et al.
PSII. Consequently, WCO2 and WPSII refer to the quantum yields
of two different quantities and their use in the same context is
potentially confusing.
The PSII ef®ciency factor, F q9 uF v9; is mathematically the
same as the widely used coef®cient of photochemical quenching, qP. One reason for not using qP is that, in the theoretical
context of the relationship described by Equation 1, F q9 uF v9 is
a factor, not a coef®cient. Another reason for avoiding the use
of qP is that it has been very widely used as an estimate for
the proportion of PSII centres in the `open' state. While this
would be reasonable if there were little or no connectivity
among PSII centres, current evidence suggests that the level of
connectivity between PSII centres is actually quite high, which
makes the relationship between the closure of PSII centres and
the ¯uorescence signal curvilinear (Joliot and Joliot, 1969). The
theoretical relationship between F q9 uF v9 and the concentration
of open PSII centres (determined from the concentration of the
primary quinone acceptor, QA) is shown in Fig. 2 and illustrates
how this curvilinearity decreases as down-regulation increases.
In order to calculate the parameters F9v uF m
9 and F q9 uF v9;
it is required that the value of F o9 is known (because
F v9ˆF m
9 F o9 ). Images of F o9 cannot be taken with the imaging
system used here (or with any other imaging system that we are
aware of). However, F o9 can be calculated using Equation 2
(Oxborough and Baker, 1997b).; images of F v9uF m
9 and F q9 uF v9
were constructed in this way.
F o9 ˆ
Fo
Fv
Fo
q
Fm F 9m
(2)
Examples of imaging and analyses of
photosynthetic activities in leaves
Induction of photosynthesis in dark-adapted leaves
It is well established that oscillations in ¯uorescence
can occur during the induction of photosynthesis when
dark-adapted leaves are illuminated (Peterson et al., 1988;
Keiller and Walker, 1990). Images of a dark-adapted
maize leaf were taken at the Fo and Fm levels of ¯uorescence, after which the leaf was exposed to a PPFD of
815 mmol m 2 s 1. Images at F 9 and F m
9 were taken at
intervals during the induction of CO2 assimilation over a
period of 1 h and images of F q9 uF 9m ; F v9uF m
9 and F q9 uF v9
constructed (Fig. 3). It should be noted that although
the resolution of these images is relatively low, in the
context of this microscope-based system, it is approximately 1000 times higher (on an area basis) than wholeleaf imaging systems previously reported. After 2 min
exposure to light, the PSII operating ef®ciency (F q9 uF 9m )
was low, and little heterogeneity was observed within
the images of F q9 uF 9m ; F v9uF m
9 or F q9 uF v9; an apparently
normal distribution of values within a narrow band about
the mean being evident for all three parameters. After
8 min in the light, the mean value of F q9 uF 9m had increased
from c. 0.07 to 0.35, and similar patterns of heterogeneity
were observed in the images of F q9 uF 9m and F q9 uF v9; but not
in the image of F v9uF m
9 : The pixel values for the images
of F q9 uF 9m and F q9 uF v9 exhibited a clear bimodal distribution, with the pixel values for the F v9uF m
9 image showing
a more normal distribution. It can also be seen that the
large increase in F q9 uF 9m that occurred between 2±8 min
was accompanied by a large increase in F q9 uF v9; but only a
very small increase in F v9uF m
9 : Clearly, this increase in the
PSII operating ef®ciency was due to an increase in the
capacity for electron transfer on the reducing side of PSII,
rather than a decrease in the level of down-regulation. In
the context of photosynthetic metabolism, the increase
in F q9 uF m
9 between 2 and 8 min, in the absence of any
Fig. 4. (A) An image of F 9q uF 9m from chloroplasts within a pair of guard cells of an attached leaf of Tradescantia albifora. (B) A re¯ected light image of
the same area. Mean values of F 9v uF 9m ; F 9q uF v9 and F 9q uF 9m are given for two chloroplasts within these guard cells. The left histogram shows the range
9 image on a scale of possible values, while the right histogram shows how these values are mapped to the colour palette.
of values within the F q9 uF m
Imaging photosynthesis of leaf cells 619
signi®cant change in F v9uF m
9 over the same period, is
indicative of an increase in the electron ¯ux through
PSII, due to an increased rate of utilization of the products of non-cyclic electron transport (NADPH and
ATP). After 1 h, the heterogeneity had all but disappeared and a reasonably uniform distribution across the
leaf was again observed for all three parameters.
Photosynthesis in stomatal guard cells and
adjacent mesophyll tissue
The imaging system (described by Oxborough and
Baker, 1997a) allows for images of re¯ected light and
chlorophyll ¯uorescence at F 9 and F m
9 to be taken from
the same area of leaf within a short time frame (-10 s).
A potentially useful application of this feature is in the
study of the relationship between changes in stomatal
aperture and photosynthetic ef®ciency within the chloroplasts of guard and mesophyll cells. The images in Fig. 4
are from a region around a pair of guard cells of an
attached leaf of Tradescantia albifora. The mean values of
F q9 uF m
9 for the chloroplasts within these guard cells range
between 0.27 and 0.43, indicating a wide range of PSII
operating ef®ciencies within the chloroplast populations
of the cells. Two chloroplasts, with F q9 uF m
9 values of 0.40
and 0.31, have been isolated in Fig. 4. Mean values of
F q9 uF v9; and F v9uF m
9 were also calculated. What these values
reveal is that, as was the case for the induction of photosynthesis in maize leaves (Fig. 3), the heterogeneity
observed in the PSII operating ef®ciency (F q9 uF m
9 ) is
largely attributable to differences in the capacity for
electron ¯ux on the reducing side of PSII, rather than
down-regulation, since most of the difference in PSII
operating ef®ciency is due to a difference in the PSII
ef®ciency factor (F q9 uF v9) rather than the PSII maximum
ef®ciency (F v9uF m
9 ).
The third example compares the photosynthetic
activities within guard cells chloroplasts and adjacent
mesophyll cells (Fig. 5), before and after the induction of
stomatal closure through a decrease in relative humidity.
Before the humidity was decreased, the mean PSII
operating ef®ciency (F q9 uF m
9 ) within the guard cell
chloroplasts was 0.42, while the equivalent value within
the adjacent mesophyll cells was signi®cantly higher at
0.53. Decreasing the relative humidity from 56% to 9%
resulted in closure of the stoma within the ®eld of view
(and presumably a decrease in cI) and resulted in a substantial decrease in the mean values of F q9 uF m
9 ; to 0.30
within the guard cells and 0.41 within adjacent mesophyll
cells. Although there was no clear difference in the way
that the guard cell and mesophyll cell chloroplasts
responded to closure of the stoma, these data clearly
illustrate the way in which high resolution imaging of
chlorophyll ¯uorescence can be used to distinguish the
photosynthetic performance of closely adjacent cells.
Concluding remarks
It is clear, from the examples presented above, that highresolution imaging of PSII operating ef®ciency, through
the ¯uorescence parameter F q9 uF m
9 ; provides a powerful
tool for identifying differences in the photosynthetic
performance of different plant tissues, cells and individual
chloroplasts within intact leaves. Also, analyses of images
of F v9uF m
9 and F q9 uF v9 allow for an evaluation of the extent
to which differences in F q9 uF m
9 among samples are due to
differences in the maximum ef®ciency of PSII photochemistry anduor differences in the capacity for photochemistry. In the two examples shown in Figs 3 and 4,
most of the heterogeneity observed in the images of
F q9 uF m
9 could be attributed to differences in the capacity
for photochemistry at PSII (which is re¯ected in the
images of F q9 uF v9). In most situations, the assimilation of
CO2 is the main sink for the reducing power that is
provided by PSII photochemistry. It therefore seems
Fig. 5. Images of F 9 (A, D), F 9q uF 9m (B, E) and re¯ected light (C, F) from
chloroplasts within a pair of guard cells of an attached leaf of Commelina
communis. The histograms relate to the data in (B) and (D); the left of
each pair of histograms shows the range of values within each image on
a scale of possible values, while the right shows how these values are
mapped to the colour palette. Data have been mapped to the palette in
such a way as to emphasize the differences in F 9q uF 9m between the two
conditions.
620
Baker et al.
Fig. 6. Image of re¯ected light (A) and luciferase activity (B, C) from an Arabadopsis plant, after 30 min of light stress at a PPFD of
1000 mmol m 2 s 1. Both images were taken (using a 16 mm lens) with the same type of Peltier-cooled camera that is used with the microscope-based
imaging system described in this paper. The re¯ected light image was taken over 100 ms, under normal room lighting, while the image of luciferase
activity was taken over 10 min in complete darkness. It can be seen from (C) that the luciferase activity is primarily associated with the major veins of
the leaf.
likely that the differences in PSII operating ef®ciencies
and PSII ef®ciency factors that are evident within Figs 3
and 4 are related to the supply of CO2 to different areas of
the leaf (Fig. 3) or different chloroplasts within closely
adjacent cells (Fig. 4).
A potentially important application of ¯uorescence
imaging is in understanding the response of plants to
various types of stress. It is well established that many
abiotic and biotic stresses result in a rapid modi®cation
of chlorophyll ¯uorescence characteristics of leaves associated with the inhibition of photosynthetic metabolism
and the development of photo-oxidative perturbations of
the photosynthetic apparatus. Consequently, ¯uorescence
imaging offers a non-intrusive method for the identi®cation of sites of stress in leaves. High-resolution ¯uorescence imaging provides the additional bene®t of allowing
for the detection of the early onset of stresses, when very
few cells (or even chloroplasts within individual cells) are
affected. Coupling of this technique with methods for
studying gene expression in individual cells within leaves,
such as single cell sampling and message ampli®cation,
should allow identi®cation of changes in gene expression
that result from the onset of stress in leaf cells. A particularly striking example of the potential of ¯uorescence
imaging to examine the molecular bases of photooxidative stress in leaves comes from studies of expression
of a peroxidase (APX2) gene in Arabidopsis. This gene is
expressed rapidly after the onset of severe photoinhibitory stress in the leaves (Karpinski et al., 1997). The
promoter of APX2 has been fused to the ®re¯y luciferase
reporter gene (LUC ) and has been used to transform
Arabidopsis (Karpinski et al., 1999). The APX2-LUC
fusion gene is induced in parallel with the native APX2
gene during photoinhibitory stress and this expression
has been imaged after painting leaves with luciferin to
elicit luminescence (Fig. 6; M Fryer, K Oxborough,
PM Mullineaux, NR Baker, unpublished data). It is most
striking that the gene expression in response to the high
light stress is only associated with the major veins of
the leaf. Currently, the relationships between the photosynthetic responses of the mesophyll cells and the induction of APX2 gene expression is being investigated by
parallel imaging of the chlorophyll ¯uorescence parameters, described above, and the luminescence associated
with LUC gene expression from the same leaves. Such
studies will help to resolve the signalling pathways
involved in triggering gene expression in response to
photo-oxidative stresses.
Imaging photosynthesis of leaf cells 621
Acknowledgements
The imaging studies presented were supported by grants to NRB
and JILM from the Biotechnology and Biological Sciences
Research Council. We are grateful to Mike Fryer and Phil
Mullineaux for assistance with generating the images presented
in Fig. 6.
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