Title page
Title:
Is chloroplast movement in tobacco plants influenced systemically after local illumination or
burning stress?
Running Title:
Systemic chloroplast movement and local abiotic stress
Full names of authors:
Jan Nauš, Monika Rolencová, Vladimíra Hlaváčková
Addresses:
Laboratory of Biophysics, Department of Experimental Physics, Palacký University, tř.
Svobody 26, 771 46 Olomouc, Czech Republic
Corresponding author:
Vladimíra Hlaváčková
email: [email protected]
telephone: +420585634179
facsimile: +420585225737
Abstract
Chloroplast movement has been studied in many plants mainly in relation to the local light,
mechanical or stress effects. Here we investigated possible systemic response of chloroplast
movement to local hight light or burning stress in tobacco plants (Nicotiana tabacum cv.
Samsun). Chloroplast movement was measured using two independent methods: SPAD 502
Chlorophyll meter method and a method of collimated transmittance at selected wavelength
(676 nm). We have used a sensitive periodic movement of chloroplasts in high or low (2000
or 50 µmol. m-2 s-1 PAR, respectively) cold white light with period of 50 or 130 min.
Measurements were performed in the irradiated area, in the non-irradiated area of the same
leaf or in the leaf located on the stem below the irradiated or burned one. No significant
changes in systemic chloroplast movement in non-irradiated parts of the leaf and in the nontreated leaf were detected. Our data indicate that chloroplast movement in tobacco is
dependent dominantly on the intensity and spectral composition of the incident light and on
the local stimulation and state of the target tissue, no systemic signal is strong enough to
evoke detectable systemic response in chloroplast movement in distant untreated tissues of
tobacco plants.
Key words:
burning, chloroplast movement, irradiance, systemic response, tobacco
Abbreviations:
ABA – abscisic acid, DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea, HL – high light, LL
– low light, MV – methylviologen, PAR – photosynthetically active radiation
Financial support:
The project was supported by grant from the Ministry of Education of the Czech Republic,
No. MSM 6198959215.
Introduction
Higher plants respond to changing environmental factors affecting either the whole
plants (changing temperature, water deficit, excess light intensity) or the part (local response)
of a plant body (wounding) by initiating various defence-related processes. These processes
include e.g. the accumulation of defence-related proteins, changes in respiration, stomatal and
photosynthetic apparatus responses and also chloroplast movement. Important characteristics
of self-defence responses of plants are their velocity and ubiquity. Fast (minutes to hours)
responses to injurious factors have been detected in the site of injury and in distant regions
(systemic response) at the tissue-, cellular- and molecular- levels in various plants (Herde et
al. 1996; Baldwin et al. 1997; Rakwal et al. 2002; Koziolek et al. 2004; Hlaváčková et. al.
2006). These findings suggest that a signal moves from the injured tissue to the distant
untreated parts of the plants and leads to systemic changes.
Precise control of organelle positioning is important for plant responses at the cellular
level to environmental conditions and stresses (Nagai 1993; Wada and Suetsugu 2004). In
particular, chloroplast photo-movement is one of the responses observed in cells of many
species (including tobacco plants used in our study, Augustynowicz et al. 2001) and occurs
throughout the plant kingdom (Wada et al. 2003). Chloroplasts move towards the illuminated
area under weak light conditions, while they move away from the area when the light is too
strong. The accepted interpretation of the ecological role of these responses is to optimize
light harvesting for photosynthesis. Kasahara et al. (2002) reported that chloroplast avoidance
movement has a protective role against photoinhibition of photosynthesis in Arabidopsis.
Jeong et al. (2002) concluded that a movement of a large number of smaller chloroplasts
(rather than a few enlarged chloroplasts in transgenic tobacco) in the wild-type tobacco is
important for both efficient light utilization under low light and a better protection from the
high light induced photoinhibition. The movement of chloroplasts under strong light to
anticlinal walls (called also an avoidance response) decreases usually the amount of light
absorbed by the leaf (Brugnoli and Björkman 1992) and hence leads to a lower photosynthetic
rate measured with the whole leaf. Lechowski (1974) reported reduction of photosynthesis in
Ajuga reptans by even 50 % in the anticlinal position of chloroplasts. Sinclair and Williams
(2001) have shown that upon movement of chloroplasts to anticlinal walls the rate of oxygen
evolution decreased by about 22 %, energy storage efficiency increased, however the light
absorption in the leaf (absorptance) has fallen by about 26 %. Chloroplast movement also
belongs to photoprotective strategy of CAM plants subjected to severe water stress (Kondo et
al. 2004). This hypothesis is further supported by the fact that treatment of leaf segments of
CAM plants by abscisic acid (accumulated in plants mainly during water stress) induced
chloroplast clumping in the leaf cells under light (Kondo et al. 2004). Moreover, Sato et al.
(1999, 2003a) discovered a new type of chloroplast movement induced by mechanical
stimulation in pteridophytes and bryophytes. The response has been designated as chloroplast
“mechano-relocation movement”. Mechano-relocation movement is quite fast (the time
required to reach the maximum level in the response was 30 min - 2h, Sato et al. 2003b) and
requires the influx of external Ca2+, most likely through a stretch-activated channels located in
the plasma membrane (Sato et al. 2003b). Thus, it seems that localized deformation of a cell is
essential for directional chloroplast movement in an early step of mechano-signaling
response.
It was reported that local wounding of plants induce hydraulic pressure surges
transmitted rapidly in the xylem that triggers changes in the activity of membrane-located
mechano-sensitive channels or pumps in living cells (Mancuso 1999; Stahlberg et al. 2006),
leading to changes in the ion fluxes across their plasma membranes and, thus in the apoplastic
electrical potential. On the basis of results of Sato et al. (2003b) and electrophysiological
study of Stoelzle et al. (2003), primary function of photoreceptor phototropin is regulation of
Ca2+ influx that leads to physiological responses including chloroplast movement. Moreover,
Wada et al. (1993) suggested local changes in membrane properties, including transient
modulation of the membrane potential to be the earliest steps in signal transduction leading to
the chloroplast movement. Whether the potential changes could affect chloroplast movement
systemically is a question to be answered.
In our previous studies, we observed that fast electrical signal (induced by a
propagating hydraulic signal through activation of mechano-sensitive stretch-activated
channels) may trigger systemic stomatal and photosynthetic responses of tobacco plants and
short-term chemical defence-related (accumulation of abscisic and jasmonic acid) signaling
pathways in tobacco (Hlaváčková et al. 2006; Hlaváčková and Nauš 2007). As chloroplast
movement is closely linked to photosynthetic efficiency, its induction by cell deformation
(activation of mechano-sensitive stretch-activated channels) and ABA treatment is plausible
and the same photoreceptors (phototropin 1 and 2) were found for controlling of chloroplasts
and stomata movements (Briggs and Christie 2002), one could expect systemic changes in
chloroplast movement after local wounding simultaneously with the above mentioned
photosynthetic, stomatal and electrical responses. The hypothesis of systemic responses of
chloroplast movement and thus existence of possible systemic signal was supported by study
of Kagawa and Wada (1999). In a fern Adiantum capillus-veneris, microbeam irradiation at a
high fluence rate not only triggered movement of chloroplasts out of the illuminated area, but
also caused movement of distant chloroplasts towards the illuminated region (Kagawa and
Wada 1999). Thus, in Adiantum part of the signal can be transferred from the irradiated area
in the centre of the cell to the cell periphery. Cytoplasmic Ca2+ probably forms part of the
signalling system as chloroplast movement of Lemna trisulca was associated with small
increases in cytoplasmic Ca2+ and was blocked by antagonists of calcium homeostasis (Tlałka
and Fricker 1999). However, to our knowledges, no papers deal with relation of long-distance
intercellular systemic signals and chloroplast movements in plants.
In the presented study, we investigated if chloroplast movement in illuminated area of
leaf has some effect on chloroplast arrangement in non-illuminated area located next to the
illuminated one or in the lower leaf. Similarly, an effect of local burning on systemic
chloroplast movement in distant non-treated tissues was investigated. On the basis of our
previous results (Hlaváčková et al. 2006), we tested a hypothesis that some systemic signals
(physical or chemical) caused by local effect may change systemic chloroplast movements.
Results
Systemic reaction to illumination
We measured fast (within hours) changes in chloroplast position by the chlorophyll
meter in a leaf part kept in darkness and in a strongly illuminated part of the leaf blade. To
investigate whether the chloroplast movement in the illuminated part of the leaf blade can
induce also a concomitant movement in the shielded part of the same blade or in another leaf,
we have conducted three experiments (see Fig. 1).
To see a communication between the right and left parts of the leaf blade when
observed from the adaxial leaf side (i.e. between parts separated by the main vein), we
illuminated the left part and measured the value M on both left and right parts (Fig. 1A). In a
similar experiment the upper part of the leaf blade was illuminated and the lower part shielded
(Fig. 1B), or three quarters of the leaf blade were illuminated and one quarter shielded (Fig.
1C).
The chloroplasts in the illuminated part moved in a standard way (Fig. 1, open circles).
The relative decrease of the value M after 60 min of illumination was found for different
leaves between 63 % and 75 % of the initial value. Only a very slight tendency to decrease the
M value was found in the shielded part (Fig. 1A). The decrease of the mean value of M was
only about 5 to 10 % and could be caused by partial light transmission or conduction through
the leaf tissue (cell walls) into the non-illuminated part through the leaf tissue or by the
measuring procedure. The decrease was not significant in the statistical point of view.
Fig 1D shows the result of studies of the expected induced chloroplast movement in
other leaf than the fully illuminated one. The shielded leaf was the nearest one situated
basipetally under the illuminated one. In this case no tendency to change the M value within
one hour could be detected.
Systemic reaction to local burning
In a similar protocol to Hlaváčková et al. (2006) after setting chloroplast to the
periodic movement, after two (Fig. 2, 3) cycles and in the point of chloroplast arrangement
along periclinal cell walls (see arrows in the Figures 2 and 3), the leaf situated next to the
measured one in the apical direction was burned by a flame for 12 seconds. Fig. 2 shows the
periodic changes in the quantity M of SPAD before and after the burning, Fig. 3 shows results
of a similar measurement using the method of collimated transmittance at selected
wavelength.
We have evaluated different basic parameters of the repetitive curves (extent, maximal
rates of increase and decrease estimated in the respective inflection points). The change in
these parameters after burning was not greater than 4 %, which lies within the experimental
error. The measurements were repeated 4 times with the same results.
It can be concluded that under our experimental conditions no change in the
chloroplast periodic movement could be detected within several hours upon local burning of
other leaf.
Discussion
As has been shown by other papers, some physiological parameters of a distant leaf
can change in a fast (minutes) or slow (hours) regimes upon local stress (e.g. burning,
mechanical damage) (e.g. Wildon et al. 1992; Peña-Cortés and Willmitzer 1995). This
reaction is usually designated as systemic reaction of the plant. These are predominantly the
parameters of plant transpiration and photosynthesis (Herde et al. 1995; Koziolek et al. 2004)
at the organ level and induced changes in the gene expression (Herde et al. 1995; Peña-Cortés
et al. 1995) in the cell levels. We have shown in our previous paper that local burning may
lead to a spreading of changes in the electrical surface potential, to a systemic decrease of
transpiration, stomatal conductance and photosynthetic rate in tobacco plants (Hlaváčková et
al. 2006). These changes are followed by changes in levels of jasmonic and abscisic acids.
Local mechanical wounding may also lead to a systemic increase of chlorophyll fluorescence
non-photochemical quenching (Hlaváčková et al. 2002).
The chloroplast movement can be understood as a tendency to avoid photoinhibition
and/or a way how to better distribute or utilize the penetrating light in the plant leaf tissue. In
all these mentioned effects the light environment in the leaf is related to the photosynthetic
productivity. Because the photosynthetic parameters change systemically upon local effect,
one would expect that the movement of photosynthetic organelles - chloroplasts - would also
be influenced by the same signal (electrical, hydraulical or chemical – see Hlaváčková and
Nauš 2007) as the changes in photosynthetic parameters. Moreover, changes in the activity of
membrane-located mechano-sensitive channels (also known to be evoked after local
wounding by hydraulic pressure surges spreading in xylem, Mancuso 1999; Stahlberg et al.
2006) (Sato et al. 1999, 2003a) and exogenous ABA treatment (Kondo et al. 2004), were
reported to influence also chloroplast movement.
Our results show that under our conditions which are near to the conditions of the
plant under full sun or in the shade, there could be detected no systemic changes in the light
induced chloroplast movement in tobacco leaves upon local burning or local light excitation
within several hours. No significant systemic reaction to local light exposition found in our
case is in agreement with the results of Tlałka et al. (1999) who demonstrated that individual
chloroplasts in Lemna were able to sense and respond to highly localized illumination and
were capable of moving when their neighbours were stationary or even moving in the
opposite direction. The complete perception, transduction and effector system must have
sufficient spatial resolution to achieve this level of discrimination. One attractive hypothesis is
that part of the perception system is associated with each individual chloroplast. This could be
readily achieved because the xanthophylls are also the blue-light photoreceptors and they are
located in thylakoid membrane and chloroplast outer envelope. Tlałka et al. (1999) suggested
combination of zeaxanthin and blue light to be required for triggering chloroplast movement
responses.
We did not detect any fast changes in systemic light induced chloroplast movement in
tobacco leaves after local burning indicating that no systemic signals (physical or chemical)
operate in these responses. Our results are supported by results of Augustynowicz et al.
(2001), who published that chloroplasts may move in isolated tobacco protoplasts just by light
stimulation. This fact can exclude the main role of the moving electrical signal or other
intercellular physical (e.g. hydraulic) or chemical signals in the process of chloroplast
movement. So the changes in membrane properties observed upon excitation of phototropins
seem to be a local property of the cell, rather independent on the surrounding cells. Thus,
although several kinds of chemical (Peña-Cortés and Wilmitzer 1995; León et al. 2001) and
physical (Wildon et al. 1992; Malone 1996) signals have been implicated in the long-distance
systemic responses of photosynthesis induced by wounds, these signals seem to be
insufficient for changes in systemic light induced chloroplast movement.
The local burning of tobacco leaf leads to stomatal closure and a decrease in the rate of
photosynthesis in the distant leaves (see e.g. Hlaváčková et al. 2006). However, these
systemic physiological changes had no effect on the light induced periodic chloroplast
movement observed in our case (Fig. 2, 3). It indicates that the chloroplast movement is
within time interval of at least several hours independent on the energy supply from
chloroplasts. It may be speculated that the main energy source is in that case cellular
respiration in mitochondria.
There have been published only several papers regarding relation between chloroplast
movement and photosynthesis (Voerkel 1934; Zurzycki 1965; Lechowski 1974; Brugnoli and
Bjorkman 1992; Park et al. 1996; Slesak and Gabrys 1996; Sinclair and Williams 2001;
Gorton et al. 2003; Grabalska and Malec 2004). It may be expected that at the anticlinal
position of chloroplasts the diffusion of CO2 would be more efficient. However, this
hypothesis was not proved in leaves of Alocasia b. (Gorton et al. 2003). Furthermore, our
unpublished results showed that neither photoinhibition nor infiltration with DCMU inhibit
chloroplast movement in the tobacco leaf. Both mentioned effects inhibit photosynthesis
through inhibition of electron transport through PSII. Slesak and Gabrys (1996) reported that
inhibitors of electron transport (DCMU, MV) have no effect on chloroplast movement in
Lemna trisulca and Arabidopsis thaliana. Blue light stimulation of Arabidopsis thaliana
plants in the presence of DCMU indicated that the activity of voltage dependent calcium
channels in the cell membrane is rather controlled by blue light receptors than by
photosynthetic processes (Stoelzle et al. 2003). Only a complete inhibition of photosynthesis
blocked the chloroplast movement in Lemna trisulca (Zurzycki 1965) indicating that at least
traces of photosynthetic function in the leaf are necessary for activation of the chloroplast
moving system. A paper of Tlałka et al. (1999) is probably the only one suggesting a direct
connection between molecular mechanisms of photosynthesis and chloroplast movement.
They observed a parallel increase in zeaxanthin content (as converted from violaxanthin in the
xathophyll cycle) and chloroplast movement in a strong blue light. In summary, the
mechanism of chloroplast movement seems to be to a great degree independent on the
photosynthetic function and it is rather a robust mechanism. The dominating controlling
signal is probably evoked by the incident light, its spectral composition and intensity.
It should be noted, that on the other hand, the chloroplast arrangement can change
photosynthetic parameters (e.g. intensity of chlorophyll fluorescence, Brugnoli and Björkman
1992 or chlorophyll fluorescence spectrum, Bartošková et al. 1999). These changes however
are controlled by changes in optical properties of the tissue and might not be related to the fast
changes in the photosynthetic performance measured by Hlaváčková et al. (2006).
On the basis of our results, we suggest that chloroplast movement is dependent mainly
upon the intensity and spectral composition of light that irradiate the target tissue (Wada et al.
2003) or upon local mechanical stimulation of the target tissue (Sato et al. 1999, 2003a).
Chloroplast movement is probably regulated only locally, independently on systemic signals.
Materials and Methods
Plant material, growth conditions and stimulation
Nicotiana tabacum (L.) cv. Samsun plants (Palacký University, Olomouc, Czech
Republic) were cultivated in perlite in pots in a growth chamber Weiss-Gallenkamp
SGC.170.PFX.J (8h dark /16h light – “white light” of 70 µmol m-2 s-1 of PAR, RH 50% dark /
45% light, at temperatures 18 ºC dark /25 ºC light with one hour of linear light-rise and lightset), in Olomouc, Czech Republic from June until September 2007. The plants were fertilized
by KRISTALON solution (Hydro Agri Rotterdam, Netherlands) every week. Measurements
were performed on tobacco plants that were 4,5-5 months old, about 70 cm tall with 25 fully
developed leaves with the length of leaf blades between 5-15 cm. Measurements were carried
out on the intact leaves attached to the plant growing in the standard non-stressing conditions.
One quarter, half of the leaf or the leaf located above the measured one were
illuminated by cold white light (2000 µmol m-2 s-1 of PAR). Adaxial side of the leaves was
illuminated.
A tip of the first fully developed upper leaf of each plant selected for study (except
control plants) was burned by a flame from a burning wooden stick moved back and forth
below it for 12 s.
Chloroplast movement
Two independent methods were used for the detection of chloroplast movement.
1. The chlorophyll meter method
In the first method, we have used the commercial chlorophyll-meter SPAD 502DL
(Konica Minolta Sensing, Inc., Japan). In fact, the chlorophyll-meter has been used in an
inverse sense to its original determination. The reading of the instrument is not only
proportional to the chlorophyll content, but it is also dependent on the chloroplast
arrangement in the cell as has been already shown in several papers (Uddling et al. 2007, Hoel
and Solhaug 1998). The instrument SPAD-502 measures intensity of light transmitted through
the sample at two wavelength (650 nm and 940 nm) using light emitting diodes with
approximate half width of the emission spectrum of 15 nm and 50 nm, respectively. The
display shows a value M which is defined as:
M = log [I´(940)/I(940)] – log[I´(650)/I(650)] = log T(940) – log T(650)
where I(650) and I(940) are signals without the sample and I´(650 and I´(940) signals
with the sample and log is a common logarithm.
For practical usage it is supposed that the negative common logarithm of the
transmittance at 650 nm related to that at 940 nm is proportional to the chlorophyll content.
We have calibrated the SPAD by LI-1800 to obtain a correct value of the correcting
factor which can be introduced into the instrument. In our case, it was 4.5 approximately the
same for periclinal and anticlinal chloroplast position. This factor has been used to correct the
reading of our SPAD-502. The calibration procedure is not shown (to be published elsewhere)
and the result is not crucial for the conclusions, the change caused by the correction factor
only shifts the curves in vertical direction.
Although the relation between the relative value M (shown by the instrument) and
transmission of light through the leaf is in a logarithmic scale (Uddling et al. 2007) it can be
used for measurement of chloroplast movement. A higher value of M in our case means a
lower leaf transmittance at 650 nm versus 940 nm and a lower value of M means a higher leaf
transmittance. The value M changes upon chloroplast movement although there is no change
in chlorophyll content per leaf area.
To prove that the changes in M are caused merely by the chloroplast movement, we
used a protocol of periodic illumination of the leaf blade by high (HL = 2000 µmol. m-2 s-1
PAR) and low (LL = 50 µmol. m-2 s-1 PAR) cold white light. The light source was the Schott
KL 2500 (Schott Glas, Wiesbaden, Germany) with 8 mm light piping. The integral PAR
intensity was measured by LI-COR quantum radiometer photometer Model LI-189 (Lincoln,
Nebraska, USA). The time period was set to 50 min (20 min HL/ 30 min LL) or 130 min (60
min HL/ 70 min LL) to reach the initial parts of the saturation levels. However, other periods
or light regimes could be used. Using glass filters (Schott BG 12 or RG 1) we have checked
the well known fact that only the blue light spectral region of the incident light is effective
whereas the red one in not. However, the light in nature is predominantly the white one
reaching at sunny summer day around 2000 µmol. m-2 s-1 PAR. We have used this value as
the HL one. The parameters of the cycles are dependent on the light intensities used (results
not shown).
Typical periodic pattern in the M value is shown in Fig. 2 (left part, up to the white
thick arrow). Under standard non-stressing condition of the plant, the reproducibility of the
maximal and minimal values in the periodic pattern was greater than 98 % within 6 hours of
the measurement. The maximal value reaches a saturation value after several hours
(arrangement of chloroplasts along periclinal cell walls), we have used a time period which
has brought the chloroplast arrangement near to the saturation value. The minimal saturation
value (arrangement of chloroplasts along anticlinal cell walls) was reached in a shorter time.
To obtain a mean value, usually 4-10 points on the defined part of the leaf blade was
measured and a mean values and SD were calculated. Due to the natural heterogeneity of the
leaf (including veins), the scatter of values is of relatively high value (5 – 10 %).
The extremely high reproducibility of the periodic parameters renders this method
potential for determination of small changes in the dynamics and extent of chloroplast
movement. The difference in the M value between the two saturation levels are within about
50 % of the lowest saturation value. This relatively low change is caused by using the quantity
M proportional to the logarithm of transmittance and by detection of nearly all diffusive light
transmitted by the leaf to the diodes of SPAD.
2. Method of collimated transmittance at selected wavelength
The above mentioned sensitivity of the detection of chloroplast movement can be
enlarged by about an order (about 10 times) by changing the method to a specified one using
monochromatic detection at wavelength of maximal chlorophyll a absorption in the red region
(676 nm), fixed illuminated spot on the leaf blade and using detection of mostly collimated
light. This was achieved by constructing a home made experimental set-up.
The incident light was provided by the cold white light source Schott KL 2500 (Schott
Glas, Wiesbaden, Germany) equipped with the light guide (8 mm diameter). The selected leaf
spot was put into a soft clip with and opening of 4,5 mm diameter. The necessary condition to
reach good results was a very soft pressure of the clip on the leaf tissue. When using a more
strong pressure of the leaf, the local spot can increase its temperature and loose water, which
may strongly change the chloroplast motion (see e.g. Brugnoli and Björkman 1992; Walczak
and Gabryś 1980, also our unpublished results) or direct effect of mechanical pressure on
chloroplast movement cannot be excluded (Sato et al. 1999, 2003a). The mechanical effect
was not a subject of this study.
The transmitted light is conducted by a lightguide to the Spectroradiometer LI-1800
(LI-COR Lincoln, Nebraska, USA). The LI-1800 was set to 676 nm (spectral slit width 6 nm).
The entering end of the light guide is fixed in the leaf clip 8 mm under the leaf blade. The
diameter of the bundle of optical fibers in the lightguide is 3.5 mm. The tube leading to the
light guide is of black color. This ensures that the incident light on the light guide is
predominantly collimated, most of diffusive light being absorbed by the black walls of the
tubing and does not reach the light guide. The degree of collimation can be characterized by
the angle 25 o of the cone formed by the light beams incident from the center of the leaf spot
on the light guide. About 1/25 part of the transmitted light is detected mostly of the collimated
character. Comparison of the amount of collimated to diffusive light was measured using light
above 800 nm and the integrating sphere LI-1800-12S.
The signal measured with the leaf in the clip was divided by the signal detected in the
same arrangement without the leaf. Thus a transmittance Tc
at 676 nm of the partly
collimated light has been obtained. If compared with the preceding method (SPAD), in
addition to much greater sensitivity, several other differences should be mentioned. The
curves measured in Tc are “inverted” to those measured in M, the maximum in M corresponds
to the minimum in Tc and vice versa. Whereas the value M takes into account the
transmittance at 940 nm, this is not done in the estimation of Tc.
Acknowledgements
We thank to Dr. Pavel Krchňák for help in the adaptation of home-made method.
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Figure legends
Fig. 1. Effect of strong light illumination on the chloroplast movement measured with the
SPAD method on the illuminated (open circles) and non-illuminated (full squares) areas or
leaves. Illuminated were: (A) the left half of the leaf blade, (B) the basal half of the leaf blade,
(C) three quarters of the leaf blade, (D) the whole leaf and the measurement were performed
on the illuminated one and the leaf located below it. Cold white light of 2000 µmol m-2 s-1 of
PAR was used. Mean values ± SD, n=8.
Fig. 2. Light induced periodic chloroplast movement in tobacco leaf measured with the SPAD
method before and after local burning (white thick arrow) of an upper leaf. Cold white light:
ON – 2000 µmol m-2 s-1, OFF – 50 µmol m-2 s-1. A tip of the upper leaf was burned (white
thick arrow) 260 min after beginning of the measurement performed on the leaf located below
the burned one. Two independent experiments are shown. Mean values ± SD, n=4.
Fig. 3. Light induced periodic chloroplast movement in tobacco leaf detected with the partly
collimated light method before and after local burning (white thick arrow) of an upper leaf.
Cold white light: ON – 2000 µmol m-2 s-1, OFF – 50 µmol m-2 s-1. A tip of the upper leaf was
burned (white thick arrow) 100 min after beginning of measurement on the leaf located below
the burned one. Two independent experiments with the same results were performed, one
representative curve is shown.
Fig. 1.
Authors : Jan Nauš, Monika Rolencová, Vladimíra Hlaváčková
Fig. 2.
Authors : Jan Nauš, Monika Rolencová, Vladimíra Hlaváčková
Fig. 3.
Authors : Jan Nauš, Monika Rolencová, Vladimíra Hlaváčková