Environmental and Experimental Botany 61 (2007) 74–84
Plasmodesmata density in vascular bundles in leaves of C4
grasses grown at different light conditions in respect to
photosynthesis and photosynthate export efficiency
Paweł Sowiński a,b,∗ , Anna Bilska b , Katarzyna Barańska a ,
Jan Fronk c , Paweł Kobus d
a University of Warsaw, Institute of Botany, Miecznikowa 1, 02-096 Warszawa, Poland
Plant Breeding and Acclimatization Institute, Plant Biochemistry and Physiology Department, Radzików, 05-870 Błonie, Poland
c University of Warsaw, Institute of Biochemistry, Miecznikowa 1, 02-096, Warszawa, Poland
d Warsaw Agricultural University, Department of Econometrics and Informatics, Nowoursynowska 159, 02-787 Warszawa, Poland
b
Received 31 March 2006; received in revised form 12 February 2007; accepted 31 March 2007
Abstract
The study was conducted with young plants of four species of C4 grasses of three photosynthetic sub-types: Panicum miliaceum (NAD-ME),
Panicum maximum (PEP-CK), and Zea mays and Digitaria sanguinalis (both NADP-ME) with the aim to verify the hypothesis that light growth
conditions affect the density of plasmodesmata connecting the photosynthetically active chlorenchymatous Kranz mesophyll (KMS) and bundle
sheath (BS), as well as vascular parenchyma (VP) cells, and that the density of the plasmodesmata limits the efficiency of photosynthesis and
photosynthate export from the leaf.
The most important ultrastructural difference between the LL (grown at 50 ␮mol quanta m−2 s−1 ), ML (grown at 200 ␮mol quanta m−2 s−1 ) and
HL (grown at 1000 ␮mol quanta m−2 s−1 ) plants was the increase, with increasing growth illumination, in the density of plasmodesmata connecting
KMS and BS cells and, to a lesser extent, of those between BS and VP cells. This tendency was observed for all C4 grasses tested, although the
magnitude of the reaction was species-specific, with the weakest and highest increase noted for P. miliaceum and D. sanguinalis, respectively. The
maximum net photosynthesis rate (measured at saturating photon flux) was much higher in HL than in ML plants (except for P. miliaceum) and
it was correlated very well with the density of the KMS/BS plasmodesmata. The photosynthate export capacity of the plants, as characterized by
the time of 14 C-photosynthate transfer into the transport path, the fraction of newly synthesized photosynthates exported from the leaf, and the
transport speed in the leaf blade showed that ML plants were source-limited and HL plants were sink-limited.
Apparently, C4 grasses are able to adjust their photosynthetic apparatus to light growth conditions by changing the number of plasmodesmata
connecting cells of Kranz mesophyll, bundle sheath and vascular parenchyma in proportion to the intensity of illumination, so that for plant grown
at high illumination the transport of photosynthates ceases to be a bottleneck limiting the efficiency of photosynthesis.
© 2007 Elsevier B.V. All rights reserved.
Keywords: C4 photosynthesis; Light growth conditions; Plasmodesmata density; Symplasmic transport; Photosynthate export; Vein ultrastructure
1. Introduction
C4 type photosynthesis is a morpho-physiological syndrome
by which plants are able to concentrate carbon dioxide at the
site of Rubisco action, thus avoiding the waste of photorespi-
∗ Corresponding author at: University of Warsaw, Institute of Botany,
Miecznikowa 1, 02-096,Warszawa, Poland. Tel.: +48 22 55 43 920; fax: +48
22 55 43 910.
E-mail address: [email protected] (P. Sowiński).
0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2007.03.002
ration. C4 type photosynthesis is related to a massive exchange
of intermediates between Kranz mesophyll (KMS) and bundle
sheath (BS) cells (Leegood, 2000). Mesophyll cells, the site of
primary carbon assimilation (PCA), export products of phosphoenolpyruvate carboxylation, i.e. malate or aspartate, to bundle
sheath cells, where these compounds are decarboxylated and
introduced into the Calvin cycle for primary carbon reduction
(PCR). The way of decarboxylation depends on the sub-type of
C4 photosynthesis: NADP-malic enzyme (NADP-ME), NADmalic enzyme (NAD-ME), or PEP-carboxykinase (PEP-CK).
After reduction, a fraction of the assimilated carbon moves
P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
back as pyruvate from BS to KMS, for phosphoenolpyruvate
(PEP) regeneration. Moreover, phosphoglyceride (PGA) and
triosephosphates (TP) are also shuttled to KMS (Furbank and
Foyer, 1988).
The route of the exchange of C4 photosynthetic intermediates between KMS and BS cells is somewhat debatable. The
view that it goes solely through plasmodesmata seems to predominate, chiefly because the suberin lamella in walls between
these two types of cells is believed to preclude apoplasmic transport (Hattersley, 1987; Hattersley and Browning, 1981). There
are, however, observations showing apoplasmic movement of
a fluorescent dye from vein to KMS in several C4 species,
both dicotyledonous and monocotyledonous (Eastman et al.,
1988a,b). Moreover, plants of the NAD-ME sub-type of C4 photosynthesis have no suberin lamella in walls between KMS and
BS. On the other hand, a positive relationship has been found
between the number of plasmodesmata in leaves of a given
species and its net photosynthesis rate of C4 grasses (Botha,
1992), confirming the importance of symplasmic transport for
C4 photosynthesis. Also, the export of sucrose from the leaf
may go, at least partially, by means of symplasmic transport. In
species which synthesise sucrose in KMS, it is transported symplasmically on the distance of at least three cells: KMS–BS–VP,
before being loaded into phloem. The crucial role of BS/VP
plasmodesmata in the export of photosynthates from leaves finds
strong support in studies of a maize mutant, SXD-1 (Russin et
al., 1996), in which plasmodesmata at the BS/VP interface are
occluded by callose (Botha et al., 2000), resulting in sucrose
export arrest. All these data lead to the conclusion that the rates
of C4 photosynthesis and photosynthate export depend on the
number and conductivity of plasmodesmata.
Plasmodesmata linking KMS, BS and VP cells in C4 grasses
have been characterized both structurally (Botha, 1992, 2005;
Evert et al., 1977; Robinson-Beers and Evert, 1991) and functionally (Weiner et al., 1988). KMS/BS plasmodesmata are
simple channels having in some cases putative sphincters at
one or both sides, with size exclusion limit (SEL) of about
850 Da. One aspect of the plasmodesmata linking KMS/BS/VP
has attracted only limited attention so far, i.e. the changes in the
number of these plasmodesmata in response to environmental
factors. We recently showed (Sowiński et al., 2003) that in maize
the density of plasmodesmata linking Kranz mesophyll cells
and bundle sheath cells as well as bundle sheath and vascular
parenchyma cells was strongly increased at a sub-optimal temperature of growth compared to control plants. In plants grown at
the optimal temperature assimilate movement strongly depends
on the actual temperature of leaves. The observed increase of the
number of plasmodesmata linking KMS–BS–VP cells in plants
grown at a moderately low temperature apparently obviated that
restriction. It seems that acclimation of maize photosynthesis
to non-lethal low temperature growth conditions may lead to
changes in the physical properties of the metabolite exchange
path between photosynthetic and other cells responsible for
exporting assimilates from the leaf.
Another factor which strongly determines the adaptation
of C4 plants to a particular environment is light. C4 plants
prefer full sunlight, i.e. light with the intensity above 1000 ␮
75
mol quanta m−2 s−1 , and are almost absent from shade environments with the light intensity less than 10% of full sunlight
(Sage and Pearcy, 2000). The aim of the present study was to
verify the hypothesis that light growth conditions affect the density of plasmodesmata connecting the photosynthetically active
chlorenchymatous cells of KMS and BS and BS with VP and that
the density of the plasmodesmata limits the efficiency of photosynthesis and photosynthate export from the leaf. In studies with
nectarine trees (Wang and Huang, 2003), plasmodesmal densities between BS and VP, VP/VP, CC/VP and CC/SE cells was
lower in plants grown under low light than in those grown at high
light conditions. On the other hand, in studies comparing symplasmic and apoplasmic phloem loaders (Amirad et al., 2005),
the density of plasmodesmata linking BS and intermediary cells
did not vary in plants grown under different light conditions. No
similar studies have been done for C4 plants until now.
Our study conducted with young plants of four species of
C4 grasses: Panicum miliaceum (NAD-ME), Panicum maximum (PEP-CK), and Zea mays and Digitaria sanguinalis (both
NADP-ME) shows that the poor photosynthetic capacity of C4
grasses grown at low light irradiances might be related to the
low density of plasmodesmata connecting KMS/BS/VP cells.
2. Material and methods
2.1. Plant material and growth conditions
Young plants of four C4 grasses were used: P. miliaceum
(NAD-ME), P. maximum (PEP-CK), D. sanguinalis (syn. Panicum sanguinale) and Zea mays var. Olenka (both NADP-ME).
Kernels of P. miliaceum, D. sanguinalis and Z. mays were
obtained from the Plant Breeding and Acclimatization Institute, Radzików. Kernels of P. maximum were obtained from
Queensland Agricultural Seeds Pty, Ltd., Toowooba, Qld, Australia. Kernels were germinated in sand and then plants were
transferred to pots containing Knop’s nutrient solution supplemented with Hoagland’s micro-nutrients. Both germination
and further growth were conducted in a growth chamber
under the photoperiod of 14 h/10 h and day/night temperature
regime of 24 ◦ C/22 ◦ C, under the light irradiance of 50 (low
light, LL), 200 ␮mol quanta m−2 s−1 (medium light, ML) or
1000 ␮mol quanta m−2 s−1 (high light, HL), until the third (Z.
mays, D. sanguinalis, P. miliaceum) or fourth (P. maximum) leaf
was fully developed. The growth of LL plants was conducted
under fluorescent tubes and that of ML and HL plants under
mercury lamps.
2.2. Density of plasmodesmata
Leaf blade samples taken from the middle region of intact,
fully developed leaf were fixed in 2.5% glutaraldehyde in 0.1 M
phosphate buffer (pH 7.3) for 4 h at room temperature and postfixed with 1% osmium tetroxide for 2 h at room temperature. In
some experiments, 0.5% tannic acid was added to the fixative to
visualize plasmodesmata sphincters (Olesen, 1979). After dehydration (ethanol 10–100%) the material was embedded in Epon
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P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
and polymerised for 24 h at 60 ◦ C. Ultrathin (80 nm) sections
were cut with a diamond knife on a Leica Ultracut ultramicrotome and stained with uranyl acetate and lead citrate. Sections
were examined with a transmission electron microscope (model
JEM-1200EX; JEOL, Japan).
Small and intermediate bundle sheaths were studied because
they are involved in assimilate loading (Evert et al., 1996).
Plasmodesmata crossing more than half of the cell wall thickness were counted directly under the electron microscope with
the magnification of 50,000× (5000× TEM and 10× optical
magnification). The plasmodesmata density was calculated per
1 ␮m of a vein, taking into account the proportionality constant (Gunning, 1978), which was 1/(t + 1.5R), where t is the
section thickness (80 nm) and R is the average radius of plasmodesmata (20 nm). Samples were collected in six independent
experiments. All together, 15–20 bundles were analyzed per
species per experimental variant. Sets of three to four vascular bundles on separate sections were obtained from separate
leaves, which resulted in high randomization of the data set.
2.3. Dimensions of vascular bundles and bundle sheaths
The circumference of small veins and bundle sheaths and
interveinal distances were estimated from scanned microphotographs with the use of a custom-made dedicated software.
The number of bundles analysed per treatment was 25–31.
2.4. Net CO2 assimilation rate and chlorophyll a
fluorescence
Photosynthesis and chlorophyll fluorescence were studied in
the youngest fully developed leaf, in the same region as that
studied for plasmodesmata number. The measurements were
performed between 3 and 6 h after the light in the growth chamber was switched on. The maximal quantum yield of PS II
electron transport (Fv/Fm) was measured with a fluorometer
FMS-1 (Hansatech, Great Britain). Photosynthesis was measured with a closed type gas-exchange system using an infrared
gas analyser (AirTECH 2500-P, Gazex, Poland).
Before measurements of Fv/Fm, plants were dark adapted
for 30 min at room temperature. To assess the light response
of photosynthesis to increasing light intensity, photosynthetic photon flux series of 100, 240, 650, 1200 and 1500 ␮
mol quanta m−2 s−1 were performed with plants adapted for at
least 15 min to each photon flux.
The experiments were repeated three times using three plants
per species per experimental variant.
2.5.
14 C-assimilate
transport
In this study, three transport parameters were evaluated.
These parameters were: the time taken by 14 C-assimilate to
appear in the transport path (AT, a measure of photosynthate
transfer rate into the phloem loading zone), the fraction of incorporated radioactivity exported from the leaf (RL, a measure of
phloem loading rate) and the transport speed in the leaf blade.
These values were evaluated by means of an in vivo method elab-
orated earlier for studies of 14 C-assimilate transport in maize
with the use of proportional counters detecting Bremsstrahlung
radiation originating from interaction of 14 C ␤ particles with
leaf tissue (Sowiński et al., 2003, and references therein).
The transport studies were performed in the youngest,
fully developed leaf between 3 and 7 h after the lights had
been switched on in the growth chamber. Measurements were
conducted at 100, 200, 650 ␮mol quanta m−2 s−1 or 200 and
650 ␮mol quanta m−2 s−1 for plants grown at 200 or 1000 ␮mol
quanta m−2 s−1 , respectively. After 30 min of adaptation, 14 CO2
(1.85 MBq) was fed into a small area in the middle of the leaf.
In order to follow the 14 C-assimilate “wave” four detectors were
mounted along the leaf blade. The first detector measured the
radioactivity of the feeding area and allowed us to establish the
starting time point of 14 C-assimilate export. AT was estimated
as the time necessary for the label to move between the first and
the second detector (located 2 cm below the first one) minus the
time of label movement between the second and the third detector (located 2 cm below the second one). The fourth detector
allowed the measurement of transport speed in the leaf blade.
The time intervals between the moments when the radioactivity
measured by a given detector had reached half of the respective
maximum were taken into considerations. The RL was estimated
as the fraction of incorporated radioactivity exported from the
feeding area.
The measurements were repeated 3–5 times per species per
experimental variant in three independent experiments.
2.6. Statistics
The significance (p < 0.05) of the differences in the linear
dimensions of both leaves and vascular bundles was tested
by analysis of variance using STATISTICA PL (StatSoft). In
the case of light curves of photosynthesis, appearance time,
radioactivity exported and transport speed in leaf blade standard
deviations were calculated.
In the case of plasmodesmata density, the standard assumption of normal, non-discrete distribution of variables could not
be met, therefore a bootstrap technique (Efron, 1982; Sowiński
et al., 2003) was used to perform the statistic test (p < 0.05).
3. Results
3.1. Anatomical adjustment of leaves to different light
intensities
Both the anatomy and ultrastructure of leaves were highly
dependent on the light growth conditions. Some changes
were obvious: C4 plants grown at low light (LL plants,
50 ␮mol quanta m−2 s−1 ) intensities were much smaller and had
narrower leaves than plants grown at the high light (HL plants,
1000 ␮mol quanta m−2 s−1 ) intensity (Table 1). LL plants were
generally weak and mechanically susceptible. The interveinal
distances (Table 1), which followed the general pattern found
among C4 subtypes, being shorter in PEP-CK and NADP-ME
compared to NAD-ME plants (Dengler et al., 1994; Ohsugi,
P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
77
Table 1
Anatomical characteristics of leaves of four C4 grasses: P. miliaceum (NAD-ME), P. maximum (PEP-CK), D. sanguinalis and Z. mays (NADP-ME) grown at various
light intensities
Light intensity (␮mol
quanta m−2 s−1 )
P. miliaceum NAD-ME
P. maximum PEP-CK
D. sanguinalis NADP-ME
Z. mays NADP-ME
Leaf width
(mm)
Inter-veinal
distance (␮m)
Leaf width
(mm)
Inter-veinal
distance (␮m)
Leaf width
(mm)
Inter-veinal
distance (␮m)
Leaf width
(mm)
Inter-veinal
distance (␮m)
50
200
1000
8.6a
13.7b
18.2c
199.8a
213.6a
151.2b
5.3a
7.4b
9.7c
178.2a
134.9b
120.8b
5.5a
7.2b
14.8c
119.8a
120.9a
159.7b
10.4a
14.2b
16.0c
101.7a
122.2a
105.9a
Significant differences (p < 0.05) within columns, as estimated by analysis of variance, are indicated by different letters.
1989), decreased with increasing growth light intensity in P.
miliaceum (NAD-ME) and P. maximum (PEP-CK), increased
in D. sanguinalis (NADP-ME) and did not change in Z. mays
(NADP-ME). The circumference of veins did not vary among
plants grown at different light conditions, there was, however,
a slight but significant increase in bundle sheath circumference
in both P. miliaceum and D. sanguinalis HL plants compared to
both LL and ML (200 ␮mol quanta m−2 s−1 ) plants (Table 2).
Except for maize, HL plants had more and larger starch grains
in chloroplast of KMS and BS cells (in D. sanguinalis) or in BS
cells only (in P. miliaceum and P. maximum; Fig. 1).
The density of KMS/BS plasmodesmata was proportional
to the light intensity during plant growth (Tables 3A and 3B).
The strongest dependence was noticed for D. sanguinalis and
the lowest one for P. miliaceum. HL grown plants of P. miliaceum, P. maximum and D. sanguinalis also showed higher
density of plasmodesmata connecting BS and VP compared to
LL and ML plants. No differences were found for plasmodesmata connecting vascular parenchyma cells to one another,
except for D. sanguinalis, where HL plants showed a higher
VP/VP plasmodesmata density (Tables 3A and 3B).
The density of plasmodesmata between VP, CC and SE apparently did not vary for plants grown at different light regimes (data
not shown), although statistical analysis was not performed for
these interfaces, due to the very low number of plasmodesmata
linking some cell types, e.g. companion cells to bundle sheath
or vascular parenchyma cells.
3.2. Photosynthesis competence of leaves acclimated to
different light growth conditions
The photosynthesis competence was evaluated by means of
the light curve of net photosynthesis rate (Fig. 2) and the Fv/Fm
coefficient (Fig. 3). It could be measured only in ML and HL
plants, since LL plants of all species studied were not hard
enough to be mounted in the experimental chamber without
serious mechanical injuries.
In ML plants, the light curve of net photosynthesis
rate showed a plateau at the photosynthetic photon flux
of 650 ␮mol quanta m−2 s−1 , except for P. miliaceum plants,
which demonstrated a net photosynthesis rate increase up to
1200 ␮mol quanta m−2 s−1 . The maximum net photosynthesis
rates were about 20 ␮mol CO2 m−2 s−1 in P. maximum, D. sanguinalis and Z. mays and 25 ␮mol CO2 m−2 s−1 in P. miliaceum.
In contrast to the ML plants, in the HL ones the net photosynthesis rate increased up to 1500 ␮mol quanta m−2 s−1 , again
with the exception of P. miliaceum, where the light curve of
photosynthesis followed the same pattern as in ML plants.
The maximum net photosynthesis rates were about 35, 42 and
38 ␮mol CO2 m−2 s−1 in P. maximum, D. sanguinalis and Z.
mays, respectively, but only about 27 ␮mol CO2 m−2 s−1 in P.
miliaceum. That species also demonstrated a slight decrease in
the Fv/Fm coefficient in HL plants suggesting inhibition of PSII
of leaves grown at 1000 ␮mol quanta m−2 s−1 (Fig. 3).
3.3. 14 C-photosynthate export from leaves grown at
different light conditions
To characterize the export of photosynthates from leaves three
parameters were used, which were obtained from the kinetics
of radioactivity movement along the leaf blade from the site of
14 CO feeding (Table 4). These parameters were the time neces2
sary for 14 C-photosynthates to be transferred into the transport
path, the fraction of currently fixed assimilates exported from
the leaf, and the transport speed.
As it was in the case of photosynthesis measurements,
the transport studies were conducted only for ML and HL
plants, because of the mechanical susceptibility of LL plants.
Table 2
Circumference of bundle sheaths and veins in leaves of four C4 grasses grown at various light intensities
Light intensity (␮mol
quanta m−2 s−1 )
50
200
1000
Circumference (␮m)
P. miliaceum
P. maximum
D. sanguinalis
Z. mays
Bundle sheath
Vein
Bundle sheath
Vein
Bundle sheath
Vein
Bundle sheath
Vein
263.1ab
240.4a
291.8b
107.2a
82.5a
119.5a
250.7a
292.7a
300.7a
44.7a
59.9a
73.5a
142.0a
153.2a
231.7b
55.1a
60.8a
92.4b
183.0a
211.5a
222.3a
78.1a
78.8a
81.7a
Significant differences (p < 0.05) within columns, estimated by analysis of variance, are indicated by different letters.
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P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
Fig. 1. Sections taken from the middle part of leaf blade of the third or fourth, fully developed leaves of C4 plants grown at two light intensities: (A, C, E, G, I,
K, M and N) 200 ␮mol quanta m−2 s−1 and (B, D, F, H, J and L) −1000 ␮mol quanta m−2 s−1 . (A–H) Transverse sections of bundles, (I–N) longitudinal sections
of plasmodesmata linking, (I–M) Kranz mesophyll (KMS) and bundle sheath (BS) cells or (N) bundle sheath and vascular parenchyma (VP) cells. During fixation,
0.5% tannic acid was added to visualise sphincters. (A and B) P. maximum (PEP-CK), HL plants compared to ML plants show longer KMS cells resulting in thicker
leaves, thicker epidermis (EP) cell walls and more and larger starch grains in both KMS and BS chloroplasts, bars = 10 ␮m. (C and D) P. miliaceum (NAD-ME), HL
plants, compared to ML plants show bigger KMS cells resulting in thicker leaves, have thicker epidermis cell walls, their BS chloroplasts are much bigger and fill
almost whole BS cells, BS chloroplasts of HL plants contain much more and larger starch grains than ML plants, bars = 20 ␮m. (E and F) Z. mays (NADP-ME),
HL plants compared to ML plants, show bigger epidermis cells with thicker cell walls, bars = 20 ␮m. (G and H) D. sanguinalis (NADP-ME), HL-plants compared
to ML-plants, show much bigger epidermis cells with thicker cell walls, both types of chloroplasts contain more and larger starch grains (G) bar = 10 ␮m and (H)
bar = 20 ␮m. (I and J) P. maximum, KMS/BS plasmodesmata have sphincters on both KMS and BS side and a constriction in the middle region, HL plants compared
to ML plants, show enlargement of both sphincters, bars = 100 nm. (K and L) P. miliaceum, KMS/BS plasmodesmata show no constriction in the middle region,
apparently because of a lack of suberin layer, HL plants compared to ML plants show dilation of the cytoplasmic sleeve in the middle region, bars = 100 nm. (M) Z.
mays (and D. sanguinalis, no distinct differences were noticed between these two species in respect to KMS/BS plasmodesmata), KMS/BS plasmodesmata posses
sphincter only on the KMS side, no difference between ML plants and HL plants was noticed, bars = 100 nm. (N) P. maximum (and other C4 grasses tested, no distinct
differences were noticed among these species in respect to BS/VP plasmodesmata) BS/VP plasmodesmata, HL plants do not vary from ML plants in respect to this
type of plasmodesmata ultrastructure, bars = 100 nm.
P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
79
Table 3A
Density of plasmodesmata, expressed as the number of plasmodesmata per micrometer of vein, between mesophyll (KMS), bundle sheath (BS), and vascular
parenchyma (VP) cells in leaves of four C4 grasses grown at various light intensities
Light intensity (␮mol
quanta m−2 s−1 )
50
200
1000
Density of plasmodesmata (␮m−1 of vein)
P. miliaceum
P. maximum
D. sanguinalis
Z mays
KMS–BSa BS–VP
VP–VP KMS–BS
BS–VP
VP–VP KMS–BS
BS–VP
VP–VP KMS–BS BS–VP
VP–VP
1358a
3045b
4102c
143a
168a
165a
60a
62a
228b
29a
64a
38a
56a
88a
326b
4a
10b
53c
22a
20a
30a
92a
428b
326b
338a
574b
1190c
196a
477b
1386c
284a
559b
913c
90b
59a
130c
Significant differences (p < 0.05) within columns, as estimated by bootstrap technique (Efron, 1982; Sowiński et al., 2003), are indicated by different letters.
a Cell interface.
The measurements were done at photon fluxes of 100, 200
and 650 ␮mol quanta m−2 s−1 for ML plants and at 200 and
650 ␮mol quanta m−2 s−1 for HL plants. Higher photosynthetic
photon fluxes could not be obtained in the experimental system
used for the transport studies.
In ML plants, regardless of the interspecific differences in a
given parameter, a higher photon flux used at the time of measurement resulted in shortening of the time necessary for the
14 C-photosynthate to move into the transport path, and brought
about a decrease in the fraction of radioactivity exported from
leaves and an increase in transport speed. No such tendency
was found for HL plants, except for shortening of the time
necessary for the 14 C-photosynthate to move into the transport path. In general, the transport parameters found for HL
plants were similar to those of ML plants, particularly if one
compares the values obtained at the photosynthetic (i.e. that
during measurements) photon flux close to the growth light
conditions, i.e. 200 ␮mol quanta m−2 s−1 for ML plants and
650 ␮mol quanta m−2 s−1 for HL plants. The only exception was
P. miliaceum: if compared to ML plants, in HL plants of that
species the photosynthates moved slower along the leaf blade.
This demonstrates a less efficient transport in HL than in ML P.
miliaceum.
4. Discussion
The reported study was undertaken to find out whether
the acclimation of C4 photosynthesis to different light growth
conditions might be achieved by adjustment of the density
of plasmodesmata linking KMS/BS/VP cells. The rationale
for such a hypothesis was our recent finding that the density of the mentioned plasmodesmata increases in maize leaves
in response to sub-optimal growth temperatures (Sowiński
et al., 2003). That observation showed that the density of
plasmodesmata connecting cells responsible for photosynthesis (KMS, BS) and those responsible for photosynthate
export (BS, VP) might vary depending on environmentallydetermined demand. The demonstration of such a dependence
in C4 species representing different photosynthesis sub-types
grown at different light conditions, a crucial factor determining C4 plant distribution on Earth, would give strong support
to the existence of a previously unknown, intriguing mechanism of C4 plant acclimation to changeable environmental
conditions.
As one might expect, the vigor of the C4 grasses tested in
this study was very poor when they were grown at low light
(LL plants, 50 ␮mol quanta m−2 s−1 ). Leaves were small, narrow and very delicate, resulting in susceptibility to breaking
(even under its own weight) and other mechanical injuries. ML
plants (grown at 200 ␮mol quanta m−2 s−1 ) performed much
better. Even under such, rather weak, light conditions, D. sanguinalis and P. miliaceum were able to flower in several weeks.
All grasses tested demonstrated the best vigor at HL conditions
(1000 ␮mol quanta m−2 s−1 ). Generally, the performance of the
species used in this study supported the widespread opinion that
shaded environments with a light intensity less than 10% of full
sunlight (assumed as higher than 1000 ␮mol quanta m−2 s−1 )
exclude C4 plants (Sage and Pearcy, 2000). The very fast development of D. sanguinalis and P. miliaceum might reflect their
adaptation to temperate climate, where they should complete
their life cycle in a very short time. P. miliaceum and D. sanguinalis are crop and weed, respectively, originated in warm
climate but adapted to Central Europe conditions by avoidance
mechanisms.
Table 3B
Density of plasmodesmata, expressed as the number of plasmodesmata per mm2 of leaf, between mesophyll (KMS), bundle sheath (BS), and vascular parenchyma
(VP) cells in leaves of four C4 grasses grown at various light intensities
Light intensity
(␮mol quanta m−2 s−1 )
50
200
1000
Density of plasmodesmata (106 /mm2 of leaf)
P. miliaceum
P. maximum
D. sanguinalis
Z. mays
KMS–BS
BS–VP
KMS–BS
BS–VP
KMS–BS
BS–VP
KMS–BS
BS–VP
7.0
14.3
27.2
0.5
2.0
2.2
1.9
4.2
9.8
0.3
0.4
1.9
1.7
4.0
8.7
0.8
0.8
2.1
2.8
4.6
8.6
0.9
0.5
1.2
Values calculated from plasmodesmata density per micrometer of vein (Table 3A) and interveinal distance (Table 1).
80
P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
Fig. 2. The effects of increasing light intensity on net photosynthesis in the third or fourth, leaf of four C4 grasses: P. maximum (PEP-CK), P. miliaceum (NAD- ME),
D. sanguinalis (NADP-ME) and Z. mays (NADP-ME) grown at two light intensities: 200 ␮mol quanta m−2 s−1 (solid line) and 1000 ␮mol quanta m−2 s−1 (dashed
line). Values are means (±S.D.) of nine plants.
Ultrastructurally, the most important difference between the
LL, ML and HL plants was the increase in the density of plasmodesmata connecting KMS and BS cells and, to a lesser extent,
of those between BS and VP cells (Tables 3A and 3B). The above
tendency was observed for all C4 grasses tested, although the
magnitude of the reaction was species-specific, with the weakest
and strongest dependence noted for P. miliaceum and D. sanguinalis, respectively. The increase in the density of KMS/BS
plasmodesmata at higher growth light intensities was not caused
by changes in the number of KMS and BS cells nor was it due
to changes in the cell wall surface between mesophyll and bundle sheath cells. The number of KMS cells increased by only
about 20% in HL plants of all tested species, as compared to LL
and ML plants, while the number of BS cells did not change
at all (data not shown). As concerns the cell interface, only
HL plants of P. miliaceum demonstrated a significant increase
of bundle sheath circumference (Table 2). In D. sanguinalis,
where the bundle sheath circumference also increased with light
growth conditions (the increase factor of HL plants versus LL
plants ∼1.5), the density of KMS/BS plasmodesmata increased
much more (ca. seven-fold for HL versus LL plants). P. maximum and Z. mays grown under different light conditions did
not show any difference in the bundle sheath circumference
(Table 2). The dependence of plasmodesmata density on light
growth conditions, was especially strong if the plasmodesmata
density was expressed as their number per square mm of the leaf
(i.e. with interveinal distance taken into considerations), particularly for KMS/BS plasmodesmata, but also – to a lower extent
– for the BS/VP ones (Table 3B). Thus one may conclude that
light growth conditions determine the density of the symplasmic network between PCA and PCR cells, ensuring an improved
efficiency of the exchange of photosynthates at high light.
On the basis of the above finding, one should expect a higher
photosynthetic competence of HL plants compared to ML plants
(and, of course, to LL plants as well, but these could not be
assayed due to their weakness). This was indeed so, as the maximum net photosynthesis rate (measured at a saturating photon
flux) was much higher in HL than in ML plants (except for
79.0 ± 11.2
89.0 ± 7.8
70.3 ± 9.0
71.0 ± 11.9
5.1 ± 1.0
4.8 ± 1.4
85.3 ± 7.5
75.3 ± 7.3
64.7 ± 11.0
49.9 ± 10.0
7.0 ± 0.7
5.0 ± 0.6
58.3 ± 8.5
54.9 ± 9.7
7.9 ± 0.5
7.3 ± 0.7
Plants grown at 1000 ␮mol quanta m−2 s−1
Photon flux during measurements (␮mol quanta m−2 s−1 )
200
8.6 ± 0.4
49.0 ± 6.5
54.3 ± 5.7
650
7.9 ± 0.9
45.6 ± 3.5
47.4 ± 6.8
Data expressed as mean ± S.E.
54.0 ± 5.7
60.6 ± 10.3
77.3 ± 10.9
46.0 ± 9.0
41.0 ± 8.0
38.7 ± 5.6
8.2 ± 0.8
7.0 ± 1.1
6.8 ± 1.3
Plants grown at
Photon flux during measurements (␮mol quanta m−2 s−1 )
100
7.7 ± 0.7
72.0 ± 5.0
42.0 ± 5.7
200
7.5 ± 0.5
61.0 ± 9.0
66.7 ± 14.7
650
7.1 ± 0.1
48.7 ± 7.6
99.0 ± 14.6
40.0 ± 5.1
41.0 ± 4.3
94.0 ± 17.8
105 ± 14.8
112 ± 15.4
6.8 ± 0.2
5.7 ± 0.9
3.9 ± 1.0
52.3 ± 4.9
66.6 ± 9.7
108 ± 12.0
64.0 ± 8.0
53.5 ± 6.7
54.0 ± 7.9
5.1 ± 1.0
4.2 ± 1.1
4.5 ± 0.4
Rl
AT
Tl
Rl
AT
Tl
Rl
200 ␮mol quanta m−2 s−1
AT
81
76.0 ± 8.0
73.0 ± 12.0
69.0 ± 5.4
Tl
Rl
AT
Tl
Z. mays
D. sanguinalis
P. maximum
P. miliaceum) and it correlated very well with KMS/BS plasmodesmata density calculated per square mm of the leaf: both
values increased by approximately 100% with the change in
light growth conditions from 200 to 1000 ␮mol quanta m−2 s−1 .
Thus, it seems that the maximal photosynthate load per plasmodesma could be approximately constant under different light
growth conditions. Additionally, the role of BS/VP plasmodesmata in photosynthetic competence should be pointed out, since
their density increased in HL plants even more strongly than the
density of KMS/BS plasmodesmata, as compared to ML plants.
The only exception was P. miliaceum, where both the BS/VP
plasmodesmata density and the maximal net photosynthesis rate
were the same in HL and ML plants. These plasmodesmata are
responsible for the symplasmic transport of sucrose to VP cells
before the apoplasmic step of phloem loading. So, their number may determine the export of sucrose from a leaf, which in
HL plants of P. miliaceum is less efficient than in other studied
species (see also discussion on photosynthate transport below).
In general, the HL plants confirmed to the widespread opinion
that C4 plants do not exhibit light saturation at photon fluxes at or
below those of full sunlight (Pearcy and Ehleringer, 1984), while
the ML plants behaved differently, showing light saturation at
the fairly low photosynthetic photon flux of 650 ␮E m−2 s−1 . It
should be noted that C4 plants grown under natural light, but of
an intensity much lower than in the field, also show saturation
at a photon flux much lower than full sunlight (Burzyński and
Lechowski, 1983; Usuda et al., 1985), just like the ML plants
used in our study. On the other hand, maize plants grown in the
field (Usuda et al., 1985; Jompuk et al., 2005) showed a light
curve of photosynthesis very much like that of HL plants in our
P. miliaceum
Fig. 3. Maximum quantum yield of PSII (Fv/Fm) of four C4 grasses—P. maximum (PEP-CK), P. miliaceum (NAD-ME), D. sanguinalis (NADP-ME) and
Zea mays (NADP-ME) grown at two light intensities: 200 ␮mol quanta m−2 s−1
(empty bars) and 1000 ␮mol quanta m−2 s−1 (black bars). Values are means
(±S.D.) of nine plants.
Table 4
Transport parameters: time taken for 14 C-photosynthates to move into the transport path (AT, min), the fraction of current 14 C-photosynthates exported from leaves (Rl, percent of total radioactivity incorporated),
transport speed in leaf blade (Tl, cm min−1 ) in four C4 grasses grown at various light intensities
P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
82
P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
study (Fig. 2). It then follows that C4 plants attain full competence of photosynthesis only when grown at light intensities
close to those of full sunlight. We believe that this maximal
competence is prevented, at least in part, under lower light
growth conditions by the inefficient exchange of photosynthetic
intermediates due to the underdeveloped symplasmic network
between KMS and BS cells.
The export of photosynthates from leaves of C4 plants
depends on the ongoing photosynthesis (Grodzinski et al., 1998),
therefore it was reasonable to study the transport processes in
plants differing in their photosynthetic competence following
growth under different light conditions. The additional rationale
for that came from the observation that in addition to the density
of KMS/BS plasmodesmata, also the density of BS/VP plasmodesmata increased in HL plants compared to LL and ML plants.
Plasmodesmata crossing this cell interface have been shown to
play a crucial role in the export of photosynthates from maize
leaves (Botha et al., 2000; Russin et al., 1996).
To study the photosynthate export an in vivo method developed earlier for maize, based on the “pulse-chase” approach,
was used. The method allows the study of individual successive
steps of photosynthate transport: from the site of synthesis to
the phloem loading zone, phloem loading, and transport along
the leaf blade. The export of photosynthates from leaves of C4
plants of different photosynthetic sub-types was studied before
(Leonardos and Grodzinski, 2000), but the system used then
allowed the authors to estimate only the overall efficiency of
transport since steady state export from leaves was studied.
The “pulse-chase” approach used here allowed us to find previously unknown relations between transport characteristics and
C4 photosynthesis sub-types, mostly in the time taken for 14 Cphotosynthates to appear in the transport path, AT, which varied
among the species tested. The AT was much longer in P. miliaceum and P. maximum, representing the NAD-ME and the
PEP-CK sub-types, respectively, than in Z. mays and in D. sanguinalis, both of the NADP-ME sub-type. The differences in
AT (an overall measure of carbon transfer between PCA, PCR
and conducting tissues in C4 plants) found among the tested
species may reflect the much more complicated way of carbon
transport in NAD-ME and PEP-CK photosynthesis compared to
NADP-ME photosynthesis, including the participation of mitochondria, besides chloroplasts, in the former two photosynthetic
pathways. Two other transport characteristics, i.e. the fraction
of currently produced photosynthates exported from leaves and
transport speed did not show any particular dependence on the
C4 photosynthesis sub-type. They were related to the size of
plants at the developmental stage they were studied at, being the
highest in Z. mays and the lowest in P. maximum. One should
interprete the above differences between the C4 sub-types with
caution. Our experiments have sampled a very limited number
(one or two) of representatives of the three C4 sub-types; therefore we cannot rigorously show that the clear-cut differences
seen in the transport characteristics are indeed sub-type related,
and not simply species-specific.
The ML and HL plants of most species studied here did not
vary distinctly in respect to transport kinetics, particularly if one
compares values obtained at the photosynthetic photon flux close
to the growth light conditions, i.e. 200 ␮mol quanta m−2 s−1 for
ML plants and 650 ␮mol quanta m−2 s−1 for HL plants. The only
exception was P. miliaceum, in which both the exported fraction
of currently fixed photosynthates and the transport speed in the
leaf blade were lower in HL compared to ML plants. In effect,
in this species the export of photosynthates from HL leaves was
impaired, as visualized by strong accumulation of starch grains
in chloroplasts (Fig. 1), resulting also in slight photoinhibition
as manifested in a decrease in Fv/Fm (Fig. 3). It seems that the
restricted photosynthate export could be related to structural limitations, since the density of plasmodesmata linking the BS and
VP cells did not increase in HL compared to ML P. miliaceum,
as did in the other species investigated here (Tables 3A and 3B),
thus confirming the importance of BS/VP plasmodesmata for
sucrose export from the leaf (Botha et al., 2000; Russin et al.,
1996). Additionally, the lower export of photosynthates from
leaves of HL-grown plants of P. miliaceum could be related to
sink limitation resulting in an inhibition of phloem loading.
In general, regarding photosynthate export, the most important difference between plants grown at high and low light
conditions was that the ML plants strongly responded to changes
in photosynthetic photon flux, while the HL plants did not. In
the ML plants, an increase of photosynthetic photon flux led
to shortening of the time taken by photosynthates to appear in
the transport path and an increase of the transport speed along
the leaf blade, with a slight decrease in the fraction of current
photosynthates exported from the leaf. Stimulation of transport
speed by light was found earlier by Troughton et al. (1977) in
maize. No such clear dependence was observed in HL plants,
except for a decrease of AT with increasing photon flux. Leaves
growing in an environment which stimulates their source activity
maintain high level of sucrose in the vacuole or other compartments, thus buffering the transport pool, so no clear relationship
between photosynthesis and export could exist. As it was argued
by Wardlaw (1990), a positive correlation between carbon export
and sucrose content in the leaf during photosynthesis might be
expected only when sucrose buffering is small, e.g. under low
light level.
Two conclusions might be drawn from the present study.
First, the known poor adaptation of C4 plants to shaded environments might be due to insufficient development of the
plasmodesmata network connecting PCA and PCR tissues,
beside other mechanisms discussed in the literature (Sage and
Pearcy, 2000; Das, 2004). For example, in such source-limited
plants, this ultrastructural bottleneck might be a reason of ineffective exploitation of sunflecks, an important source of solar
energy in shaded environments. Second, C4 grasses are able to
adjust their photosynthetic apparatus to high light growth conditions by changing the number of plasmodesmata connecting
Kranz mesophyll cells and bundle sheath cells. In such sunny
environments, C4 plants seem to be sink-limited.
The mechanism of the proposed modification of plasmodesmata density is not clear. The simplest explanation would
be that the KMS/BS and BS/VP plasmodesma are secondary
ones (Cooke et al., 2000; Dengler and Taylor, 2000; Ding and
Lucas, 2000), and their number increases in response to a higher
demand for metabolite exchange of the respective cells. How-
P. Sowiński et al. / Environmental and Experimental Botany 61 (2007) 74–84
ever, no symptoms of secondary plasmodesmata formation have
been found in the developing maize leaf (Evert et al., 1996) and
Botha (2005) suggest that KMS/BS plasmodesmata in grasses
are secondarily modified. Another possibility is that their number is determined at some very early stage of leaf development.
If this is correct, it would be a similar phenomenon to the known
linkage between sink activity and size of vasculature, where the
sink pre-adapts the vasculature size to the “anticipated” peak
demand of assimilates at the very early stages of development,
during the cell division phase (Morris, 1996, and literature cited
therein). In such a case, however, other factors linking the growth
conditions and plasmodesmata number should exists than the
demand for the exchange of metabolites.
Regardless of the mechanism of the formation of KMS/BS
plasmodesmata, the phenomenon is intriguing, since it might
determine the adaptation of C4 plants to extreme conditions
other than full sunlight, e.g. to high temperatures and/or elevated
carbon dioxide level, in which conditions C4 photosynthesis
with the massive exchange of intermediates between Kranz mesophyll and bundle sheath cells is stimulated.
Acknowledgements
The authors wish to thank an anonymous reviewer for suggestions that have greatly improved the clarity of the paper. This
work was partially supported by grant KBN 3P04C04325.
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