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Aquatic Botany 72 (2002) 5566

Physiological and anatomical characterisation


of Phragmites australis leaves
Marisa Antonielli a, , Stefania Pasqualini a , Paola Batini a ,
Luisa Ederli a , Angelo Massacci b , Francesco Loreto b
a

Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Universita degli studi di Perugia,


Borgo XX Giugno 74-06121 Perugia, Italy
b CNR-Istituto di Biochimica ed Ecofisiologia Vegetali, Monterotondo Scalo, Rome, Italy
Received 10 November 2000; received in revised form 26 July 2001; accepted 1 October 2001

Abstract
The anatomy, biochemistry and physiology of Phragmites leaves have been investigated. Biochemical and physiological measurements indicate that Phragmites australis leaves have a C3
mechanism of carbon fixation. However, structural and ultra-structural observations of young
leaves are more reminiscent of a C4 -like anatomy. In addition, chloroplasts apparently fully competent for photochemical and biochemical reactions were in both the mesophyll and in the bundle
sheath cells of young leaves. The activity of the principal enzyme involved in carbon metabolism,
ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was high under ambient conditions
(59.8 and 64.2 mol m2 s1 , respectively in mature and young leaves). In contrast, phosphoenolpyruvate carboxylase activity was low in both mature and young leaves (7.8 and
8.1 mol m2 s1 , respectively) Despite the high Rubisco activity, the rate of photosynthesis of
Phragmites leaves on a leaf area basis was low. We investigated if resistances to CO2 entry in the
leaves could limit photosynthesis. However, stomatal and mesophyll resistances to CO2 diffusion
in Phragmites leaves were comparable to those of terrestrial plants and did not restrict intercellular
CO2 concentration significantly. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Bundle sheath anatomy; Diffusive resistance to CO2 ; Leaf anatomy; PEPcase; Photosynthesis;
Phragmites australis; Rubisco

1. Introduction
Phragmites australis is a perennial, emergent aquatic plant with the widest geographical distribution of any flowering plant (Tucker, 1990). Aquatic plants cope with oxygen
Corresponding author. Tel.: +39-075-5856408; fax: +39-075-5856425.
E-mail address: antmfisveg@unipg.it (M. Antonielli).

0304-3770/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 3 7 7 0 ( 0 1 ) 0 0 2 2 0 - 0

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M. Antonielli et al. / Aquatic Botany 72 (2002) 5566

deprivation in the water medium by carrying out a series of morphological and ultrastructural modifications in their organs. There have been many studies on the strategy used to
avoid anoxia of submerged Phragmites parts (e.g. Armstrong and Armstrong, 1990; Armstrong et al., 1996; Brix et al., 1996). These works stressed the importance of convective
gas flow through the influx culm-rhizome-efflux culm system, but did not address the gas
exchange at leaf lamina level. Gas-exchange studies offer a valuable opportunity to study in
vivo carbon metabolism and oxygen evolution through photosynthesis and water relations
(Laisk and Loreto, 1996).
The carbon metabolism of Phragmites seems to be ambiguous. PEP carboxylase activity
and Rubisco/PEP ratio are apparently similar to those of C4 plants (Rintamaki and Aro,
1985; Zheng et al., 2000), but the optimum temperature for photosynthesis is low compared
to that of C4 plants (Ondok and Gloser, 1978). The objective of this study was to characterise unambiguously, through a combination of anatomical, physiological and biochemical
measurements, the carbon metabolism of P. australis leaves.
2. Materials and methods
Anatomical, biochemical and physiological analyses were carried out on mature completely expanded and young (25 days after leaf lamina unfolding) leaves of P. australis
(cav.) Trin. ex Steud. collected at eight different locations around the Trasimeno lake (central
Italy).
2.1. Anatomical observations
Preliminary, 12 mm thick sections were hand made and immediately fixed in 3% glutaraldehyde in a phosphate buffer for 3 h. Samples were then washed three times for 15 min
in a phosphate buffer 0.1 M at pH 7.2, and post-fixed in 1% OsO4 . At this stage, samples
were prepared for inclusion in epoxy resin by dehydration steps in increasing concentrations of ethanol. A pre-inclusion in freshly prepared resin at 35 C (4 h) was followed by
the final inclusion in freshly prepared resin (24 h at 35 C and 72 h at 60 C). The inclusion
steps were separated by washing in a phosphate + sodium cacodylate buffer. The resin
was made by Araldite Hardner (15 ml), Araldite M (5 ml), Epon 812 (4.3 ml) and Fastner (0.4 ml). Semi-thin (12 m) sections were cut with an ultramicrotome (U2 Reichter)
equipped with a glass blade. These sections were stained with toluidine blue and observed
by light microscopy. Thin (600800 ) sections were cut with the microtome equipped with
a diamond blade. These sections were layered on a thin colloidal film on a thin metal net.
Contrast was achieved by adding uranile acetate and an aqueous solution of lead nitrate
before observation with a transmission electron microscope (TEM 400 T, Philips).
2.2. Biochemical measurements
2.2.1. Rubisco activity
Leaf discs (3.5 cm2 ) were rapidly frozen and ground to a powder under liquid nitrogen.
The 2 cm3 of extraction buffer (100 mM bicine pH 7.8, 20 mM MgCl2 , 5 mM dithiothreitol

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(DTT), 1 mM ethylendiaminetetracetic acid (EDTA), 10 mM NaHCO3 , 2% (w/v) PVPP


(polyvinyl polypyrrolidone) and 0.02% (w/v) bovine serum albumin (BSA) was added
to the powder and the homogenate was centrifuged at 13,000 g for 2 min. The 10 l of the
supernatant were added to 980 l of a solution made by 50 mM Bicine (pH 8), 1 mM EDTA,
15 mM MgCl2 , 20 mM NaCl, 9.2 mM DTT, 9.2 mM NaHCO3 , 4.6 mM phosphocreatin,
0.5 mM ATP, 0.04 mM NADH, 47 phosphoglycerate kinase units, 47 glyceraladehyde-3phosphate dehydrogenase units, 1.3 phosphocreatin kinase units. The mixture assay was
incubated for 3 min at 25 C and the reaction was started by adding 30 mM RuBP. The
Rubisco activity was measured spectrophotometrically by determining the absorbance at
340 nm. For other details see Sharkey et al. (1991).
2.2.2. PEP-case activity
Leaf discs were ground and extracted as for Rubisco determination. The extraction buffer
contained 50 mM TrisHCl (pH 7.6), 2 mM EDTA, 5 mM MgCl2 , 0.02% BSA (w/v), 1 mM
phenylmethylsulfonyl fluoride (PMSF), 5 mM DTT, 2% (w/v) PVPP. After centrifugation
at 13,000 g for 8 min, the supernatant (100 l) was added to a buffer made by 100 mM
HEPES-NaOH (pH 8), 10 mM MgCl2 , 10 mM NaHCO3 , 0.2 mM NADH, 1.5 malic dehydrogenase units, 2 mM PEP and maintained at 30 C. The PEP-case activity was determined
spetrophotometrically by determining the absorbance at 340 nm.
2.2.3. Carbonic anhydrase (CA) activity
Half a gram of leaf tissue was ground in liquid nitrogen with a 5 ml buffer made by
100 mM TrisHCl (pH 8.3), 1 mM EDTA, 10 mM mercaptoethanol, 0.1% Triton X100. The
homogenate was centrifuged at 5,000 g for 5 min and the supernatant (0.5 ml) was added to
3 ml of 25 mM Veronal buffer (pH 8.2, at 6 C). At this stage 2 ml of CO2 -saturated distilled
water was added to start the reaction which proceeded by continuously shaking the solution.
The CA activity was determined following the pH reduction from 8 to 6.4 according to the
method of Wilbur and Anderson (1948).
2.3. Physiological measurement
2.3.1. CO2 and H2 O gas-exchange
Photosynthesis, stomatal conductance, intercellular CO2 concentration, electron transport rate, and transpiration were determined by infrared gas analysis with a laboratory
instrumentation (Li-Cor 6262) previously described (Loreto et al., 1994) on plants collected at the Trasimeno lake and grown for a season in a pond close to the institute in Rome.
The plants were grown in 20-L pots and were transported to the laboratory every time they
were used for physiological measurements. All measurements were done on at least three
mature leaves and three young leaves of different plants. A portion (4.9 cm2 ) of intact leaf
was inserted in the gas exchange cuvette and exposed to saturating (1500 mol photons
m2 s1 ) irradiance and to two different gas conditions: ambient air allowing both photosynthesis and photorespiration (350 mol mol1 CO2 , 20% O2 , 80% N2 ) or low oxygen
air (400 mol mol1 CO2 , 2% O2 , 98% N2 ) inhibiting photorespiration and stimulating
photosynthesis. Leaf temperature was maintained at 25 C and air relative humidity was set
at 40% throughout the experiments.

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2.3.2. Fluorescence measurements


Chlorophyll fluorescence was detected with a modulated fluorometer (Walz) vertically
inserted over the gas exchange cuvette simultaneously with gas exchange determination.
The leaves were dark-adapted (30 min) to measure the PSII quantum yield or regularly illuminated to measure the electron transport rate. Comparison between the electron transport
rate measured by fluorescence and that calculated by gas exchange yielded the mesophyll
conductance and the chloroplast CO2 concentration (Loreto et al., 1992; Loreto et al., 1994).

3. Results
3.1. Leaf anatomy
Transverse sections of mature P. australis leaves examined by light microscopy showed
a mesophyll formed by 1215 layers of cells (Fig. 1a). The external part of the mesophyll

Fig. 1. Light micrographs of transverse semi-thin sections of (a) mature, bar = 200 m and (b) young,
bar = 140 m leaves of P. australis. BC: bulliform cell; BS: bundle sheath; MC: mesophyll cell; ST: stomata; XV: xylem vessel; PV: phloem vessel.

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59

is made by several layers of tightly appressed cells and have limited intercellular spaces
on both leaf sides. The internal part of the mesophyll consisted of irregularly shaped cells,
larger than peripheral cells and separated by large intercellular spaces. Stomata were present
on both leaf surfaces and were more numerous in the abaxial surface. On the adaxial leaf
surface, numerous and turgid bulliform cells extend from the leaf surface to the centre of
the mesophyll. The vascular bundles were surrounded by a double bundle-sheath. The inner
sheath, termed mestom sheath by Schwendener (1890), consisted of thick-walled cells, and
the outer sheath of thin-walled cells with chloroplasts. At level of the vascular bundle,
clusters of fibers which interrupt the chlorophyllian parenchyma were observed.
The structure of young leaves is quite different from that of mature leaves (Fig. 1b). The
mesophyll of young leaves consists of only 45 layers of cells and around the vascular
bundles a single thin-walled sheath with chloroplasts is visible (Fig. 1b). The fibrous cells
are few and thin-walled, and the bulliform cells are not well distinguishable.
The examination of transverse sections of P. australis mature leaves by electron microscopy showed that the large intercellular spaces are often associated with invaginations
of the cell walls (Fig. 2a and b). Inside the mesophyll cells, chloroplasts, rich of osmophilic
globules, are appressed to the cell walls and cover most of them (Fig. 2b). Small vacuoles,
many mitochondria and peroxisomes are visible in the mesophyll cells (Fig. 2b). In young
leaves, where the mechanical tissues are limited, a sheath of parenchimatous cells surrounding the vascular bundles is clearly observed. These cells are rich of chloroplasts (Fig. 3a).
Chloroplasts have a complete structure, with well developed and stacked grana, and show
starch depositions and osmophilic globules (Fig. 3b).
3.2. Biochemical and physiological measurements
A high activity of Rubisco was found both in young and mature Phragmites leaves
(Table 1). In contrast, PEP-case activity was low. The ratio Rubisco/PEP-case was about 8.
The activity of carbonic anhydrase was typical of C3 plants.
Table 1
Biochemical (activities of Rubisco, PEP-case and CA, and ratio between Rubisco and PEP-case) and physiological
(photosynthesis, stomatal and mesophyll conductance, intercellular and calculated chloroplastic CO2 concentrations) characteristics of mature and young Phragmites leaves maintained in ambient conditions (21% O2 , 350 ppm
CO2 )a
Mature
m2 s1 )

Rubisco (mol CO2


PEP-case (mol CO2 m2 s1 )
Rubisco/PEP-case
CA (mol CO2 min1 g1 FW)
Photosynthesis (mol CO2 m2 s1 )
Stomatal conductance (mol CO2 m2 s1 )
Intercellular CO2 concentration (mol mol1 )
Mesophyll conductance (mol CO2 m2 s1 )
Chloroplastic CO2 concentration (mol mol1 )
a

Mean (n = 6) S.E. is shown.

59.8 5.1
7.8 1.0
7.7
78.0 4.0
14.1 1.6
0.19 0.02
275 20
0.26 0.05
220 30

Young
64.2 8.8
8.1 1.3
7.9
75 4.5
15.2 2.2
0.22 0.04
269 25
0.28 0.07
225 28

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M. Antonielli et al. / Aquatic Botany 72 (2002) 5566

Fig. 2. Electron micrographs of transverse ultra-thin sections from mature leaves of P. australis: (a) bar = 6.5m;
(b) bar = 2 m. C: chloroplast; N: nucleous; W: wall.

When expressed per unit leaf area, photosynthesis of Phragmites leaves at ambient CO2
was rather low (Table 1). Conductances to CO2 diffusion (stomatal and mesophyll) were also
low and similar to those of terrestrial plants. Diffusion resistances decreased the concentrations of CO2 in the intercellular spaces and in the chloroplasts with respect to the external
(ambient) concentration. Nevertheless, we calculated that at least 200 mol mol1 of CO2
should be available in the chloroplasts, a concentration much higher than the compensation
point between photosynthesis and photorespiration.

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61

Fig. 3. Electron micrographs of transverse ultra-thin sections from young leaves of P. australis: (a) bar = 1.5 m:
mesophyll at level of bundle sheath; (b) bar = 0.5 m: chloroplast of a bundle sheath cell. BS: bundle sheath cell;
G: grana; LG: lipid globules; MC: mesophyll cell; S: starch; W: wall.

Photosynthesis decreased in both mature and young leaves as CO2 was reduced below
ambient, reaching the compensation point at a CO2 concentration of about 50 mol mol1
(Fig. 4). Photosynthesis of Phragmites leaves saturated at intercellular CO2 concentrations
between 300 and 400 mol mol1 . At ambient CO2 , exposure of mature leaves to low O2
(2%) resulted in a 25% stimulation of photosynthesis (Fig. 4a). Photosynthesis stimulation
was almost absent if O2 was lowered while maintaining leaves at high CO2 concentration.

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Fig. 4. Photosynthesis response to changes of intercellular CO2 concentration (solid symbols) and to low O2 (2%,
open symbols) at ambient and high CO2 concentrations in P. australis (a) mature and (b) young leaves maintained
at a leaf temperature of 25 C and exposed to a light intensity of 1000 mol m2 s1 . Mean (n = 6) S.E. is
shown.

4. Discussion
Our aims were to characterise through a combination of anatomical, biochemical and
physiological measurements the mechanism of carbon fixation and oxygen evolution in
Phragmites leaves.

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Mature and young leaves have different anatomy in Phragmites. The mesophyll of mature leaves is characterised by a cells tightly appressed and by a lack of intercellular spaces.
Therefore, the aerenchyma is only present in the submerged organs, such as roots and
rhizome (data not shown), whereas the leaf blade show a typical xeromorphic structure.
The structure of mature leaves have the anatomical features of C3 grasses (Esau, 1965),
with large vascular bundles surrounded by two sheaths, the outer of which with chloroplasts. In young leaves, on the other hand, the bundle sheath is formed by a single layer
of cells, with numerous chloroplasts, an anatomical feature typical of C4 plants (Hatch
et al., 1975; Dengler and Nelson, 1999). In C4 plants the bundle sheath chloroplasts are
however agranal in NADP-ME type and do not perform efficient photochemistry (Kanai
and Edwards, 1999). We wanted to know if this was also the case in young leaves of Phragmites. Our ultra-structural observations, however, show that chloroplasts of Phragmites
bundle sheath are apparently functional since they have a complete structure. Thus, they
may be photochemically active and may independently evolve oxygen, an important feature
in oxygen-limited environments.
In some aquatic plant species, the C4 traits develop only when they emerge from the water
(Ueno et al., 1988) or when they grow in dense stands (Bowes and Salvucci, 1984). Rintamaki and Aro (1985) reported that PEP-case activity and Rubisco/PEP ratio of P. australis
were similar to those of C4 plants. Our biochemical and physiological data, however, suggest
a C3 photosynthetic metabolism in P. australis leaves. For example, the Rubisco/PEP-case
activity measured ratio is well within the range (421) found for C3 species and not as low
as for C4 species (0.050.56) (Leegood, 1993).
Photorespiration is suppressed in C4 plants. In Phragmites we found a photorespiration
typical of C3 plants in both young and mature leaves. In fact, the compensation point was
reached at a CO2 concentration of about 50 mol mol1 , as expected in C3 plants at leaf
temperatures of about 25 C (Brooks and Farquhar, 1985). Exposure to low O2 suppresses
photorespiration in C3 plants. Consequently, photosynthesis at low O2 is enhanced in C3
plants while is not affected in C4 plants. Photosynthesis stimulation at low O2 was evident
in Phragmites leaves maintained at ambient CO2 (Fig. 4). This further suggests that Kranz
anatomy of young leaf does not result in the C4 pathway (Raghavendra, 1980). It may
be possible that plant species with a C4 metabolism exist within the genus Phragmites,
or that Phragmites sp. have a C3 C4 metabolism. Many C3 C4 intermediate species have
been found with granal chloroplasts in the bundle sheath cells and a remarkable rate of
photorespiration (Holaday and Chollet, 1984). Activities of the enzymes involved in the
C4 cycle (e.g. PEP-case) are often low in C3 C4 intermediates (Monson and Rawsthorne,
2000). However, C3 C4 intermediates have a compensation point intermediate between
that of C4 and C3 plants (Monson and Rawsthorne, 2000), while Phragmites compensation
point, as already reported, was typical of C3 plants. Moreover, the isotopic discrimination
against C18 OO of Phragmites leaves seem to be within the range of C3 plants (Gillon and
Yakir, 2000). On this basis, we consider it unlikely that C3 C4 intermediate metabolism
occurs in Phragmites.
The photosynthetic rates of Phragmites leaves that we observed were similar to those
of many terrestrial C3 plants but very low with respect to many C4 plants producing an
equivalent biomass (Massacci et al., 1996). Photosynthesis is often limited by reduced CO2
acquisition in terrestrial species (Loreto et al., 1994). We wanted to determine whether

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the anatomy of Phragmites leaves resulted in a limitation of CO2 acquisition and, as a


consequence, in a limitation of photosynthesis. We calculated that at ambient external CO2
(350 mol mol1 ), the CO2 concentration in the chloroplasts was lowered by diffusion
resistances to about 220 mol mol1 in Phragmites leaves. The ratio between internal and
external CO2 concentrations (about 0.7) is typical for herbaceous leaves of C3 plants (Evans
and Loreto, 2000) and does not suggest any greater CO2 limitations at the chloroplast than
that found in other C3 species. Errors in the estimation of mesophyll conductance can
account for 30 mol mol1 with respect to the reported average value of chloroplast CO2
concentration but this should not influence significantly CO2 limitations to photosynthesis
(Loreto et al., 1992).
It has been shown that photosynthesis of species with low mesophyll conductance benefits
from CO2 concentrations above ambient and does not reach saturation until very high
concentrations are reached (Loreto et al., 1992). Because of the low CO2 in the chloroplasts,
photosynthesis of species with low mesophyll conductance should also benefit when O2
cannot compete with CO2 for Rubisco and when a high content of carbonic anhydrase
facilitates CO2 diffusion across leaf membranes and chloroplast stroma (Poincelot, 1972;
Cowan, 1986; Badger and Price, 1994). However, photosynthesis of Phragmites leaves
reached saturation at relatively low intercellular CO2 concentrations (<400 mol mol1 ).
The stimulation of photosynthesis by low O2 concentration (+1525%) and the activity of
CA were within the range observed in C3 plant species with average diffusion resistances.
It should be noted that a high in vitro activity of Rubisco under ambient conditions was
found both in young and mature leaves. Thus, there is a potential to improve photosynthetic
performances if the limitations were removed. Perhaps the content of nitrogen is low in
Phragmites dense stands grown in natural environments and, consequently, the Rubisco
content is not sufficient to sustain high photosynthetic rates.
In summary, our anatomical, biochemical and physiological measurements characterised
a C3 mechanism of carbon fixation in P. australis leaves. Particularly relevant is however the
anatomy of young leaves, where the bundle sheath chloroplasts are apparently competent
both photochemically and biochemically and may therefore be involved in oxygen production. Rubisco activity is high under ambient conditions, and resistances to CO2 diffusion of
Phragmites leaves are comparable to those of terrestrial plants. The low photosynthesis of
Phragmites leaves on a leaf area basis is therefore not explained by our studies, but these
short-term leaf measurement do not limit the rapid growth of Phragmites stands in nature.

Acknowledgements
We are grateful to Professor Hamelyn Jones for proof-reading the manuscript and Paola
Pinelli for helping with gas-exchange measurements.

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