ISSN 1408-3671 
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izdajate! j/pu bi isher 
Društvo biologov Slovenije 
ISSN 1408-3671 
UDK 57(497.4) 
~ACTA 
~ BIOLOGICA 
SLOVENICA 
VOL. 46 ŠT. 2 LJUBLJANA 2003 
prej/formerly BIOLOŠKI VESTNIK 
izdajatelj/publisher 
Društvo biologov Slovenije 
Acta Biologica Slovenica 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2 : 1- 60 
Glasilo Društva biologov Slovenije - Joumal of Biological Society of Slovenia 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2 : 3 - 1 O 
Sprejeto (accepted): 2003-10-30 
Non-foliar photosynthesis and its contribution to the 
overall carbon balance of plants 
Guido ASCHAN and Hardy PFANZ 
Institute of Applied Botany, University of Duisburg - Essen, 45117 Essen, FRG 
lntroduction 
Abstract. In addition to the green leaves, commonly considered as the main 
sources of photosynthate production, higher plants can potentially use nearly all 
vegetative and reproductive structures to perform photosynthetic CO
2 
assimilation. 
Green leaves, stems and green sterile flower organs, optimized for light harvesting 
and photosynthetic performance, are characterized by net photosynthetic assimilation 
utilizing mainly the atmospheric carbon dioxide. In contrast, chlorophyll-containing 
bark and wood tissue, most fruit, root and fertile flower organs are primary sub-
ordinated to non-photosynthetic functions, but typically perform an effective intemal 
CO
2 
recycling using the respiratory released CO
2
• Non-foliar photosynthesis, either 
manifested as positive net photosynthesis or internal CO
2 
refixation is regarded as 
an important strategy of additional carbon acquisition. The main strategies of 
additional carbon acquisition by non-foliar chlorophyllous organs are illustrated, 
presenting selected examples developed in reproductive as well as in vegetative plant 
structures. 
Keywords: floral photosynthesis, aerial roots, orchids, Helleborus, chlorophyll 
fluorescence 
Abbreviations: Chl = chlorophyll 
The visual impression of higher plants is typically dominated by green leaves as the main 
photosynthetic organ, but a closer look on herbaceous or even woody plants reveals a variety of 
non-foliar vegetative and moreover reproductive structures which contain chlorophyll and are thus 
potentially able to conduct photosynthetic CO
2 
assirnilation. 
The positive net photosynthesis by green stems or flowers contrasts remarkably to the exclusive 
recycling of respiratory released CO
2 
in fleshy fruits or bark tissues (BLANKE & LENZ 1989, PFANZ & 
AL. 2002). Independent of positive net photosynthesis or interna! CO
2 
refixation there is no doubt, 
that photosynthesis of chlorophyllous tissues other than leaf mesophyll will both partially pay for 
their own carbon requirements and thus positively contribute to the overall carbon gain of plants. 
Since most existing information on the CO
2 
exchange and carbon gain of higher vascular plants 
refers to Jeaves, data on the carbon budgets of stems, roots or fruits are almost entirely lacking (e.g. 
in epiphytes, see Zon & HIETZ 2001). Therefore, we intend to illurninate the importance of non-
4 Acta Biologica Slovenica, 46 (2), 2003 
foliar photosynthesis as a strategy of additional carbon acquisition, presenting selected examples 
evolved in reproductive as well as in vegetative plant structures. While the photosynthetic activity 
of stem and bark tissues is mentioned elsewhere in this issue (see also PFANZ & AscHAN 2001, PFANZ 
& AL. 2002), we are focussing here on representative case studies of flora! and root photosynthesis. 
Methods 
Carbon fixation in non-foliar plant organs has typically -been measured using gas exchange or 
14C-uptake techniques, but both methods may underestimate photosynthetic rates (e.g. BLANKE & 
LENZ 1989). Changes in extemal CO
2 
often not adequately reflect CO
2 
uptake in photosynthesis and 
in particular the amount of intemally-generated photosynthetically fixed CO
2 
because of a low 
epidermal and cuticular permeability (cf. PFANZ & AscHAN 2001). Especially on bulky green fruit, 
root or stem material, chlorophyll fluorescence techniques are advantageous, because photosynthetic 
performance doesn' t depend on wether the CO
2 
fixed originates extemally, or intemally via respiratory 
processes (see SMILLIE 1992). 
Chlorophyll fluorescence measurements on intact leaves and non-foliar organs were performed 
with a chlorophyll fluorometer PAM-210 (Walz, Effeltrich, Germany) under laboratory conditions 
(20°C). 
Instant light-response curves of the effective quantum yield (Y = Af</Fm') were obtained using 
the light-curve prograrnme of the PAM (Run 10). After determination of maximum quantum yield a 
5 min pre-illumination period at moderate light intensity (setting 3 corresponding to 120 µmol 
photons m·2s·1) provide a stationary fluorescence leve! of the samples. Then the actinic light intensity 
is decreased to setting 1 (60 µmol photons m·2s·1l for 5 min, before a saturation light pulse is applied 
to obtain Y and ETR. The apparent rate of photosynthetic electron transport of PSII (ETR) was 
calculated as ETR = Af</Fm' x PFD x 0.5 x 0.84 (ScHREJBER & AL. 1995). Within the following 20 
min light intensity is increased stepwise with illumination period s of 2 min and subsequent saturation 
pulses until setting 10 (1250 µmol photons m·2s·1l is reached. 
In situ gas-exchange measurements on plant organs were conducted with a portable porometer 
system (Ll-6400, Li-Cor Inc., Lincoln, USA). Photosynthetic light response curves were obtained 
under constant climatic conditions (20°C, 75 % relative humidity) and a controlled CO
2 
supply (350 
ppm). 
Statistical analysis, calculations and curve fittings performed using Sigma Plot 5 .O (SPSS Science 
Software). 
Results and Discussion 
I. Carbon acquisition by reproductive structures 
In the early developmental stages, petals of most flowering plants are green due to the presence 
of chlorophyll, whereas mature petals often are whitish or brightly coloured, because Chl is either 
absent or masked by other pigments. Therefore, photosynthetic activity was typically observeči in 
visibly green petals (see DuECKER & ARmITI 1968; Vu & AL. 1985; SALOPEK-SONDI & AL. 2000), such 
as developed by the deciduous perennial Green Hellebore (Helleborus viridis). The photosynthetic 
light response of Green Hellebore sepals and leaves is studied by the comparative use of Chl 
G. Aschan, H. Pfanz: Non-foliar photosynthesis and its contribution to the ... 5 
fluorescence as well as in situ gas-exchange rneasurernents. The sepals achieved maximum electron 
transport rates of about 75 % of mature leaves at moderate to high PFDs, as determined by Chl 
fluorescence measurements (<lata not shown). In contrast, the maximum net CO
2
-exchange rates of 
Hellebore sepals (2.3 µmol CO
2 
m-2 s·1) were less than one fourth of the leaves (10.6 µmol CO
2 
m 2 s·1) 
(Fig. 1 ). This difference is possibly explainable by the 70-80% lower stomatal density of the sepals 
in comparison to the Hellebore leaves. 
12 -..... 1 
tn -----
(':I 10 
E 
N 8 o o leaf - 6 o / sepal E ... 
::::1. 4 ..... 
CI) 
C) 2 -----C: 
ta 
.C: o (.) 
>< q, 
-2 N o o 
-4 
o 500 1000 1500 2000 
PFD [µmol photons m-2s-1] 
Fig. 1: Photosynthetic light response curves of Green Hellebore (Helleborus viridis) leaves (circles) 
and sepals (triangles). Measurements were performed in spring under constant climatic conditions 
(20°C, 75 % relative hurnidity) and a controlled CO
2 
supply (350 ppm) using a CO
2
-porometer. 
Means with s.d. are presented; n = 12-14. 
The sepals of the germane Christmas Rose (Helleborus niger) are whitish at anthesis and get 
gradually green (in shaded plants) or pinkish-red (in sun-exposed plants) during seed ripening. 
Maximum electron transport rates (ETR) of green sepals reached slightly lower values about 60% 
of the leaves ' rates at medium PFDs (ca. 600 µmol photons m·2s· 1), as assessed by Chl fluorescence 
measurements, The maximum electron transport rates of red sepals, coloured by anthocyan-like 
pigments, were clearly reduced to about one fourth of the respective leaf rates. As expected, Helleborus 
leaves use the quantum energy 1.5 fold more efficient than the green sepals, whereas the red ones 
need three times more quantum energy to transport the same amount of electrons within PSII (see 
ASCHAN & PFANZ 2003). 
Using another method (0
2 
gas-exchange) SALOPEK-SONDI & AL. (2000) reported photosynthetic 
capacities of greenish Helleborus niger sepals about 40-50 % of those of the green leaves. In greenish 
Helleborus sepals one third of the leaf Chl content (390 mg m·2, n = 6) was found, whereas red 
6 Acta Biologica Slovenica, 46 (2), 2003 
sepals contained only about 80 mg m·2 Chl. Also for orchid bracts Chl contents about one third of 
the respective leaves were obtained (e.g. Spiranthes cernua: ANTLFINGER & WENDEL 1997), whereas 
those of the pure white petals only amounted to 4 %. 
The pendulous, bell-shaped flowers of the Spring Snowflake (Leucojum vernum) have six equal 
white petals each tipped with an emerald green spot at the top. While the white areas of the petals 
are not photosynthetically active, even these small greenish spots achieve about 25 % of the ETR of 
the respective leaves (Fig. 2). 
Remarkably, similar photosynthetic activity was obtained for the shorter inner petals of the 
white-flowering Snowdrop (Galanthus nivalis), which are also marked with a terminal green spot 
(data not shown). 
140 
CI) -"' 120 l.. 
t:: 
o 
C. 100 u, 
C 
~ 80 -C e 60 -(J CI) 
"i 40 
CI) 
> 
~ 
20 "' "i 
l.. f 
o 
o 
----------
/ 
/ 
/ 
/ /'/ • leaf ,, ... green pe a spo 
200 400 600 800 1000 1200 
PFD [µmol photons m·2s·11 
1400 
Fig. 2: Photosynthetic light response curves of leaves (circles) and green peta! spots (triangles) of 
Spring snowflake (Leucojum vernum). Relative electron transport rate (ETR), calculated as ETR = 
0.5 x 0.84 x PFD x "F/Fm" (e.g. SCHREIBER ET AL. 1995), against photon flux density (PFD). 
Measurements were performed using a PAM-Fluorometer. Means with s.d. are presented; n = 9-15. 
In general, photosynthetic rates of flowers vary widely between less than 1 to 170 nmol CO
2 
g·1 
DW s·1 or 30--,200 µmol CO
2 
g·1 Chl s·1, many perennials having lower values than those of annuals 
(see HEILMEIER & WHALE 1987). Even the CO
2
-assimilation of Lilium anthers at 100 µmol m·2 s·1 
represents 73 % of the respective leaf CO
2
-fixation (CLEMENT & AL. 1997a), whereas the CO
2 
fixation 
rate of green Cymbidium flower was only around 10 % of the leaf (DUEKER & ARDIITI 1968). Tab. l 
summarizes the photosynthetic rates ofdifferent green flowering plant species in comparison to the 
resp. leaf rates. 
G. Aschan, H. Pfanz: Non-foliar photosynthesis and its contribution to the ... 7 
Table 1: Floral and leaf net photosynthetic rates of green flowering plants. 
Species Flora! Leaf Author 
photosynthesis [µmol m·2s·1] 
Helleborus viridis (Ranunculaceae) 2.3 (sepals) 10.6 own measurements 
Aciphylla glaucescens (Apiaceae): 11.0 13.5 HOGAN & AL. (1998) 
spines 
Helleborus niger (Ranunculaceae) 295 (sepals) 592-741* SALOPEK-SONDI & 
AL. (2000) 
Lilium hvb. enchantment (Liliaceae) 2.3 (anther)l.8 (tepals) 3.2 QEMENT & AL. 
(1997 a: b) 
Ranunculus adoneus (Ranunculaceae) -3.42 (young) 14.0-18.1 GALEN & AL. (1993) 
-0.25 (fully expanded) 
3.45 (petals abscised) 
Spiranthes cernua (Orchidaceae) 2.5 (flower), 3.7 (bud) 9.2 ANTLFINGER & WENDEL 
(1997) 
* in (µmol 0
2 
h·1g·1 DW). 
Flora! photosynthesis is considered to be an important additional source for assimiJates within 
usuall y heterotrophic inflorescences and may thus provide a significant po rti on of the C requirement 
of reproduction. Reproductive structures could gain up to 60 % of their totaJ carbon requirement, 
either acquired by flora! or fruit photosynthesis ( e.g. BAzzAZ & AL. 1979, WEISS & AL. 1988, MARCELIS 
& HOFMAN-EJJER 1995). 
II. Carbon acquisition by non-foliar vegetative organs 
Non-foliar vegetative photosynthetic organs couJd represent the primary assimilate source, as 
often realized in stems (NILSEN 1995, PFANZ & AscHAN 2001) and occasionally in roots, e.g. in 
Jeafless and nearly stemless orchid species (BENZING & AL. 1983, HEW & AL. 1984). Generally 
characterized by the Jack of Jeaves as well as of stomates, the aerial roots of epiphytes ( orchids, 
bromeJiads, aroids) and vines, the green pneumatophores of mangroves or the stilt roots of some 
paJms often contain chlorophyll and exhibit a well deveJoped photosynthetic capability. In epiphytic 
orchids the chJorophyllous parenchymaJ tissues usually were masked by a spongy whitish Jayer, the 
ve/amen radicum, but the growing apex of the root is commonJy green. AeriaJ root chJorophyll of 
some orchid species was found to range between 9 % and 55 % of respective Jeaf Chl contents, 
proportionateJy up to 89 % reJated to the fresh weight (AscHAN & PFANZ 2003; see aJso BENZING & 
Orr 1981). 
The roots of some orchid species are sufficiently autotrophic to either maintain themseJves or 
contribute substantially to their needs (e.g. Dvcus & KNuosoN 1957). However, most green orchid 
roots (and stems) show nonet photosynthesis (HEw & AL. 1984; BENZING & PocKMAN 1989), because 
of their stout structure, their Jack of a reguJated gas exchange and their predominant role for absorption 
and storage (BENZING & AL. 1983). 
Regarding these functionaJ properties of roots, the recycling of internally respired CO
2 
is 
considered as the main task of green, photosynthesizing root tissues. Preliminary gas-exchange 
measurements with aerial roots of two orchid species reveaJed CO
2 
refixation rates ranging from 49 % 
(Doritis pulcherrima coerulea) to 67 % (Vanda spec. data not shown), whereas nonet photosynthesis 
was detected. In earJy gas-exchange experiments with the Warburg apparatus a reduction of respiratory 
CO2 evoJution by aeriaJ orchid roots is shown under high illumination (Dvcus & KNuosoN 1957). 
8 Acta Biologica Slovenica, 46 (2), 2003 
Based on these <lata of four different orchid species we re-calculated root-internal CO2-refixation 
rates between 40 and 50%. 
Nevertheless, the photosynthetic light response of aerial orchid roots could be satisfactorily 
demonstrated using Chl fluorescence techniques. The chlorophyllous aerial roots of two selected 
orchid species achieved maximum electron transport rates between 55% (Doritis pulcherrima 
coerulea) and 65% (Vanda spec.) of mature leaves at PFDs between 600 and 1250 µmol photons m·2 s·1• 
The aerial roots use the incident light energy only about 40% less effective than the orchid leaves, as 
shown by the slightly different initial slopes of the respective light curves (Fig. 3). 
In spite of rather similar photosynthetic performance, the area-related Chl content of these two 
orchid roots differed considerably between 20% (Doritis pulcherrima coerulea) and 50% (Vanda 
spec.) of the respective leaf contents. Calculated on a unit Chl basis, electron transport rates in 
aerial roots thus exceed those of leaves. 
100 ------------------------, 
• leaf 
~ aerial root 
80 ------- -------- ----------! 
----
o ------,.---...-------.--------"""T"---....-----t 
o 200 400 600 800 1000 1200 1400 
Fig. 3: Photosynthetic light response curves of Jeaves (circles) and aerial roots (triangles) of the 
orchid Doritis pulcherrima coerulea. Relative e!ectron transport rate (ETR), calculated as ETR = 
0.5 x 0.84 x PFD x "F/Fm' (e.g. SCHREIBER ET AL. 1995), against photon flux density (PFD). 
Measurements were performed using a PAM-Fluorometer. Means with s.d. are presented; n=9-16. 
A few leafless and (almost) stemless orchids photosynthesize only via flower spikes, stems 
(leafless Vanilla, leafless Taeniophyllum) or exclusively via green roots (e.g. the essentially shootless 
Polyradicion: BENZING ET AL. 1983; ghost orchid (Polyrrhiza sp.); Microcoelia smithii: DVEKER & 
G. Aschan, H. Pfanz: Non-foliar photosynthesis and its contribution to the ... 9 
ARDITI! 1968; Chiloschista usneoides: CocKBURN ET AL. 1985). Such a strong reduction of the 
vegetative body is thought to enhance nutrient economy as an adaptation to extreme epiphytic life 
(BENZING & OTI 1981). Besides an evident refixation of respiratory C0
2
, the remaining green non-
foliar organs obviously have to fulfil autotrophic functions. 
Concluding remarks 
Higher flowering plants could potentially use almost all vegetative and reproductive organs to 
perform photosynthetic C0
2 
assimilation, 
non-foliar photosynthesis is either manifested as positive net photosynthesis, as demonstrated 
here for green flowers, or intemal C0
2 
refixation, typically found in chlorophyllous roots, 
green flowers or other photosynthetic parts associated with the inflorescence frequently conduct 
net photosynthetic C0
2 
assimilation comparable to the leaves and thus may supply a significant 
fraction of the total carbon and energy costs of reproduction, 
aerial roots of orchids perform an efficient intemal C0
2 
recycling, compensating for up to two 
third of the respiratory released C0
2 
and reaching electron transport rates about 60 % of the 
respective leaves. 
Literature 
ANTLFINGER A. E. & L. F. WENDEL 1997: Reproductive effort and floral photosynthesis in Spiranthes 
cernua (Orchidaceae). Am. J. Bot. 84: 769-780. 
AscHAN G. & H. PFANZ 2003: Non- foliar photosynthesis - a strategy of additional carbon acquisition. 
Flora 198: 81-97. 
BAZZAZ F. A., R. W. CARLSON & J. L. HARPER 1979: Contribution to reproductive effort by 
photosynthesis of flowers and fruits. Nature 279: 554-555. 
BENZING D. H. & D. W. OTI 1981: Vegetative reduction in epiphytic Bromeliaceae and Orchidaceae: 
its origin and significance. Biotropica 13 (2): 131-140. 
BENZING D. H. & W. T. PocKMAN 1989: Why do nonfoliar green organs of leafy orchids fail to 
exhibit net photosynthesis? Lindleyana 4: 53-60. 
BENZING D. H., W. E. FRIEDMAN, G. PETERSON & A. RENFROW 1983: Shootlessness, velamentous roots, 
and the pre-eminence of Orchidaceae in the epiphytic biotope. Am. J. Bot. 70: 121-133. 
BLANKE M. M. & F. LENZ 1989: Fruit photosynthesis - a review. Plant Cell Environ. 12: 31-46. 
CLEMENT C., P .. M1scHLER, M. BURRUS & J. C. AuDRAN 1997 A: Characteristics of the photosynthetic 
apparatus and C0
2
-fixation in the flower bud of Lilium. I. Corolla. Int. J. Plant Sci. 158: 
794-800. 
CLEMENT C., P. M1scHLER, M. BURRUS & J. C. AuDRAN 1997B: Characteristics of the photosynthetic 
apparatus and C0
2
-fixation in the flower bud of Lilium. II. Anther. Int. J. Plant Sci. 158: 
801-810. 
CocKBURN W., GoH C. J. & P. N. AvADHANI 1985: Photosynthetic carbon assimilation in a shootless 
orchid, Chiloschista usneoides (DON) LDL. Plant Physiol. 77: 83-86. 
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2
-fixation by green Cymbidium (Orchidaceae) 
flowers. Plant Physiol. 43: 130-132. 
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HEILMEIER H . & D. M . WHALE 1987: Carbon dioxide assimilation in the flowerhead of Arctium. 
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HOGAN K. P., M. B. GARCIA, J. M. CHEESEMAN & M . D. LovELESS 1998: Inflorescence photosynthesis 
and investment in reproduction in the dioecious species Aciphylla glaucescens (Apiaceae ). 
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the overall carbon gain. An eco-pysiological and ecological approach. Progress in Botany 
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357-364. 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2 : 11 - 15 
Sprejeto (accepted): 2003-10-30 
Investigation of Plant Surfaces with Environmental Scanning Electron 
Microscopy (ESEM®) - A Comparison with Conventional SEM 
Dagmar KOLB, Edith STABENTHEINER* 
Institute of Plant Physiology, Karl - Franzens University Graz, SchubertstraBe 51, 
A-8010 Graz, Austria 
Introduction 
*corresponding author (e-mail: edith.stabentheiner@uni-graz.at) 
Abstract. Environmental scanning electron microscopy (ESEM) enables the 
investigation of untreated and watercontaining material without preparation with 
the benefit of SEM (depth of focus and three dimensional imaging of surfaces with 
a high resolution). Conventional SEM (CSEM) usually requires tirne consuming 
fixation, drying and coating of samples. Their surlace structures may be altered by 
this procedure. For comparison a large number of plant samples was observed with 
both methods. Using CSEM, secretion products or mucilagineous coatings may be 
removed and dynamic processes cannot be observed. However, the samples can be 
investigated several times. In contrast, ESEM allows the observation of 
watercontaining, native surfaces and this method is the only possibility to watch 
dynamic processes in the SEM. However, using ESEM the plant material is very 
sensitive to beam damages because of the lack of the protecting metall layer -
necessary for non-conducting surlaces in CSEM and dehydration cannot be prevented 
completely. In summary, ESEM will not compete with CSEM but it will establish 
oneself as a valuable and essential supplement in studying plant surlaces. 
Key words: environmental scanning electron microscopy (ESEM), conventional 
scanning electron m.icroscopy (CSEM), plant surlaces 
Scanning electron microscopy (SEM) became an indispensable tool in studying plant surlaces. 
For conventional SEM (CSEM) biological samples usually have to be fixed, dehydrated and coated 
(ROBINSON & al. 1987, DYKSTRA 1992). 
The environmental SEM (ESEM) allows the observation of many types of specimens without 
subjecting them to conventional preparation techniques (DANILATOS 1993). This is possible because 
of a pressure limiting aperture with high vacuum maintained in the beam-generating and- focusing 
part of the column (- 104 Pa in the gun area), while low vacuum (up to 102- 103 Pa) is tolerated in the 
specimen chamber (BozzoLA & RussEL 1992, DYKSTRA 1992, DANILATOS 1993). The secondary 
electrons em.itted from the sample collide with water molecules in the chamber so as to produce 
additional electrons and positive ions. The positive ions are attracted to the sample surlace and 
eliminate charging artefacts. This ionization process results in a proportional cascade amplification 
12 Acta Biologica Slovenica, 46 (2), 2003 
of the original SE signal which is detected by a special gaseous secondary electron detector (DANILATOS 
1993, TAr & TANG 2001). Asa consequence, unfixed and uncoated samples - even those containing 
considerable amount of water - can be investigated by SEM. 
To compare CSEM and ESEM various plant samples were investigated on the one hand after 
fixation, drying and coating and on the other hand without any preparation at differing ESEM 
conditions. 
Material and Methods 
A large number of different plant samples was investigated using the following microscope 
conditions (KOLB 2002): 
CSEM: conventional SEM with high vacuum (- 10·4 Pa) in the chamber; sample preparation: 
chemical fixation (e.g., glutaraldehyde), dehydration, critical point drying with CO
2 
as drying agent, 
sputtercoating with gold (ROBINSON & al. 1987). 
ESEM: a) sample temperature 5°C (Peltier cooling stage); gaseous secondary electron detector; 
chamber pressure 133-930 Pa; relative humidity: up to 100 % (Table l); no sample preparation. b) 
samples at room temperature (without cooling); gaseous secondary electron detector; chamber 
pressure 133-670 Pa; relative humidity < 20 % (Table l); no sample preparation. c) large field 
gaseous secondary electron detector (pressure limiting aperture with wider diameter than a) and b); 
pressure in the charnber maximally 133 Pa; relative humidity < 1 O % (Table 1 ); no sample preparation. 
All samples (CSEM, ESEM) were mounted on aluminium stubs with double sided conductive 
tape and were investigated at different microscope conditions with a Philips XL30 ESEM using an 
acceleration voltage of 20 kV. 
Table 1: Values show chamber pressure in Pa, corresponding relative humidity (%) at sample 
temperatures (0 C) from 0° to room temperature (25°C). 
RH 100 % 80 % 60 % 40 % 20 % 10 % 
Temperature 
oo 612 480 360 239 120 67 
50 865 692 519 346 173 93 
100 1224 971 732 492 239 120 
15° 1702 1357 1024 678 346 173 
20° 2328 1862 1397 931 466 239 
25° 3152 2527 1889 1264 625 319 
Results and Discussion 
The environmental scanning electron microscope (ESEM) allows the observation of many types 
of specimens without subjecting them to conventional preparation techniques because it allows the 
introduction of a gaseous environment in the specimen chamber (DANILATOS 1993, TAr & TANG 
2001) . To compare CSEM and ESEM a great many different plant samples were investigated on the 
one hand after fixation, drying and coating and on the other hand without any preparati on at differing 
ESEM conditions. 
All samples - without any restrictions - could be investigated with conventional SEM (CSEM) 
using chemical fixation followed by dehydration, drying and coating procedure (Figs. 1-3). Leaves 
and shoots of plants are easy to handle during preparation. Very small samples, e.g. unicellular 
algae (Fig. 1, Micrasterias sp.) were attached to cover slips with poly-L-lysine prior to the preparati on 
procedure (ROBINSON & al. 1987). Generally, no artefacts due to beam damage or due to insufficient 
D. Kolb, E. Stabentheiner: Investigation of Plant Surfaces with Environmental Scanning .. . 13 
electrical (charging effects) and/or thermal conductivity occurred. Samples can be stored for a long 
tirne under appropriate conditions (dry and clean atmosphere) and can be investigated as often as 
necessary (CRANG 1988, DYKSTRA 1992). 
14 Acta Biologica Slovenica, 46 (2), 2003 
Figs. 1-3 CSEM samples. Fig. 1: Micrasterias sp. bar= 50 µm; Fig. 2: a stigma of Setcreasea 
purpurea with its stigmatic hairs bar = 200 µm; Fig.3: a preparation artefact of a calcareous crust 
surface of Saxifraga kolenitiana bar= 10 µm; Figs. 4--10 ESEM samples. Fig. 4: a pollen grain 
interaction with stigmatic hairs bar= 50 µrn; Fig. 5: a turgeszent trichome of Lycopersicon esculentum 
bar= 20 µm; Fig. 6: a native state of Micrasterias sp. bar= 50 µm ; Fig.7: a stigma of Setcreasea 
purpurea with mucus bar == 100 µm; Fig. 8: tbe native state of a calcereous crust of Saxifraga 
kolenitiana bar = 20 µm; Fig. 9: water droplets on the surface of Drosera rotundifolia bar== 100 µm; 
Fig.10: an example of surface debydration and cbarging effects of Melissa officinale bar== 20 µm. 
In contrast, ESEM allows tbe observation of biological samples in tbeir natura! state and witbout 
coating (Figs. 4--8; DANILATOS 1993, TAI & TANG 2001, Y AXLEY & al. 2001). Typically, the first steps 
in sample preparation for CSEM are fixation and dehydration (ROBINSON & al. 1987). However, 
these steps often result in a removal of surface coatings (CRANG 1988). In Fig. 7 tbe mucilaginous 
coating on a stigma of Setcreasea purpurea can be observed investigating fresh samples (ESEM), 
whereas this coating is removed after preparation for CSEM (Fig. 2). However, the stigmatic hairs 
are hidden on the fresh surface and can only be investigated in detail after sample preparation. So 
both methods complement one another. A well known artefact due to dehydration is sbrinkage of up 
to 40 % ofthe original volume (CRANG 1988). This can be clearly demonstrated bere wben comparing 
the sample with (Fig. 2, bar== 200 µm) and without (Fig. 7; bar== 100 µm) preparation. Only ESEM 
allows the investigation of tbe close interactions between pollen grains and stigmatic hairs of Hibiscus 
sp. (Fig. 4). 
Besides the removal ofcoatings surface deposits can be modified. In Fig. 8 the native state of 
tbe calcareous crust on leaves of Saxifraga kolenitiana can be observed wbile in Fig. 3 tbe structure 
of tbe crust was altered due to sample preparati on. 
Investigating biological samples containing a considerable amount of water a cooling stage 
belps to control the temperature of tbe specimen and thus the relative humidity, whicb is a strong 
function of the temperature (Table 1; DANILATOS 1993). Cooling enables tbe maintenance of a bigb 
relative bumidity on the sample surface (Table 1). As a consequence debydration is prevented and 
even delicate plant structures can be observed in tbeir native state for up to 60 minutes (Figs. 4--8). 
Different types of plant hairs can be easily observed without any preparation (Fig. 5). Even very 
sensitive algae as Micrasterias sp. (Fig. 6) can be investigated when sufficient water supply from a 
wet filter paper or agar is ensured. However, care bas to be taken that cbamber conditions are controlled 
in a way tbat bumidity on tbe sample surface is bigb enougb to stop dehydration but not too bigb to 
produce water droplets on tbe surface making it invisible (Fig. 9). The following conditions tumed 
out to be optimal for the investigation of wet samples: 5°C sample temperature and approximately 
640 Pa vapour pressure. Tbese conditions are very sirnilar to those found by TAI & TANG 2001. The 
possibility to control relative bumidity enables tbe direct investigation of dynamic processes on 
plant surfaces, e.g. debydration and rebydration cycles as it was done investigating the swelling 
bebaviour of cellulose fibres (JENKINS & D0NALD 1997). 
However, if not undisturbed samples ( e.g., wbole leaves) but sliced samples are used dehydration 
can not be completely prevented. Shrinkage and cbarging are tbe consequence (Fig. 10). 
ESEM witbout cooling results in quite low relative bumidities on the sample surface (Table 1). 
Many plant structures, especially tbose equipped with thick celi walls and cuticles, can be investigated 
in tbis mode giving tbe same results as cooling tbe samples. Investigation tirne, bowever, is mucb 
shorter and debydration occurs much faster. 
In contrast to CSEM, wet samples can only be used once - for further investigations a new 
sample is necessary. Tbe samples are more easily damaged by beam current and accelerating voltage 
since no coating is present that ensures sufficient tbermal conductivity and stabilization of the surface 
(CRANG 1988). Anotber disadvantage of ESEM is a reduced field of view at lower magnifications 
D. Kolb, E. Stabentheiner: Investigation of Plant Surfaces with Environmental Scanning ... 15 
due to the pressure limiting aperture (Fig. 9). This can be overcome using the large field gaseous 
secondary electron detector (FEI). However, due to the wider diameter of the aperture the maximal 
chamber pressure is restricted to 133 Pa and asa consequence the relative humidity on the sample 
surface is rather low (Table 1). So the same restrictions for fresh samples as stated above goes for 
this mode of ESEM. The mentioned restrictions are not applied to dry and stable samples (e.g., 
wood, insects). 
Conclusions 
ESEM represents a step forward in the instrumentation of electron microscopy and it allows 
access to areas of research not previously possible (DANILATos 1993). However, it will not compete 
with CSEM but it will establish oneself as a valuable and essential supplement in studying plant 
surfaces. 
Literature 
BozzoLA J. J. & L. D. RussEL 1992: Electron Microscopy. Jones and Bartlett Publishers, Boston-
London. 
CRANG R.F.E. 1988: Artifacts in specimen preparation for scanning electron microscopy. In: Crang 
R. F. E. & K. L. Klomparens (eds.): Artifacts in Biological Electron Microscopy, Plenum 
Press, New York - London pp. 107-129. 
DANILATOS G. D. 1993: Introduction to the ESEM instrument. Microscopy research and Technique 
25: 54-361. 
DYKSTRA M. J. 1992: Biological Electron Microscopy. Plenum Press, New York-London. 
KoLB D. 2002: Untersuchungen an frischem und fixiertem Pflanzenmaterial mit Hilfe des 
Rasterelektronenmikroskops, Diplomarbeit, Karl - Franzens - Universitat Graz. 
JENKINS L. M. & A. M. DoNALD 1997: Use ofthe Environmental Scanning Electron Microscope for the 
Observation of the Swelling Behaviour of Cellulosic Fibres, Scanning 19: 92-97. 
ROBINSON D. G., U. EHLERS, R. HERKEN, B. HERRMANN, F. MAYER &F.-W. ScH0RMANN 1987: Methodes 
of Preparation for Electron Microscopy, An Introduction for the Biomedical Sciences, 
Springer - Verlag Berlin Heidelberg New York. 
TAI S. S. W. & X. M. TANG 2001: Manipulating Biological Samples for Environmental Scanning 
Electron Microscopy Oberservation. Scanning 23: 267-272. 
YAXLEY J. L., W. JABLONSKI, J.B. Rmo. 2001: Leaf and flower development in pea (Pisum sativum 
L.): mutants cochleata and unifoliata. Annals of Botany 88: 225-234. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2 : 17 - 20 
Sprejeto (accepted): 2003-10-30 
Inter- and intracellular distribution of modulated glutathione in 
plant tissues 
Maria MULLER, Bernd ZECHMANN, Andreja URBANEK & Gtinther ZELLNIG 
Institute of Plant Physiology, University of Graz, SchubertstraBe 51, 8010 Graz, Austria 
e-mail: maria.mueller@uni-graz.at 
Introduction 
Abstract. In the present study we want to summarize <lata dealing with the inter-
and intracellular distribution of glutathione in different plant tissues under various 
environmental conditions. Monochlorobimane fluorescence of reduced glutathione 
is described for light microscopical investigations and allows distinguishing between 
glutathione in cytoplasm and in the nucleus. Further, a specific antibody, which 
recognizes both the reduced and oxidized form of glutathione, was used in order to 
demonstrate glutathione in different cell compartments. The labelling showed 
different glutathione concentrations in the cell compartments with a high staining 
intensity in mitochondria. 
Keywords: Glutathione, immunolabelling, monochlorobimane, plant tissues, 
ultrastructure 
Glutathione (GSH) is considered to have a broad spectrum of functions in plants. It is a cellular 
protectant, signal substance and a major reservoir of non-protein reduced sulphur in plants. 
Glutathione is present in all organs, with different concentrations in the organs or within the same 
organ at different developmental stages (FOYER & RENNENBERG 2000), but thus far little is known 
about the GSH distribution in cellular compartments. Intercompartmental variations in glutathione 
concentration are supposed to be crucial in signalling processes. NocroR & al. (2002) discuss varying 
concentrations of glutathione in chloroplasts, which are reported between 8 % and 50 % of the leaf 
glutathione depending on the isolation media and the methods. 
Our study presents an overview of a histochemical and an immunocytochernical method for the 
determination of inter- and intracellular glutathione contents in plant tissues. 
Material and methods 
Plant material 
Epidermal cells of Allium cepa L. as well as leaves of Cucurbita pepo L. were used as described 
elsewhere (MDLLER & al. 1999 a, 2002 b) . 
18 Acta Biologica Slovenica, 46 (2), 2003 
Monochlorobimane (BmCl) labelling of thiols (modified according to MOLLER & al. 1999 a, 
2002 a). 
A stock solution of BmCl (Molecular Probes) was diluted in phosphate buffer, pH 7 .2, 
immediately prior to use to obtain the required concentration of 50 mM BmCl. All plant material 
was exposed to BmCI from 5 to 20 minutes (depending on the used material). After removing the 
staining solution, the samples were washed with buffer to remove excess staining solution. Single 
cells and single cell layers were investigated immediately under the fluorescence microscope. 
Immunogold-analysis (modified according to M0LLER & al. 2002 b) 
Small sections of the plant material were fixed according to a standard fixation protocol (cf. 
ZELLNIG & al. 2000) or in 0.5 % glutaraldehyde/2.5 % paraformaldehyde, dehydrated in a graded 
series of ethanol and embedded in LR-White resin. Ultrathin sections were incubated with an anti-
glutathione rabbit polyclonal lgG (Signature Immunologics, lnc.) followed by a 10 nm gold-labelled 
secondary antibody (British Biocell lnt.). The antiserum was shown to react selectively with 
glutathione, although it does not differentiate between the reduced and oxidized forms of glutathione. 
To confirm the selectivity of the immunolabelling in the present set of experiments, the tissue sections 
were either incubated with the primary antibody, which was exposed to 5 mM of GSH prior to the 
application, followed by the secondary antibody or treated with the secondary antibody alone. Both 
treatments resulted in a negative immunolabelling. 
Results and discussion 
The technique of histochemical glutathione tracing by monochlorobimane (BmCl) in fluorescence 
microscopy was developed for single cell layers (Allium epidermis - M0LLER & al. 1999 b) and for 
suspension ce]l cultures (root protoplasts of Allium - M0LLER & al. 1999 a). The method relies on 
conjugation of BmCl to GSH within intact tissues resulting in a fluorescent GS-bimane conjugate 
(Fig. 1). The GS-bimane fluorescence levels are well known as an indicator of GSH in living 
marnmalian tissues and are also described for plants (FRICKER & al. 2000, MEYER & FRICKER 2000, 
MEYER & al. 2001, SANCHEz-FERNANDEZ & al. 1997). A photoactivation of fluorescence from non-
conj11gated BmCI after UV excitation, as previously mentioned by FRICKER & al. (2000) was not 
observed. Further data dealed with the manipulation of the intensity of the fluorescence that is 
observed after modification of the glutathione status of the cells, either by application of oxidants 
(M0LLER & al. 1999 c) or by inhibiting glutathione synthesis by buthionine sulphoximine (M0LLER 
& al. 1999 a). For the tissue- and subcellular imaging of glutathione concentrations in sections of 
intact root tissues (Brassica oleracea), where the glutathione contents were manipulated by H
2
S 
fumigation, the staining procedure had to be adjusted (MOLLER & al. 2002 b). In the roots of Brassica, 
a sulphur demanding plant, the H
2
S treatment resulted in an increased fluorescence in the meristem 
cells, implying an enhanced glutathione content in the cytoplasm as well as in the nucleoplasm. 
These results showed the sensitivity of this method, making it possible to distinguish in the glutathione 
contents not only between different tissues but also in different cell compartments at the light 
microscopic level. 
With this background, it was of interest to collect more precise data on GSH localization and 
GSH concentrations in the cells by using transmission electron microscopy. The subcellular 
localization of glutathione was investigated with an indirect immunogold-labelling method. On all 
investigated cell sections gold particles were present after the immunoreaction, but the labelling 
intensity varied over a wide range between the compartments. Very low intensities of gold labelling 
were found in peroxisomes and the ER; high intensities could be detected in the mitochondria (Fig. 
2). The density of gold particles in chloroplasts and especially in the nuclei was lower (Fig. 2) than 
expected from either biochemical data (NocroR & al. 2002) or from staining with bimane derivates 
(M0LLER & al. 2002 b). 
M. Mtiller, B. Zechmann, A. Urbanek & G. Zellnig: Inter- and intracellular distribution of modulated... 19 
p 
cw 
Figure 2: Part of a mesophyll cell of Cucurbita pepo L. Electron micrograph showing the distribution 
of GSH in various compartments after immunolabelling; gold particles are present in the chloroplast 
(P), the nucleus (N) and in an increased number in the mitochondria (M). Bar= 1 mm. 
Conclusions 
We can conclude that with the use of the above-described methods it is possible to obtain data 
about glutathione concentrations in different compartments by light and electron rnicroscopy. But 
there is stili a need of quantification analyses of the total pool of glutathione and till now no systematic 
investigations ofthe subcellular expression of glutathione, and the distribution ofy-glutamylcysteine, 
the immediate precursor of glutathione, were done with plant tissues. Therefore the investigations 
have to be continued and intensified in this field. 
20 Acta Biologica Slovenica, 46 (2), 2003 
Literature 
FRICKER M. D., M. MAY, A. J. MEYER, N. SHEARD, & N. S. WHITE 2000: Measurement of glutathione 
levels in intact roots of Arabidopsis. J. Microsc. 198: 162-173. 
FoYER C. H. & H. RENNENBERG 2000: Regulation of glutathione synthesis and its role in abiotic and 
biotic stress defence. In: Brunold C., H. Rennenberg, L.J. De Kok, I. Stulen, J. C. Davidian 
(eds.): Sulfur Nutrition and Sulfur Assimilation in Higher Plants, Paul Haupt Publishers, 
Berne, pp. 127-153. 
MEYER A. J. & M. D. FRICKER 2000: Direct measurement of glutathione in epidermal cells of intact 
Arabidopsis roots by two photon laser scanning microscopy. J. Microsc. 198: 174-181. 
MEYER A. J., M. J. MAY & M. FRICKER 2001: Quantitative in vivo measurement of glutathione in 
Arabidopsis cells. Plant J. 27: 67-78. 
M0LLER M., M. TAusz & D. GRILL 1999A: Histochemical tracing of glutathione by fluorescence 
microscopy and image analysis system in living plant cells. In: DENKE A., K. DORNISCH, F. 
FLEISCHMANN, J. GRABMANN, I. HEISER, S. HIPPELI, W. OBwALD & H. ScHEMPF ( eds.): Different 
Pathways through Life - Biochemical Aspects of Plant Biology and Medicine, pp. 189-
197. 
M0LLER M., M. T AUSZ, H. GurrENBERGER & D. GRILL l 999B: Histochemical localization of glutathione 
in plant tissues: A comparison oftwo fluorescence staining methods. Phyton (Horn, Austria) 
39: 69-74. 
M0LLER M., M . TAusz, A. WoNISCH & D. GRILL 1999c: Effects of an oxidizing agent (hydrogen 
peroxide) on the glutathione system in epidermal cells of Allium cepa L. investigated by 
histochemical staining. Free Rad. Res. 31: 121-127. 
MDLLER M., L.J. DE KoK, W. WEIDNER &_ M. TAusz 2002A: Differential effects of H
2
S on cytoplasmic 
and nuclear thiol concentrations in different tissues of Brassica roots. Plant Physiol. 
Biochem. 40: 585-589. 
MDLLER M., B. ZECHMANN, M. TAusz & G. ZELLNIG 2002B: Subcellular distribution of glutathione -
a high resolution immunogold analysis in leaves of pumpkin (Cucurbita pepo L.). In: 
Davidian J. C., D. Grili, L.J. De Kok, I. Stulen, M. J. Hawkesford, E. Schnug, & H. 
Rennenberg (eds.) : Sulfur transport and assimilation in plants - Regulation, interaction 
and signalling, Backhuys Publishers, Leiden, pp.295-297. 
NocTOR G., L. GOMEZ, H. VANACKER & C. H. FOYER 2002: Interactions between biosynthesis, 
compartmentation and transport in the control of glutathione homeostasis and signalling. 
J. Exp. Bot. 53: 1283-1304. 
SANCHEZ-FERANDEZ R., M. D. FRICKER, L. B. CoRBEN, N. S. WHITE, N. SHEARD, C. J. LEAVER 1997: 
Celi proliferation and hair tip growth in the Arabidopsis root are under mechanistically 
different forms of redox control. Proc. Natl. Acad. Sci. USA 94: 2745-2750. 
ZELLNIG G., M . TAusz, B. PEŠEC, D. GRILL & M . M-OLLER 2000: Structural and ultrastructural changes 
in root cells of spruce trees (Picea abies (L.) Karst.) caused by exogenously applied 
glutathione. Protoplasma 212: 227-235. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2: 21 - 28 
Sprejeto (accepted): 2003-10-30 
Ozone biomonitoring using tobacco, Nicotiana tabacum "BelW3" 
Edith STABENTHEINER1*, Andrea GROSS', Gerhard SOJA2, Dieter GRILU 
1Institute of Plant Physiology, Karl- Franzens University Graz, SchubertstraBe 51 
A-8010 Graz, Austria 
Introduction 
2Forschungszentrum Seibersdorf, A-2444 Seibersdorf 
*corresponding author (e-mail: edith.stabentheiner@uni-graz.at) 
Abstract. The ozone sensitive tobacco cultivar BelW3 (Nicotiana tabacum L.) 
was used to monitor ozone pollution for one vegetation period (beginning of May 
till end of September) in Graz, a city of approximately 240 000 inhabitants in the 
south of Austria. The plants were exposed for 14 days in standardized shaded 
exposition racks. The insensitive tobacco cultivar BelB was used as a control. The 
leaf necroses characteristic for ozone damage were well correlated with ozone dose 
(AOT 40) with the exception of the first exposition period in May. The reliability of 
the evaluation of visible symptoms on tobacco leaves was confirmed by computer-
aided image analysis. It was possible to identify the spatial and temporal ozone 
distribution within a project area. 
Key words: Ozone biomonitoring, Nicotiana tabacum "BelW3", leaf damage, 
AOT40 
Bioindication perrnits a qualitative and quantitative assessment of the effect of anthropogenic 
and natura! influences using suitable indicator organisms which react to environmental influences 
by changing their vital functions and/or chernical composition (VDI 1999). In the case of air-quality 
control the use of bioindicator plants is an appropriate means to detect and monitor air pollution 
effects (KLUMPP & al. 2002). 
The ozone sensitive tobacco cultivar BelW3 produces characteristic lesions as a response to 
ozone exposure (HEGGESTAD 1991, KRUPA & al. 1993, Noucm 2002) and this visible foliar response 
is widely used in ozone monitoring projects (e.g., ARNDT & al. 1987, HEGGESTAD 1991, KLuMPP & al. 
2002, Noucm 2002). 
A monitoring study with plants as bioindicators was perforrned in Graz, a city of approximately 
240 000 inhabitants in the south of Austria. The results of monitoring ozone using tobacco, Nicotiana 
tabacum "BelW3", are presented here. Apart from gathering information on air quality in different 
areas of the city the reliaqility of the visual estimation and the dependence of leaf damage on the 
ozone dose was investigated. 
22 Acta Biologica Slovenica, 46 (2), 2003 
Materials and Methods 
Bioindicator plants were exposed 1996 on 11 sites in the city area of Graz (Fig. 1). The first 
exposition started 711t of May and the last exposition ended 24th of September. 
The cultivar BelW3 was used as an ozone sensitive indicator. Tobacco cultivar BelB, an ozone 
tolerant variety, was used as a control. 6-8 weeks old plants were exposed on shaded and slightly 
elevated exposition racks in self watering assemblies for 14 days (ARNDT & al. 1987, VDI 2000, 
GROSS 2001). Then the percentage of damaged leave area was visually estimated and classified into 
damage classes (Tab. 1). The damage class of one plant resulted from adding up the damage classes 
of all leaves (average 8 leaves) and dividing by the number ofleaves. The mean damage class of one 
site was the mean of all exposed plants (2-4). 
On 4 sites (sites 1, 3, 5, 6) continuous measurements of ozone concentrations (half hour average 
means) were available and were used to determine the AOT 40 value (ppb.h; 00:00-24:00). 
The reliability of the evaluation of visible symptoms on tobacco leaves was confirmed by 
computer-aided image analysis (Optimas 5.2). Percentage of injury was visually estimated and the 
leaves classified into damage classes (Tab. 1). Then the leaves were scanned, analysed and also 
classified into damage classes. 
Other bioindicators (Phaseolus vulgaris L., Trifolium subterraneum L., Medicago sativa L. and 
Lolium multiflorum Lam.) were also exposed to monitor the effect and the distribution on NOx, SO
2 
and the accumulation of heavy metals (DuGAUQUIER & al. 1997, GRoss 2001, STABENTHEINER & al. 
2002). 
This presentation only focuses on monitoring ozone. 
Table 1: Damage classes (Nicotiana tabacum 'BelW3 ' ) based on the percentage of foliar surface 
affected (according to ARNDT & al. 1987) 
Damage class Percentage affected leaf area 
o o 
0-2 
2 2-5 
3 5-10 
4 10-25 
5 25-60 
6 60-100 
E. Stabentheiner, A. Gross, G. Soja, D. Grili : Ozone Biomonitoring using tobacco ... 23 
Figure 1: Site map of Graz with the city limits, the river Mur crossing the city from north to south, 
the motorways and the 11 exposition sites 
Results and Discussion 
The leaves of tobacco, Nicotiana tabacum 'Be!W3' showed the typical leaf necroses due to 
ozone (HEGGESTAD 1991 , VDI 2000). The characteristic bifacial lesions were clearly defined areas 
on the leaves and were identical on the upper and the lower leaf surface. The lesions were characterized 
by a clear border between turgescent epidermal cells of the healthy parts of the leave and collapsed 
cells of the lesions (Fig. 2). They are due to lyses of parenchymatic cells and as a consequence both 
epidermal cell layers were close together (Gi.JNTHARDT-GOERG 1996). 
Distinct age dependence in the development of the lesions could be observed with only slight 
damage of the young leaves and more severe damage of the older leaves (Tab. 2). 
24 Acta Biologica Slovenica, 46 (2), 2003 
The reliability of the evaluation of visible symptoms on tobacco leaves was confirmed by 
computer-aided image analysis. Percentage of injury was visually estimated and classified. The 
leaves were than scanned and analysed using image analysis software, the results were also classified 
into damage classes. Visual estimation of leaf damage was sufficiently accurate (n = 73, Spearman 
R = 0.76, p < 0.001). Colour information was absolutely essential for correct threshold settings 
using the image analysis software. Using black and white images only visual estimation was not in 
good agreement with computer analysis (DELLA MEA & al. 1997). Visual estimation is much less 
tirne consuming and it will not be replaced by image analysis methods in the near future. However, 
for the purpose of standardization a uniform, simple and reliable method based on image analysis 
would be very welcome. 
Figure 2: SEM micrograph of a leaf of Nicotiana tabacum 'Be!W3'; characteristic lesion due to 
ozone; horizontal diameter of the lesion = 1.85 mm 
E. Stabentheiner, A. Gross, G. Soja, D. Grili: Ozone Biomonitoring using tobacco ... 25 
Table 2: Dcpcndencc of damagc c.:la s (Nicotiana rabacum · BelW3 ') on lhe leaf age: mean ± standard 
deviation; 1 = oldest leaf, 9 = youngest leaf. 
Age class n Damage class 
1 110 2.29 ± 1.32 
2 323 2.60 ± 1.26 
3 455 2.50 ± 1.40 
4 489 2.00 ± 1.43 
5 489 1.35 ± 1.43 
6 462 0.82 ± 1.15 
7 392 0.44 ± 0.83 
8 309 0.18 ± 0.50 
9 201 0.11 ± 0.39 
At sites 1, 3, 5 and 6 (Fig. 1) tobacco plants were exposed at sites where also continuous ozone 
measurements were performed. The mean damage class per site was correlated with the AOT 40 
value (ppb.h) of the exposition period ( 14 days) for all evaluation periods (Fig. 3). A high ozone 
dose corresponded with high leaf damage as was also reported from some other studies (KRUPA & al. 
1993, KosTKA-RrcK 2002). However, in spite of average ozone concentrations in beginning of May 
the leaves only showed below-average damage (box of 4 values in Fig. 3). Obviously, the reaction 
rnechanism in developing leaf necroses also depends on other factors and not only ozone 
concentrations. Vapour pressure deficit is one important environmental factor in modulating the 
ozone response (BENTON & al. 2000). 
During the whole exposition period the typical leaf necroses could be observed depending on 
the site. Highest damage on single leaves (damage class 6) occurred in August. The overall results 
for the whole exposition period can be seen in Tab. 3. 
At the same tirne tobacco plants were exposed in a slightly different methodological approach 
(exposition on the ground, exposure tirne: 4 weeks) within the scope of an international project with 
participation of eight European cities (DuoAUQUIER & al. 1997, STABENTHEINER & al. 2002). The 
lowest leaf damage due to ozone could be observed in the industrialized cities Lille (France) and 
Charleroi (Belgium) and highest leaf damage in the ltalian cities Modena, Bologna and Florenz. 
The results of Graz were comparable to that of Niirnberg (Germany) and Tampere (Finland) and 
ranged in the centerfield (DuGAUQUIER & al. 1997). 
26 Acta Biologica Slovenica, 46 (2), 2003 
Table 3: Damage class of tobacco varieties BelW3 and BelB of all sites (compare Fig. 1) for the 
whole exposition period (14-days exposure, beginning ofMay till end of September); mean ± standard 
deviation 
Site BelW3 Be!B 
1 1.44 ± 0.46 0.31 ± 0.23 
2 1.11 ± 0.57 0.22 ± 0.13 
3 1.53 ± 0.59 0.29 ± 0.20 
4 0.90 ± 0.39 0.32 ± 0.15 
5 1.05 ± 0.44 0.24 ± 0.12 
6 1.11 ± 0.47 0.15 ± 0.07 
7 1.20 ± 0.56 0.23 ± 0.14 
8 1.21 ± 0.36 0.27 ± 0.12 
9 1.07 ± 0.38 0.24 ± 0.11 
10 1.44 ± 0.62 0.30 ± 0.16 
11 1.08 ± o.so 0.21 ± 0.06 
3 r----------------~---------------, 
-; 
'i 2 
m - •• • • • 
• • 
• • 
------ • • 
• • 
• 
• 
. ------• • __:-----• 
--------• 
-------_____ ___,-
1 . . . . 
o ~I _.____-=--=--=--=--='---------
o 1000 2000 3000 4000 5000 6000 7000 
AOT40 (ppb.h) 
Figure 3: Scatter plotof AOT 40 values of ozone (ppb.h; 14 days) against damage class of Nicotiana 
tabacum 'Be!W3'; n = 33, Spearman R = 0.422, p = 0.014; the marked box represents the mean 
damage class values of the 4 sites for the first exposition period beginning middle of May; correlation 
without these values: n = 29, Spearman R = 0.51, p = 0.005. 
E. Stabentheiner, A. Gross, G. Soja, D. GriJI: Ozone Biomonitoring using tobacco ... 27 
Though the differences between the exposition sites in the city area are not very distinct (Tab. 3) 
it was possible to identify areas differing in ozone-depending darnage. The highest damage class 
could be observed at sites on the elevated outskirts in the east (10) and northeast (3) of Graz. The 
plants on the SchloBberg (1), an elevated site near the city centre also showed high leaf darnages. 
The sites with the lowest leaf damage due to ozone (4, 5, and 9) were situated near main traffic 
routes. The darnage on leaves of Be!B was always very low and no distinct site differences could be 
observed. 
Conclusions 
The tobacco cultivar Be!W3 is a sensitive and reliable bioindicator for ozone. Though the indicator 
is very sensible its reaction is representative also for the reaction of native plants (BuNGENER & al. 
1999). It is possible to identify the spatial and temporal ozone distribution within a project area. A 
strict compliance with guidelines (e.g., VDI 2000) and the further development of standardized 
evaluation methods using computer-aided image analysis will increase the acceptance at a national 
and international leve!. 
Acknowledgements 
The help of the following people and public sectors is gratefully acknowledged: municipal 
authorities of Graz, Referat fiir Luftgtiteiiber-wachung, Miele Company and Hr. Tiichler (permission 
of indicator exposure), Dr. Guttenberger (help with image-analysis). Part of the work was supported 
by the European Commission. 
Literature 
ARNDT U., W. NOBEL & B. SCHWEIZER 1987: Bioindikatoren: Mčiglichkeiten, Grenzen und neue 
Erkenntnisse. Ulmer Verlag Stuttgart. 
BENTON J., J. FUHRER, B.S. GrMENO, L. SKARBY, D. PALMER-BR0WN, G. BALL, C. RoADKNIGHT & G. 
MrLLS 2000: An international cooperative programme indicates the widespread occurrence 
of ozone injury on crops. Agriculture, Ecosystems and Environment 78: 19-30. 
BuNGENER P., G.R. BALLS, S. NussBAUM, M. GEISSMANN, A. GRuB & J. FuHRER 1999: Leaf injury 
characteristics of grassland species exposed to ozone in relation to soil moisture and vapour 
pressure deficit. New Phytologist 142: 271-282. 
DELLA MEA M., G.L. CALZONI & N. BAGNI 1997: Evaluation of ozone injury in Nicotiana tabacum cv. 
Bel-W3 with computerised image analysis. Fresenius Environmental Bulletin 6: 475-480. 
DuoAUQUIER F., R. IMPENS, R. PAUL & E. DELCARTE 1997: Bio-indication of air quality in urban areas 
- European Pilot Project. Fina! Report, Synthesis, October 1997; Coordination S.A. Agrer. 
GRoss A. 2001: BIOINDIKATION IN GRAZ 1996: Diploma work, University of Graz. 
G0NTHARDT-GOERG M. 1996: Different response of ozone to tobacco, poplar, birch and alder. Journal 
of Plant Physiology 148: 207-214. 
HEGGESTAD H.E. 1991: Origin ofBel W3, Bel C and Bel B tobacco varieties and theiruse as Indicators 
of ozone. Environrnental Pollution 74: 264-291. 
KLUMPP A., G. KLuMPP, W. ANSEL & A. F0MIN 2002: European network for assessment of air quality 
by the use of bioindicator plants - the first year of EuroBionet. In: Klumpp A. , A. Fonin, 
28 Acta Biologica Slovenica, 46 (2), 2003 
G. Klumpp & W. Ansel (eds): Bioindication and air quality in European cities - research, 
application, communication, Verlag Giinter Heimbach, Stuttgart, pp. 37-55. 
KosrKA-RrcK R. 2002: Ozone biomonitoring in a local network around an automotive plant. KLuMPP 
A. , A. FoNIN, G. KLuMPP & W. ANSEL (eds.): Bioindication and air quality in European 
cities - research, application, communication, Verlag Giinter Heimbach, Stuttgart, pp. 243-
248. 
KRUPA S.V., W.J. MANNING & M. NosAL 1993: Use of tobacco cultivars as biological indicator of 
ambient ozone pollution: An analyses of exposure-response relationship. Environmental 
Po!lution 81: 137-146. 
Novem I. 2002: Plants as bioindicators of air pollutants. In: OMASA K., H. SAJI, S. YoussEFIAN & N. 
KoNoo (eds.): Air pollution and plant biotechnology - prospects for phytomonitoring and 
phytoremediation. Springer Verlag, Tokyo, pp. 41-60. 
SrABENTHEINER E., A. GRoss, G. SOJA & D. GRILL 2002: Bewertung der Luftgiite in Graz mit Hilfe 
von Pflanzen als Bioindikatoren. Mitteilungen Naturwissenschaftlicher Verein Steiermark 
132: 181-193. 
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Luftverunreinigungen auf Pflanzen (Bioindikation) - Ermittlung und Beurteilung der 
phytotoxischen Wirkung von Ozon und anderen Photooxidantien - Verfahren der 
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Ingenieure. VDI/DIN-Handbuch Reinhaltung der Luft, Band la. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2: 29 - 35 
Sprejeto (accepted): 2003-10-30 
Organization of interphase microtubules and actin filaments in spruce 
callus cells after glutathione treatment 
Andreja URBANEK, Bernd ZECHMANN, Giinther ZELLNIG & Maria MULLER 
Institute of Plant Physiology, Karl-Franzens-University Graz, SchubertstraBe 51, 
8010 Graz, Austria. E-mail: andreja.urbanek@stud.uni-graz.at 
Introduction 
Abstract. Changes in the distribution of microtubules (MT) and actin filaments 
were examined in suspension-cultured spruce cells [Picea abies (L.) ~RST.] that 
were exposed to different concentrations (500 and 1000 µM) of exogenously applied 
reduced glutathione (GSH). Using fluorescence microscopy the MT were visualized 
with monoclonal anti-tubulin antibodies and actin filaments were stained with 
rhodamin labelled phalloidin (RLP). GSH-treated callus cells showed modifications 
on the form and arrangement of both cytoskeletal elements, when compared to the 
control. 
Keywords: Glutathione, spruce callus cells, microtubules, actin filaments 
Abbreviations: GSH reduced glutathione; MT microtubules, RLP rhodamin 
labelled phalloidin; DAPI 4', 6-Diamidino-2-phenyl-indol-dihydrochlorid; 
The plant cytoskeleton is a membrane-associated structure, which is essential in a variety of cell 
processes like celi growth and differentiation, celi division and cytoplasmatic streaming (VOLKMANN 
& BALUŠKA 1999, DAVIES 2001). Callus cells are commonly used for fundamental studies of the 
cytoskeieton, as they show dynamic changes in their form and arrangement in response to 
environmental perturbations (S!VAGURU & al. 1999, BARLOW & BALUŠKA 2000). 
GSH is a significant member of low molecular weight thiols and is ubiquitous to plants and 
animals (FoYER & RENNENBERG 2000). The influence of exogenously applied GSH on plant sulfur 
metabolism and its involvement in defence reactions were previously studied (WINGATE & al. 1988, 
SANCHEZ-FERNA.NDEZ & al. 1997, ZELLNIG & al. 2000, M0LLER & al. 2001). Positive effects on root 
growth through cell division rate were observed on Arabidopsis (SANCHEZ-FERNANDEZ & al. 1997). 
On the other hand, elevated concentrations of GSH in plant tissues are reported to be deleterious. 
Transformed tobacco lines with elevated GSH-biosynthesis capacity showed increased oxidative 
stress, stunted phenotypes and leafnecrosis (CREISSEN & al. 1999). Furthermore, a decrease in mitotic 
index, increased chromosomal aberrations and alterations in the ultrastructure were reported in 
different spruce tissues after GSH-treatment (ZELLNIG & al. 2000, MDLLER & al. 2001). 
30 Acta Biologica Slovenica, 46 (2), 2003 
To clarify, whether alterations of the cytoskeleton are responsible for the observed GSH-induced 
chromosomal aberrations, the effects of exogenously applied GSH on the arrangement of the MT 
and actin filaments were investigated in spruce callus cells during the interphase. 
Material and methods 
Plant material and GSH-treatment 
Callus cultures of Picea abies (L.) ~RST. were established from cotyledons by using solid MS 
medium (MURASHIGE & SKOOG 1962) with ingredients according to M0LLER & al. (2001) [3 % sucrose, 
1 % agar, 1-naphthalenacetic acid (3mg/I) and 6-benzylaminopurine (lmg/l), pH 5.8) . The callus 
cultures were cultivated in a growth chamber (21 °C, 12 h photoperiod, light intensities between 33 
and 43.7 µmol m·2s·1) and were sub-cultured on fresh MS medium every two weeks. 
For our experiments callus tissue was divided into small pieces (500 ± 50 mg fresh weight) and 
transferred to flasks containing 50 ml sterile liquid MS medium supplemented with the same hormone 
composition as described above and with either 500 µM GSH, 1000 µM GSH orno GSH (control). 
Four flasks of suspension culture were used for each treatment. The GSH treated material and 
controls were maintained for 48 hours under the same light and temperature regimen as the cultures 
on solid medium and remained unshaken to make sure that the GSH was not oxidized. This procedure 
was repeated five times and about 600 callus cells were recorded. 
Fluorescence microscopy 
Indirect immunofluorescent labelling of a.-tubulin: Control samples and GSH-treated material 
were processed according to the method described by W1cK & al. ( 1981) and APOSTOLAKOS & GALATIS 
(1999). Briefly, callus tissue was fixed in 4 % paraforrnaldehyde in a MT stabilising buffer (MSB: 
50 mM PIPES in 0.1 M KOH, 5 mM EGTA, 2 mM MgSO
4
, pH 6.8) for 45 minutes at room 
temperature (RT), washed in MSB, and digested in 2 % cellulase and 1 % macerozyme in MSB for 
45 minutes at RT. The samples were then washed in MSE for 30 minutes and extracted with 2 % 
Triton X-100 in phosphate-buffered saline (PBS, pH 7,3) for 120 minutes. After washing in PBS, 
squashed cells were incubated with the primary antibody (monoclonal mouse IgG 1 anti- a-tubulin 
clone B-5-1-2, Sigma) diluted 1:150 in PES and blocked in 1 % bovine serum albumine for 40 
minutes in a moist chamber at 37°C. The specimen were washed severa! times in PBS and incubated 
with the fluorochrom-labelled secondary antibody [alexa fluor 488 goat anti-mouse IgG (H+L) 
conjugate, Molecular Probes] diluted 1 :30 in PES for 60 minutes at 37°C in a moist chamber. After 
extensive washing in PBS, sections were mounted on slides in an antifade agent (Citifluor PBS 
solution AF3, Gropl). 
Staining of actin filaments: For actin filaments staining a modified method according to 
OLYSLAEGERS & VERBELEN (1998) was used. The plant material was incubated on slides in actin 
buffer (100 mM PIPES in 0.2 M KOH, 10 mM EGTA, 5 mM MgSO
4 
and 0.2 M mannitol, pH 6.9), 
containing 2 % glycerol and 66 nM RLP for 1 hour. 
Staining of nuclei: Additionally, after the labelling of MT and actin filaments selected material 
was stained by applying 5 ml of a DAPI solution (4', 6-Diamidino-2-phenyl-indol-dihydrochlorid, 
10 mg DAPl/ml distilled water) to 100 ml of corresponding buffer. 
A. Urbanek, B. Zechmann, G. Zellnig & M. Miiller: Organization of interphase microtubules and ... 31 
Equipment: A Zeiss Axioskop equipped with a 100 W mercury are lamp was used to obtain 
digital images with a 3-chip-colour video camera (Sony DXC 930 P with Sony-control-system), a 
frame grabber (ITI MFG-3M-V, Imaging Technology Inc., with variable scan module AM-CLR-VP 
and colour recording module AM-CLR-VP). Optimas 6.5.1 (BioScan Corp.) was used as image 
analysis software. Fluorescence images were obtained through a Plan-Neofluar 63x dry objective 
(n. a., 0.95) and a Plan-Apochromat l00x oil immersion objective (n. a., 1.4). 
MT were investigated with a 450--490 nm excitation and 520 nm emission filter block. Rhodamine 
labelled actin was visualised with a 546/12 nm excitation and 590 nm emission filter block. 
Chromosome labelling was obtained at 365/12 nm excitation and the fluorescence was imaged 
through a 397 long pass filter. 
Results 
In GSH-exposed callus cells alterations in the organization of interphase MT were observed. In 
ali callus cell types treated with 500 µM and 1000 µM GSH the cortical MT appeared as shortened 
and tortuous structures (Fig. 1 a). Additionally, MT showed severe alterations in form of tubulin-
positive dots in cortical cell areas and in association with the nuclei at both glutathione concentrations 
(Fig. 1 b). In contrast, such abnormal structures were not observed in control cells. They exhibited 
an intact cortical MT network characterized by arrays, which showed different organization depending 
on the cell form. In oblique callus cells with polar celi extension the MT were observed throughout 
the celi cortex arranged perpendicular to the cell growing axis and parallel to each other (Fig. 1 c). 
Whereas, disorganized cortical MT were observed in spherical callus cells of control material. In all 
types of control cells MT were also found extending from the nucleus towards the plasma membrane 
(Fig. 1 d). Furthermore, they formed a thick network around plastids connecting with other organelles 
and the cell membrane. 
Similar impacts of the GSH-treatment (500 µM and 1000 µM) were observed on cortical actin 
filaments of interphase cells as already described on MT. The actin filaments remained as small, 
tortuous fragments or bright fluorescent dots in the cell cortex. Mostly only a diffuse fluorescence 
was noticed. The transvacuolar actin cables could not be recognised. The actin baskets around the 
nuclei persisted, however, they were amorphous in appearance and only seldom observed as arrays 
that emerged from the nucleus to the plasma membrane. They ended blind in the cytoplasm (Fig. 1 
e). Only a diffuse fluorescence was seen around the plastids; an anchoring of plastids at the plasma 
membrane was lacking. On the other hand, control callus cells showed very well preserved cortical 
actin filaments (Fig. 1 f). Also transvacuolar actin cables and actin arrangements in the association 
with nucleus and organelles were clearly evident within control cells. 
32 Acta Biologica Slovenica, 46 (2), 2003 
Figure 1: (a-d) Arrangement of MT in GSH-treated (1000 µM GSH) spruce callus cells (a, b) and 
control callus cells (c, d) during interphase. (a) Shortened and tortuous MT (arrow) observed in the 
cell cortex of GSH-treated callus cells. (b) Cells with tubulin-positive dots in cortical cell area and 
in association with the nucleus (arrow). (c) A cortical MT network with arrays arranged parallel to 
each other and perpendicular to the cell-growing axis. (d) MT radiation from the nucleus (arrow) 
towards the plasma membrane in oblong callus cell. 
Distribution of actin filaments in GSH-treated (1000 µM GSH) callus cells (e) and in control callus 
celi (f). (e) Amorphous actin-positive fluorescence was noticed in association with nucleus (arrow) 
(staining with DAPI). (f) Thin cortical filaments visualized in control callus celi. Bars = 10 µm. 
A. Urbanek, B. Zechmann, G. Zellnig· & M. Mi.iller: Organization of interphase microtubules and ... 33 
Discussion 
The study of the cytoskeleton in spruce callus cells reported in this paper bas revealed the 
appearance of modified MT and actin filaments after GSH-treatment. Since cytoskeletal elements 
are involved in rnitotic cycle (NICK 1999), the disruption of MT and actin filaments could be related 
to recently reported chromosomal darnages in GSH treated cell tissues (ZELLNIG & al. 2000, M0LLER 
& al. 2001). After GSH-treatment shortened and tortuos MT were observed in spruce callus cells 
during the interphase. Moreover, GSH-treated cells were noticed, containing tubulin-positive dots 
in the cortical celi area. A similar phenomenon was described in colchicin treated cells of Vigna 
sinensis (APOSTOLAKOS & al. 1990) and in cryptogein and oligogalacturonide treated cells of Nicotiana 
tabacum (BINET & al. 2001). 
Interphase cells of control spruce material, showed an intact cortical MT network delineating 
the plasma membrane (NrcK 1999). In callus cells, which undergo ce!l differention in a polar manner, 
parallel MT arrays arranged perpendicular to the cell-growing axis were found to be typical. Whereas, 
in spherical callus cells cortical MT are laying unorganized, as previously described in suspension 
culture cells of Solanum tuberosum (CoLLINGS & EMONS 1999). 
In GSH-treated spruce material similar changes in arrangement of cortical actin filaments were 
observed as described on cortical MT network of interphase cells. Alterations were also visible on 
transvacuolar actin cables, nucleus- and plastid-associated filaments. They were observed in form 
of short stretches or were noticed as diffuse fluorescent structures. Arrays emerging from the nucleus 
to the plasma membrane were seldom observed. Otherwise, control cal!us cells were characterized 
by very well preserved actin bundles, which were similar to actin organizations previously described 
in other papers (JUNG & WERNICKE 1991, CLEARY 1995, LAZZARO 1996, DE RUIJTER & EMONS 1999, 
K.ANDASAMY & MEAGHER 1999). 
The arrangement of the cytoskeleton in the interphase is of great importance in a variety of celi 
processes including ce!l growth and celi morphogenesis (KosT & al. 1999, VoLKMANN & BALUŠKA 
1999, DAVIES 2001). Based on our results, we could postulate that GSH-induced disruption of cortical 
MT and MF network in spruce ca!lus cells caused limited celi elongation or disabled a cleary polar 
cell elongation. Furthermore, the alterations on nucleus-associated actin network, found in the present 
study, may indicate possible abnormalities during the celi division, since actin filaments are involved 
in the transport of the nucleus to the celi centre prior to the cell division (NICK 1999). 
Conclusion 
Although GSH is an essential substance in plant metabolism, our experiments demonstrated 
that the plant cytoskeleton reacts sensitively to exogenously applied GSH. Severe alterations in the 
form and arrangement of the cytoskeleton in GSH-treated spruce callus were observed during 
interphase. 
The impacts of GSH-treatment on interactions arnong cytoskeletal elements and the correlation 
between changes in patterns of actin filarnents and MT require further investigations. 
34 Acta Biologica Slovenica, 46 (2), 2003 
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CREISSEN G., J. FIRMIN, M. FRYER, B. KULAR, N. LEYLAND, H. REYNOLDS, G. PASTOR!, F. WELLBURN, N. 
BAKER, A. WELLBURN & P. MuLLINEAUX 1999: Elevated glutathione biosynthetic capacity 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2 : 37 - 41 
Sprejeto (accepted): 2003-10-30 
The distribution and propagation of Zucchini yellow mosaic virus 
(ZYMV) within Styrian pumpkin plants 
Bemd ZECHMANN, Renate FUCHS, Giinther ZELLNIG, Maria MULLER 
University of Graz, Institute of Plant Physiology, Schubertstrasse 51, A-8010 Graz, Austria 
E-mail: bemd.zechmann@stud.uni-graz.at, Fax: +43/316/380-9880 
Introduction 
Abstract. The distribution and propagation of Zucchini yellow mosaic virus 
(ZYMV) within Styrian pumpkin plants ( Cucurbita pepo L. subsp. pepo var. styriaca 
GREB.) was studied for one week starting 6 hours after ZYMV-inoculation using 
transmission electron microscopy (TEM). By negative staining, virus particles were 
found within the cotyledons and its petioles four days after the inoculation and within 
the whole plant seven days after the infection. Two weeks after ZYMV-inoculation 
the cytoplasm of infected leaf and s tem cells showed typical features of ZYMV like 
cylindrical inclusions and proliferated endoplasmatic reticulum. No ZYMV-induced 
modifications were found within root cells. 
Keywords: Zucchini yellow mosaic virus (ZYMV), Styrian pumpkin, Cucurbita 
pepo L., negative staining, transmission electron microscopy, TEM. 
Zucchini yellow mosaic virus (ZYMV) is a Potyvirus and infects a large variety of economically 
important cucurbit plants worldwide like zucchini squash (Curcubita pepo), cucumber (Cucumis 
sativus), cantaloupe ( Cucumis meto), watermelon ( Citrullus lanatus) and various species of pumpkin 
(WANG & al. 1992, DESBIEZ & LEC0Q 1997). Since 1997, severe epidemics of ZYMV-disease in 
Styria (Austria) have caused of yearly crop losses in Styrian pumpkin (Cucurbita pepo L. subsp. 
pepo var. styriaca GREB.) production of up to 60 % (WEBER 1998, RIEDLE-BAUER & al. 2002). 
Symptoms caused by ZYMV on Styrian pumpkin plants are yellowing, leaf deformations, stunting, 
color alterations and deformations of the fruits which make them unmarketable. The leaves develop 
a yellow mosaic and often show dark green blisters. 
Within the cells of ZYMV-infected plants like zucchini squash, cucumber etc. the virus induces 
scroll elements of cylindrical inclusions (pinwheels) which do not contain virions, vesicles containing 
fibrillous material and cytoplasmic inclusions of proliferated endoplasmatic reticulum (LISA & al. 
1981, LESEMANN & al. 1983, DESBIEZ & LECOQ 1997). In the present study the distribution and 
propagation of ZYMV within two weeks of old Styrian pumpkin plants was studied starting 6 hours 
after inoculation over a period of one week. Additionally the impact of ZYMV on the ultrastructure 
was documented two weeks after the inoculation when plants developed severe symptoms of ZYMV-
disease. The movement of viruses similar to ZYMV within plants is well documented. It is assumed 
38 Acta Biologica Slovenica, 46 (2), 2003 
that after the infection the virus goes through replication and moves symplastically through 
plasmodesmata from cell to cell until it reaches sieve elements. Systernic infection of the host is 
then performed by the virus through the phloem transport system (CARRINGTON & al. 1996, DERRICK & 
NELSON 1999, CHENG & al. 2000). However little information is available about the propagation and 
distribution of ZYMV within Styrian pumpkin plants. Therefore the transmission electron rnicroscope 
(TEM) was used to study the distribution and propagation of ZYMV over a period of one week 
starting 6 hours after inoculation and to document ultrastructural alterations induced by the virus 
within Styrian pumpkin plants. 
Material and Methods 
Plant material 
Styrian pumpkin seeds (Cucurbita pepo L. subsp. pepo var. styriaca GREB.) received from 
Saatzucht Gleisdorf (Plant Breeding Company, Gleisdorf, Austria) were germinated on a humid 
Perlite cloth. One week old seedlings were replanted in pots filled with soil and transferred into 
separated growth chambers with a photoperiod of 12 hours (PhAR 400-700 nm). Day and night 
temperatures were 22 °C and 18 °C, respectively, the relative humidity was 70 %. The plant material 
was kept at 100 % relative water content. Cotyledons were infected with the sap of ZYMV-infected 
plant material before the first foliage leaves emerged. Therefore lg of infected Styrian pumpkin 
material (leaves or mesocarp) was homogenated in 1 ml of a 1 %-K
2
HPO
4 
solution (pH 6.5). After 
applying celite (Cebeda, CSR) to the homogenate, the inoculum was spread out on the cotyledons 
of the seedlings. Mock inoculation was performed on control plants by spreading out the buffer with 
celite but without the inoculum onto the cotyledons. 
Negative staining 
Starting 6 hours after inoculation, negative staining was performed on at least three plants every 
day over a period of one week using different plant parts (cotyledons, leaves, stems and roots) in 
order to study the distribution and propagation of ZYMV within the plant. Therefore crude sap of 
infected Styrian pumpkin material was applied on top of a carbon coated copper grid ( 400-mesh) 
for five minutes, and subsequently the grid was washed in a 0.06 M phosphate buffer. Then the 
depositions on the grid were stained with 2 %-phosphotungstic acid in phosphate buffer (pH 6.5) for 
two rninutes. The grids were then air-dried and observed with the TEM. 
Chemical fixation 
To study the impact of ZYMV on the ultrastructure of Styrian pumpkin plants, infected plant 
material showing severe symptoms of ZYMV-disease was prepared for TEM. Therefore leaves and 
roots of infected and control Styrian pumpkin plants were harvested separately two weeks after 
ZYMV-inoculation. Small pieces of the samples were fixed in 3 %-glutaraldehyde in 0.06 M 
phosphate buffer at pH 7 .2 for 90 minutes at room temperature (RT). Postfixation was carried out in 
1 % osmium tetroxide in 0.06 M phosphate buffer pH 7.2 for 90 minutes at RT. Dehydration was 
performed in increasing concentrations of acetone (50 % to 100 %) and propylene oxide before the 
samples were embedded in Agar 100 epoxy resin. Ultrathin sections were post-stained with lead 
citrate and uranyl acetate before they were observed in a Philips CMlO TEM. 
B. Zechmann, R. Fuchs, G. ZeJlnig, M. Mtiller: The distribution and propagation of Zucchini ... 39 
Results 
Filamentous, elongated virus particles were detected by negative staining for the first tirne only 
in symptom free cotyledons and its petiole 4 days after ZYMV-inoculation. The particles were 750 
nm in length and 11 nm in width (Fig. lb). The virus did not spread out of the cotyledons until the 
7m day after the infection when it was found in the cotyledons, stems, the first leaves and in roots. 
No visible symptoms of ZYMV-disease were observed on the plants at this tirne. However two 
weeks after inoculation infected plants developed severe symptoms of ZYMV-disease (yellowing, 
stunting, blistering, leaf-deformations). At this tirne the cytoplasm of infected leaf and stem cells 
contained a large amount of cylindrical inclusions, which were found to be organized as long tubular 
bundles if cut longitudinally and as scrolls and pinwheels if cut transversely. Besides that, proliferated 
endoplasmatic reticulum (ER) was observed frequently throughout the infected cells of leaves and 
stems (Fig. la, c). Virus particles were not found in plasmodesmata. No virus-induced modifications 
were observed within root cells although virus particles were detected there by negative staining. 
In control plants no virions, cylindrical inclusions nor virus-induced modification were found. 
Discussion 
Viruses move within the plant from cell to cell (short distance movement) and after entering the 
vascular system over long distances to systemically infect the whole plant (CARRINGTON & al. 1996, 
CHENG & al. 2000). Whereas the virus moves from cell to cell in rather slow rates of approximately 
one celi per 2 hours (M1sE & AHLQUIST 1995, SzEcs1 & al. 1999), vascular movement occurs at rates 
of centimeters per hour (NELSON & VAN BEL 1998). 
In the present study the distribution and propagation of ZYMV within Styrian pumpkin plants 
was investigated, over a period of one week starting 6 hours after inoculation, by negative staining 
using TEM. Four days after inoculation, the virions were detected within the cotyledons and their 
petioles. Seven days after the infection the virus was detected in ali parts of the plants, including 
roots. These results lead to the assumption that after the virus reaches the phloem of the host it 
moves up and down within the plant at the same tirne. Our results are similar to what has been 
reported recently for Tobacco mosaic virus, which has been found moving up and down within the 
phloem of the stem seven days after the infection of the 2nd leaf (CHENG & al. 2000). 
Two weeks after ZYMV-inoculation the cytoplasm of infected Styrian pumpkin leaf and stem 
cells showed severe ultrastructural changes like cylindrical inclusions and proliferated ER, which 
were similar to what was found in other ZYMV-infected cucurbit plants (LISA & al. 1981, LESEMANN 
& al. 1983, DESBIEZ & LEcOQ 1997). Virus particles, which had the same average size described in 
the literature for ZYMV (LISA & al. 1981) were detected by negative staining in leaves, cotyledons, 
stems and roots depending on the infection state. However, no ZYMV-induced ultrastructural 
modifications occurred within root cells. It seems that ZYMV-related ultrastructural changes within 
the plant are restricted to stem and leaf cells only. 
40 Acta Biologica Slovenica, 46 (2), 2003 
Figure 1: Transmission electron micrographs. Bars: a = 1 µrn; b = 250 nrn, c = 500 nm. a) Zucchini 
yellow mosaic virus (ZYMV)- infected mesophyll cell two weeks after the inoculation showing a 
chloroplast (C), cylindrical inclusions and proliferated endoplasrnatic reticulurn (arrows) within the 
cytoplasm. b) Particles of ZYMV detected in the sap of infected leaf-material by negative staining 
one week after inoculation. c) Infected mesophyll cell two weeks after the ZYMV-inoculation showing 
cylindrical inclusions in various forrnations including pinwheels (arrow). 
Conclusion 
After entering the host ZYMV needs about one week to infect the whole plant. Although the 
virus needs quite a long tirne to spread through the first infected leaf, it rnoves quite fast after it 
enters the phloern to systernically infect the whole plant. This leads to the conclusion that a rapid 
movement and distribution of viruses within plants is limited by the short distance movement (cell 
to cell) rather than the long distance movement through the phloem transport system. 
B. Zechmann, R. Fuchs, G. Zellnig, M. Miiller: The distribution and propagation of Zucchini ... 41 
Acknowledgement 
This research was partly supported by a research scholarship to B. Z. of the Karl-Franzens-
University of Graz. The authors wisb to thank Dr. Imtraud Thaler for helpful suggestions and advices 
during the studies. 
Literature 
CARRINGTON J. C., K. D. KAsscHAU, S. K. MAHAJAN & M. C. SCHAAD 1996: Cell-to-cell and long-
distance transport ofviruses in plants. Plant Cell 8: 1669-1681. 
CHENG N. H. , C. L. Su, S. A. CARTER & R. S. NELSON 2000: Vascular invasion routes and systemic 
accumulation pattems of tobacco mosaic virus in Nicotiana benthamiana. Plant J. 23 (3): 
349-362. 
DERRICK P. M. & R. S. NELSON 1999: Plasmodesmata and long distance virus movement. In: van Bel 
A. J. E. & W. J. P. van Kesteren (eds.): Plasmodesmata. Structure, Function, Role in Cell 
Communication. Springer Verlag, Berlin, pp. 315-339. 
DESBIEZ C. & H. LECOQ 1997: Zucchini yellow mosaic virus. Plant Pathol. 46: 809-829. 
LESEMANN D. E., K. M. MAKKOUK, R. KbNIG, & E. N. SAMMAN 1983: Natural infection of cucumbers 
by zucchini yellow mosaic virus in Lebanon. Phytopathol. Z. 108: 304-313. 
LrsA V., G. BoccARDO, G. D' AGOSTINO, G. DELLAVALLE & M. D ' AQrnuo 1981: Characterization of a 
potyvirus that causes zucchini yellow mosaic. Phytopathol. 71: 667-672. 
M1sE K. & P. AHLQUIST 1995: Host-specificity restriction by bromovirus cell-to-cell movement protein 
occurs after initial cell-to-cell spread of infection in nonhost plants. Virol. 206: 276-286. 
NELSON R. S. & A. J. E. VAN BEL 1998: The mystery of virus trafficking into, through and out of the 
vascular tissue. Prog. Bot. 59: 476-533. 
RIEDLE-BAUER M ., B. SuAREZ & H. J. REINPRECH[ 2002: Seed transmission and natura] reservoirs of 
Zucchini yellow mosaic virus in Cucurbita pepo var. styriaca. J. Plant Dis. Prot. 109 (2): 
200-206. 
SzEcsr J., X. S . DrNG, C. O. LrM, M. BENDAHMANE, M. J. CHo, R. S. NELSON R. S. & R. N. BEACHY 
1999: Developement of tobacco mosaic virus infection sites in Nicotiana benthamiana. 
Mol. Plant-Microbe Internet. 12: 143-152. 
WANG H. L. , D. GoNSALVES, R. PROVVIDENTJ & T. A. ZrTTER 1992: Comparative biological and 
serological properties of four strains of zucchini yellow mosaic virus. Plant Dis. 76: 530-
535. 
WEBER J. 1998: Kiirbisanbau nur mit virusfreiem Saatgut. Der Fortschrittliche Landwirt 3: 10-11. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2 : 43 - 47 
Sprejeto (accepted): 2003-10-30 
Diurnal variation of chloroplast fine structures of spinach 
Giinther ZELLNIG, Andreas PERKTOLD 
University of Graz, Institute of Plant Physiology, Schubertstr. 51 , A-8010 Graz, Austria 
Fax: ++43/316-380-9880; e-mail: guenther.zellnig@uni-graz.at 
lntroduction 
Abstract. Complete chloroplasts and their fine structures were quantitatively 
analysed by means of ultrathin serial sections and digital image analysis in order to 
obtain precise ultrastructural data about diurnal adaptations of the organelles. During 
the daily course the average chloroplast volume increased from 31 µm3 in the moming 
to 44 µm3 in the evening. In the same tirne the absolute volumes of starch, thylakoids 
and plastoglobuli also increased, though to different extents. These observed 
differences in the diumal behaviour of this organelles are essential for a precise 
evaluation of naturally occurring or stress induced changes in chloroplasts. 
Keywords: Chloroplasts, serial sections, ultrastructure, volume, spinach 
Chloroplasts are highly specific organelles of plant cells carrying out severa! important 
biosynthetic processes involved in photosynthesis. Investigations of their complex fine structural 
intemal organisation need the use of ultrathin sections and transmission electron microscopy. Applying 
this technique usually leads to a characterization of the condition of cell organelles in a descriptive 
manner only. Different environmental factors are known to affect the ultrastructure of cell organelles 
in general, but plastids were investigated most intensively because alterations or damages were 
noted very early and/or mainly in these organelles (FoRSCHNER & al. 1989, RANTANEN & al. 1994, 
HoLOPAINEN & al. 1996). However, the evaluation of the condition of this organelle usually based on 
the information of a limited number of ultrathin sections. Exact quantitative data resulting from 
ultrastructural analyses are stili rare though quantitative morphological analyses proved to be more 
reliable and highly valuable for a more detailed assessment of cellular changes or ultrastructural 
adaptations of organelles (ZELLNIG & PERKTOLD 1999, WHEELER & FAGERBERG 2000, REY & al. 2000, 
GRJFFIN & al. 2001, R0ZAK & al. 2002). 
In this research complete mesophyll chloroplasts of young spinach leaves were investigated by 
means of ultrathin serial sectioning, electron microscopy and digital image analysis in order to 
register the daily periodic ultrastructural adjustrnents in the architecture and the amount of structures 
inside the organelle. 
44 Acta Biologica Slovenica, 46 (2), 2003 
Material and methods 
Pot cultures of Spinacia oleracea L. were grown under defined conditions in a climate chamber 
with a temperature of 23 °C during daytime and l 7°C ovemight with an average relative humidity of 
60 %. The photosynthetic active radiation (PhAR) was adjusted at a level of 900 µmol m·2 s·1, 
distance was 1.5 m. Twilight started at 8 a.m. in 30 minutes from 0-900 µmol m·2 s·1 and the light 
decreased at 8 p.m. 
Sections were taken from the middle part of young leaves (size about 4 cm) at 07.00 h (early 
moming), 13.00 h (midday) and 19.00 h (evening). The sections were fixed in 3 % cacodylate 
buffered glutaraldehyde, pH 7.2, for 90 min. After rinsing in buffer the sections were postfixed in 1 % 
OsO
4 
for 90 min, dehydrated in a graded series of ethanol followed by propylenoxide and embedded 
in Agar 100 epoxy resin. 
Serial ultrathin sections (100 nm) were cut with a Reichert Ultracut S ultramicrotome, stained 
with lead citrate and uranyl acetate and viewed with a Philips CM 10 transmission electron 
microscope. 
3D measurements 
Preliminary investigations are essential in order to exclude 3D reconstructions of untypical 
chloroplasts. Therefore two leaves were taken from two selected plants of every sample. A ribbon of 
about 80 serial sections of palisade parenchyma cells was cut of every leaf. TEM micrographs were 
used for 2D measurements. Four arbitrary selected sectioned areas of chloroplasts in section 1, 30, 
and 60 of every ribbon (= 12 areas per ribbon, 48 areas per sample) were investigated by TEM and 
measured by computer to get characteristic data of fine structures. The results of these preliminary 
investigations served for the selection of the sample with the most characteristic chloroplasts according 
to the calculated mean values for each sample. 
The TEM micrographs were digitised by a scanner and the sectioned areas of interest were 
measured with an image analysis system (Optimas 6.5., Bio Scan) and exported to the software 
program Excel. The volumes were calculated by the sum of sectioned areas multiplied by the thickness 
of the sections. The digitised micrographs were pixel images, which were transformed semi-
automatically by a computer program (Corel Trace 10), or by hand (Corel Draw 10), into 
vectorgraphics. 3D reconstructions were created by the program Carrara Studio 1.0 (Softline). For 
each sample 6 complete palisade parenchyma chloroplasts and details of them were measured and 
3D reconstructed. 
The statistical analyses were performed by using the software package Origin (OriginLab 
Corporation, USA). Differences between morning and evening samples were evaluated using two 
sample t-test. 
Results 
The investigated chloroplasts show a homogeneous distribution of thylakoids ~nd plastoglobuli 
without any noticeable diurnal variation, starch is present in form of a varying number of grains in 
the single chloroplasts (Fig. 1). During the day the total chloroplast volume increases from 31 µm3 
in the morning samples to 44 µm3 in the evening. This increase is associated with an increase in the 
absolute values of ali interna! chloroplast structures (cf. Fig. 2). A similar trend is also present, 
when the data are related to relative values. In this case the starch content increases significantly 
from 12 % of the chloroplast volume in the moming to 27 % in the evening. This change is connected 
with a decrease in the thylakoid and stroma volumes and a significant increase in the total volume of 
the plastoglobuli in the evening samples (cf. Fig. 3). 
G. Zellnig, A. Perktold: Diumal variation of chloroplast fine structures of spinach 
p 
E 
45 
Figure 1: Three-dimensional reconstruction of a spinach palisade parenchyma chloroplast showing 
the thylakoid system (T), starch grains (S), plastoglobuli (P) and the envelope (E) . Sample taken at 
07.00 h. 
e 
:i. 
C: 
o 
00 
o 
CD 
·;~ 
E 
::i o 
>o 
N 
o 
mean values of volumes 
5.ltotal velurne 
rithylakoids 
• plastoglobuli 
+--- --------------------, • starch 
• siroma 
43,80 
early morning midday evening 
Figure 2: Mean volumes and standard deviation of complete palisade parenchyma chloroplasts and 
their interna! fine structures during the daily course (n = 6 for each sampling tirne). 
46 
Cllo 
E'° 
::r 
o 
> 
-o J!I .., 
o -'o 
~~ 
63.90 
early morning 
Acta Biologica Slovenica, 46 (2), 2003 
relative volumes 
60.32 
midday 
r.ithylakoids 
• plastoglobuli 
evening 
Figure 3: Mean values and standard deviation of the relative values of fine structures of chloroplasts 
during the daily course. Significance was calculated from randomly sectioned chloroplasts for the 
morning and evening samples (n == 48 for each sample). 
*, ** and *** indicate values that differ significantly from the control at P < O.OS, P < 0.001, and 
P < 0.0001, respectively; P > O.OS not significant (ns). 
Discussion 
The investigated spinach chloroplasts clearly showed diurnal fine structural adaptations and 
changes. During the day the starch content increased due to the photosynthetic C0
2 
fixation resulting 
in the synthesis of transitory starch inside the chloroplasts. The higher starch content is the main 
reason for a significant increase in the volumes of the chloroplasts in the evening, though all other 
structures also showed increasing values. Interestingly, two-dimensional measurements of spinach 
chloroplasts did not reveal a significant diurnal net change in the grana size and number (RozAK & 
al. 2002). The thylakoid membrane is a very dynamic system even in mature chloroplasts, rapidly 
adapting to changes in light conditions or performing long term adaptation due to a number of 
environmental factors (cf. VoTHKNECHT & WESTHOFF 2001). Our investigations demonstrated changes 
in the thylakoid system during the day, which are supposed to be caused not only by an increasing 
light intensity. An adaptation to changes in light intensity were already reported for the thylakoid 
surface area in sunflower (WHEELER & FAGERBERG 2000) and the grana size and number in spinach 
chloroplasts (RoZAK & al. 2002). These changes occurred within minutes, thus light intensity is not 
supposed to be the main reason for the observed increase in the thylakoid system in the evening 
samples. In addition to the thylakoid system also the plastoglobuli content showed a more than 
tenfold increase in the evening samples. Plastoglobuli are regarded as a kind of storage particles for 
thylakoid components being involved in the forrnation and degeneration of the thylakoid membrane 
(cf. KEssLER & al. 1999). A diurnal change in the plastoglobuli content and a simultaneous increase 
of both the thy lakoid membranes and the plastoglobuli during the day has not been reported yet and 
has to be investigated in more detail in further experiments. 
G. Zellnig, A. Perktold: Diurnal variation of chloroplast fine structures of spinach 47 
Conclusion 
Palisade parenchyma chloroplasts of spinach show quantitative cbanges and adaptations of their 
fine structures during the daily course, which have to be considered in connection with investigations 
and measurements of this organelle. 
Acknowledgement 
This work was supported by the FWF (P13614-BIO, P-15374). 
Literature 
FoRSCHNER W., V. SCHMITT & A. Wrw 1989: lnvestigations on the starch content and ultrastructure 
of spruce needles relative to the occurrence of Novel forest decline. Bot. Acta. 102: 208-
221. 
GRIFFIN K.L., O.R. ANDERSON, M.D. GASTRICH, J.D. LEWIS, G. LIN, W. SCHUSTER, J .R. SEEMANN, D.T. 
TrssuE, M.H. T uRNBULL & D. WHITEHEAD 2001: Plant growth in elevated CO
2 
alters 
mitochondrial number and chloroplast fine structure. PNAS 98: 2473-2478. 
HOLOPAINEN T., s. ANTTONEN, V. PALOMAKI, P. KAINULAINEN & J.K. HOLOPAINEN 1996: Needle 
ultrastructure and starch content in scots pine and norway spruce after ozone fumigation. 
Can. J. Bot. 74: 67-76. 
KEssLER F., D. ScHNELL & G. BLOBEL 1999: Identification of proteins associated with plastoglobules 
isolated from pea (Pisum sativum L.) chloroplasts. Planta 208: 107-113. 
RANTANEN L., V. PALOMAKI, A.F. HARRISON, P.W. LucAs & T.A. MANSFIELD 1994: Interactions between 
combined exposure to SO
2 
and NO
2 
and nutrient status of trees: effects on nutrient content 
and uptake, growth, needle ultrastructure and pigments. New Phytol. 128: 689-701. 
REY P., B. GJLLET, S. R•MER, F. EYMERY, J. MASSIMINO, G. PELTIER & M. KuNTZ 2000: Over-expression 
of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid 
ultrastructure and plant development upon stress. Plant J. 21: 483-494. 
RozAK P.R., R.M. SEISER, W.F. W ACH0LTZ & R.R. WrsE 2002: Rapid, reversible alterations in spinach 
thylakoid appression upon cbanges in light intensity. Plant Cell Environ. 25: 421-429. 
VOTHKNECHT U .C. & P. WesrH0FF 2001 : Biogenesis and origin of thylakoid membranes. Biochim. 
Biophys. Acta 1541: 91-101. 
WHEELER W.S. & W.R. FAGERBERG 2000: Exposure to low levels of photosynthetically active radiation 
induces rapid increases in palisade cell chloroplast volume and thylakoid surface area in 
sunflower (Helianthus annuus L.). Protoplasma 212: 38-45. 
ZELLNIG G. & A. PERKTOLD 1999: Plant organelles analyzed by ultrathin serial-sections. Phyton 39: 
65-68. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2: 49 - 54 
Sprejeto (accepted): 2003-10-30 
Light-modulation of bark photosynthesis in birch (Betula pendula Roth.) 
Christiane WITIMANN and Hardy PFANZ * 
Institute of Applied Botany, University of Duisburg-Essen, FRG 
*Corresponding address: 
Univ.-Prof. Dr. H. Pfanz, Institut fiir Angewandte Botanik, Universitat Essen, Universitatsstr. 5 
D-45117 Essen; Tel.: 0201-183-2153; Fax: 0201-183-4219; e-mail: hardy.pfanz@uni-essen.de 
Introduction 
Abstract. Bark photosynthesis has been shown to be an effective mechanism 
for stem-intemal refixation of respiratory CO
2
• In young birch trees (Betula pendula 
Roth.) this function is clearly modulated by the prevailing light intensity regime. 
Although positive net photosynthesis was not found in intact birch twigs, apparent 
twig respiration was reduced upon illumination by 65 % in high-light grown birches 
and even more in shade grown trees (81 %). Compared ona unit area basis the bark 
chlorenchyma contained up to 55% of the chlorophyll of the concomitant !eaves 
when grown under 100 % sunlight and even 66 % when trees were grown under 
low-light (20 % of full sunlight). Light penetration through the periderm of birch 
twigs and branches is age-dependent and ranges in control trees from roughly 24 % 
of the incident sunlight in recent-year twigs to 1-3 % in 5-year-old main stems. 
Peridermal light transmittance was also changed by the light intensity regime. An 
additional light-reducing peridermal layer present in control trees was not found in 
shade-grown birches. It was shown that CO
2
-refixation is not limited to the light-
exposed outer parts of tree crowns. Our results show that also inner branches of 
trees are well adapted to function as a rather efficient system to prevent respiratory 
carbon loss. 
Key words: Betula, bark photosynthesis, light intensity, carbon fluxes, CO
2
-
refixation 
In nearly all trees, woody shrnbs and bushes chlorophyll-containing tissues can be found in the 
inner bark !ayers. These chlorenchymes are able to photosynthetically reduce the flux of respiratory 
CO
2 
to the atmosphere and in parallel to evolve thylakoid-bome 0
2 
(PFANZ et al. 2002, PFANZ & 
AscHAN 2000), a process that bas been termed „CO
2
-refixation" or alternatively „corticular 
photosynthesis" (SPRUGEL & BENECKE 1991, NILSEN 1995, PrLARSKI 1995). 
Plant productivity depends on the balance between photosynthetic carbon assimilation and the 
expenditure of fixed carbon by respiration (EDWARDS ET al. 1981 , W ARING & RuNNING 1998). Equi valent 
gains in whole plant carbon assimilation can be made by increasing the rate of carbon uptake from 
50 Acta Biologica Slovenica, 46 (2), 2003 
the atmosphere (i.e by the stimulation of leaf photosynthesis) or by decreasing respiratory carbon 
losses (i.e through bark photosynthesis; CERNUSAK & MARSHALL 2000). In different deciduous trees 
(e.g. Fagus sylvatica, Populus tremula) stem-intemal refixation of CO2 in young twigs and branches 
may compensate for 60-90 % of the potential respiratory carbon loss (Prr.ARsKI 1995, WITTMANN Er AL. 
2001, AscHAN Er AL. 2001). The light-driven stem-intemal refixation of carbon dioxide may therefore 
be an important measure to optirnize carbon fluxes at the whole-tree (or even whole-canopy) leve!. 
Light is one of the major environmental factors regulating plant productivity. There is plenty of 
knowledge on the effects of different light on the carbon gain of Ieaves, but information on the light-
dependency of bark photosynthesis or on the carbon budgets of twigs and branches is still scarce. To 
illuminate the influence of the light environment on the photosynthetic activity of the inner 
bark cells we grew young birch trees (6-year-old) in distinct light environments (20 % and 100 % of 
full sunlight) and studied the effectiveness and the probable adaptive light dependency of stem-
intemal carbon refixation as well as related morphological and cyto-chemical parameters. 
Materials and Methods 
Plant material 
Six-years-old birch trees (Betula pendula L.) were grown outside in 20 l plastic containers 
under sufficient nutrition (Einheitserde Typ T, Balster, Germany) and water supply, realised by 
periodic fertilisation with Osmocote (Bayer, Germany) and daily irrigation. In early spring 1999, 20 
trees were shaded with a tentlike construction, covered with a light-reducing net tissue (light 
permeability about 20 % of incident sunlight); the other cluster (control, 100 %) was exposed to 
natura! sunlight conditions. The net structure of the shading tissue as well as the open front and back 
of the tent enabled permanent air circulation, avoicling overheating during hot summer days. 
PFD transmittance 
The quantity and quality of light (photon flux density ; PFD) transmitted through the peridermal 
layers of birch twigs were studied in 2001. According to PFANZ (1999) tissues were removed with a 
cork borer and placed for 5 min in isotonic solution. The wet bark samples were sandwiched between 
two microscopy slides and tightly adjusted over an opening in a black plastic plate towards a quantum 
sensor (model Ll-190SA, Li-Cor) connected with a quantum radiometer (Li-250, Li-Cor, Lincoln, 
Neb., USA). PFD transrnission was measured by illurninating it with a 100 W quartz halogen lamp 
(Xenophot HLX 64625, Osram, Germany) equipped with an infrared filter (NIR filter ST 931619/ 
KB, Balzers, Lichtenstein) to avoid warrning the samples and producing approximately 1000 µmol 
m·2 s·1 (see Pfanz 1999). Relative PFD transmission was calculated as a percentage of the incident 
PFD directly above the sample surface. 
Photosynthetic pigment determination 
Disks from leaves or from bark were removed by a calibrated cork borer (5 mm in diameter) and 
placed in 80 % (v/v) dimethyl sulfoxide (DMSO). Pigment extraction required approximately 2 h at 
65°C in the dark. To avoid acidification and a concomitant phaeophytinisation of the chlorophylls, 
20 mg Mgz(OH)2CO3 was added . Finally, extract absorbances were measured with a 
spectrophotometer (UV 160, Shimadzu, Japan) and pigment contents calculated according to standard 
equations (Wellburn 1994). 
C. Wittmann and H. Pfanz: Light-modulation of bark photosynthesis in birch (Betula pendula Roth.) 51 
Gas-exchange measurement 
Photosynthetic performance of intact twigs and leaves was studied with a portable gas-exchange 
system (model LI-6400, Li-Cor Inc., Lincoln, USA). To avoid possible wound respiration only 
intact twig internodes or single mature leaves stili attached to the twigs were selected for the gas-
exchange measurements. Dark respiration was measured after a 30-min shading period of the 
respective plant parts within the cuvette. Subsequent light response curves were conducted under 
constant climatic conditions (20°C, 50-55% relative humidity) and a controHed CO
2 
supply (350 
ppm). High-irradiance CO2-exchange rates were first measured after a 30-rnin period oflight exposure 
to 2000 µmol photons m-2 s·1• At least ten independent light response curves were assessed for each 
type of leaf or twig intemode. 
Curve fittings and calculations of all parameters were done using Sigma Plot 5.0 (SPSS Science 
Software). Light saturation of CO2-assirnilation was defined at 90% of maximum photosynthetic 
rate (e.g. von WILLERT ET AL. 1995). Stem internat CO2-re-fixation rates were calculated as the 
difference between high-irradiance maximum CO
2
-exchange rate and dark respiration. 
Results and Discussion 
PFD transmittance 
Transrnittance of light (qualitatively and quantitatively) through the periderm clearly depends 
on thickness, cellular structure and optical properties of the outer bark layers (VoGELMANN 1993, 
PFANZ & AscHAN 2000). With advancing age of the twigs the thickness of the peridermal layers 
increases; as a result peridermal PFD transmission is reduced. 
Light penetration through the periderm ofbirch twigs ranged from ca. 24 % in recent-year twigs 
to 1-3 % in 5-year-old main stems. Growth under limiting light conditions greatly influenced 
peridermal PFD transmittance. A normally formed additional peridermal layer was not found under 
low light growth conditions. Thus, the peridermal layers of birch twigs permit a relatively higher 
light flux under shaded conditions. 
Pigment content 
Compared on a unit area basis the bark chlorenchyma contained up to 55 % of the chlorophyll 
content of concomitant leaves in high-light grown trees and even 66 % in trees grown under low-
light conditions. It is well known that shade-leaves optimise the effectiveness of light absorption by 
an increased pigment density per unit leaf area and this strategy holds also for twigs. 
The chlorophyll alb ratio in birch chlorenchyma ranged between 2.1 and 2.7. These results are 
in good agreement with those described for other deciduous species, such as Fagus sylvatica (1.8, 
LARCHER ET AL. 1988), Populus tremuloides (2.7, KHAROUK ET AL. 1955), Syringa vulgaris (PILARSKI 
1999), and Betula pendula (KAuPPI 1991). In general, shade adapted cells within a leafhave a lower 
chlorophyll alb ratio than cells adapted to high light ( e.g. LICHTENTHALER ET AL. 1981, ANDERSON & 
52 Acta Biologica Slovenica, 46 (2), 2003 
OsMOND 1987; THERASHIMA & HIKoSAKA 1995). The lower chlorophyll alb ratio of the bark 
chlorenchyma (as compared to the leaves) is easily explained by the hidden location behind the cork 
layers. 
Table 1: Cardinal points of the light response curves of Betula pendula twigs cultivated under different 
growth light regimes (n = 10; +/- SD) 
relative light during cultivation [% sunlight] 20% 100 % 
Age of the twig organ current-year current-year 
max. net photosynthesis rate [µmol C0
2 
m·2 s·'] -0.27 ± 0.05 -0.98 ± 0.12 
stem-internal photosynthesis 
1 
[µmol C0
2 
m·2 s·'] 1.07 ± 0.05 1.79±0.12 
saturated PFD 
2 
[µmol photons m·2 s·1] 250 339 
dark respiration rate (µmol C0
2 
m·2 s·1] -1.34 ± 0.04 -2.77 ± 0.10 
Age of the twig organ 1-year-old 1-year-old 
max. net photosynthesis rate [µmol C0
2 
m·2 s·1] -0.16 ± 0.04 -0.44 ± 0.10 
stem-internal photosynthesis 
1 
[µmol C0
2 
m·2 s·'] 0.69 ± 0.04 0.98 ± 0.10 
saturated PFD 
2 
[µmol photons m·2 s·'] 250 334 
dark respiration rate [µmol C0
2 
m·2 s· 1] -0.85 ± 0.04 -1.42 ± 0.09 
1 
stem-internal or "hidden" photosynthesis (Wittmann et al. 2001) was calculated as the difference between 
high-irradiance maximum C0
2
-exchange rate and dark respiration. 
2 
saturated PFD [µmol photons m·2 s·1] was defined at 90 % of maximum photosynthetic rate (e.g. von 
Willert et al. 1995); calculations were done using Sigma Plot 5.0 (SPSS Science Software). 
CO2-exchange of twigs and leaves 
Birches revealed typical hyperbolic light response curves for both, mature leaves and intact twig 
segments. 
The measured cardinal points of the light response curves of leaf and twig photosynthesis are 
given in Tab. l. Besides changes in net photosynthetic rates and apparent dark respiration, distinct 
changes in stem-intemal photosynthesis were observed (cf. WrTIMANN ET AL. 2001). Stem-intemal 
CO
2
-refixation was calculated as the difference between the maximum CO
2
-fixation rate and dark 
respiration. Furthermore, the light saturation of twig photosynthesis clearly differed, being around 
340 µmol photons m·2 s·1 in high-light-grown twigs and 250 µmol photons m·2 s·1 in the shade-grown 
variant (Tab.l). According to LARCHER ET AL. (1994), light saturation of deciduous shade leaves is 
around 200-500 µE m-2 s·1; corticular photosynthesis is thus performed by extremly shade adapted 
chloroplasts (as indicated above by the low chl alb ratios). This fact is even more corroborated when 
photosynthesis of chloroplasts of the tree's pith or wood is studied (PFANZ ET AL. 2002). 
C. Wittmann and H. Pfanz: Lig:ht-modulation of bark photosynthesis in birch (Betula pendula Roth.) 53 
90 
- 80 = ~ o 70 ...... .... 
~ 
...... - ~ i. 60 = .... o C. .... r,.i 
50 ...... ~ 
~ i. 
~ ~ 40 = i. ~ ~ 
i. "C 30 1 M ~ o o u ~ 20 = ....... 
10 
o 
20% sunlight 100% sunlight 
Preliminary calculations revealed that in birch grown under full sunlight around 65 % of the 
respired CO
2 
can be refixed within the twigs. With 81 % corticular photosynthesis is even more 
pronounced under shaded conditions (Fig.!). 
Figure 1: CO
2
-refixation rates of Betula pendula twigs (current- and 1-year-old) cultivated under 
different light intensity regimes (means with SE, n = 10). 
Conclusions 
Although twig and branch photosynthesis rarely result in positive net CO
2
-fixation, quite a high 
portion of the respired mitochondrial carbon dioxide is immediately re-used within the tree skeleton 
and thus contributes substantially to the overall carbon balance and productivity of trees. In young 
twigs of tree crowns the respiratory CO
2 
losses are more efficiently reduced than in older branch 
parts. Yet, CO
2
-refixation is not restricted to the light-exposed outer parts of the canopy. Our results 
show that the light use efficiency of inner branches is relatively higher. More morphometrical data 
are needed to elucidate the quantitative effect of bark photosynthesis on the C-budget of trees. 
Acknowledgements 
We would like to thank Gudrun Friesewinkel, Sabine Kuhr and Christa Kosch for technical 
assistance. Warm thanks also to Dipl. Umweltwiss. Jens Ahting and Maja Humar (Ljubljana). 
54 Acta Biologica Slovenica, 46 (2), 2003 
Literature 
ANDERSON J.M., OsMOND C.B. 1987: Shade-sun responses: compromises between acclimation and 
photoinhibition. In: Kyle D.J., Osmond C.B., Arntzen C.J. (eds.) Photoinhibition. Elsevier, 
Amsterdam, pp 1-38. 
AscHAN G., WrTIMANN C., PFANZ H. 2001: Age-dependent bark photosynthesis of aspen twigs. Trees 
15: 431-437. 
CERNUSAK L.A., MARSHALL J.D. 2000: Photosynthetic refixation in branches of western white pine. 
Funct. Eco!. 14: 300-311. 
KAurrr A. 1991: Seasonal fluctuations in chlorophyll content in birch stems with special reference 
to bark thickness and light transmission, a comparison between sprouts and seedlings. 
Flora 185: 107-125. 
KHAROUK V.I. , MIDDLETON E.M., SPENCER S.L., RocK B.N., WILLIAMS D.L. 1995: Aspen bark 
photosynthesis and ist significance to remote sensing and carbon budget estimate in the 
boreal ecosystem. Water Air Soil Pollut. 82: 483-497. 
LARCHER W. 1994: Okophysiologie der Pflanzen: Leben, Leistung und Stressbewaltigung der Pflanzen 
in ihrer Umwelt. 5th edn. Ulmer, Stuttgart, Germany. 
LARCHER W., LOTZ C., NAGELE M., BooNER M. 1988: Photosynthetic functioning and ultrastructure 
od chloroplasts in stem tissue of Fagus sylvatica. J. Plant. Physiol. 132: 731-737. 
LICHTENTHALER H.K. , BuscHMANN C., D6LL M., FrETZ H.-J., BAcH T., KozEL U., MEIER D., RAHMSDORF 
U. 1981: Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-
light and low-light plants and of sun and shade leaves. Photosynthesis Res. 2: 115-41. 
NILSEN E .T. 1995: Stem photosynthesis - extent, patterns and role in plant carbon economy. In: 
Gartner B. (ed.) Plant stems: physiology and functional morphology. Academic Press, San 
Diego,pp. 223-240. 
PFANZ H. 1999: Photosynthetic performance of twigs and stems of trees with and without stress. 
Phyton 39: 29-33 . 
PFANZ H., AscHAN G. 2000: The existence of bark and stem photosynthesis and its significance for the overall 
carbon gain: an eco-physiological and ecological approach. Progress in Botany 62: 477-510. 
PFANZ H., AscHAN G., LANGENFELD-HEYSER R., WITTMANN C., LoosE M. 2002: Ecology and ecophysiology 
of tree stems - corticular and wood photosynthesis. Naturwissenschaften 89, 147-162. 
PILARSKI J. 1995: Participation of stems and leaves in photosynthetic assimilation of C0
2 
in lilac 
(Syringa vulgaris L.). Photosynthetica 31: 585-592. 
PILARSKJJ. 1999: Gradient of photosynthetic pigments in the bark and leaves oflilac (Syringa vulgaris 
L.). Acta Physiologicae Plantarum 21: 365-373. 
SPRUGEL D .G., BENECKE U. 1991 : Measuring woody-tissue respiration and photosynthesis. In: LASSOIE 
J.P., HlNCKLEY T.M. (eds.) Techniques and approaches in forest tree ecophysiology. CRC, 
Boca Raton, pp. 329-355. 
TERASHIMA I., HJKoSAKA K. 1995: Comparative ecophysiology of leaf and canopy photosynthesis. 
Plant Cell Environ 18: 1111-1128. 
VoGELMANN 1993: Plant tissue optics. Annu. Rev. Plant Physiol. Mol. Biol. 44: 231-251. 
WARING R.H. & RuNNING S.W. 1998: Forest Ecosystems: Analysis at multiple scales. Academic 
Press, San Diego, CA. 
WELLBURN A.R. 1994: The spectral determination of chlorophylls a and b, as well as total carotenoids, using 
various solvents with spectrophotometers of differentresolution. J. Plant. Physiol. 144: 307-313. 
WITIMANN C., AscHAN G., PFANZ H. 2001: Leaf and twig photosynthesis of young beech (Fagus 
sylvatica) and aspen (Populus tremula) trees grown under different light intensity regimes. 
Basic Appl. Ecol. 2: 145-154. 
VoN WJLLERT D.J., MATYSSEK R., HERPPICH W. 1995: Experimentelle Pflanzenokologie. - Georg 
Thieme Verlag, Stuttgart, New York. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 2: 55 - 60 
Eff ect of sucrose nutrition on the histological structure of carob shoot 
cultures 
Branka VINTERHALTER1, Dragan VINTERHALTER: 
1Institute for Biological Research, 29. novembra 142, Belgrade, Yugoslavia 
2Faculty of Science, Zmaja od Bosne 33-35, Sarajevo, Bosnia and Herzegovina 
Introduction 
Abstract. Analysis of carob (Ceratonia siliqua L.) shoot cultures grown on 2 
and 8 % sucrose in light and darkness (etiolation) showed marked differences in 
histological structure. Etiolated, dark grown shoot cultures were hypolignified on 
both sucrose concentrations. They had highly reduced sclerenchyma and xylem 
production, endodermis with Casparian bands and absence of lenticel formation. In 
light grown cultures shoots in transection showed nearly concentric ring of 
sclerenchyma ( outer) and xylem (inner) ring and the absence of Casparian bands in 
endodermal cells. Increased sucrose nutrition promoted lignification and efficiently 
prevented lenticel hypertrophy characteristic for low sucrose concentration. 
Key words: carob, in vitro, sucrose, light, etiolation, lenticel hypertrophy, 
lignification, hypolignification, Casparian bands 
Effect of sucrose on the growth of plant in vitro cultures bas been subject of numerous studies. 
In most species cultured in vitro optimal concentration of sucrose is 2-3 %. Apart from nutritive 
role sucrose has been recently attributed a regulatory role since changes in sucrose concentration 
can serve in gene activation and signal sensing (KocH & al., 1992, MITRA & al. 1995, SMEEKENS & 
RooK 1997). Increased sucrose nutrition can induce morphogenetic effects as for instance axillary 
bud formation in the rooting stage of Dracaenafragrans (VINTERHALTER & VINTERHALTER 1997). In 
carob sucrose together with light regulates leaf size (VINTERHALTER & VINTERHALTER 2001). It is well 
documented that sucrose stimulates xylogenesis in callus of angiosperms (WETMORE & RIER 1963) 
and in intemodes of Coleus (RIER & BESLOW 1967). WARREN WILSON & al. (1994) confirmed the 
stimulatory effect of exogenous sucrose on xylogenesis. However, the optimal sucrose concentration 
was species dependant. In letuce pith explants it was at 0.2 % and in tobacco pith explants at 3.0 % 
sucrose. 
Carob shoot cultures grown on low sucrose concentrations (2 % sucrose or less) exhibit lenticel 
hypertrophy a rare physiological disorder of in vitro cultures described also in Populus euphratica 
(LLEDO & al. 1995). Histological investigation showed that lenticels appear only on hypolignified 
intemodes (VINTERHALTER & al. 1997). Increased sucrose nutrition promotes the growth of carob 
shoot cultures (VrNTERHALTER 1998) and at the same tirne decreases the frequency of lenticel 
hypertrophy. Another treatment in which lenticel hypertrophy in carob was found to be absen was 
56 Acta Biologica Slovenica, 46 (2), 2003 
cultivation in the darkness which results in fom1ation of etiolated shoot cultures (VINTERHALTER & 
VINTERHALTER 2002). 
In the present study we investigated the histological structure of carob shoot cultures grown in 
conditions of high and low sucrose nutrition both in the light and darkness. We were particulary 
interested in a possible connection between lignification and lenticel hypertrophy. 
Material and methods 
Carob shoot cultures were established and maintained on MuRASH!GE & SKoOG (1962) medium 
with 2.25 µM BA and 0.5 µM IBA as previously described (VINTERHALTER & VINTERHALTER 2001). 
Treatments consisted ofmedia supplemented with 2 % (low) and 8 % (high) sucrose in the light and 
darkness. The light 46.5 µmol m·2s·1 was provided by cool white fluorescent lamps. All treatrnents 
lasted for 35 days. The material for histological study was prepared and stained according to JoHANSEN 
(1940). Shoots were fixed in 1 : 1 : 18 formalin-acetic acid- ethanol (FAA) embedded in the paraffine 
and cut on a rotary microtome in the 8-10 µm thick sections. Sections were double stained with 
safranine-light green or safranine-wasser blau. Presence oflignine was demonstrated in fresh material 
using band sections stained either with an acidified phloroglucinol or aniline sulphate. Hand sections 
of etiolated tissues were stained with Lugol-s reagent (JJK) in combination with acidified 
phloroglucinol or safranine. 
Results 
Experiments conducted in the light: At the end of subculture, shoot explants, initially consisting 
of an apical hud and 2-3 intemodes develop 5-8 new intemodes. Intemodes of the initial explant 
become basal intemodes whilst apical and central intemodes develop in the current subculture. 
Basal intemodes submersed into the medium produce a callus which appears in the endodermis and 
replaces ali cortical and surface tissues developing in a form of pseudo-cortex. 
Apical intemodes in transection have characteristic 5 !obed structure and maturation of vascular 
tissues in not yet complete. Central intemodes are circular in transection and have typical mature 
structure with well differentiated vascular tissues and two massive circumferential rings of lignified 
cells, outer sclerenchyma and inner xylem ring. Difference in color (sclerenchyma is more orange) 
indicates possible difference in lignine composition. Epidermal and subepidermal cells are red from 
accumulation of anthocyanin and resins. 
Basal intemodes of cultures grown on 2 % sucrose, typically undergo lenticel hypertrophy with 
massive surface proliferation of phelloderm cells. Inner tissues of such intemodes show severe 
hypolignification, meaning that the two massive rings of lignified cells (sclerenchyma and xylem) 
are reduced or missing. In conditions of low sucrose concentration lenticel hypertrophy is therefore 
always associated with hypolignification. The analyses of hand sectioned fresh material showed 
that in the earliest stages of lenticel hypertrophy sclerenchyma ring is ruptured or missing (Fig. 1). 
Frequency of lenticel hypertrophy significantly decreases if cultures are grown on the media 
with increased sucrose concentration (Fig. 5). Accumulation of anthocyanins and resins in epidermal 
and subepidermal cells is stimulated same as abundant formation of amyloplasts in all parenchymatous 
cells. In central and basal intemodes sclerenchyma ring is well developed and there is a massive 
formation of both phloem and xylem (Figs. 3, 4). 
B. Vinterhalter, D. Vinterhalter: Effect of sucrose nutrition on the histological structure ... 57 
Figure 1: Early stage of lenticel development on 2 % sucrose in light, basal intemodes, bar= 60 µm. 
Hand section of fresh material stained with Lugol and safranine. Sclerenchyma ring absent, xylem 
organised in srna! groups, prominet lenticel (hl). 
Figure 2: Transection of a dark grown etiolated shoot, 2 % sucrose, bar = 22 µm. Hand section of 
fresh material stained with Lugol and safranine. Endodermal cells with Casparian bands (cb), 
sclerenchyma ring absent and replaced with large, thin-walled parenchymatous cel!s. 
Figure 3: Transection of a basal intemode on 8 % sucrose in light, bar = 60 µm. Paraffine section 
stained with safranine- light green. Typical heavy lignified structure, lenticels absen. Epidermis and 
subpeidermis rich with anthocyanins an resins, circumferential sclerenchyma ring (se), phloem, 
cambal layer and a massive layer of lignified xylem (xy). 
Figure 4: Transection of a basal intemode on 8 % sucrose in light, bar = 22 µm. Hand section of 
fresh material stained with Lugol and safranine. Endodermis with amyloplast (starch sheet, ss), well 
deveoped sclerenchyma (se), phloem (fl) and xylem (xy) . 
58 Acta Biologica Slovenica, 46 (2), 2003 
Experiments conducted in the darkness: After a short transition period cultures became 
etiolated. Paraffine sections of etiolated cultures which were soft and spongy were successfully 
replaced with hand sectioned fresh material. 
Etiolation induced drastic changes in the shoot structure. Etiolated cultures are in general highly 
hypolignified. In the darkness no lignine is produced and rings of lignified sclerenchyma and xylem 
elements (characteristic for light conditions) are never formed. Xylem elements are rare and 
sclerenchyma layer is completely absent, replaced by large parenchymatic cells laying inner to the 
endodermis. Characteristic feature of this endodermal parenchyma are cells with Casparian bands 
which form a continuous or nearly continuous ring around the central cylinder (Fig. 2). 
Hypertrophied lenticels were never observed to appear in etiolated shoot cultures irrespectively of the 
concentration of sucrose in the medium. However, etiolated cultures transferred to light quick:ly became de-
etiolated and in favorable conditions (low sucrose in the medium) lenticel hypertrophy re-appeared. 
Fig 
-41) 
"'O 
o 
C ... ., -C 
., 
M ., 
.0 
C 
o 
>-
.C 
0. 
o ... -... ., 
0. 
>-
.C 
., 
o -C 
41) 
...J 
100 
90 
80 
70 
60 
50 
40 
30 
20 
10 
o 
o.o 2 4 6 8 10 12 14 16 
Sucrose, X 
Number of hypertrophied 
lenticels per internode 
0 0-2 ~ 3-10 ~ 10-30 • 30 > 
Figure 5: Effect of sucrose concentration on the number of hypertrophied lenticels formed on the 
first intemode. 
B. Vinterhalter, D. Vinterhalter: Effect of sucrose nutrition on the histological structure ... 59 
Discussion 
Hand sectioning of fresh material proved to be an exellent method for rapid investigation of 
lignified tissues in carob shoot cultures grown both in light and darkness. In etiolated shoot cultures 
it enabled us to observe Casparian bands. 
ARZEE & al. (1977) found that in carob seedlings phellogen formation starts at the end of the 
first year. In carob shoot cultures tissue differentiation is very fast and certain conditions may induce 
premature formation and hypertrophy of lenticels which we consider as a physiological disorder. 
What are the factors which induce this phenomenon? 
Our first studies showed that lenticel hypertrophy is connected to cytokinin concentration 
(VINTERHALTER & al. 1992) and restricted ventilation (VINTERHALTER & VINTERHALTER 1992). Absence 
of ventilation triggered lenticel formati on but only if cytokinins were present in the medi um, otherwise 
leaves were sheadeed and cultures perished. 
In some species of trees lenticel hypertrophy is a physiological adaptation to anoxia induced by 
flooding and mediated by ethylene (TANG & KozLOWSKI 1984). Process includes synthesis of ethylene 
precursor (ACC) in submersed tissues, its translocation through the vascular tissues and conversion 
into ethylene in aerated tissues above the line of submersion. 
Although lenticel hypertrophy in vitro has been described only in two species, Ceratonia siliqua 
(VINTERHALTER & AL. 1992) and Populus euphratica (LLEDO & al. 1995) there are notions that it is a 
more widespread phenomenon. Is it possible that in vitro conditions mimic flooding in some plants 
species? In carob lenticel hypertrophy always appears on the basal intemode, first intemode above 
the surface of the medium and than it spreads acropetally. 
Our studies showed that lenticel hypertrophy is affected by sucrose nutrition and light. High 
sucrose nutrition decreases frequency of lenticel hypertrophy whilst growth in darkness (etiolated 
cultures) completelly prevents it (VINTERHALTER 1998, VINTERHALTER & VINTERHALTER 2002). In both 
cases there are changes in the histological structure which affects the abundance of cells with lignified 
walls as reported here. In the first case lenticel hypertrophy does not occur whilst sclerenchyma ring 
is present. Sclerenchyma ring is a barrier which efficiently prevents lateral movement of water in 
stems. It seems that undeveloped sclerenchyma ring characteristic for low sucrose concentrations is 
the factor which stimulates lenticel hypertophy and which the high sucrose nutrition prevents. In the 
second case lenticel hypertrophy is absent although etiolated shoots contain no lignified cells. Here 
sclerenchyma ring although absent is functionally replaced with another barrier, ring of cells with 
Casparian bands which are known to restrict lateral translocation of water (KoLDA 1937). We thus 
suppose that lenticel hypertrophy in carob may be prevented by conditions which limit laterni 
translocation of water and solutes in shoots. 
Literature 
ARZEE T., E. ARBEL & L. COHEN 1977: Ontogeny of periderm and phellogen activity in Ceratonia 
siliqua L. Bot. Gaz. 138 (3): 329-333. 
JoHANSEN D.A. 1940: Plant microtechnique. McGraw-Hill. New York & London, 523 pp. 
KoCH K. E., K. D. NoLTE, E.R. DuKE, D. R. McCARTHY & W. T. AVIGNE 1992: Sugar levels modulate 
differential expression of maize sucrose synthase genes. Plant Cell 4: 59-69. 
KowA A. 1937: Zur Anatomie etiolierter und periodisch belichteter Pflanzen und iiber die Wirkung 
nachtriiglicher Kulturam Lichte. Bieh. Bot. Centralb. Abt. A., 57 (3): 319-380. 
LLEDO M.D., M.B.CRESPO & J.B. AMo-MARco 1995: The role of cytokinins and ethylene inhibitors 
on lenticel hypertrophy generation and ethylene production in in vitro cultures of Populus 
euphratica Olivier. Israel Journal of Plant Sciences 43: 339-345. 
60 Acta Biologica Slovenica, 46 (2), 2003 
MITRA S., K. SuzuKJ-Funr & K NAKAMURA 1995: Sugar-inducible expression of a gene for ±-amylase 
in Arabidopsis thaliana. Plant Physiol. 107: 895-904. 
MuRASHIGE T. & F. SKooG 1962: A revised medium for rapid growth and bioassays with tobacco 
tissue cultures. Physiol. Plant. 15: 473-497. 
RrnR J.P. & D.T. BESLOW 1967: Sucrose concentration and differentiation of xylem in callus. Bot. 
Gaz. 128: 73-77. 
SMEEKENS S. & F. RooK 1997: Sugar sensing and sugar mediated signal transduction in plants. Plant 
Physiol. 113: 293-304. 
TANG Z.C. & T.T. KozwwsK1 1984: Ethylene production and morphological adaptation of woody 
plants to flooding. Can. J. Bot. 62: 1659-1664. 
V!NTERHALTER B. 1998: Uticaj svetlosti i saharoze na rastenje i graDu izdanaka rogač (Ceratonia 
siliqua L.) u kulturi in vitro. Ph.D. thesis. Univ. Belgrade, 134 pp. 
V1NTERHALTER D., D. GRUBIŠIC, D. Bomvic-CvETIC & S. BUDIMIR 1992: Lenticel hypertrophy in shoot 
cultures of Ceratonia siliqua L. Plant Celi Tiss.Org.Culture 31: 111-114. 
VINTERHALTER D. & B. VJNTERHALTER 1992: Factors affecting in vitro propagation of carob ( Ceratonia 
siliqua L.). Arch. Biol.Sci. 44: 177-186. 
VINTERHALTER D. & B. VINTERHALTER 1997: Micropropagation of Dracaena Species. Biotechnology 
in Agriculture and Forestry Vol. 40, High-Tech and Micropropagation VI, editor Y.P.S. 
Bajaj , Springer, 131-146. 
VINTERHALTER B., D. VINTERHALTER & M. NEšKovic 1997: Effect of sucrose nutrition and light on the 
histological structure of carob (Ceratonia siliqua L.) shoot cultures. Procc. 3n1 ICFWST 
Conference, Belgrade-Goč, 37-42. 
VINTERHALTER B. & D. VrNTERHALTER 2001: Effect of irradiance, sugars and nitrogen on leaf size of 
in vitro grown Ceratonia siliqua L. Biologia Plantarum 44:185-188. 
VJNTERHALTER D. & B. VINTERHALTER 2002: Etiolated, dark grown shoot cultures and light emitting 
diodes - New approaches in plant biotechnology. In" Plant Physiology in the New Millenium 
(eds):. Quarrie S., Krstic B, Janjic V. Yugoslav Soc. Plant Physiol. Belgrade, 95-99. (ISBN 
86-7384-011-2). 
WARREN WILSON J.W., L.V. ROBERTS, P.M.W. WARREN WILSON & P.M„GRESSHOFF 1994: Stymulatory 
and inhibitory effects of sucrose concentration on xylogenesis in lettuce pith explants; 
possible mediation by ethylen biosynthesis. Ann. Bot. 73: 65-73 . 
WETMORE R.H. & J.P. RIER 1963: Experimental induction ofvascular tissues in callus of angiosperms. 
Amer.J.Bot. 50: 418-430. 
NAVODILA AVTORJEM 
l. Vrste prispevkov 
a) ZNANSTVENI ČLANEK je celovit opis originalne raziskave in vključuje teoretični pregled 
tematike, podrobno predstavljene rezultate z diskusijo in sklepe ter literatumi pregled: shema 
IMRAD (Introduction, Methods, Results And Discussion). Dolžina članka, vključno s tabelami, 
grafi in slikami, ne sme presegati 15 strani; razmak med vrsticami je dvojen. Recenzirata ga dva 
recenzenta. 
b) PREGLEDNI ČLANEK objavi revija po posvetu uredniškega odbora z avtorjem. Število 
strani je lahko večje od 15. 
c) KRATKA NOTICA je originalni prispevek z različnih bioloških področij (sistematike, bio-
kemije, genetike, mikrobiologije, ekologije itd.), ki ne vsebuje podrobnega teoretičnega pregleda. 
Njen namen je seznaniti bralca s preliminarnimi ali delnimi rezultati raziskave. Dolžina na sme 
presegati 5 strani. Recenzira ga en recenzent. 
d) KONGRESNA VEST seznanja bralce z vsebinami in sklepi pomembnih kongresov in po-
svetovanj doma in v tujini. 
e) DRUŠTVENA VEST poroča o delovanju slovenskih bioloških društev. 
2. Originalnost prispevka 
Članek, objavljen v reviji Acta Biologica Slovenica, ne sme biti predhodno objavljen v drugih 
revijah ali kongresnih knjigah. 
3. Jezik 
Teksti naj bodo pisani v angleškem jeziku, izjemoma v slovenskem, če je tematika zelo lokalna. 
Kongresne in društvene vesti so praviloma v slovenskem jeziku. 
4. Naslov prispevka 
Naslov (v slovenskem in angleškem jeziku) mora biti kratek, informativen in razumljiv. Za 
naslovom sledijo imena avtorjev in njihovi polni naslovi (če je mogoče, tudi štev. faxa in e-mail). 
5. Izvleček - Abstract 
Podati mora jedrnato informacijo o namenu, uporabljenih metodah, dobljenih rezultatih in 
zaključkih. Primerna dolžina za znanstveni članek naj bo približno 250 besed, za kratko notico pa 
100 besed. 
6. Ključne besede - Keywords 
Število naj ne presega 10 besed; predstavljati morajo področje raziskave, obravnavane v članku. 
Člankom v slovenskem jeziku morajo avtorji dodati ključne besede v angleškem jeziku. 
7. Uvod 
Nanašati se mora le na tematiko, ki je predstavljena v članku ali kratki notici . 
8. Slike in tabele 
Tabele in slike (grafi, dendrogrami, risbe, fotografije idr.) naj v članku ne presegajo števila 10, 
v članku naj bo njihovo mesto nedvoumno označeno. Ves slikovni material naj bo oddan kot fizični 
original (fotografija ali slika). Tabele in legende naj bodo tipkane na posebnih listih (v tabelah naj 
bodo le vodoravne črte). Naslove tabel pišemo nad imi, naslove slik in fotografij pod njimi. Naslovi 
tabel in slik ter legenda so v slovenskem in angleškem jeziku. Pri citiranju tabel in slik v besedilu 
uporabljamo okrajšave (npr. Tab. 1 ali Tabs. 1-2, Fig. 1 ali Figs. 1-2; Tab. 1 in Sl. 1). 
9. Zaključki 
Članek končamo s povzetkom glavnih ugotovitev, ki jih lahko zapišemo tudi po točkah . 
10. Povzetek - Summary 
Članek, ki je pisan v slovenskem jeziku, mora vsebovati še obširnejši angleški povzetek. Velja 
tudi obratno. 
11. Literatura 
Uporabljene literaturne vire citiramo med tekstom. Če citiramo enega avtorja, pišemo ALLAN 
(1995) ali (ALLAN 1995), če sta dva avtorja (ThINAJSTIC & FRANJIC 1994), če je več avtorjev (PuLLIN 
& al. 1995). Kadar navajamo citat iz večih del hkrati, pišemo (HoNSIG-ERLENBURG & al. 1992, 
WARD 1994a, ALLAN 1995, PuLLIN & al. 1995). V primeru, če citiramo več del istega avtorja, 
objavljenih v enem letu, posamezno delo označimo s črkaini a, b, c itd. (W ARD 1994a,b ). Če navajamo 
dobesedni citat, označimo dodatno še strani: TOMAN (1992: 5) ali (TOMAN 1992: 5-6). Literaturo 
uredimo po abecednem redu, začnemo s priimkom prvega avtorja, sledi leto izdaje in naslov članka, 
mednarodna kratica za revijo (časopis), volumen poudarjeno, številka v oklepaju in strani. Npr.: 
HoNsro-ERLENBURG W., K. .KRAINER, P. MILDNER & C. WIESERR 1992: Zur Flora und Fauna des 
Webersees. Carinthia II 182/102 (1): 159-173. 
TRINAJSTIC I. & J. FRANJIC 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) 
M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253-256. 
WARD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10--15. 
WARD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10-15. 
Knjige, poglavja iz knjig, poročila, kongresne povzetke citiramo sledeče: 
ALLAN J. D. 1995: Stream Ecology. Structure and Function ofRunning Waters, 1st ed. Chapman & 
Hall, London, 388 pp. 
PULLIN A. S., I. F. G. McLEAN & M. R. WEBB 1995: Ecology and Conservation of Lycaena dispar: 
British and European Perspectives. In: PuLLIN A. S. (ed.): Ecology and Conservation of 
Butterflies, 1st ed. Chapman & Hall, London, pp. 150--164. 
TOMAN M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s po-
svetovanja DZVS, Gozd Martuljek, pp. 17. 
12. Format in oblika članka 
Članek naj bo poslan v elektronski obliki v Microsoft Word formatu (doc) ali kot obogateno 
besedilo (rtf) v pisavi "Times New Roman CE 12" z dvojnim medvrstnim razmakom in levo poravnavo 
ter s 3 cm robovi naA4 formatu. Odstavki naj bodo med seboj ločeni s prazno vrstico. Naslov članka 
in poglavij naj bodo pisani krepko in v velikosti pisave 14. Vsa latinska imena morajo biti napisana 
ležeče. V besedilu navedemo uporabljene nomenklatume vire. Tabele in slike so posebej priložene 
tekstu. Glavnemu uredniku je potrebno oddati original, dve kopiji in disketni zapis v elektronski 
obliki na disketi 3,5", CD-romu ali kot priponko elektronske pošte (slednjega odda avtor po oprav-
ljenih strokovnih in jezikovnih popravkih). 
13. Recenzije 
Vsak znanstveni članek bosta recenzirala dva recenzenta (en domači in en tuji), kratko notico 
pa domači recenzent. Avtor lahko v spremnem dopisu predlaga tuje recenzente. Recenziran članek, 
ki bo sprejet v objavo, popravi avtor. Po objavi prejme 50 brezplačnih izvodov. V primeru zavrnitve 
se originalne materiale vrne avtorju skupaj z negativno odločitvijo glavnega urednika. 
INSTRUCTIONS FOR AUTHORS 
l. Types of Articles 
a) SCIENTIFIC ARTICLES are comprehensive descriptions of original research and include a 
theoretical survey of the topic, a detailed presentation of results with discussion and conclusion, 
and a bibliography according to the IMRAD outline (Introduction, Methods, Results, and 
Discussion). The length of an article including tables, graphs, and illustrations may not exceed 
fifteen (15) pages; lines must be double-spaced. Scientific articles shall be subject to peer review 
by two experts in the field. 
b) REVIEW ARTICLES will be published in the joumal after consultation between the editorial 
board and the author. Review articles may be longer than fifteen (15) pages. 
c) BRIEF NOTES are original articles from various biological fields (systematics, biochemistry, 
genetics, microbiology, ecology, etc .) that do not include a detailed theoretical discussion. Their 
aim is to acquaint readers with preliminary or partial results of research. They should not be longer 
than five (5) pages. Brief note articles shall be subject to peer review by one expert in the field. 
d) CONGRESS NEWS acquaints readers with the content and conclusions of important 
congresses and seminars at home and abroad. 
e) ASSOCIATION NEWS reports on the work of Slovene biology associations. 
2. Originality of Articles 
Manuscripts submitted for publication in Acta Biologica Slovenica should not contain previously 
published material and should not be under consideration for publication elsewhere. 
3. Language 
Articles and notes should be submitted in English, or as an exception in Slovene if the topic is 
very local. As a rule, congress and association news will appear in Slovene. 
4. Titles of Articles 
Titles (in Slovene and English) must be short, informative, and understandable. The title should be 
followed by the name and full address of the author (and if possible, fax number and e-mail address). 
5. Abstract 
The abstract must give concise information about the objective, the methods used, the results 
obtained, and the conclusions. The suitable length for scientific articles is approximately 250 words, 
and for brief note articles, 100 words. 
6. Keywords 
There should be no more than ten ( 1 O) keywords; they must reflect the field of research covered 
in the article. Authors must add keywords in English to articles written in Slovene. 
7. Introduction 
The introduction must refer only to topics presented in the article or brief note. 
8. Illustrations and Tables 
Articles should not contain more than ten (10) illustrations (graphs, dendrograms, pictures, photos 
etc.) and tables, and their positions in the article should be clearly indicated. All illustrative material 
should be provided as physical originals (photographs or illustrations). Tables with their legends 
should be submitted on separate pages (only horizontal lines should be used in tables). Titles oftables 
should appear above the tables, and titles of photographs and illustrations below. Titles of tables and 
illustrations and their legends should be in both Slovene and English. Tables and illustrations should 
be cited shortly in the text (Tab. 1 or Tabs. 1-2, Fig. 1 or Figs. 1-2; Tab. 1 and Sl. 1). 
9. Conclusions 
Articles shall end with a sumrnary of the main findings which may be written in point form. 
10. Summary 
Articles written in Slovene must contain a more extensive English sumrnary. The reverse also applies. 
11. Literature 
References shall be cited in the text. If a reference work by one author is cited, we write ALLAN 
(1995) or (ALLAN 1995); if a work by two authors is cited, (TRINAJSTic & FRANnc 1994); if a work 
by three or more authors is cited, (PuLLIN & al. 1995); and if the reference appears in several works, 
(HoNSIG-ERLENBURG & al. 1992, WARD 1994a, ALLAN 1995, PULLIN & al. 1995). If several works by 
the same author published in the same year are cited, the individual works are indicated with the 
added letters a, b, c, etc.: (WARD 1994a,b). If direct quotations are used, the page numbers should 
be included: TOMAN (1992: 5) or (TOMAN 1992: 5---6). 
The bibliography shall be arranged in alphabetical order beginning with the surname of the first 
author followed by the year of publication, the title of the article, the intemational abbreviation for the 
journal (periodical), the volume (in bold print), the number in parenthesis, and the pages. Examples: 
HoNSIG-ERLENBURG W., K. KRAINER, P. MILDNER & C. WIESER 1992: Zur Flora und Fauna des We-
bersees. Carinthia II 182/102 (1) : 159-173. 
TRINAJSTic I. & J. FRANnc 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) 
M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253-256. 
WARD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10-15. 
WARD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10-15. 
Books, chapters from books, reports, and congress anthologies use the following forms : 
ALLAN J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & 
Hall, London, 388 pp. 
PULLIN A. S., I. F. G. McLEAN & M. R. WEBB 1995: Ecology and Conservation of Lycaena dispar: 
British and European Perspectives. In: PULLIN A. S. (ed.): Ecology and Conservation of 
Butterflies, 1st ed. Chapman & Hall, London, pp. 150-164. 
TOMAN M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s po-
svetovanja DZVS, Gozd Martuljek, pp. 17. 
12. Format and Form of Articles 
Articles should be send as Microsoft Word document (doc) or rich text format (rtf) using 
"Times New Roman CE 12" font with double spacing, align left and margins of 3 cm on A4 pages. 
Paragraphs should be separated with an empty line. The title and chapters should be written bold in 
font size 14. All scientific names must be properly italicized. Used nomenclature source should be 
cited. Tables and illustrations shall accompany the texts separately. The original manuscript, two 
copies, and an electronic copy ona 3.5" computer diskette, on CD-ROM or by e-mail must be 
given to the editor-in-chief. All articles must be proofread for professional and language errors 
before submission. 
13. Peer Review 
All Scientific Articles shall be subject to peer review by two experts in the field (one Slovene 
and one foreign) and Brief Note articles by one Slovene expert in the field. Authors may nominate 
a foreign reviewer in an accompanying letter. Reviewed articles accepted for publication shall be 
corrected by the author. Authors shall receive fifty (50) free copies of the joumal upon publication. 
In the event an article is rejected, the original material shall be retumed to the author together with 
the negative_deterrnination of the editor-in-chief.