PHYSIOL. PLANTARUM 70: 664-672.

Copenhagen 1987

Ribulose-l,5-bisphosphate carboxylase activity and influence of air

pollution in spruce

Manfred Weidner and Monika Kraus

Weidner, M. and Kraus, M. 1987. Ribulose-1,5-bisphosphate carboxylase activity

and influence of air pollution in spruce. - Physiol. Plantarum 70: 664-672.

Methods were established, whieh render possible a simultaneous determination of ri-

bulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity and chlorophyll
content of Norway spruce (Picea abies Karst.) needles from a detergent-containing
aqueous crude extract. Spruce RuBP carboxylase was tentatively characterized with
regard to kinetie properties. Recovery experiments employing purified wheat RuBP
carboxylase proved quantitative extraction of the enzyme from spruce foliage. F'ive
timber stands consisting of 35-62 years old spruce, two of which exhibited the typical
symptoms of recent spruce decline, were compared. For the needle generations 1 to 4
the enzyme aetivities as well as chlorophyll and protein concentrations were deter-
mined. The results do not indicate an involvement of RuBP carboxylase in spruce de-
cline.
Additional key words - Chlorophyll, Picea abies, spruce decline.

M. Weidner (corresponding author) and M. Kraus, Botanisches Inst. der Utiiv. Koln,
Gyrhofstrasse 15, D-5000 Koln 41, West Germany.

The lack of information about conifer mesophyll en-

Introduction
Increasing interest is focussed on the process of pho-
tosynthesis in conifers, because of the deteriorating ef-
fects of airborne pollution on assimilatory structures
and functions. Photosynthetic suppression is conside-
red an element in the process of forest dieback, ob-
served at present in many areas in Germany and else-
where (Keller 1978, Lichtenthaler and Buschmann
1983, Elstner and Osswald 1984). One possible target of
the airborne pollutants - i.e. SO2, nitrogen oxides, O,,
free radicals etc. - is the enzymological apparatus of
photosynthetic COj fixation and especially RuBP car-
boxylase, the entrance enzyme of the Calvin cycle (Zie-
gler 1972, Wellburn et al. 1976, Gezelius and Hallgren
1980, Pell and Pearson 1983, Parry et al. 1984). How-
ever, earlier results on inhibition of RuBP carboxylase
activity and on decreased levels of fraction I protein
caused by SO^ and O, have mostly originated from stud-
ies on angiosperm assimilatory tissue and thus do not
necessarily warrant conclusions with regard to the re-
cent and complex plant physiological syndrome of
spruce decline.
zymes is partly due to high concentrations of phenolic
compounds (i.e. tannins), released from membrane ves-
icles during the process of homogenization and extrac-
tion and acting as potent enzyme inhibitors (Soikkeli
1978). Some of these difficulties have been overcome
and RuBP carboxylase activity has been assayed re-
cently in extracts from Pinus silvestris (Gezelius and
Hallen 1980, Gezelius 1986) and other coniferous trees
(Beadle et al. 1983), but not from adult Picea abies.
Employing the non-ionic detergent Triton X-100 and
insoluble PVP (Loomis and Bataille 1966) we succeeded
in developing a rapid and reliable method for the ex-
traction and determination of carboxylating activity of
RuBP carboxylase from Norway spruce needles, where
the reference parameter "chlorophyll concentration"
was measured concomitantly from the same crude aque-
ous extract. RuBP carboxylase activities are presented
for needles of different age and different origin. Five lo-
cations in the field were chosen in order to compare
spruce stands, which either exhibited the symptoms ty-
pical for the recent forest decline syndrome (as it is ex-
pressed in Picea abies) or consisted of apparently unaf-

Received 9 March, 1987; revised 16 April, 1987

Phvsiol. Plantarum 7(1. 1987

664
 fected trees which did not exhibit flecking, bronzing or
necrosis of the needles.
Abbreviations - CMC, critical micellar concentration; DTT, di-
thiothreitol; GAPDH, glyceraldehyde phosphate dehydroge-
nase; HI and H2, harvest 1 and 2, respectively; PGK, phos-
phoglycerate kinase; PMSF, phenylmethylsulfonylfluoride;
PVP, polyvinylpyrrolidone; RuBP, ribulose-'l,5-bisphosphate.
Materials and methods
Plant material
Branches from Norway spruce trees, Picea abies (L.)
Karsten, were harvested in the field and sectioned into
side branches of different age: The spruce trees were
classified healthy or sick and one first order side branch
was gathered from about 5 m above ground level and
sectioned. Needles were then collected in an unselective
(bias-free) manner: From trees classified as "injured",
needles exhibiting chlorotic tips, discoloration etc. were
not favoured; on the other hand these kind of needles
were not discarded when found on assumedly healthy
trees. From the side branches, the needle generations
1-4 were separated. Needles were gathered either by
dipping the branch sections into liquid nitrogen, where-
upon the needles could easily be stripped, or - in the
field - they were cut off with scissors, sampled,
weighted and frozen in liquid nitrogen for transporta-
tion to the laboratory.
Extraction procedure
In general, samples of 1 g fresh weight were powdered
under addition of 1 g quartz sand (analytical grade) in a
precooled mortar in liquid nitrogen. The deep-frozen
pulverized needles were transferred into a second ice-
cold mortar, which contained the extraction medium.
The extraction medium, pH 7.2 (18 ml) was composed
of 100 mM Tris-HCI, 1.5 mM EDTA. 1.5 mM sodium
thioglycolate, 5 mM D-isoascorbic acid, 1.5 mM MgCl,,
2.5% (v/v) Triton X-100, 10% (v/v) glycerol and insol-
uble PVP (2 g prewashed Polyclar AT; Serva, Heidel-
berg, FRG). The extraction medium was prepared the
day before use and stored at 4°C in order to allow for
maximum hydration of the PVP. The frozen needle
powder was rapidly mixed with the extraction medium
and thoroughly blended and ground for 3 min. The re-
sulting slurry was centrifuged (20 min, 40 000^). This
and the following analytical procedures were carried out
at 4°C. The volume of the supernatant - the crude en-
zyme extract - was measured and two 1.5 ml-aliquots
were freed from traces of suspended/floating material
by a short second centrifugation step in a table top cen-
trifuge (Beckman Microfuge B, 8 700^, 1 min). Activ-
ity of RuBP carboxylase, protein and chlorophyll were
assayed directly in this purified supernatant.
The residual extract volume, enclosed in the volu-
minous pellet of the first centrifugation step was deter-
mined by weighing the sediment before and after drying
•43 Physiol. Plantarum 70. 1987
at 80°C for 48 h. The difference in weight, expressed as
ml (assuming 1 g = 1 ml), was added to the supernatant
volume, thus obtaining a true total volume of the ex-
tract. On basis of this value the data were calculated.
Assay of RuBP-carboxylase (EC 4.L1.39) activity
Preincubation (= activation) and assay temperature was
30°C. The assay mixture (260 fxl) contained: 210 mM
Tris-HCI, pH 8.3, 7.7 mM DTT, 7.7 mM MgCU, 100
mM NaH'-'CO, (3.7-11.1 kBq), 2.3 mM Na,-RuBP
(Sigma, Deisenhofen, FRG) and 60 |il enzyme extract.
The exact amount of radioactivity employed was routin-
ely determined by counting l()-|il aliquots of the
NaH'''CO3 solution. The enzyme was preincubated for 5
min in the reaction mixture prior to the addition of
RuBP, which was used subsequently to start the car-
boxylation reaction. Blanks contained no RuBP and 120
|xl Tris-HCI buffer instead of 110 \x\. After 8 min the re-
action was terminated by adding 200 pil 33% acetic acid.
In order to quantitatively remove non-incorporated
'"•CO,, two aliquots (150 (xl) of each acidified sample
(460 [il) were transferred into 6-ml scintillation vials and
dried under an infrared-lamp. The residue was redis-
solved in 200 ^l 10% acetic acid, placing the vials on a
shaker for 1 h. Finally, scintillation fluid (2 ml; Super-
solve - Zinsser, Frankfurt, FRG) was added and acid-
stable radioactivity was counted. In some experiments a
coupled assay for RuBP carboxylase activity was em-
ployed with PGK/GAPDH (NADH) as auxiliary en-
zymes. The assay mixture (1.3 ml) consisted of 140 mM
Tris-HCI, pH 8.1, 7.7 mM MgCU, 7.7 mM DTT, 30 mM
NaHCO,, 4 mM ATP (adjusted to pH 7.5), 0.26 mM
NADH (adjusted to pH 7.5), 10 ^1 PGK/GAPDH (0.1
mg ml"' enzyme-mix, Boehringer. Mannheim. FRG), 1
mM Na4-RuBP and 300 ^il enzyme extract. Preincuba-
tion and start of the reaction were as described above.
Absorbance at 366 nm was recorded. The true concen-
tration of the purchased RuBP preparation was control-
led routinely, employing a purified RuBP carboxylase
preparation (Weidner and Fehling 1985).
Determination of chlorophyll and protein concentrations
Aliquots (200 |al) of the crude extract (purified super-
natant, see above) were made 80% with respect to ace-
tone (analytical grade). The precipitate was sedimented
by centrifugation (8 700 g, 1 min) and total chlorophyll
concentration was measured in the supernatant accord-
ing to Arnon (1949).
The protein concentration was determined according
to Bradford (1976). Aliquots (1000 |il) of the crude ex-
tract (purified supernatant, see above) were mixed with
100 III cold 10% TCA. The protein was precipitated
(8700 g. 1 min), the pellet rinsed once with 1000 ^il 5%
TCA and recentrifuged. The sediment was resuspended
in 500 \i\ 1% NaOH. Aliquots (100 pil) were thoroughly
mixed with the Coomassie reagent (5 ml). Absorbance
665
 General sampling scheme
Harvest 1 Harvest 2
VI^ [I-VUB II-VIIQ* II-VUD [I-VIl£* 1
^ • • ' ^ Y Y Y Y
1 2 3 i 4 i 1 2 3 A
1 1 1
5g5g 5g5g
I I I
5g 5g
I I
5g 5g 5g 5g i 4
Y vv
15g 15g

^ ^ / / \ \ ^ \
Ig Ig Ig 1g Ig Ig Ig Ig Ig Ig 1g Ig

' I. "- - ^iHV-VU

_—-—
r' + • • •
*^11A1 ^2 [A] '^31A] Y 4[/

^AlHarvil ^ilHarvil ^AlHarv2] ^4[Harv.21

' _ _

XA X^
Fig. 1. Sampling scheme for field harvest of spruce needle samples. Details of the procedure are given in the text. Geographic lo-
cations: A) Arnsberg. Ruhr-valley, northern Sauerland (8°5'E. 5r22.3'N, altitude: 260 m above sea level). Age (of spruce stand):
31 years; Fl (= harvest): 2 and 18 Sep. 1985. B) Hilchenbach, ridge of the Rothaargebirge (8°12'E/50°55'N/640 m). Age: 35 years;
H:'2()Aug./16Sep. 1985. C*) Lammersdorf, northwestern Eifel (6°17'E/50°39.5'N/560 m). Age: 68 years; H: 23 July/12 Sep. 1985.
D) MiJnstereifel. northeastern Eifel (6°37'E/50°31.5'N/48() m). Age: 35 years; H: 5 Aug./17 Sep. 1985. E*) Veldrom, western
slope of the Eggegebirge (8°57'E/51°5()'N/400 m). Age: 45 years; H: 11 Sep. 1985. The "*'" indicates spruce stands with visible
damage symptoms as described in the text. Soil- and climatic data are given by Kraus (1986. Diplomarbeit, University of Koln
Koln, FRG).

at 595 nm was read after 5 min. BSA (fraction V,
Sigma, Deisenhofen, FRG) served as a protein stan-
dard.
Enzyme, chlorophyll and protein measurements were
run in duplicate from each extract. Mean values are
based on 4-8 extractions, in field experiments 8 extrac-
tions were always made. Significance was tested at the
5"/> level throughout.
Outline of field experiments
The sampling and randomization scheme for the field
experiments is depicted in Fig. 1. Samples were taken
twice (harvest 1 and 2) from each spruce stand between
June and September. Five locations were chosen (A, B,
C*, D, E*) and at each location 6 trees were selected
and marked. The 6 trees were divided in two subgroups,
comprising 3 trees each. From each tree, 5-g portions of
needles were harvested. Needle generations 1 (current
needles), 2, 3, and 4 were included. The needle portions
of trees I-III and IV-VI, respectively, were mixed thor-
oughly to make up two 15-g samples from which then 8
1-g samples were taken at random. These were frozen in
liquid nitrogen and analyzed later on. The analytical
data were combined to give the mean values X (e.g.
^4(1-111)^ mean of needle generation 4, trees I, II, III).
The two X-values were combined to form Y for all 6
trees, provided and X,iv_vii were not significantly
different on the 95 %-level (2P = 0.05). Thus, Y are the
mean values for each needle generation and location
(e.g. Y4,^): mean value of needle generation 4, location
A). The Y-values of needle generations 2, 3, 4 were
combined in order to obtain an average Z value of
RuBP carboxylase activity etc. for each spruce stand,
either for harvest 1 (H 1) or H 2. The first needle gener-
ation was omitted here for reasons specified below. Fi-
nally, the two Z-values were combined in order to form
one general mean value X for each harvesting location
(e.g. X,,,). provided the two Z-values were not signifi-
cantly different on the 95% level. On basis of these val-
ues the locations A, B, C*, D and E* are compared in
Tab. 4. Further, the 4 needle generations are compared.
In this case the Z values for the different needle gener-
ations (e.g. Z4,Hi) and Z4(n^)), comprising all 5 locations,
were combined to give the general mean value X for
each needle generation (e.g. X4). On basis of these val-
ues conclusions about the influence of needle age on
RuBP carboxylase activity etc. were drawn (Tab. 3).
This rather elaborate sampling scheme was executed,
because no experiences were available as to the biologi-
cal variability of samples from 35- to 60-year-old spruce
trees. Therefore we had to make sure that data were
pooled only after a detailed statistical evaluation had
been carried out. Hence, each step was accompanied by
statistical treatment, employing the software pro-
gramme Abstat (Anderson-Bell 1982, Canon City. CO,

666 Phvsiol. Plantarum 70. 1987

Tab. 1. Effects on RuBP carboxylase of various supposedly shown in Fig. 2. A concentration of 2.5% Triton X-100,
protective compounds included in the extraction medium. The as apphed routinely, is on the safe side and still does not
constituents of the extraction buffer are listed in Materials and
methods. Experiment 1 was performed, omitting Triton X-KM), cause any enzyme or pigment destruction. Table 1 docu-
insoluble PVP and glycerol. In experiment 2b-2f the com- ments the protection effect by Polyclar AT. The other
pounds listed were added to the extraction medium used in ex- compounds listed, known to protect against enzyme in-
periment 2a. The effect of insoluble PVP without addition of activation by phenolic compounds, did not further en-
Triton X-100 is shown in Fig. 2.
hance the activity of the enzyme. However, without ad-
Composition of RuBP carboxylase dition of Triton X-100 Polyclar AT yielded only ca 15%
extraction medium activity, of the RuBP carboxylase activity (Fig. 2).
nmol CO2 ml ' min ' Using purified RuBP carboxylase from wheat leaves
(Weidner and Fehling 1985) as an internal standard, we
1. Extraction buffer 0,2 could demonstrate that the level of enzyme activity, at-
2. a) Extraction buffer -1- Triton X-lOO
(2.5% w/v) + insoluble PVP tainable by the extraction procedure used here agrees
(Polyclar AT; 10%, w/v) 13,9 well with the in vivo total RuBP carboxylase activity
b) -(- soluble PVP 13 000 (10%, w/v) 15.1 present in the spruce needles: The mixing experiment
c) -1- BSA (1%, w/v) 12.8 (Tab. 2) yielded complete recovery of the added wheat
d) + glyeerol (10%, v/v) 17.3 enzyme activity. Therefore, activity values presented
e) + PEG 200 (10%. v/v) 17.1
f) -\- proline (1%, w/v) 16.2 here are indeed valid, provided the stability properties
of wheat and spruce RuBP carboxylase are similar. A
reduction of maximum specific activity by endopro-
teases, which strongly bind to RuBP carboxylase during
the extraction procedure, cannot be excluded, however
USA) for executing two-way analysis of variance. For t- (Rosichan and Huffacker 1984, Servaites 1985).
tests and F-tests the 95% level of significance was ap- Extract stability with regard to RuBP-carboxylase ac-
plied throughout. The investigation was carried out in tivity was rather poor. Even at storage temperature of
summer 1985. 0°C, carboxylation rates declined with an estimated
half-life of ca 2 h. Whether endoproteolysis (Garcia-
Martinez and Moreno 1986) or other compounds, toxic
to RuBP carboxylase, caused this activity loss is un-
Results clear. The protease inhibitor PMSF proved ineffective
in stabilizing spruce RuBP carboxylase (results not
RuBP-carboxylase assay shown). In conclusion, it was imperative to minimize
A realistic yield of RuBP-carboxylase activity could and standardize the time intervals of the extraction and
only be extracted from spruce needles in the presence of assay procedures.
insoluble PVP and Triton X-100 (Tab. 1). The efficiency Linearity of the carboxylation reaction with time was
of the non-ionic detergent Triton X-100 to extract RuBP maintained for ca 8 min although a slightly higher re-
carboxylase and solubilize chlorophyll quantitatively is action rate could occur within the first min (Fig. 3). The

Fig. 2. Extraction of RuBP

carboxylase and chlorophyll
from spruce needles by
increasing concentrations of
Triton X-100. Other
constituents of the
extraction medium: see
Materials and methods. 0.5 1 1.5 2.5
Means ±SD. n = 4. TRITON X-1Q0 1%1

4.T Phvsiol. Plantarum 7t). 19X7 667

Tab. 2. Mixing experiment for testing RuBP carboxylase re-
covery. A known amount of purified wheat RuBP carboxylase
was added to spruce needles prior to homogenization (1). Con-
currently, spruce and wheat RuBP carboxylase were assayed
separately (2 and 4). From these data the recovery of wheat en-
zyme activity and of extractable protein (fraction 1-protein)
was calculated (3). Also given is %-recovery (5). Enzyme ac-
tivities were measured with PGK/GAPDH as auxilliary en-
zymes.
Source of enzyme RuBP carboxylase Protein
activity, content,
nmol CO, ml"' ' mg ml '
1) wheat + spruce measured 8 100 0.85
2) spruce measured 5 700 0.70
3) wheat calculated I minus 2 2 400 0.15
4) wheat measured 2 200 0.18
5) 3 expressed as percentage of 4 109% 83%
slow-down of the reaction rate was probably not due to
substrate deprivation or product inhibition, since pu-
rified wheat RuBP carboxylase exhibited linearity for 30
min under the same conditions. When added to the
spruce extract, however, wheat carboxylase was also
unstable, indicating the presence of an inhibitory com-
pound in the spruce extract. None of the compounds
listed in Tab. 1 could extend the linear period.
Optimization of test conditions required an evalu-
ation of the dependence of the carboxylation reaction
on substrate concentrations, pH and temperature. The
kinetic data are summarized in Fig. 4. The kinetics for
HCO3 (CO2) was sigmoidal as indicated by a Hill-con-
stant of n^ = 1.33 (Fig. 4a and inset). This anomaly is
observed frequently with RuBP carboxylase: At low bi-
carbonate (CO,) concentrations the enzyme is not fully
activated when - as in our case - preincubation takes
place at the same concentrations as used subsequently
in the assay (Lorimer et al. 1977). By extrapolation
from V^.,j a value for an apparent K^ of 7.8 mM can be
roughly estimated for bicarbonate, which equals 0.14
mM for CO, [Umbreit et al. 1951; pK' (30°C) = 6.348].
Thus, in routine tests 100 mM bicarbonate (as applied)
ensures saturating conditions and the enzyme presum-
edly was in the fully activated state. Michaelis-Menten
kinetics were obtained for RuBP (at pH 8.3, 100 mM bi-
carbonate), yielding an apparent K^-value of 0.43 mM
(Fig. 4b).
The pH optimum (at 7.7 mM Mg'^) was at pH 8.1-8.3
(Fig. 4c). The apparent free enthalpy of activation (Ar-
rhenius E^) was determined as 76 kJ mol"' (Fig. 4d).
Temperature stability extended to ca 30°C.
Preliminary experiments with different needle gener-
ations showed that current needles, collected about 1
month after emergence, exhibited only 35% of the
RuBP carboxylase activity found in older needles when
compared on a chlorophyll basis. While it is possible
that carboxylating activity is substantially lower in vivo
668
in those immature needles, it can not be excluded that
inhibitory compounds are distributed unequally in nee-
dles of different age. Whether or not the extraction pro-
cedures are also appropriate for young needles was
therefore tested in another mixing experiment. Current
needles did not affect RuBP-carboxylase activity of ol-
der needles or vice versa when both were co-homo-
genized and extracted (results not shown).
Field experiments
With regard to the measurements of RuBP carboxylase
activities in spruce foliage of mature timber stands we
had two objectives, a) To gather representative data
about the enzyme levels in Picea abies needles as a func-
tion of needle age; b) to compare RuBP carboxylase ac-
tivities (as well as protein and chlorophyll concentra-
tions) in spruce needles collected at different locations
either exhibiting the typical symptoms of spruce decline
or not. At 2 out of 5 harvesting places (C* and E*) the
trees were visibly injured: Yellow-brown discoloration
("bronzing") of the abaxial, sun-exposed leaf surface of
older needles could be observed, the discoloration fre-
quently turned into necroses and subsequent needle
loss. Now and then needle tips showed signs of be-
ginning chlorosis. In general, tree-tops were damaged
or dead. However, the symptoms were heterogenously
spread in the timber stands and also within single trees,
the degree of impairment being variable and inconsist-
ent. At 3 other locations, the spruce trees did not show
visible damage (A and D) or were only sporadically af-
fected (B).
A comparison of the needle generation 1 to 4 (Tab. 3)
10 15 20 25 30
Time (min)
Fig. 3. Linearity of the carboxylation reaction employing crude
RuBP carboxylase from spruce. Enzyme activities were meas-
ured with PGK/GAPDH as auxilliary enzymes. Recordings of
single runs.
Physiol. Plantarum 7(1. 1987
Fig. 4. Kinetic
characterization of spruce (0)6
RuBP carboxylase. (r = 250-
coefficients of regression; n CD / „ 0 - r=:0.999 /-
= 4). a) Substrate saturation ^ 2
curve for HCO; (at 2.15
-iOO -0
mM RuBP, 7.7 mM MgCl, J-1
and pH 8.3). The apparent
Kn,-value was calculated ^-2 "/
from S(v ,,) of the o nH = 1.33
tu
sigmoidaTcurve. Inset: Hill- '^(Vmax/2)=
plot, b) Lineweaver-Burke y 7.8-10"3M -^ -3 -2
plot for RuBP (at 100 mM
HCO:, 7.7 mM MgCl, and -^ 5-10"' 10-3 10-3 10-2 5-10-^ 10" 7.5 8.0 8.5 9.0
pH 8.3). c) pH-curve of pH
RuBP carboxylase activity in
Tris-HCI buffers, d)
Temperature dependence of
RuBP carboxylase activity;
Arrhenius-plot.
2.5 X ' ' _'

log Bq
\
2.0 -
•^ 1 9
1.5 1

30 20 10\ J

3.1 3.2 3.3 3./. 3.5 3.6

yielded the following results. The CO, assimilation po-
tential of 2- to 4-year-old spruce needles was quite simi-
lar, as far as RuBP carboxylase activity on a chlorophyll
basis was concerned. However, when the data were ex-
pressed on a fresh weight or protein basis RuBP car-
boxylase activity of the second needle generation was
about 20% lower than that of 3- and 4-year-old needles.
The difference was almost statistically significant on the
95% level. The age-dependent changes in chlorophyll
content followed the same pattern as fresh weight- and
protein-based enzyme activities with respect to the dif-
ference between 2-year-old needles and those of gener-
ations 3 or 4. In current immature needles (= needle
generation 1) however, there was a discrepancy be-
tween chlorophyll and protein content on one side and
RuBP carboxylase activity on the other: while both of
the former parameters reached about 65% of the final
value, the carboxylation potential amounted to only
about 25% of the enzyme activity found in older nee-
dles. Thus the build-up of RuBP carboxylase activity
lagged markedly behind net chlorophyll and protein
synthesis.
Table 4 shows a compilation of the results where the
field data were processed in order to evaluate the differ-
ences between the harvesting locations. RuBP carboxyl-
ase activities from all 5 timber stands are rather close,
regardless of whether the data are expressed on a fresh
weight or on a chlorophyll basis. Additionally, the ref-
erence system "extractable protein" has also been in-
cluded, although it may be questioned whether it is
really an adequate basis of reference in case of RuBP
carboxylase: Considering the fact that fraction I protein
amounts to ca 50% of the total soluble leaf protein
(Huffacker 1982), somewhat exaggerated one could say

Tab. 3. RuBP carboxylase activity, chlorophyll content and protein content as a function of needle age. Included are the needle
generations 1 to 4 (year of emergence: 1985-1982). The data for the 5 locations investigated (see Fig. 1) were averaged in order to
obtain the mean ±SD (n = 8) values shown here.

Parameter Needle generation

RuBP carboxylase activity,

nmol CO, (g FW) ' mm ' 2 370 ± 1 110 7 620 ±3 000 9 500 ±4 270 9 800 ±4 640
nmol CO, (mgchi) ' min 2 180 ± 750 5 400 ±2 180 5 860 ±2 800 5 640 ±2 560
nmol CO; (mg protein) min 72 ± 35 168 -t- 52 203 4- 62 197 + 65
Total chlorophyll, mg (g FW) ' l.09± 0.14 1.42 ± 0.11 1.64 ± 0.08 1.72 + 0.12
Extractable protein, mg (g FW)" 33 ± 5.7 45 4- 4.3 48 4- 6.9 49 4- 7.1

Phvsiol. Plantiirum 711. 1987 669

Tab. 4. RuBP carboxylase activity, chlorophyll content and protein content of spruce needles harvested at 5 locations (see Fig 1)
The data for needle generations 2 to 4 (year of emergenee: 1984-1982) were averaged in order to obtain the mean ±SD(n = 8) val-
ues shown here.

Parameter Geographic location

A D

RuBP carboxylase activity,

nmol CO, (gFW)"' min ' 7.590 ± 700 8 610 ± 450 12 440 ±3 510 7 670 ±1610 8 110 +1680
nmol CO, (mg chl) ' min ' 4 750 ± 330 5 580 ± 570 7 810 ±1190 4 760 ± 460 4 900 ± 450
nmol CO, (mg protein) ' min" 170 ± 13 187 ± 9 208 ± 35 193 ± 46 164 ± 17
Total chlorophyll, mg (g FW) ' 1.58± 0.07 1.58± 0.21 1.60± 0.17 1.61± 0 21 1 62± 0 21
Extractable protein, mg (g FW)" 45 ± 2 48 ± 3 54 ± 5 41 ± 2 50 ± 5

that RuBP carboxylase activity is being brought in rela-
tion to itself. In any case, no difference can be re-
cognized between the supposedly healthy timber stands,
where Picea abies hardly exhibits visual symptoms of
deterioration (A, B and D) and those, which show vari-
ous stages of spruce decline (C*, E*). The values for C*
are somewhat higher but the difference is not statis-
tically significant. Effects of ecological factors, like ex-
posure to air pollutants, on enzyme levels are of di-
agnostic value only if they emerge in advance of visual
injury. Hence, the data presented here indicate that a
reduction of the amount of RuBP carboxylase did not
precede visual symptoms of spruce needle damage as an
"invisible injury" (Heath 1980).
With regard to the extractable protein content, again
C* showed the highest value, although not significantly
different from the others. Therefore, on a protein basis
the C*-enzyme activity was in the normal range. The
chlorophyll content of the spruce foliage was very simi-
lar for all 5 locations. This indicates that a moderate
bronze discoloration, the most commonly observed dis-
ease symptom, observed on a part of the needles of gen-
erations 3 and especially 4 is not a manifestation of
chlorophyll degradation.
Discussion
The success in obtaining a comparably high yield of
RuBP carboxylase activity from Norway spruce needles
is based on the one hand on two constituents of the ex-
traction medium and on the other hand on the fact that
tissue homogenization and enzyme extraction were sep-
arated. Insoluble PVP (Polyclar AT) binds polyphenolic
compounds like tannins, which are abundant in conifer
assimilatory tissue (Loomis and Battaille 1966, Soikkeli
1978) thus protecting the non-denatured state and pre-
venting precipitation of cellular proteins. Competition
between hydrogen bonding of the polyphenols to the
native proteins and to the PVP takes place (Loomis and
Battaile 1966). Hence, a large excess of PVP over pro-
tein is essential. The separation of the homogenization
670
from the extraction procedure serves the same purpose:
In the deep-frozen needle powder no protein-polyphe-
nol interaction occurs. During subsequent thawing,
PVP is effective immediately, because the time interval
until the tannins are accessible to Poiyclar AT is mini-
mized. This also applies to the protection of the native
protein conformation by sulfhydryl reagents and other
antioxidants. Furthermore, the combination of deep-
freezing and use of detergent eliminates the necessity to
break up the chloroplasts by the strong mechanical
force of a high-revolution homogenizer, which inev-
itably generates heat which may effect enzyme activity.
The polyoxyethylene compound Triton X-100, a non-
ionic detergent, is characterized by a low CMC
(3x10" M) and large micelles. Although enzymes are
not normally inactivated by non-ionic detergents pos-
sessing polyoxyethylene or sugar/sugar alcohol groups
(Hjelmeland and Chrambach 1984) it still remains to be
proven whether the specific activity and the kinetic
properties are identical in "free" enzyme and enzyme
possibly contained in mixed micelles.
The RuBP carboxylase activities reported for conifer
needles from various species are mostly lower than
ours. On a chlorophyll basis, Gezelius (1975) found
1 260-1 580 nmol CO, (mg chl)"' min"' for young seed-
lings of Pinus silvestris. Beadle et al. (1983) found be-
tween 1 740 and 3 080 nmol CO, (mg chl)"' min"' for a
selection of 9 different species of coniferous trees, in-
cluding Sitka spruce. However, in a more recent investi-
gation Gezelius and Hallen (1980) obtained values,
which are similar to ours: ca 5 000 nmol COj (mg chl)"'
min"'.
The enzyme activities given here were measured dur-
ing June-September. Probably they represent the maxi-
mum level attainable in the course of a year, and corre-
spond to the annual maximum of photosynthesis (Fry
and Phillips 1977), of structural organization of chloro-
plasts (Senser et al. 1975, Soikkeli 1978) and of thy-
lakoidal pigments (Oquist et al. 1978). Annual fluctua-
tions of RuBP carboxylase activity in Pinus silvestris
seedlings (Gezelius and Hallen 1980) point in the same
direction.
The kinetic data obtained for a crude preparation of
Physiol. Platltarum 70. 19H7
Norway spruce RuBP carboxylase are within the normal
range. The values for the half saturation constants ( =
apparent K J for RuBP and HCO3 of 0.43 and 7.8 mM
(= 0.14 mM CO2) respectively, at pH 8.3 (pH optimum)
and 7.7 mM Mg'* characterize the crude RuBP car-
boxylase preparation as the low-affinity form of the en-
zyme (Miziorko and Lorimer 1983). Gezelius (1975)
and Beadle et al. (1983) reported similar values for
RuBP carboxylase from other conifers. Gezelius and
Hallgren (1980), however, observed a K„, (CO,) = 27
|iM for properly activated RuBP carboxylase from pine.
Relatively low rates of CO2 fixation have been repor-
ted for conifer needles, ranging from about 0.5 to 1.5
^imol CO2 (g fresh weight)"' min"' (Zelawsky et al.
1973, Fry and Phillips 1977). Recent investigations give
higher rates of 5 to 10 |xmol CO, (g fresh weight)"'
min"' (recalculated from Lange et al. 1986) for 15- to
20-year-old Picea abies. These values are in good agree-
ment with our data (Tabs 3 and 4). No such agreement is
observed, however, when the in vivo photosynthetic ca-
pacity of different needle generations is compared with
their in vitro RuBP carboxylase activity. Generally, the
rates of photosynthesis decrease with increasing needle
age (Freeland 1952, Dickman and Kozlowsky 1968,
Lange et al. 1986). Mature current needles exhibit the
highest assimilation rates. In contrast, the RuBP-car-
boxylase activity of current needles is relatively low,
even 2-year-old needles may not have reached the maxi-
mal activity (Tab. 3). This discrepancy is not easily un-
derstood. Stomatal resistance may be increased in older
needles due to an enhanced accumulation of cuticular
wax particles in the external air cavities. As a result, the
CO, supply might be insufficient and the rates of pho-
tosynthesis could decrease (Ludlow and Jarvis 1971). In
addition, a relative excess of RuBP over CO, at high
light intensities and concurrent CO, shortage could give
rise to substrate inhibition, when RuBP binds to the en-
zyme prior to CO, (Dailey and Criddle 1980, Vu et al.
1984). Assuming that the in vitro RuBP carboxylase ac-
tivity in the current needle generation still suffices to ac-
count for the in vivo rates of photosynthesis observed,
one could speculate that 2- to 4-year-old needles contain
more RuBP carboxylase, because the then reduced in-
ternal CO, level can only be fully utilized when more
enzyme molecules are available to compensate for the
desreased substrate saturation level of the enzyme as
governed by K^, (CO,).
RuBP carboxylase may be involved in several differ-
ent ways in suppression of photosynthesis in Picea abies
needles by air pollution. However, only those effects of
environmental stress can be recognized, which cause a
change in the amount of enzyme or which irreversibly
inactivate it. In vitro estimations of RuBP carboxylase
activity, carried out under conditions of substrate and
cofactor saturation are not adequate, though, to obtain
reliable information about the actual in vivo carboxyla-
tion rates - unless the in situ metabolic concentrations
of all reactants and effectors are known as well.
Phvsiol. Plantarum 70. 1987
Low levels of atmospheric pollutants like SO,, O, and
reactive radicals supposedly inhibit photosynthetic elec-
tron transport thus affecting the pH-gradient and Mg-"^
efflux across the thylakoid membranes. Furthermore,
uncoupling of photophosphorylation and photobleach-
ing of the chlorophyll complexes may occur (Hallgren
1978, Heath 1980, Wellburn et al. 1981). The resulting
suppression of the alkalization of the stroma, the lack of
ATP (and subsequently RuBP) and perhaps of Mg-*
may secondarily affect RuBP carboxylase activity in
situ. However, only when the chloroplasts are irrever-
sibly damaged the in vitro activity of RuBP carboxylase
would reflect this impairment. Our negative results indi-
cate that such a severe deterioration of chloroplast func-
tion is not part of the spruce decline at the locations in-
vestigated here.
Photosynthetic CO, fixation may be affected directly
by inhibition of RuBP carboxylase by SO^". either com-
petitively, as shown by Ziegler (1972) for spinach RuBP
carboxylase or non-competitively in case of Pinus silves-
tris (Gezelius and Hallgren 1980). However, even at ex-
treme ambient SO, levels SO, certainly does not inhibit
photosynthesis by this mechanism of inhibition, because
the Kj-values are at ca 5-20 mM and thus several orders
of magnitude beyond the range of heavily polluted air.
O, concentrations in the range of 500 (ig m"' give a sig-
nificant reduction in the quantity of RuBP carboxylase
extracted from alfalfa foliage (Pell and Pearson 1983).
However, any direct effect of O, on RuBP carboxylase
can probably also be excluded for the timber stands in-
vestigated here, since the ambient O, concentrations at
C* and E* and even more so at the other locations, are
ca one order of magnitude lower (with peak values of
about 160 |ig O, mg"^).
It has been postulated that sublethal doses of multiple
stress may lead to premature senescence of conifer nee-
dles (Mohr 1984, Ziegler 1986). Loss of RuBP carboxyl-
ase as an integral part of the senescence process is ob-
served (Huffacker 1982) concomitantly with chlorophyll
degradation and deterioration of thylakoids (Elstner
and Osswald 1984). The result of the present investi-
gation - no significant decrease of RuBP carboxylase in
affected trees - indicates that the air pollution-induced
senescence phenomena observed in Norway spruce
probably do not (or at least not always) follow the nor-
mal physiological and biochemical patterns of leaf se-
nescence.
Acknowledgements - Gabriele Schweyen assisted in harvesting
the field samples. Valuable advice was obtained from Dr W.
Knabe (Landesanstalt fur Okologie. Landschaftsentwicklung
und Forstplanung. Nordrhein-Westfalen) and from Dr B. Prinz
(Landesanstalt fur Immissionsschutz. Nordrhein-Westfalen)
with regard to the selection of the field sites. Many local forest
management authorities were helpful in giving access to spe-
cific timber stands. Financial support was given by the Minister
fur LJmwelt. Raumordnung und Landwirtschafl des Landes
Nordrhein-Westfalen. To all individuals and institutions who
contributed to this work we want to express our gratitude.
671

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