Weidner 1987
Weidner 1987
Weidner 1987
Copenhagen 1987
M. Weidner (corresponding author) and M. Kraus, Botanisches Inst. der Utiiv. Koln,
Gyrhofstrasse 15, D-5000 Koln 41, West Germany.
^ ^ / / \ \ ^ \
Ig Ig Ig 1g Ig Ig Ig Ig Ig Ig 1g Ig
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, trees, provided and X,iv_vii were not significantly
Sigma, Deisenhofen, FRG) served as a protein stan- different on the 95 %-level (2P = 0.05). Thus, Y are the
dard. mean values for each needle generation and location
Enzyme, chlorophyll and protein measurements were (e.g. Y4,^): mean value of needle generation 4, location
run in duplicate from each extract. Mean values are A). The Y-values of needle generations 2, 3, 4 were
based on 4-8 extractions, in field experiments 8 extrac- combined in order to obtain an average Z value of
tions were always made. Significance was tested at the RuBP carboxylase activity etc. for each spruce stand,
5"/> level throughout. 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
Outline of field experiments
(e.g. X,,,). provided the two Z-values were not signifi-
cantly different on the 95% level. On basis of these val-
The sampling and randomization scheme for the field ues the locations A, B, C*, D and E* are compared in
experiments is depicted in Fig. 1. Samples were taken Tab. 4. Further, the 4 needle generations are compared.
twice (harvest 1 and 2) from each spruce stand between In this case the Z values for the different needle gener-
June and September. Five locations were chosen (A, B, ations (e.g. Z4,Hi) and Z4(n^)), comprising all 5 locations,
C*, D, E*) and at each location 6 trees were selected were combined to give the general mean value X for
and marked. The 6 trees were divided in two subgroups, each needle generation (e.g. X4). On basis of these val-
comprising 3 trees each. From each tree, 5-g portions of ues conclusions about the influence of needle age on
needles were harvested. Needle generations 1 (current RuBP carboxylase activity etc. were drawn (Tab. 3).
needles), 2, 3, and 4 were included. The needle portions This rather elaborate sampling scheme was executed,
of trees I-III and IV-VI, respectively, were mixed thor- because no experiences were available as to the biologi-
oughly to make up two 15-g samples from which then 8 cal variability of samples from 35- to 60-year-old spruce
1-g samples were taken at random. These were frozen in trees. Therefore we had to make sure that data were
liquid nitrogen and analyzed later on. The analytical pooled only after a detailed statistical evaluation had
data were combined to give the mean values X (e.g. been carried out. Hence, each step was accompanied by
^4(1-111)^ mean of needle generation 4, trees I, II, III). statistical treatment, employing the software pro-
The two X-values were combined to form Y for all 6 gramme Abstat (Anderson-Bell 1982, Canon City. CO,
log Bq
\
2.0 -
•^ 1 9
1.5 1
30 20 10\ J
yielded the following results. The CO, assimilation po- value, the carboxylation potential amounted to only
tential of 2- to 4-year-old spruce needles was quite simi- about 25% of the enzyme activity found in older nee-
lar, as far as RuBP carboxylase activity on a chlorophyll dles. Thus the build-up of RuBP carboxylase activity
basis was concerned. However, when the data were ex- lagged markedly behind net chlorophyll and protein
pressed on a fresh weight or protein basis RuBP car- synthesis.
boxylase activity of the second needle generation was Table 4 shows a compilation of the results where the
about 20% lower than that of 3- and 4-year-old needles. field data were processed in order to evaluate the differ-
The difference was almost statistically significant on the ences between the harvesting locations. RuBP carboxyl-
95% level. The age-dependent changes in chlorophyll ase activities from all 5 timber stands are rather close,
content followed the same pattern as fresh weight- and regardless of whether the data are expressed on a fresh
protein-based enzyme activities with respect to the dif- weight or on a chlorophyll basis. Additionally, the ref-
ference between 2-year-old needles and those of gener- erence system "extractable protein" has also been in-
ations 3 or 4. In current immature needles (= needle cluded, although it may be questioned whether it is
generation 1) however, there was a discrepancy be- really an adequate basis of reference in case of RuBP
tween chlorophyll and protein content on one side and carboxylase: Considering the fact that fraction I protein
RuBP carboxylase activity on the other: while both of amounts to ca 50% of the total soluble leaf protein
the former parameters reached about 65% of the final (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.
that RuBP carboxylase activity is being brought in rela- from the extraction procedure serves the same purpose:
tion to itself. In any case, no difference can be re- In the deep-frozen needle powder no protein-polyphe-
cognized between the supposedly healthy timber stands, nol interaction occurs. During subsequent thawing,
where Picea abies hardly exhibits visual symptoms of PVP is effective immediately, because the time interval
deterioration (A, B and D) and those, which show vari- until the tannins are accessible to Poiyclar AT is mini-
ous stages of spruce decline (C*, E*). The values for C* mized. This also applies to the protection of the native
are somewhat higher but the difference is not statis- protein conformation by sulfhydryl reagents and other
tically significant. Effects of ecological factors, like ex- antioxidants. Furthermore, the combination of deep-
posure to air pollutants, on enzyme levels are of di- freezing and use of detergent eliminates the necessity to
agnostic value only if they emerge in advance of visual break up the chloroplasts by the strong mechanical
injury. Hence, the data presented here indicate that a force of a high-revolution homogenizer, which inev-
reduction of the amount of RuBP carboxylase did not itably generates heat which may effect enzyme activity.
precede visual symptoms of spruce needle damage as an The polyoxyethylene compound Triton X-100, a non-
"invisible injury" (Heath 1980). ionic detergent, is characterized by a low CMC
With regard to the extractable protein content, again (3x10" M) and large micelles. Although enzymes are
C* showed the highest value, although not significantly not normally inactivated by non-ionic detergents pos-
different from the others. Therefore, on a protein basis sessing polyoxyethylene or sugar/sugar alcohol groups
the C*-enzyme activity was in the normal range. The (Hjelmeland and Chrambach 1984) it still remains to be
chlorophyll content of the spruce foliage was very simi- proven whether the specific activity and the kinetic
lar for all 5 locations. This indicates that a moderate properties are identical in "free" enzyme and enzyme
bronze discoloration, the most commonly observed dis- possibly contained in mixed micelles.
ease symptom, observed on a part of the needles of gen- The RuBP carboxylase activities reported for conifer
erations 3 and especially 4 is not a manifestation of needles from various species are mostly lower than
chlorophyll degradation. 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
Discussion selection of 9 different species of coniferous trees, in-
cluding Sitka spruce. However, in a more recent investi-
The success in obtaining a comparably high yield of gation Gezelius and Hallen (1980) obtained values,
RuBP carboxylase activity from Norway spruce needles which are similar to ours: ca 5 000 nmol COj (mg chl)"'
is based on the one hand on two constituents of the ex- min"'.
traction medium and on the other hand on the fact that The enzyme activities given here were measured dur-
tissue homogenization and enzyme extraction were sep- ing June-September. Probably they represent the maxi-
arated. Insoluble PVP (Polyclar AT) binds polyphenolic mum level attainable in the course of a year, and corre-
compounds like tannins, which are abundant in conifer spond to the annual maximum of photosynthesis (Fry
assimilatory tissue (Loomis and Battaille 1966, Soikkeli and Phillips 1977), of structural organization of chloro-
1978) thus protecting the non-denatured state and pre- plasts (Senser et al. 1975, Soikkeli 1978) and of thy-
venting precipitation of cellular proteins. Competition lakoidal pigments (Oquist et al. 1978). Annual fluctua-
between hydrogen bonding of the polyphenols to the tions of RuBP carboxylase activity in Pinus silvestris
native proteins and to the PVP takes place (Loomis and seedlings (Gezelius and Hallen 1980) point in the same
Battaile 1966). Hence, a large excess of PVP over pro- direction.
tein is essential. The separation of the homogenization The kinetic data obtained for a crude preparation of
670 Physiol. Platltarum 70. 19H7
Norway spruce RuBP carboxylase are within the normal Low levels of atmospheric pollutants like SO,, O, and
range. The values for the half saturation constants ( = reactive radicals supposedly inhibit photosynthetic elec-
apparent K J for RuBP and HCO3 of 0.43 and 7.8 mM tron transport thus affecting the pH-gradient and Mg-"^
(= 0.14 mM CO2) respectively, at pH 8.3 (pH optimum) efflux across the thylakoid membranes. Furthermore,
and 7.7 mM Mg'* characterize the crude RuBP car- uncoupling of photophosphorylation and photobleach-
boxylase preparation as the low-affinity form of the en- ing of the chlorophyll complexes may occur (Hallgren
zyme (Miziorko and Lorimer 1983). Gezelius (1975) 1978, Heath 1980, Wellburn et al. 1981). The resulting
and Beadle et al. (1983) reported similar values for suppression of the alkalization of the stroma, the lack of
RuBP carboxylase from other conifers. Gezelius and ATP (and subsequently RuBP) and perhaps of Mg-*
Hallgren (1980), however, observed a K„, (CO,) = 27 may secondarily affect RuBP carboxylase activity in
|iM for properly activated RuBP carboxylase from pine. situ. However, only when the chloroplasts are irrever-
Relatively low rates of CO2 fixation have been repor- sibly damaged the in vitro activity of RuBP carboxylase
ted for conifer needles, ranging from about 0.5 to 1.5 would reflect this impairment. Our negative results indi-
^imol CO2 (g fresh weight)"' min"' (Zelawsky et al. cate that such a severe deterioration of chloroplast func-
1973, Fry and Phillips 1977). Recent investigations give tion is not part of the spruce decline at the locations in-
higher rates of 5 to 10 |xmol CO, (g fresh weight)"' vestigated here.
min"' (recalculated from Lange et al. 1986) for 15- to Photosynthetic CO, fixation may be affected directly
20-year-old Picea abies. These values are in good agree- by inhibition of RuBP carboxylase by SO^". either com-
ment with our data (Tabs 3 and 4). No such agreement is petitively, as shown by Ziegler (1972) for spinach RuBP
observed, however, when the in vivo photosynthetic ca- carboxylase or non-competitively in case of Pinus silves-
pacity of different needle generations is compared with tris (Gezelius and Hallgren 1980). However, even at ex-
their in vitro RuBP carboxylase activity. Generally, the treme ambient SO, levels SO, certainly does not inhibit
rates of photosynthesis decrease with increasing needle photosynthesis by this mechanism of inhibition, because
age (Freeland 1952, Dickman and Kozlowsky 1968, the Kj-values are at ca 5-20 mM and thus several orders
Lange et al. 1986). Mature current needles exhibit the of magnitude beyond the range of heavily polluted air.
highest assimilation rates. In contrast, the RuBP-car- O, concentrations in the range of 500 (ig m"' give a sig-
boxylase activity of current needles is relatively low, nificant reduction in the quantity of RuBP carboxylase
even 2-year-old needles may not have reached the maxi- extracted from alfalfa foliage (Pell and Pearson 1983).
mal activity (Tab. 3). This discrepancy is not easily un- However, any direct effect of O, on RuBP carboxylase
derstood. Stomatal resistance may be increased in older can probably also be excluded for the timber stands in-
needles due to an enhanced accumulation of cuticular vestigated here, since the ambient O, concentrations at
wax particles in the external air cavities. As a result, the C* and E* and even more so at the other locations, are
CO, supply might be insufficient and the rates of pho- ca one order of magnitude lower (with peak values of
tosynthesis could decrease (Ludlow and Jarvis 1971). In about 160 |ig O, mg"^).
addition, a relative excess of RuBP over CO, at high It has been postulated that sublethal doses of multiple
light intensities and concurrent CO, shortage could give stress may lead to premature senescence of conifer nee-
rise to substrate inhibition, when RuBP binds to the en- dles (Mohr 1984, Ziegler 1986). Loss of RuBP carboxyl-
zyme prior to CO, (Dailey and Criddle 1980, Vu et al. ase as an integral part of the senescence process is ob-
1984). Assuming that the in vitro RuBP carboxylase ac- served (Huffacker 1982) concomitantly with chlorophyll
tivity in the current needle generation still suffices to ac- degradation and deterioration of thylakoids (Elstner
count for the in vivo rates of photosynthesis observed, and Osswald 1984). The result of the present investi-
one could speculate that 2- to 4-year-old needles contain gation - no significant decrease of RuBP carboxylase in
more RuBP carboxylase, because the then reduced in- affected trees - indicates that the air pollution-induced
ternal CO, level can only be fully utilized when more senescence phenomena observed in Norway spruce
enzyme molecules are available to compensate for the probably do not (or at least not always) follow the nor-
desreased substrate saturation level of the enzyme as mal physiological and biochemical patterns of leaf se-
governed by K^, (CO,). nescence.
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 Acknowledgements - Gabriele Schweyen assisted in harvesting
environmental stress can be recognized, which cause a the field samples. Valuable advice was obtained from Dr W.
Knabe (Landesanstalt fur Okologie. Landschaftsentwicklung
change in the amount of enzyme or which irreversibly und Forstplanung. Nordrhein-Westfalen) and from Dr B. Prinz
inactivate it. In vitro estimations of RuBP carboxylase (Landesanstalt fur Immissionsschutz. Nordrhein-Westfalen)
activity, carried out under conditions of substrate and with regard to the selection of the field sites. Many local forest
cofactor saturation are not adequate, though, to obtain management authorities were helpful in giving access to spe-
cific timber stands. Financial support was given by the Minister
reliable information about the actual in vivo carboxyla- fur LJmwelt. Raumordnung und Landwirtschafl des Landes
tion rates - unless the in situ metabolic concentrations Nordrhein-Westfalen. To all individuals and institutions who
of all reactants and effectors are known as well. contributed to this work we want to express our gratitude.
Edited by C. Larsson