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Investigations on the content indole-3-acetic spruce needles of healthy and damaged trees of various sites
]. Plant Pbysiol. Vol. 141. pp. 615 -620 (1993)
Investigations on the Content of Indole-3-acetic Acid in Spruce Needles of Healthy and Damaged Trees of Various Sites A.
WESSLER
and A.
WILD
Institute of General Botany, Johannes Gutenberg University, Saarstr. 21, 6500 Mainz, Germany Received September 9,1992 . Accepted December 15, 1992
Summary
Surveys were made on the concentration of indole-3-acetic acid (IAA) in spruce trees of various sites. The concentrations of the hormone were specified with a radioimmunoassay after the needle extract had been purified in several steps. The content of IAA in needles of damaged trees was distinctly lower than the content in needles of healthy trees. Another feature was the annual rythm of the IAA content with the maximum of concentration in June. A distinct diurnal course with highest level of concentration during the early afternoon hours was also observed. The diurnal rhythm of the damaged trees was clearly lower. The supposition is that the lower IAA content is responsible for a premature needle abscission.
Key words: Picea abies, indole·3-acetic acid, annual rhythm, diurnal course, novel forest decline, radioimmunoassay. Abbreviations: BHT = buty1hydroxytoluene; IAA = indo1e-3-acetic acid; PBS = phosphate buffered saline; RIA = radioimmunoassay; MEST = middle european summer time.
Introduction
Since the end of the seventies, damage to forest trees has appeared in Central Europe, which is now designated as «novel forest decline» (BML, 1984-1991). Although early discussions on forest damage had brought up the speculation that changes in hormones are also responsible for the symptoms of new forest damage, there are less or no definite statements on alterations in the hormone system of damaged trees (Christmann and Frenzel, 1987; Schwanzenberg et al., 1988). In most cases a decrease in indole-3-acetic acid (IAA) and cytokinin concentration is to be expected in stress loaded trees, whereas the concentration of the antagonistic working hormones abscisic acid and ethylene is mostly higher during the influence of stress Qohnson, 1987). The surveys on alteration of hormones, as a contribution to clarify cause and pathogenesis of novel forest decline, require a basic characterization of the hormone system of the affected tree species. © 1993 by Gustav Fischer Verlag, Stuttgart
IAA is the most imponant native auxin in plants. The relevance of IAA in connection with control of senescence and abscission has repeatedly been reponed (Addicott, 1970; Leshem et al., 1986; Ke and Sa1tveit, 1988). Symptoms of novel forest decline like premature loss of leaves or needles and early aging may be connected with disturbances in the balance of IAA metabolism. It was the aim of the present study to determine the quantity of IAA concentration in order to clarify regulation processes in damaged trees_ With the assistance of a specific and sensitive radioimmunoassay combined with several purification steps, investigations of spruce trees at different sites with similar immission charge and comparable climatic conditions were able to be carried out. Phenotypically undamaged and damaged trees were compared with each other and the interdependence between the IAA concentrations and the corresponding tree- and location-specific damage classes were reviewed. Special attention was given to the annual and diurnal dynamics of the IAA contents, as deviations in ref-
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A. WESSLER and A. WILD
erence to these influences are to be expected, especially in the analysis of IAA (Dangar and Basu, 1984). The present study on the IAA content of spruce trees is part of a research project characterizing the damage of trees with the help of physiological, biochemical and cytomorphological parameters (Benner et aI., 1988; Dietz et al., 1988; Wild et al., 1988, 1990; Forschner et al., 1989; Hasemann and Wild, 1990; Wild and Tietz-Siemer, 1992). Materials and Methods
Description of the sites The investigations described below were performed over a period of two years, 1989 and 1990, at several natural sites located in BadenWiirttemberg, Nordrhein-Westfalen and Rheinland-Pfalz in the Federal Republic of Germany. In 1989, samples were collected between April and November in order to get information about changes in IAA levels during the vegetation period. In 1990, diurnal courses of the content of IAA were investigated. Climatic and immission data were registered continuously. At all sites there were high ozone concentrations in summer whereas the levels of the air pollutants S02 and N0 2 were relatively low during the whole test period. Detailed descriptions of the sites have been presented by Wild et al. (1990), Tenter and Wild (1991), Tietz and Wild (1991) and Richter and Wild (1992).
Hattgenstein site This site is located in the western part of the Hunsriick mountains in Rheinland-Pfalz at about 660 m above sea level (Idar-Oberstein Forestry Office, Hattgenstein Forest District, Division 257 b2). The spruce trees were between 25 and 30 years old. The damaged trees showed partial needle loss and yellowing of the upper part of the older needles (mountainous yellowing). The harvest date of the diurnal concentration course was September 7, 1989.
Freudenstadt site The site is located at 820 - 830 m above sea level near Freudenstadt in the northern Black Forest (Freudenstadt Forest Office, Vordersteinwald Forest District, Division III/12 a\ at Schollkopf). At this site two different stands of spruce trees were examined. The trees were between 45 and 50 years old. The trees of one stand were highly damaged (damage classes 2 and 3). These trees were compared with an adjacent stand of apparently relatively symptomfree spru~e trees (damage classes 0 and 1). The general type of tree damage at this site is called «Montane Vergilbung» (mountainous yellowing of spruce) (Siefermann-Harms, 1990). The harvest dates in 1989 were April 19, June 28, August 30 and November 8, and in 1990 the needles used for the diurnal course were examined on August 2.
Glindfeld and Velmerstot site The Glindfeld site is located in the Rothaargebirge (Glindfeld Forestry Office, Kiistelberg Forest District, Division 62 c6) about 630 m above sea level. The spruce plantations under investigation consist of trees that are about 50 years old. They belonged to the damage class 0 and 1, but gradual detoriation could be seen through needle loss and chlorosis during the last year. In 1989, mixed samples of six trees were harvested four times a year (April 25, June 20, September 5 and November 14). On June 12, 1990, needles of an undamaged tree were
harvested in order to get information about the diurnal course of IAA-concentrations. The spruce plantation under investigation at the Velmerstot site is located in the southern part of the Teutoburger Wald called Eggegebirge (Paderborn Forestry Office, Kempen Forest District, Division 399 B), which is about 410 m above sea level. The spruce trees at the Velmerstot site were about 55 years old. The trees examined were described as damaged, belonging to damage classes 2 and 3. However, it is noteworthy that for the last three years a distinct recovery phase of the plantation could be detected. Sampling took place on April 25, June 19, September 4 and November 13 in 1989 and on June 11 in 1990.
Materials
The investigations were performed on spruce needles (Picea abies L. Karst.) of the second needle generation (needles grown in the previous year) from the sixth to the eight whorl. The samples, which were collected in oder to get information about changes in IAA levels during the vegetation period, were harvested from 11.00 h to 13.00 h (MEST). The studies were performed with the needles of 6 trees per stand in mixed samples. Samples needed for the investigation of diurnal courses were collected hourly from 7.00 h to 18.00 h (MEST) and were conducted in pairs: One symptom-free tree was compared with a more severely damaged tree. The needles were removed instantly from the twigs by stirring them in liquid nitrogen; afterwards the needles were stored at -80°C.
Methods
Extraction offree IAA from spruce needles One gram of deep frozen needles was grounded to a powder with mortar and pestle in liquid nitrogen and extracted with 50 mL icecold 80 % (v/v) methanol for several minutes. BHT was added to the methanol to prevent oxidation. The homogenate was filtered in a Biichner funnel and brought with 0.5 M HEPES-buffer, pH 8.0, to a concentration of 70 % methanol. After passing through two SEPPAK C I8 Cartridges (Fa. Waters) the methanol was removed in a rotary evaporator (Fa. Schlitt) at 35°C. The remaining aqueous phase was centrifuged for 5 min by 1000 x gto remove the insoluble components. Then, the extract was purified with high-pH buffer ethyl acetate partition. The aqueous phase was adjusted to a pH of 2.4-2.6; phenolic substances were eliminated with unsoluble polyvinylpyrrolidone and the extract was partitioned again with ethyl acetate at low pH. The remaining ethyl acetate phases were dried with N 2at 37°C and then methylated with a boron trifluoride methanol complex. The extracts were kept at - 80 °C in the dark and assayed after some days.
Radioimmunoassay (RIA) The general procedure used for the RIA has been described by Weiler (1981, 1982, 1984). Samples were measured in triplicate. The PBS-buffer consisted of 8 mM KH 2P0 4, 1.6 mM K1 HP0 4 and 150 mM NaC!, pH 7.4. The RIA-mixture per tube (MiIli-3-PE, Fa. Baker) contained 0.65 mL PBS-buffer, 0.10 mL bovine serum albumin (1: 10) and 2 ~L radioactive IAA tracer (c. 15,000dpm). After an incubation period of 2 h at 4 °C the reaction was stopped with 1 mL (NH 4)2S04 (60.16 g in 100 mL distilled water). Precipitation occurred for 20 min. After centrifugation (1500 x g) the pellets were washed with 1 mL (NH4)2S04 (30.85 g in 100 mL distilled
IAA in spruce needles in healthy and damaged trees
617
350
water), centrifuged and decanted. The pellet was dissolved in 0.2 mL water and 1.5 mL scintillation cocktail. Radioactivity was measured by liquid scintillation counting (Volckers and Wild, 1988).
300 250
General parameters
The preparation and assay of IAA-BSA-conjugates, the immunization and the specifity of the antiserum have already been described by Volckers and Wild (1988). The detection limit was 0.5 pmol IAA per assay. The measuring range was from 0.5 -1000 pmol IAA. The carboxyl-coupled IAA-elicited-antibodies gave a titer of 1 : 170 (final dilution, binding 26 % of the tracer).
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Calculation and statistical analysis
The calculation of the RIA dates was performed with a separate calibration. The logit B/Bo-data (logit B/Bo = In(B/Bo)/(100-B/Bo) was evaluated by standard dilution curves using a linear regression analysis. The measured equation data were y = -2.56 x +2.37 and the correlation coefficient was r = 0.997. Data were analyzed using a two-tailed independent Student's t test. Results were considered significantly different if ex < 0.05. Significance levels were indicated according to Sachs (1988) by asterics (* ex < 0.05, ** ex < 0.01, *** ex < 0.001). Measurements were usually repeated twice or three times per sample.
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Results With respect to the ratio fresh weight per dry weight no remarkable differences could be found between needles of undamaged and damaged spruce trees. Deviation with relation to the annual and daily courses could also not be found (data not shown). Therefore, dry weight was used as a suitable reference basis for the measurements of IAA.
100
o April
June
September
November
Fig. 1 A, B: Content of free IAA in relation to dry weight at the Freudenstadt site (A) in needles of undamaged (0) and damaged (_) trees and at the Glindfeld (0 needles of apparently healthy or slightly damaged trees) and Velmerstot sites (_ needles of more severely damaged trees) (B).
Seasonal changes of IAA concentrations In order to locate seasonal condition changes of IAA contents (free IAA) during the vegetation period, samples were taken in 1989 at different times of the year at the Freudenstadt, Glindfeld and Velmerstot sites. The seasonal condition changes of the IAA concentrations are shown in Fig. 1 A and B. In the first instance, an increase in IAA concentrations from the harvest month April to June can be observed, which is followed by a decrease in the IAA contents towards the fall. The highest IAA contents were accordingly measured at all sites in June after the start of the cambial growing period in spring.
Diurnal changes of IAA concentrations In order to determine the diurnal rhythm of IAA concentrations pair comparisons were carried out at various sites: needles of a damaged tree as well as needles of an apparently healthy tree were harvested hourly between 7.00 hand 18.00 h (MEST) and the IAA contents of these needles were determined. Figure 2A-C shows the diurnal related concentration changes of the IAA contents at the locations of Hattgenstein (Fig. 2 A), Glindfeld and Velmerstot (Fig. 2 B), and Freudenstadt (Fig. 2 C).
A clear diurnal course of the IAA content was discernable at all tested locations. The IAA concentrations of the undamaged trees in Hattgenstein and Freudenstadt showed first a decrease during the morning hours and a definite increase during the noon and early afternoon hours, which then decreased again during the late afternoon until 6.00 h. The concentration of the corresponding damaged trees showed only a weak reflection of these observed diurnal rhythms; clearly weaker noon-maxima, for example, were found. The undamaged tree at the Glindfeld site in the Rothaargebirge showed two concentration maxima on the day studied. One maximum could be observed at 9.00 h as well as one at 1s.00h. The diurnal course of the damaged tree at the Velmerstot site, however, only showed one maximum at 14.00 h, just like the trees of the other locations.
Influence of the damage class of the trees on the IAA concentrations The influence of the damage classes of the trees on the endogenous IAA contents showed the same development at all tested locations (Fig. 1; Table 1). At every harvest time in 1989 the IAA concentrations of damaged trees at Freuden-
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WESSLER
and A.
WILD
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damaged corresponding trees. At the beginning of the vegetation period the difference in IAA content at the Freudenstadt site (Fig. 1 A) was relatively low (17 %), whereas an increased loss of the IAA level of the damaged spruce trees up to 60 % was determined in the fall. Thus, a remarkable decrease of 29 % (mean value of the year, Table 1) was registered. The spruce trees of the Velmerstot location also showed clearly lower IAA concentrations than the ones at the Glindfeld site (Fig. 1 B). A remarkable decrease of 46 % (mean value of the year, Table 1) was registered. The differences are similar at the first three harvest dates (45 %, 50 % and 55 %). At the last harvest date in November the difference amounted to only 24 %. The mean values of the diurnal courses at all locations also show significant differences (Table 1). These surveys also lead to the result that IAA levels of damaged trees are much lower than those of their corresponding undamaged partner trees. At the Hattgenstein site this fact is becoming very clear, as the daily average of the damaged tree is 63 % lower than the daily average of the apparently healthy tree. At the Freudenstadt location a similar processing difference of approximately 40 % can be observed, whereas the damaged tree from the Velmerstot location only shows 17 % lower IAA concentrations compared with the undamaged tree at the Glindfeld site.
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Discussion
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harvest time Fig. 2 A-C: Diurnal courses of free lAA content (nmol. kg- 1 dry weight) in the needles of undamaged (x) and damaged (_) spruce trees at different sites (A) Hattgenstein, September 7, 1989; (B) Velmerstot/Glindfeld, June 11112, 1990; and (C) Freudenstadt, August 2,1990. Table 1: Content of free lAA (nmol· kg- 1 dry weight) in needles of damaged and healthy spruce trees at different sites. Mean values of the year 1989 and of measurements from the diurnal courses in 1989 and 1990. site
mean values of the year undamaged
damaged
Freudenstadt Glindfeld/Velmerstot
206.42 245.93
145.70 132.99
significance
*a<0.05 *a<0.05
Diurnal changes 0/ L4A concentrations
mean values of the day Hattgenstein Glindfeld/Velmerstot Freudenstadt
undamaged
damaged
375.41 164.06 101.24
140.07 136.42 60.42
Menyaila (1985) pointed out that the IAA contents in 1year-old needles (needles of this year) of Pinus sylvestris show an increase towards the summer and a decrease in the fall. At the same time a towards the IAA content opposite seasonal change of the IAA-oxidase activity can be observed (Dangar and Basu, 1984). In the frame of the presented studies similar results could be obtained. With regard to seasonal changes of IAA concentrations our results correspond with those described by Little and Savidge (1987). They correlate the seasonal differences of the auxin concentrations with the cambial growth in conifers. Cambial growth as well as changes in IAA levels are seasonal. Therefore, Littel and Savidge (1987) show that, especially in young sprouts in spring, high IAA concentrations are involved in the regulation of cambial activity (i.e. mitotic activity) and xylem development. In older sprouts and in fall the IAA contents decrease after concluding xylem development. At the same time reduced cambial growth can be observed (Savidge, 1988).
*a<0.05 *a<0.05 *a<0.05
stadt site as well as Glindfeld and Velmerstot sites were significantly lower than the IAA levels of their respective un-
A definite diurnal dynamic in regard to the IAA content was observed at all locations under investigation. Similar results of circadian rhythms were also shown by Paasch et al. (1991). The different diurnal courses of the single trees seem to be location- or tree-specific, whereas the genetic variability between the tested trees can also be the cause of such effects. The presence of an endogenous rhythm with reference to the IAA concentrations can neither be excluded.
IAA in spruce needles in healthy and damaged trees IAA is highly light-sensitive. The reduction of free IAA in the light in contrast to total IAA is explained by an increased esterification of free IAA in light (Bandurski et al., 1977). This fact leads to the question whether diurnal conditioned light-stress could be the cause of photooxidative decomposition of free IAA. Yet, the diurnal deviations do not correlate with the light-intensity at the location, as supposed up to now (data not shown). Accordingly, photooxidative destruction does not take place or is balanced by endogenous hormone transport mechanisms or by an alterating metabolism. It could not be clarified to what extent alterations of the IAA-transport within the plant are responsible for the diurnal hormone balance. Neither could an influence of air pollutants concerning the auxin-transport be verified, as described by different authors (Kremer et aI., 1987; Meyer et aI., 1987; Thein and Michalke, 1988), nor did it seem that drought confines the auxin-transport, as concurrent investigations of the osmotic potential for quantifying plant water status show (data not shown).
Influence of damage class of the trees on the LAA concentrations Comparing undamaged and damaged trees, a definite decrease in free endogenous IAA could be determined in the case of damage. Therefore, these results confirm the reports of Volckers and Wild (1988). In this context the more moderately expressed diurnal course of the damaged trees in comparison to the undamaged partner trees is worth mentioning. In the following, different interpretations are introduced: Phenolic compounds may exercise an influence on the IAA metabolism. Monophenols stimulate the activity of IAA-oxidase, whereas di- or polyphenols can impede it (Pawar and Laloraya, 1988; Hoque, 1989). Hasemann et al. (1990) and Richter and Wild (1992) proved a distinct accumulation of phenols in damaged spruce trees. Thus, the growth retarding effect of phenols could at least partly be based on the ability to oxidize IAA and in this way diminish the internal IAA concentration at the time of damage. Auxins delay senescence. The content usually decreases before or during senescence (Nooden and Leopold, 1988). Higher IAA content in needles of trees that are free of damage symptoms could therefore effect a better resistance to premature needle aging, the associated senescence and the premature needle abscission. According to Dorffling (1982), auxin produced by leaves delays abscission. Yet, Gruber (1987) showed that the abscission of needles is primarily regulated by the endogenous water status. According to this, older needles are not or often incompletely connected to the secondary xylem and therefore are exposed to a poor water supply. A blockade in the transport system, respectively an interruption of the transport ways in the cambial zone and in the zone of xylogenesis, can also be responsible for a poor water supply in the needles. These processes are mostly regulated endogenously by plant growth substances. IAA is involved in the regulation of cambial activity and xylem development (Little and Savidge, 1987, Sundberg and Little, 1987). Therefore, the decreased IAA concentrations in the case of damage, as outlined in the present studies, could be responsible for the
619
incomplete connection of the needles to the secondary xylem or for a blockade in the transport systems. The decreased IAA concentrations at damage would thus indirectly lead to a premature loss of needles. The results of the present study show a clear dependency of the IAA content to the general degree of damage of the trees. The level of the plant growth regulator IAA also shows a clear alteration in dependency of the season and the time of day. Alterations in the cell metabolism of damaged spruce trees therefore seem to lead to a change in the IAA content. Three possibilities are conceivable to discuss the principal question of the interdependence between damage and hormone level: The first one postulates a clearly coincidental and nonspecific alteration of the hormone concentrations in virtue of a disturbed metabolism, caused by damage. The second possibility points out a stress orientated metabolism as a cause of damage, which is regulated by an active alteration of hormone levels. The stress induced hormones playa role in protecting plants against stress by building up stress tolerance. The third possibility presupposes an active alteration of hormone levels as a reason for the manifested damage. The here described changes in the IAA contents presumably may be caused by damage of the cell membranes. The hormone metabolism and the translocation or the perception of the hormones seem to be unspecifically disturbed and therefore correspond to the first possibility. The alteration of the phytohormone contents are declared as a secondary and damage accompanying reaction in spruce trees. The interaction between different phytohormones are numberous, and therefore only a minimum of information is given about the physiological role and the mode of action in these processes. Acknowledgements
This study was supported by the KfK-PEF (Kernforschungszentrum Karlsruhe - Projekt Europaisches Forschungszentrum fur MaBnahmen zur Luftreinhaltung), grant no.: 88/00711 A and by the Umweltbundesamt (Federal Environmental Office) Berlin, grant no.: 10803 046/16. References
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metabolism with reference to nitrogen fixation in Pterocarpus mursupium ROXB. Biochem. PhysioI. Pflanzen 179, 359-368 (1984). DIETZ, B., I. MOORS, U. FLAMMERSFELD, W. RUHLE, and A. WILD: Investigation on the photosynthetic membranes of spruce needles in relation to the occurrence of novel forest decline. 1. The photosynthetic electron transport. Z. Naturforsch. 43c, 581-588 (1988). DORFFUNG, K.: Das Hormonsystem der Pflanzen. Georg Thieme Verlag, Stuttgart (1982). FORSCHNER, W., V. SCHMITT, and A. WILD: Investigation on the starch content and ultrastructure of spruce needles relative to the occurrence of novel forest decline. Botanica Acta 102, 208 - 221. GRUBER, F.: Das Verzweigungssystem und der Nadelfall der Fichte (Picea abies (L). Karst.) als Grundlage zur Beurteilung von Waldschaden. Berichte des Forschungszentrums WaldokosystemlWaldsterben, Reihe A, Bd. 26 (1987). HASEMANN, G. and A. WILD: The loss of structural integrity in damaged spruce needles from locations exposed to air pollution. I. Mesophyll and central cylinder. J. Phytopathology 128, 15-32 (1990). HOQUE, E.: Effects of p-hydroxyacetophenone and its glucoside on the enzymic oxidation of indole-3-acetic acid. Agric. BioI. Chern. 53,239-240 (1989). JOHNSON, J. D.: Stress physiology of forest trees: The role of plant growth regulators. Plant Growth Regulation 6, 193-215 (1987). KE, D. and M. E. SALTVEIT: Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce. Plant PhysioI. 88, 1136 -1140 (1988). KREMER, H., H. PFLANZ, and W. HARTUNG: Die Wirkung saurer Luftschadstoffe auf Verteilung und Transport pflanzlicher Wachstumsregulatoren in Laub- und Nadelblattern. Allg. Forst Zeitschrift 27/28/29,741-744 (1987). LESHEM, Y. Y., A. H. HALEVY, and C. FRENKEL: Processes and Control of Plant Senescence. Elsevier, Amsterdam (1986). LITTLE, C. H. A. and R. A. SAVIDGE: The role of plant growth regulators in forest tree cambial growth. Plant Growth ReguI. 6, 137 -169 (1987). MENYAILA, L. N.: Role of the needles in hormonal regulation of xylogenesis. Sov. Plant PhysioI. 32,95-100 (1985). MEYER, A., P. MOUER, and G. SEMBDNER: Air pollution and plant hormone. Biochem. PhysioI. Pflanzen 182, 1-21 (1987). NOODEN, L. D. and A. C. LEOPOLD (eds.): Senescence and aging in Plants. Academic Press, New York (1988). PAASCH, K., F. SCHAFER, and K.-H. NEUMANN: A circadian rhythm of concentrations of several phytohormones in carrots (Daucus carota). In: Abstracts 14th International Conference on Plant Growth Substances. IPGSA Amsterdam 21-26 July (1991). PAWAR, M. S. and M. M. LALORAYA: Effect of phenolic compounds on indole-3-acetic acid induced abscission. Biochem. PhysioI. Pflanzen 183, 75-82 (1988). RICHTER, CH. and A. WILD: Phenolic compounds in needles of Norway spruce trees in relation to novel forest decline. 1. Studies on
trees from a site in the northern Black Forest. Biochem. PhysioI. Pflanzen, 188, 305-320 (1992). SACHS, L.: Statistische Methoden: Planung und Auswertung. 6. Aufl. Springer-Verlag, Berlin, Heidelberg (1988). SAVIDGE, R. A.: Auxin and ethylene regulation of diameter growth in trees. Tree PhysioI. 4, 404-414 (1988). SCHWARTZENBERG, K. VON, K. LUTZE, and H. HAHN: Determination of cytokinin content in needles of spruce (Picea abies (L.) Karst.) by an indirect enzyme-linked immunosorbent assay. J. Plant PhysioI. 133, 529-534 (1988). SIEFERMANN-HARMS, D.: Forschungsschwerpunkt Freudenstadt. 6. Statuskolloquium des PEF vom 6.-8. Marz 1990, Kernforschungszentrum Karlsruhe - Projekt Europaisches Forschungszentrum flir MaBnahmen zur Luftreinhaltung. KfK-PEF 61, 1-10 (1990). SUNDBERG, B. and C. H. A. LITTLE: Effect of defoliation on tracheid production and the level of indole-3-acetic acid in Abies balsamea shoots. PhysioI. Plant. 71, 430-435 (1987). TENTER, M. and A. WILD: Investigations on the polyamine content of spruce needles relative to the occurrence of novel forest decline. J. Plant PhysioI. 137, 647 -654 (1991). THEIN, M. and W. MICHALKE: Bisulfite interacts with binding sites of the auxin-transport inhibitor N-l-naphthylphthalamic acid. Planta 176, 323-350 (1988). TIETZ, S. and A. WILD: Investigations on the phosphoenolpyruvate carboxylase activity of spruce needles relative to the occurrence of novel forest decline. J. Plant PhysioI. 137, 327 -332 (1991). VOLCKERS, P. and A. WILD: A specific radioimmunoassay for the determination of low quantities of indole-3-acetic acid in spruce needles of healthy and damaged trees. J. Plant PhysioI. 133, 320-324 (1988). WEILER, E. W.: Radioimmunoassay for pmol-quantities of indole-3acetic acid for use with highly stable (125J)- and CH)-IAA derivatives as radiotracers. Plant a 153,319-325 (1981). - Plant hormone immunoassay. PhysioI. Plant. 54, 230-234 (1982). - Immunoassay of plant growth regulators. Annu. Rev. Plant PhysioI. 35, 85-95 (1984). WILD, A., U. FLAMMERSFELD, 1. MOORS, B. DIETZ, and W. RUHLE: Investigation on the photosynthetic membranes of spruce needles in relation to the occurrence of novel forest decline - II. The content of QB-protein, cytochrome f and P-700. Z. Naturforsch. 43 c (Biosciences 43),589-595 (1988). WILD, A., W. FORSCHNER, and V. SCHMITT: Physiologische, biochemische und cytomorphologische Untersuchungen an immissionsbelasteten Fichten. Forschungsendbericht zum FE Vorhaben Nr. 10803046/16, im Auftrag des Umweltbundesamtes Berlin (1990). WILD, A. and S. TIETZ-SIEMER: Physiologische und cytomorphologische Untersuchungen an ungeschadigten und geschadigten Fichten im Nordschwarzwald (Freudenstadt). 8. Statuskolloquium des PEF vom 17.-19. Marz 1992, Kernforschungszentrum Karlsruhe - Projekt Europaisches Forschungszentrum flir MaBnahmen zur Luftreinhaltung. KfK-PEF 94, 47 -59 (1992).
Investigations on the Content of Indole-3-acetic Acid in Spruce Needles of Healthy and Damaged Trees of Various Sites A.
WESSLER
and A.
WILD
Institute of General Botany, Johannes Gutenberg University, Saarstr. 21, 6500 Mainz, Germany Received September 9,1992 . Accepted December 15, 1992
Summary
Surveys were made on the concentration of indole-3-acetic acid (IAA) in spruce trees of various sites. The concentrations of the hormone were specified with a radioimmunoassay after the needle extract had been purified in several steps. The content of IAA in needles of damaged trees was distinctly lower than the content in needles of healthy trees. Another feature was the annual rythm of the IAA content with the maximum of concentration in June. A distinct diurnal course with highest level of concentration during the early afternoon hours was also observed. The diurnal rhythm of the damaged trees was clearly lower. The supposition is that the lower IAA content is responsible for a premature needle abscission.
Key words: Picea abies, indole·3-acetic acid, annual rhythm, diurnal course, novel forest decline, radioimmunoassay. Abbreviations: BHT = buty1hydroxytoluene; IAA = indo1e-3-acetic acid; PBS = phosphate buffered saline; RIA = radioimmunoassay; MEST = middle european summer time.
Introduction
Since the end of the seventies, damage to forest trees has appeared in Central Europe, which is now designated as «novel forest decline» (BML, 1984-1991). Although early discussions on forest damage had brought up the speculation that changes in hormones are also responsible for the symptoms of new forest damage, there are less or no definite statements on alterations in the hormone system of damaged trees (Christmann and Frenzel, 1987; Schwanzenberg et al., 1988). In most cases a decrease in indole-3-acetic acid (IAA) and cytokinin concentration is to be expected in stress loaded trees, whereas the concentration of the antagonistic working hormones abscisic acid and ethylene is mostly higher during the influence of stress Qohnson, 1987). The surveys on alteration of hormones, as a contribution to clarify cause and pathogenesis of novel forest decline, require a basic characterization of the hormone system of the affected tree species. © 1993 by Gustav Fischer Verlag, Stuttgart
IAA is the most imponant native auxin in plants. The relevance of IAA in connection with control of senescence and abscission has repeatedly been reponed (Addicott, 1970; Leshem et al., 1986; Ke and Sa1tveit, 1988). Symptoms of novel forest decline like premature loss of leaves or needles and early aging may be connected with disturbances in the balance of IAA metabolism. It was the aim of the present study to determine the quantity of IAA concentration in order to clarify regulation processes in damaged trees_ With the assistance of a specific and sensitive radioimmunoassay combined with several purification steps, investigations of spruce trees at different sites with similar immission charge and comparable climatic conditions were able to be carried out. Phenotypically undamaged and damaged trees were compared with each other and the interdependence between the IAA concentrations and the corresponding tree- and location-specific damage classes were reviewed. Special attention was given to the annual and diurnal dynamics of the IAA contents, as deviations in ref-
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erence to these influences are to be expected, especially in the analysis of IAA (Dangar and Basu, 1984). The present study on the IAA content of spruce trees is part of a research project characterizing the damage of trees with the help of physiological, biochemical and cytomorphological parameters (Benner et aI., 1988; Dietz et al., 1988; Wild et al., 1988, 1990; Forschner et al., 1989; Hasemann and Wild, 1990; Wild and Tietz-Siemer, 1992). Materials and Methods
Description of the sites The investigations described below were performed over a period of two years, 1989 and 1990, at several natural sites located in BadenWiirttemberg, Nordrhein-Westfalen and Rheinland-Pfalz in the Federal Republic of Germany. In 1989, samples were collected between April and November in order to get information about changes in IAA levels during the vegetation period. In 1990, diurnal courses of the content of IAA were investigated. Climatic and immission data were registered continuously. At all sites there were high ozone concentrations in summer whereas the levels of the air pollutants S02 and N0 2 were relatively low during the whole test period. Detailed descriptions of the sites have been presented by Wild et al. (1990), Tenter and Wild (1991), Tietz and Wild (1991) and Richter and Wild (1992).
Hattgenstein site This site is located in the western part of the Hunsriick mountains in Rheinland-Pfalz at about 660 m above sea level (Idar-Oberstein Forestry Office, Hattgenstein Forest District, Division 257 b2). The spruce trees were between 25 and 30 years old. The damaged trees showed partial needle loss and yellowing of the upper part of the older needles (mountainous yellowing). The harvest date of the diurnal concentration course was September 7, 1989.
Freudenstadt site The site is located at 820 - 830 m above sea level near Freudenstadt in the northern Black Forest (Freudenstadt Forest Office, Vordersteinwald Forest District, Division III/12 a\ at Schollkopf). At this site two different stands of spruce trees were examined. The trees were between 45 and 50 years old. The trees of one stand were highly damaged (damage classes 2 and 3). These trees were compared with an adjacent stand of apparently relatively symptomfree spru~e trees (damage classes 0 and 1). The general type of tree damage at this site is called «Montane Vergilbung» (mountainous yellowing of spruce) (Siefermann-Harms, 1990). The harvest dates in 1989 were April 19, June 28, August 30 and November 8, and in 1990 the needles used for the diurnal course were examined on August 2.
Glindfeld and Velmerstot site The Glindfeld site is located in the Rothaargebirge (Glindfeld Forestry Office, Kiistelberg Forest District, Division 62 c6) about 630 m above sea level. The spruce plantations under investigation consist of trees that are about 50 years old. They belonged to the damage class 0 and 1, but gradual detoriation could be seen through needle loss and chlorosis during the last year. In 1989, mixed samples of six trees were harvested four times a year (April 25, June 20, September 5 and November 14). On June 12, 1990, needles of an undamaged tree were
harvested in order to get information about the diurnal course of IAA-concentrations. The spruce plantation under investigation at the Velmerstot site is located in the southern part of the Teutoburger Wald called Eggegebirge (Paderborn Forestry Office, Kempen Forest District, Division 399 B), which is about 410 m above sea level. The spruce trees at the Velmerstot site were about 55 years old. The trees examined were described as damaged, belonging to damage classes 2 and 3. However, it is noteworthy that for the last three years a distinct recovery phase of the plantation could be detected. Sampling took place on April 25, June 19, September 4 and November 13 in 1989 and on June 11 in 1990.
Materials
The investigations were performed on spruce needles (Picea abies L. Karst.) of the second needle generation (needles grown in the previous year) from the sixth to the eight whorl. The samples, which were collected in oder to get information about changes in IAA levels during the vegetation period, were harvested from 11.00 h to 13.00 h (MEST). The studies were performed with the needles of 6 trees per stand in mixed samples. Samples needed for the investigation of diurnal courses were collected hourly from 7.00 h to 18.00 h (MEST) and were conducted in pairs: One symptom-free tree was compared with a more severely damaged tree. The needles were removed instantly from the twigs by stirring them in liquid nitrogen; afterwards the needles were stored at -80°C.
Methods
Extraction offree IAA from spruce needles One gram of deep frozen needles was grounded to a powder with mortar and pestle in liquid nitrogen and extracted with 50 mL icecold 80 % (v/v) methanol for several minutes. BHT was added to the methanol to prevent oxidation. The homogenate was filtered in a Biichner funnel and brought with 0.5 M HEPES-buffer, pH 8.0, to a concentration of 70 % methanol. After passing through two SEPPAK C I8 Cartridges (Fa. Waters) the methanol was removed in a rotary evaporator (Fa. Schlitt) at 35°C. The remaining aqueous phase was centrifuged for 5 min by 1000 x gto remove the insoluble components. Then, the extract was purified with high-pH buffer ethyl acetate partition. The aqueous phase was adjusted to a pH of 2.4-2.6; phenolic substances were eliminated with unsoluble polyvinylpyrrolidone and the extract was partitioned again with ethyl acetate at low pH. The remaining ethyl acetate phases were dried with N 2at 37°C and then methylated with a boron trifluoride methanol complex. The extracts were kept at - 80 °C in the dark and assayed after some days.
Radioimmunoassay (RIA) The general procedure used for the RIA has been described by Weiler (1981, 1982, 1984). Samples were measured in triplicate. The PBS-buffer consisted of 8 mM KH 2P0 4, 1.6 mM K1 HP0 4 and 150 mM NaC!, pH 7.4. The RIA-mixture per tube (MiIli-3-PE, Fa. Baker) contained 0.65 mL PBS-buffer, 0.10 mL bovine serum albumin (1: 10) and 2 ~L radioactive IAA tracer (c. 15,000dpm). After an incubation period of 2 h at 4 °C the reaction was stopped with 1 mL (NH 4)2S04 (60.16 g in 100 mL distilled water). Precipitation occurred for 20 min. After centrifugation (1500 x g) the pellets were washed with 1 mL (NH4)2S04 (30.85 g in 100 mL distilled
IAA in spruce needles in healthy and damaged trees
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350
water), centrifuged and decanted. The pellet was dissolved in 0.2 mL water and 1.5 mL scintillation cocktail. Radioactivity was measured by liquid scintillation counting (Volckers and Wild, 1988).
300 250
General parameters
The preparation and assay of IAA-BSA-conjugates, the immunization and the specifity of the antiserum have already been described by Volckers and Wild (1988). The detection limit was 0.5 pmol IAA per assay. The measuring range was from 0.5 -1000 pmol IAA. The carboxyl-coupled IAA-elicited-antibodies gave a titer of 1 : 170 (final dilution, binding 26 % of the tracer).
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Calculation and statistical analysis
The calculation of the RIA dates was performed with a separate calibration. The logit B/Bo-data (logit B/Bo = In(B/Bo)/(100-B/Bo) was evaluated by standard dilution curves using a linear regression analysis. The measured equation data were y = -2.56 x +2.37 and the correlation coefficient was r = 0.997. Data were analyzed using a two-tailed independent Student's t test. Results were considered significantly different if ex < 0.05. Significance levels were indicated according to Sachs (1988) by asterics (* ex < 0.05, ** ex < 0.01, *** ex < 0.001). Measurements were usually repeated twice or three times per sample.
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Results With respect to the ratio fresh weight per dry weight no remarkable differences could be found between needles of undamaged and damaged spruce trees. Deviation with relation to the annual and daily courses could also not be found (data not shown). Therefore, dry weight was used as a suitable reference basis for the measurements of IAA.
100
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June
September
November
Fig. 1 A, B: Content of free IAA in relation to dry weight at the Freudenstadt site (A) in needles of undamaged (0) and damaged (_) trees and at the Glindfeld (0 needles of apparently healthy or slightly damaged trees) and Velmerstot sites (_ needles of more severely damaged trees) (B).
Seasonal changes of IAA concentrations In order to locate seasonal condition changes of IAA contents (free IAA) during the vegetation period, samples were taken in 1989 at different times of the year at the Freudenstadt, Glindfeld and Velmerstot sites. The seasonal condition changes of the IAA concentrations are shown in Fig. 1 A and B. In the first instance, an increase in IAA concentrations from the harvest month April to June can be observed, which is followed by a decrease in the IAA contents towards the fall. The highest IAA contents were accordingly measured at all sites in June after the start of the cambial growing period in spring.
Diurnal changes of IAA concentrations In order to determine the diurnal rhythm of IAA concentrations pair comparisons were carried out at various sites: needles of a damaged tree as well as needles of an apparently healthy tree were harvested hourly between 7.00 hand 18.00 h (MEST) and the IAA contents of these needles were determined. Figure 2A-C shows the diurnal related concentration changes of the IAA contents at the locations of Hattgenstein (Fig. 2 A), Glindfeld and Velmerstot (Fig. 2 B), and Freudenstadt (Fig. 2 C).
A clear diurnal course of the IAA content was discernable at all tested locations. The IAA concentrations of the undamaged trees in Hattgenstein and Freudenstadt showed first a decrease during the morning hours and a definite increase during the noon and early afternoon hours, which then decreased again during the late afternoon until 6.00 h. The concentration of the corresponding damaged trees showed only a weak reflection of these observed diurnal rhythms; clearly weaker noon-maxima, for example, were found. The undamaged tree at the Glindfeld site in the Rothaargebirge showed two concentration maxima on the day studied. One maximum could be observed at 9.00 h as well as one at 1s.00h. The diurnal course of the damaged tree at the Velmerstot site, however, only showed one maximum at 14.00 h, just like the trees of the other locations.
Influence of the damage class of the trees on the IAA concentrations The influence of the damage classes of the trees on the endogenous IAA contents showed the same development at all tested locations (Fig. 1; Table 1). At every harvest time in 1989 the IAA concentrations of damaged trees at Freuden-
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damaged corresponding trees. At the beginning of the vegetation period the difference in IAA content at the Freudenstadt site (Fig. 1 A) was relatively low (17 %), whereas an increased loss of the IAA level of the damaged spruce trees up to 60 % was determined in the fall. Thus, a remarkable decrease of 29 % (mean value of the year, Table 1) was registered. The spruce trees of the Velmerstot location also showed clearly lower IAA concentrations than the ones at the Glindfeld site (Fig. 1 B). A remarkable decrease of 46 % (mean value of the year, Table 1) was registered. The differences are similar at the first three harvest dates (45 %, 50 % and 55 %). At the last harvest date in November the difference amounted to only 24 %. The mean values of the diurnal courses at all locations also show significant differences (Table 1). These surveys also lead to the result that IAA levels of damaged trees are much lower than those of their corresponding undamaged partner trees. At the Hattgenstein site this fact is becoming very clear, as the daily average of the damaged tree is 63 % lower than the daily average of the apparently healthy tree. At the Freudenstadt location a similar processing difference of approximately 40 % can be observed, whereas the damaged tree from the Velmerstot location only shows 17 % lower IAA concentrations compared with the undamaged tree at the Glindfeld site.
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Discussion
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harvest time Fig. 2 A-C: Diurnal courses of free lAA content (nmol. kg- 1 dry weight) in the needles of undamaged (x) and damaged (_) spruce trees at different sites (A) Hattgenstein, September 7, 1989; (B) Velmerstot/Glindfeld, June 11112, 1990; and (C) Freudenstadt, August 2,1990. Table 1: Content of free lAA (nmol· kg- 1 dry weight) in needles of damaged and healthy spruce trees at different sites. Mean values of the year 1989 and of measurements from the diurnal courses in 1989 and 1990. site
mean values of the year undamaged
damaged
Freudenstadt Glindfeld/Velmerstot
206.42 245.93
145.70 132.99
significance
*a<0.05 *a<0.05
Diurnal changes 0/ L4A concentrations
mean values of the day Hattgenstein Glindfeld/Velmerstot Freudenstadt
undamaged
damaged
375.41 164.06 101.24
140.07 136.42 60.42
Menyaila (1985) pointed out that the IAA contents in 1year-old needles (needles of this year) of Pinus sylvestris show an increase towards the summer and a decrease in the fall. At the same time a towards the IAA content opposite seasonal change of the IAA-oxidase activity can be observed (Dangar and Basu, 1984). In the frame of the presented studies similar results could be obtained. With regard to seasonal changes of IAA concentrations our results correspond with those described by Little and Savidge (1987). They correlate the seasonal differences of the auxin concentrations with the cambial growth in conifers. Cambial growth as well as changes in IAA levels are seasonal. Therefore, Littel and Savidge (1987) show that, especially in young sprouts in spring, high IAA concentrations are involved in the regulation of cambial activity (i.e. mitotic activity) and xylem development. In older sprouts and in fall the IAA contents decrease after concluding xylem development. At the same time reduced cambial growth can be observed (Savidge, 1988).
*a<0.05 *a<0.05 *a<0.05
stadt site as well as Glindfeld and Velmerstot sites were significantly lower than the IAA levels of their respective un-
A definite diurnal dynamic in regard to the IAA content was observed at all locations under investigation. Similar results of circadian rhythms were also shown by Paasch et al. (1991). The different diurnal courses of the single trees seem to be location- or tree-specific, whereas the genetic variability between the tested trees can also be the cause of such effects. The presence of an endogenous rhythm with reference to the IAA concentrations can neither be excluded.
IAA in spruce needles in healthy and damaged trees IAA is highly light-sensitive. The reduction of free IAA in the light in contrast to total IAA is explained by an increased esterification of free IAA in light (Bandurski et al., 1977). This fact leads to the question whether diurnal conditioned light-stress could be the cause of photooxidative decomposition of free IAA. Yet, the diurnal deviations do not correlate with the light-intensity at the location, as supposed up to now (data not shown). Accordingly, photooxidative destruction does not take place or is balanced by endogenous hormone transport mechanisms or by an alterating metabolism. It could not be clarified to what extent alterations of the IAA-transport within the plant are responsible for the diurnal hormone balance. Neither could an influence of air pollutants concerning the auxin-transport be verified, as described by different authors (Kremer et aI., 1987; Meyer et aI., 1987; Thein and Michalke, 1988), nor did it seem that drought confines the auxin-transport, as concurrent investigations of the osmotic potential for quantifying plant water status show (data not shown).
Influence of damage class of the trees on the LAA concentrations Comparing undamaged and damaged trees, a definite decrease in free endogenous IAA could be determined in the case of damage. Therefore, these results confirm the reports of Volckers and Wild (1988). In this context the more moderately expressed diurnal course of the damaged trees in comparison to the undamaged partner trees is worth mentioning. In the following, different interpretations are introduced: Phenolic compounds may exercise an influence on the IAA metabolism. Monophenols stimulate the activity of IAA-oxidase, whereas di- or polyphenols can impede it (Pawar and Laloraya, 1988; Hoque, 1989). Hasemann et al. (1990) and Richter and Wild (1992) proved a distinct accumulation of phenols in damaged spruce trees. Thus, the growth retarding effect of phenols could at least partly be based on the ability to oxidize IAA and in this way diminish the internal IAA concentration at the time of damage. Auxins delay senescence. The content usually decreases before or during senescence (Nooden and Leopold, 1988). Higher IAA content in needles of trees that are free of damage symptoms could therefore effect a better resistance to premature needle aging, the associated senescence and the premature needle abscission. According to Dorffling (1982), auxin produced by leaves delays abscission. Yet, Gruber (1987) showed that the abscission of needles is primarily regulated by the endogenous water status. According to this, older needles are not or often incompletely connected to the secondary xylem and therefore are exposed to a poor water supply. A blockade in the transport system, respectively an interruption of the transport ways in the cambial zone and in the zone of xylogenesis, can also be responsible for a poor water supply in the needles. These processes are mostly regulated endogenously by plant growth substances. IAA is involved in the regulation of cambial activity and xylem development (Little and Savidge, 1987, Sundberg and Little, 1987). Therefore, the decreased IAA concentrations in the case of damage, as outlined in the present studies, could be responsible for the
619
incomplete connection of the needles to the secondary xylem or for a blockade in the transport systems. The decreased IAA concentrations at damage would thus indirectly lead to a premature loss of needles. The results of the present study show a clear dependency of the IAA content to the general degree of damage of the trees. The level of the plant growth regulator IAA also shows a clear alteration in dependency of the season and the time of day. Alterations in the cell metabolism of damaged spruce trees therefore seem to lead to a change in the IAA content. Three possibilities are conceivable to discuss the principal question of the interdependence between damage and hormone level: The first one postulates a clearly coincidental and nonspecific alteration of the hormone concentrations in virtue of a disturbed metabolism, caused by damage. The second possibility points out a stress orientated metabolism as a cause of damage, which is regulated by an active alteration of hormone levels. The stress induced hormones playa role in protecting plants against stress by building up stress tolerance. The third possibility presupposes an active alteration of hormone levels as a reason for the manifested damage. The here described changes in the IAA contents presumably may be caused by damage of the cell membranes. The hormone metabolism and the translocation or the perception of the hormones seem to be unspecifically disturbed and therefore correspond to the first possibility. The alteration of the phytohormone contents are declared as a secondary and damage accompanying reaction in spruce trees. The interaction between different phytohormones are numberous, and therefore only a minimum of information is given about the physiological role and the mode of action in these processes. Acknowledgements
This study was supported by the KfK-PEF (Kernforschungszentrum Karlsruhe - Projekt Europaisches Forschungszentrum fur MaBnahmen zur Luftreinhaltung), grant no.: 88/00711 A and by the Umweltbundesamt (Federal Environmental Office) Berlin, grant no.: 10803 046/16. References
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trees from a site in the northern Black Forest. Biochem. PhysioI. Pflanzen, 188, 305-320 (1992). SACHS, L.: Statistische Methoden: Planung und Auswertung. 6. Aufl. Springer-Verlag, Berlin, Heidelberg (1988). SAVIDGE, R. A.: Auxin and ethylene regulation of diameter growth in trees. Tree PhysioI. 4, 404-414 (1988). SCHWARTZENBERG, K. VON, K. LUTZE, and H. HAHN: Determination of cytokinin content in needles of spruce (Picea abies (L.) Karst.) by an indirect enzyme-linked immunosorbent assay. J. Plant PhysioI. 133, 529-534 (1988). SIEFERMANN-HARMS, D.: Forschungsschwerpunkt Freudenstadt. 6. Statuskolloquium des PEF vom 6.-8. Marz 1990, Kernforschungszentrum Karlsruhe - Projekt Europaisches Forschungszentrum flir MaBnahmen zur Luftreinhaltung. KfK-PEF 61, 1-10 (1990). SUNDBERG, B. and C. H. A. LITTLE: Effect of defoliation on tracheid production and the level of indole-3-acetic acid in Abies balsamea shoots. PhysioI. Plant. 71, 430-435 (1987). TENTER, M. and A. WILD: Investigations on the polyamine content of spruce needles relative to the occurrence of novel forest decline. J. Plant PhysioI. 137, 647 -654 (1991). THEIN, M. and W. MICHALKE: Bisulfite interacts with binding sites of the auxin-transport inhibitor N-l-naphthylphthalamic acid. Planta 176, 323-350 (1988). TIETZ, S. and A. WILD: Investigations on the phosphoenolpyruvate carboxylase activity of spruce needles relative to the occurrence of novel forest decline. J. Plant PhysioI. 137, 327 -332 (1991). VOLCKERS, P. and A. WILD: A specific radioimmunoassay for the determination of low quantities of indole-3-acetic acid in spruce needles of healthy and damaged trees. J. Plant PhysioI. 133, 320-324 (1988). WEILER, E. W.: Radioimmunoassay for pmol-quantities of indole-3acetic acid for use with highly stable (125J)- and CH)-IAA derivatives as radiotracers. Plant a 153,319-325 (1981). - Plant hormone immunoassay. PhysioI. Plant. 54, 230-234 (1982). - Immunoassay of plant growth regulators. Annu. Rev. Plant PhysioI. 35, 85-95 (1984). WILD, A., U. FLAMMERSFELD, 1. MOORS, B. DIETZ, and W. RUHLE: Investigation on the photosynthetic membranes of spruce needles in relation to the occurrence of novel forest decline - II. The content of QB-protein, cytochrome f and P-700. Z. Naturforsch. 43 c (Biosciences 43),589-595 (1988). WILD, A., W. FORSCHNER, and V. SCHMITT: Physiologische, biochemische und cytomorphologische Untersuchungen an immissionsbelasteten Fichten. Forschungsendbericht zum FE Vorhaben Nr. 10803046/16, im Auftrag des Umweltbundesamtes Berlin (1990). WILD, A. and S. TIETZ-SIEMER: Physiologische und cytomorphologische Untersuchungen an ungeschadigten und geschadigten Fichten im Nordschwarzwald (Freudenstadt). 8. Statuskolloquium des PEF vom 17.-19. Marz 1992, Kernforschungszentrum Karlsruhe - Projekt Europaisches Forschungszentrum flir MaBnahmen zur Luftreinhaltung. KfK-PEF 94, 47 -59 (1992).