ORIGINAL ARTICLE

Alexander Christmann ? Jacqueline ChristmannPetra Schiller ? Burkhard Frenzel

Phytohormones in needles of healthy and declining silver fir

(Abies alba Mill.) : I. Indole-3-acetic acid

Received: 4 May 1995 / Accepted: 29 August 1995

AbstractmLevels of indole-3-acetic acid (IAA) were deter-mined in needles from silver fir (Abies albaMill.) trees inthe northern Black Forest. IAA was quantified by gaschromatography (GC) as 1-heptafluorobutyryl-IAA-meth-ylester (HFB-IAA-ME) using electron capture detection.Prior to GC analysis, extensive purification of needleextracts was performed employing two HPLC steps. Peakidentity of HFB-IAA-ME was confirmed by combined gaschromatography-mass spectrometry in selected samples.Levels of IAA in needles belonging to different needleage-classes exhibited a cyclic seasonal pattern with highestconcentrations in winter and lowest levels in spring whenbud-break occurred. Such a cyclic seasonal pattern of IAAlevels was also observed in needles from declining fir treesor fir trees suffering from a strong sulfur impact (S-impact)in the field due to a local SO2 source. Levels of IAAincreased with increasing needle age. This age dependencyof IAA concentrations was most pronounced in late autumnwhen IAA levels were high and nearly disappeared inspring when IAA levels reached their minimum. In needlesfrom declining fir trees or fir trees suffering from a strongS-impact in the field, IAA levels hardly increased withincreasing needle age. It is suggested that in healthy treeshigh levels of IAA protect older needles from abscissionand that the considerable losses of older needles of declin-ing fir trees or of fir trees under S-impact are a consequenceof the low levels of IAA found in older needles of suchtrees.

Key wordsmIndole-3-acetic acid? Abies? Needle senes-cence? Forest decline? SO2

Introduction

Among the different tree species suffering from forestdecline in Germany, silver fir (Abies albaMill.) is stillaffected most (Moosmayer 1992). Similar silver fir declinesseem to have appeared in former times, which are welldocumented since the nineteenth century (Kandler 1992).However, it is difficult to demonstrate that the causes of thesuccessive declines were always the same.

While declines of other forest tree species are also norecent phenomenon (cf. Demorlaine 1927; Frenzel 1985;Kandler 1992), silver fir growing in Central Europe seemsto be more sensitive to biotic and abiotic stressors, probablydue to a reduced genetic variation at the northern boundaryof the natural geographic distribution of this species (Lar-sen 1989). Thus with this species it should be less difficultto establish possible relationships between presumed causesof forest decline and changes in tree physiology.

In contrast to other species affected, silver fir exhibitsonly one, clearly defined damage type (ForschungsbeiratWaldscha¨den/Luftverunreinigungen 1989). This damagetype is characterized by (1) needle losses starting at olderparts of the branches, (2) increment reduction, (3) bendingdown of first order twigs and (4) formation of the so-calledstork’s nest caused by a reduction in apical growth (Frenzel1985). These morphological symptoms are a consequenceof (1) premature senescence and abscission of needles, (2) alower number of cell divisions in the cambial region, (3)reduced formation of tension wood and (4) a reduced apicaldominance. In all of these aspects of plant growth, the planthormone indole-3-acetic acid (IAA) may play a significantrole (cf. Dorffling 1982; Little and Savidge 1987).

We have studied levels of IAA in needles of silver fir,because changes in needle IAA levels might well affect thewhole tree due to the commonly accepted view that IAA isproduced mainly in young leaf organs (Davies 1995) andreaches other parts of the trees by basipetal transport(Lomax et al. 1995).

Since visual appearance of the trees may not correspondto the true physiological state, we tried to define the health

Trees (1996) 10: 331–338 Springer-Verlag 1996

A. Christmann ( ) ? J. Christmann? P. Schiller? B. FrenzelInstitut fur Botanik, Universita¨t Hohenheim (210), D-70593 Stuttgart,Germany

status of the trees by studying parameters other thanhormone levels like gas exchange and ultrastructure ofmesophyll cells (Christmann 1993; Frenzel et al. 1987).By repeatedly measuring these parameters on the sametrees, we were further able to monitor changes of treehealth status which helped us to consider our results asconsequences of dynamic processes in declining trees.

Materials and methods

Trees and experimental sites

Fifteen silver fir (Abies albaMill.) trees between 30 and 140 years oldand with different degrees of damage were investigated at fourexperimental sites situated in the northern Black Forest (see Christ-mann 1993 for the location of the sites and for further details).

Pollution climate

Sulfur impact (S-impact), i.e. impact of SO2 and of the ions formedfrom SO2 after it dissolves in water, was exclusively present on theeastern slope of the Sto¨ckerkopf site due to a local SO2 source[Baumbach et al. 1988; Gliemeroth 1993; Adam 1986 (ForstlicheVersuchs- und Forschungsanstalt Baden-Wu¨rttemberg, Abteilung Bo-tanik und Standortskunde, data presented in Frenzel et al. 1987 andChristmann 1993)]. There, half-hour values exceeding 375 nmol mol–1

were measured during high pressure periods when inversion layersformed in the valley during the night (Baumbach et al. 1988). Sulfurcontents of needles increased with needle age and reached maximumcontents in 5-year-old needles of spruce [Picea abies(L.) Karst.,seventh whorl] of 94–153µmol total S g–1 dry weight (dw;3000–4900µg total S g–1 dw; Adam 1986), which is comparable toconcentrations found by Pfanz and Beyschlag (1993) in spruce needlesfrom the heavily polluted Erzgebirge. According to Baumbach andBaumann (1990), ozone concentrations at our sites presumably corre-spond to those of the nearby site Scho¨llkopf: 50–75 nmol mol–1

(monthly averages) in the summer months with peak levels (half-hour values) of up to 125 nmol mol–1 (Baumbach and Baumann 1990).Due to negligible concentrations of nitrogen oxides in the air (Baum-bach and Baumann 1991) and of ammonium ions in precipiation in thenorthern Black Forest (Evers 1985), these two pollutants should be ofless importance at our sites.

Plant nutrition

The nutritional status of spruce and fir trees growing at our sites wasinvestigated by the Forstliche Versuchsanstalt Baden-Wu¨rttemberg(Forstliche Versuchsanstalt Baden-Wu¨rttemberg 1991, personal com-munication; Adam 1986; data presented in Christmann 1993). Apartfrom trees of the Sto¨ckerkopf site, the needles of the experimental treesexhibited no deficiencies in N, P, S, K, Ca, Mn, Al, Zn and Fe.Magnesium content of needles varied with degree of damage. Sincetrees used in our study showed no pathological changes in needlephloem (Christmann 1993), magnesium deficiency can be ruled out forthese trees (cf. Fink 1988). On the eastern slope of the Sto¨ckerkopf site,just in front of the sulfur emittent, sulfur contents of needles wereelevated whereas a strong potassium deficiency was also found in theseneedles. Trees growing on the western slope had no elevated sulfurconcentrations in their needles, but were poorly supplied with nitrogen.

Sampling of needles

At each sampling date needles belonging to different needle age-classes were taken early in the morning from one twig belonging tothe 7th to 12th whorl of the sun crown. Needles of the main twig axis

and of second order twigs belonging to different needle age-classeswere immediately frozen in liquid nitrogen after sampling and stored inliquid nitrogen until they were freeze-dried. Homogenisation of freeze-dried needles was done in a hammer-mill (Retsch, Haan, Germany)which was cooled with liquid nitrogen. Final grain size was 0.2 mm.Homogenised needles were again freeze-dried overnight to remove anytraces of water condensed during homogenisation and were finallystored at –20°C over silica gel.

Extraction and purification of plant material

Indole 3-acetic acid (IAA) was extracted from freeze-dried, groundneedles (200 mg) together with abscisic acid (AbA) under nitrogen for16 h in the dark. Samples were stirred during extraction and kept at4 °C. Extraction medium was 50 ml methanol :0.02 M phosphatebuffer pH 7.0 (4 :1, v :v) containing 0.01% (w/v) 2,6-di-t-butyl-4-methylphenol. After filtration through a Teflon filter (0.45µm),methanol was removed in vacuo at 35°C. The aqueous residue waspassed through a PVPP column (1.4× 2.5 cm) and the eluate (35 ml)was extracted with an equal volume of water-saturated t-butylmethyl-ether (TBME). The aqueous phase was then acidified with 0.4 n HCl(pH 3.0) and extracted 3 times with TBME. Organic phases werecombined and TBME removed in vacuo at 35°C. Samples were thentransferred to 5 ml Reacti-Vials with methanol and were methylatedwith a solution of diazomethane in diethylether according to Schlenckand Gellerman (1960). Normal phase HPLC was performed aftermethylation with a Gynkotek HPLC System (Gynkotek, Germering,Germany) using a 100× 4.6 mm Partisil 5µm column [mobile phase:hexane (A) and ethyl acetate (B); after 5 min of 5% B, 95% A: gradientto 100% B in 30 min, flow rate: 1 ml min–1]. Retention time of IAA-ME was 17.3 min. The HPLC fraction containing IAA-ME wasevaporated to dryness under reduced pressure (35°C). IAA-ME wasconverted to 1-heptafluorobutyryl-indole-3-acetic acid methyl ester(HFB-IAA-ME) with 40 µl heptafluorobutyrylimidazole in the pres-ence of 30µl pyridine (150°C, 120 min) according to Allen et al.(1982). The reaction mixture was dried under a stream of nitrogen andtransferred to a Silica Sep-Pak cartridge (Waters Chromatographie,Eschborn, Germany) with 3× 200µl ethyl acetate :hexane (5 :95; v :v).HFB-IAA-ME was eluted from the cartidge with 5 ml ethylacetate :hexane (5 :95; v :v). Further purification of HFB-IAA-MEwas achieved on a 100× 4.6 mm Partisil 5µm column [mobilephase ethyl acetate :hexane = 5:95 (v :v), flow rate: 1 ml min–1].Retention time of HFB-IAA-ME was 8 min. The HPLC fractioncontaining HFB-IAA-ME was evaporated to dryness under reducedpressure (35°C) and the sample was then dissolved in 100µl ethylacetate with 2,29,3,4,49,59-hexachlorobiphenyl (PCB 138) as internalstandard. Quantification of HFB-IAA-ME was carried out by GLC(Hewlett Packard 5890A gas chromatograph) using a 25 m fused silicacapillary column [CPMS/17001] chemical bonded phase 0.25µm,0.35 mm internal diameter (id), Perkin-Elmer, U¨ berlingen, Germany]with on-column injection and a63Ni-electron capture detector. Con-centration of HFB-IAA-ME in the injected solutions was kept withinthe linear range of the detector response. Recovery was calculated bymeans of an internal standard [1-(14C)-IAA] which was added at thebeginning of the extraction.

GC-MS analysis

Samples were injected splitless onto a fused silica capillary column(AT-1 chemical bonded phase 0.25µm, 0.35 mm id × 30 m, Helilex)installed in a MFC500 gas chromatograph (Fisons instruments) linkedto a QMD 1000 mass spectrometer (Fisons instruments). Electronenergy was 70 eV. Identification of HFB-IAA-ME was done in theselected ion monitoring mode.

Protein determination

Total protein was determined in freeze-dried, ground needles accordingto Christmann (1993).

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Chemicals

2,29,3,4,49,59-Hexachlorobiphenyl (PCB 138) used as an internalstandard in gas chromatography was obtained from amchro Chromato-graphie (Sulzbach/Taunus, Germany). 1-[14C]-IAA (specific activity2.28 GBq mg–1) was a gift from Prof. F. Bangerth, Institut fu¨r Obst-,Gemuse- und Weinbau, University of Hohenheim. HPLC grade sol-vents were from Carl Roth (Karlsruhe, Germany), while t-butylmethy-lether (TBME, LiChrosolv) was from E. Merck (Darmstadt, Germany).All other chemicals were purchased from Sigma Chemie (Deisenhofen,Germany).

Statistical analysis

Differences between mean values were evaluated by Mann-WhitneyU-test (Sachs 1984). The significance level was set up atP = 0.05.

Results

Methodology

With the purification steps described above, final gaschromatographic separation of HFB-IAA-ME from remain-ing impurities in extracts from fir needles was easilyperformed (cf. Fig. 1). SIM mass spectra of sample peakswith the retention time of HFB-IAA-ME corresponded tospectra of HFB-IAA-ME standard (Table 1). Thus reliablequantification of IAA was possible using GC-ECD andstandard deviation from parallel determinations was low(CV 55%). Recovery of [14C]-IAA which was added toeach sample at the beginning of the extraction procedureranged between 30 and 45%.

Seasonal variation

Current-year-needles

When sampling was done some days after bud burst, levelsof IAA were sometimes found to be elevated in current-yearneedles as compared to sampling dates later on in theseason (cf. Fig. 2) or as compared to older needles(Fig. 2). However, unlike mature needles, growing needlesexhibit considerable changes over time in the ratio ofcytoplasm to dry weight. Thus any variation of biochemicalparameters in growing needles found after relating theseparameters to dry weight might be merely attributed tochanges of the cytoplasm to dry weight ratio.

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Fig. 1mGas chromatogram (on-column injection, ECD) of the 1-heptafluorobutyryl-indole-3-acetic acid methyl ester (HFB-IAA-ME)fraction isolated from silver fir needles (Abies albaMill.) PCB 138 wasused as an internal standard to monitor the exact volume injected.HFB-IAA-ME peak corresponds to 0.3 pmol IAA. Chart speed was1 mm min–1 unless otherwise indicated

Fig. 2A, BmSeasonal trend of IAA levels in needles belonging todifferent needle age-classes from two healthy silver fir (Abies albaMill.) trees. Sampling was done in 1987 at the Schwarzmisse site (A)and in 1993 at the Ko¨nigswart site (B) in the forest district Kloster-reichenbach in the northern Black Forest on first order twigs of the suncrowns.Dotted vertical linesindicate onset of budbreak and termina-tion of current-year needle growth

Table 1mGC-MS (SIM-mode) of authentic N-heptafluorobutyryl-IAA-methyl ester (HFB-IAA-ME) standard and of fluorinated fractions afternormal phase HPLC of methylated silver fir needle extracts

Standard compound or fractionidentification

Five characteristic ions m/z(relative intensities)

Authentic HFB-IAA-ME 385(36), 326(100), 179(3),169(8), 146(2)

Methylated and fluorinated needleextracts of silver fir (Abies albaMill.)after normal phase HPLC

385(36), 326(100), 179(3),169(8), 146(3)

Hence, we related IAA levels in growing needles to acertain quantity of total protein (which should correspondto a particular quantity of cytoplasm). IAA levels incurrent-year needles were still elevated in some samples(5 samples out of 12, data not shown). Thus in growingneedles high endogenous levels of IAA only seem to bepresent during a rather short period after budbreak and fewof the samples taken after budbreak were within that period.

Mature needles

In mature needles, levels of IAA exhibited a cyclic seasonalpattern with a maximum in winter and a minimum in springjust before budbreak and during growth of young needles(Fig. 2). Concentrations of IAA in mature needles did notchange markedly while young needles grew, but increasedafter current-year needle growth had terminated (Fig. 2).

While the seasonal trend of IAA was studied in moredetail in only two trees (Fig. 2), data from nine other firtrees, from which samples were taken 3 or 4 times during ayear and from two fir trees which were sampled only twicein 1989, fitted well into this seasonal pattern (cf. Fig. 3).

This trend appeared to be unaffected by a period ofsevere water stress in summer and early autumn 1989(cf. Fig. 3).

Influence of needle age

Due to the fact that IAA levels in fir needles exhibited aseasonal trend, a comparison of IAA levels in needlesbelonging to different age-classes should be done at timesof the season which correspond to different phases of theannual IAA-cycle. So we chose sampling dates with de-

creasing, minimal and maximum levels of IAA for such acomparison.

The autumnal increase of IAA levels was more pro-nounced in older than in younger needles (Fig. 4). However,in the oldest needles of a twig, a drop in IAA levels ascompared to levels of the penultimate needle age-class wasobserved several times in late summer or autumn and oncein midsummer (Fig. 5; needles of the healthy tree weresampled in midsummer). This drop was found in trees ofdifferent health status and also in trees suffering from asulfur impact in the field (Fig. 5).

Differences among healthy and declining fir trees andfir trees suffering from a strong sulfur-impact (S-impact)in the field

The general seasonal trend of IAA levels in declining firtrees or in fir trees suffering from a strong S-impact in the

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Fig. 3mSeasonal trend of mean IAA levels in current-year-needles, 1-year-old needles and 2-year-old needles from silver fir (Abies albaMill.) trees with a different health status. Some of the trees furthersuffered from S-impact in the field. Sampling was done in 1989 whenwater availability at the sites was strongly restricted during summer[Stockerkopf site, eastern slope (S-impact) and western slope (no S-impact), forest district Klosterreichenbach in the northern BlackForest].Dashed vertical linesindicate beginning and end of the periodwith a restricted water availability at the sites

Fig. 4mLevels of IAA in needles belonging to different needle age-classes from healthy (A, B, C) or declining (D, E, F) silver fir (Abiesalba Mill.) trees. Sampling was done on first order twigs of the suncrowns before budbreak in spring (A, D), after budbreak, whilecurrent-year needles were elongating (B, E) and in autumn aftertermination of current-year needle and cambial growth (C, F) in theforest district Klosterreichenbach in the northern Black Forest. SD isnot indicated if below 0.06 nmol g–1 dw. * Difference is statisticallysignificant (P 50.05) between needle age-class from healthy ordeclining trees

field was not different from the trend observed in needlesfrom healthy trees (Fig. 3). However, in declining trees,levels of IAA no longer increased with needle age atdifferent times of the season (Fig. 4).

Among the trees which suffered from a strong S-impactin the field, both “healthy” (i. e. only affected by sulfur andnot by forest decline) and declining trees (i. e. trees affectedby both sulfur and by forest decline) were present. In latesummer and autumn, levels of IAA did not rise withincreasing needle age in two declining trees under S-impact(Fig. 6), while in two healthy trees from the same site IAAlevels still rose up to the 2-year-old needles and thendropped (Fig. 6).

Discussion

GC quantification of IAA

The method which we developed is sensitive enough toallow gas chromatographic quantification of IAA in ex-tracts from 200 mg or even less of needle dry weight with adetection limit of about 0.02 pmol IAA in 1µl injectedvolume.

The use of the HFB-derivative of IAA for a sensitivedetection of IAA in plant material has been proposed earlier(Allen et al. 1982). However, we found that after simplepurification steps like extraction with water (cf. Allen et al.1982), quantification of HFB-IAA-ME was impossible afterderivatization due to a vast number of impurities withelectron-capture properties in the derivatization mixture.However, even if such a simple purification had beeneffective in removing impurities that interfered with GCanalysis, scrupulous purification of HFB-IAA-ME wouldstill have been necessary to separate it from radiolabelleddecomposition products of the [14C]-IAA internal standard.If such radiolabelled impurities had not been removedbefore liquid scintillation counting, calculation of recovery

of the internal standard would have been incorrect(cf. Horgan 1987). We therefore purified HFB-IAA-MEby HPLC.

The whole procedure used to quantify IAA in needleextracts is more time consuming than the method employedby Volckers and Wild (1988) who were able to determineIAA levels in spruce needle extracts with a specific radio-immunoassay. However, our method offers the opportunityto determine levels of both abscisic acid (AbA) and IAA inthe same extract. During HPLC purification of IAA-ME, afraction is obtained which contains AbA-ME and which isready for gas chromatographic analysis without furtherpurification (Christmann 1993).

Seasonal variation

The seasonal trend of IAA levels found in silver fir needlescorresponds to the trend found in spruce needles by Vo¨lck-ers and Wild (1988). Since IAA levels in silver fir needleswere highest when days were short and were lowest duringlong days in the field, the seasonal variation of IAA inneedles might be controlled by photoperiod. In Scots pine, ashort-day treatment was indeed found to affect IAA levelsin the needles (Sandberg and Ode´n 1982). However, in thiscase the treatment lead to a decrease of IAA levels whilewe found highest levels of IAA in needles of silver fir undernatural short days. These contradictory results support theview that some variation does exist among different co-nifers in regulation of needle IAA levels.

The fate of IAA disappearing from the older needlesbefore budbreak ist not known. It might either be chemi-cally modified by conjugation or degradation (cf. Ernstsenand Sandberg 1988) or also be exported from the needles(cf. Sandberg and Ode´n 1982). In the latter case, other partsof the tree might be affected: IAA exported from olderneedles might reach the cambial region, initiating cambialactivity there (cf. Little and Savidge 1987). While it isgenerally believed that IAA which reaches the cambial

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Fig. 5mDrop of levels of IAA in the oldest needles present on a twig ascompared to the penultimate needle age-class. Needles were fromhealthy and declining silver fir (Abies albaMill.) trees and from firtrees suffering from a strong S-impact in the field. Sampling was donesome weeks after termination of current-year needle growth (healthytree) or after termination of cambial growth (declining trees, treessuffering from S-impact in the field) in the forest district Klosterrei-chenbach in the northern Black Forest

Fig. 6mLevels of IAA in needles belonging to different age-classesfrom silver fir (Abies albaMill.) trees suffering from a strong S-impactin the field. Trees were healthy (i.e. not affected by forest decline) ordeclining. Sampling was done after termination of current-year needlegrowth and cambial growth at the Sto¨ckerkopf site, eastern slope in theforest district Klosterreichenbach in the northern Black Forest

region originates mainly from current-year needles (cf.Little and Sundberg 1988), we suppose that in silver firtrees older needles should also be considered as a source ofIAA, and deserve to be further investigated. This idea issupported by results from the work of Baker and Allen(1987) who found that IAA was exported from old leaves ofRicinus communisthrough the phloem to sink tissues withthe flow of photoassimilates.

We could not find disturbances of the seasonal trend ofIAA levels that might have been attributed to differentenvironmental factors. Even when water availability wasrestricted during a prolonged period in summer 1989, IAAlevels in the needles were not affected (cf. Fig. 3), whileAbA levels were markedly elevated in needles of thedeclining trees (Christmann 1993). This is surprising,since inAbies balsamea, basipetal transport of [14C]-IAAwas restricted upon water stress (Little 1975). As a con-sequence, accumulation of IAA in needles might have beenexpected. However, high levels of AbA present in needlesduring water stress might prevent a rise in needle endoge-nous IAA concentrations thus counteracting the effects of areduced IAA export. While little is still known about suchphytohormone interactions in plants (cf. Matthysse andScott 1984), studies of phytohormones in flacca, a wiltymutant of tomato with low endogenous AbA levels (Talet al. 1979) suggest that in non-mutant plants, high AbAlevels might indeed prevent a rise in IAA levels.

The question of which developmental or physiologicalprocesses are triggered in the needles by the observedcyclic pattern of IAA levels cannot yet be decided. Sincemaximum levels were observed during autumn and winter,an effect on the development of cold hardiness with all theassociated changes in needle physiology (cf. Larcher et al.1985) might be possible.

In growing needles elevated levels of the growth pro-moting hormone IAA were only found during the initialgrowth phase when cell elongation was rapid, suggestingthat for further growth of these needles, high IAA levels arenot required.

Influence of needle age

Increasing levels of IAA with increasing needle age havebeen also reported for needles of spruce trees (Vo¨lckers andWild 1988). While our data from fir trees between 30 and140 years old do not support the view of Vo¨lckers and Wild(1988) that needles of older trees have a higher demand ofIAA for protection against needle abscission, we supposethat on trees of different age older needles are in generalprotected against senescence and abscission by high IAAlevels (cf. Mattoo and Aharoni 1988).

This opinion is supported by observations that in thevery oldest needles present on a twig, in late summer orautumn IAA levels were sometimes found to be lower thanin the penultimate needle age-class (Fig. 5). In silver firshedding of the oldest needles takes place in early autumn(Damsohn 1995). So probably with the drop in IAA levelsfound in such needles protection against abscission ceases

and processes are initiated that lead to needle senescenceand finally to needle abscission.

Differences among healthy and declining fir treesand fir trees suffering from a strong S-impact in the field

Due to the seasonal oscillation of IAA levels, a comparisonamong trees of different health status is best done in autumnor in early winter, when IAA levels are at their maximum.

Statistical evaluation of differences in needle IAA levelsbetween healthy and declining trees was difficult becauseseveral trees which were healthy at the beginning of ourstudies later showed pathological alterations in the para-meters used to examine tree health status and meanwhiledeveloped visible symptoms of decline.

Among trees under S-impact some variation was foundin changes of needle IAA levels with increasing needle agewhich might be attributed to the different health status withrespect to forest decline of such trees [the health status ofsuch trees was assessed using epicuticular wax structure(Guth and Frenzel 1989) and AbA levels during water stress(Christmann 1993)]. In healthy trees suffering from S-im-pact, IAA levels in the youngest needles remained unaf-fected by S-impact whereas levels in the older needles werelower than in healthy trees not impacted by sulfur. Indeclining trees under S-impact no additional effect onneedle IAA levels was observed. However, only fourtrees under S-impact had been studied, when emissions ofSO2 from the local source were markedly reduced.

Since both SO2 and the still unknown factors causingsilver fir decline can be regarded as stressors (cf. Bolhar-Nordenkampf 1989), the lowered levels of IAA found inolder needles of declining trees or trees under S-impact canbe considered as a consequence of the long-term influenceof these stressors (cf. Johnson 1987). However, the needlesseem to be able to resist these stressors for 2 or 3 years iflevels of IAA are considered. This corresponds to findingsthat the health or vitality status of fir trees cannot beascertained through different parameters if only the youngerneedles are considered (Frenzel et al. 1987; Gu¨th andFrenzel 1989; Christmann 1993) which is in contrast tothe diagnosis of damage to Norway spruce (Wild andSchmitt 1995).

Among the different parameters used by us to estimatethe actual health status of fir trees, levels of IAA in needlesof different age were found to be one of the most suitableparameters. We suppose that unlike other biochemicalindicators (cf. Wild and Schmitt 1995), levels of IAA arenot affected by comparatively short-term environmentalinfluences. Thus in fir needles they probably give a measureof vitality of the needles which is independent of short-terminfluences present at the time of sampling. Such influencesmight, in contrast, already affect other biochemical para-meters such as antioxidants or polyamines.

In older needles of declining conifers, changes in nu-merous parameters have been reported which point tosevere and lasting disturbances of needle physiology suchas a reduction in the rate of photosynthesis (Lange et al.

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1986; Wild 1987; Lautenschlager 1983; Christmann 1993),alterations in epicuticular wax structure (Gu¨th and Frenzel1989) or changes in the ATP/ADP ratio (Weidmann et al.1990). These alterations of needle physiology resemble apremature initiation of senescence processes (cf. Thimann1980) which is not surprising, because stress of varioustypes is known to change physiological processes in asimilar way to the changes caused by normal senescence(Nooden 1988).

Low levels of IAA in older needles are thus related to adecrease in general needle vitality. While our data indicatethat low levels of needle IAA probably cause prematureneedle losses of declining trees and trees under S-impact,Scheid and Do¨rffling (1989) showed that increment reduc-tion in declining trees is paralleled by low IAA levels in thecambial region. Thus in the dynamic processes that lead tosymptom development in declining trees, low levels of IAAseem to play a crucial role.

AcknowledgementsmWe thank Prof. F. Bangerth, Institut fu¨r Obst-,Gemuse- und Weinbau, Universita¨t Hohenheim, for a gift of [14C]-IAAand Dr. P. Doumas, Institut National de la Recherche Agronomiqued’Orleans for help with the GC-MS analysis. We are further gratefulfor financial support which came from the federal state department ofBaden-Wu¨rttemberg and from the European Community [grant No. 88/009/1A of “Projekt Europa¨isches Forschungszentrum fu¨r Maßnahmenzur Luftreinhaltung (PEF)”].

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