histology of magnesium-deficient norway spruce needles influen

Summary Effects of magnesium deficiency and variation innitrate to ammonium ratio on needle histology and chlorophyllconcentration were investigated in current-year and one-year-old needles of clonal Norway spruce trees (Picea abies (L.)Karst.). Six-year-old trees were grown for one year in sandculture with circulating nutrient solutions containing a suffi-cient (0.2 mM) or a limiting (0.04 mM) concentration of Mg.The nitrogen concentration was not varied (5 mM), but theNO3

−/NH4+-ratio was adjusted to 0.76 in Mg-sufficient and to

1.86, 0.76 or 0.035 in Mg-limited plants. Visible symptoms ofMg deficiency occurred only in current-year needles, indicat-ing adequate Mg nutrition before the experiment. Under con-ditions of Mg limitation, chlorophyll and Mg concentrationswere lowest in needles of trees supplied with NH4

+ as the majornitrogen source and highest in needles of trees supplied withNO3

− as the major nitrogen source. In current-year and one-year-old needles, starch accumulation induced by Mg defi-ciency was increased when NH4

+ was the major nitrogen source.The accumulation of tannin spherules in current-year needles,which occurred in response to Mg deficiency, also increasedwith decreasing NO3

−/NH4+-ratios. Deficient Mg supply caused

premature aging in tissues of the vascular bundle, as indicatedby modifications of the cambium and increased amounts ofcollapsed sieve cells. The number of collapsed sieve cells wasslightly lower in needles grown in a NH4

+-dominated nutrientregime than in needles grown in a NO3

−-dominated nutrientregime. We conclude that NH4

+ was not directly toxic to Norwayspruce trees at the applied concentrations. However, effects ofMg deficiency were considerably greater in an NH4

+-dominatednutrient regime than in a NO3

−-dominated nutrient regime.

Keywords: chlorophyll, magnesium deficiency, needle histol-ogy, Picea abies, starch accumulation.

Introduction

Nitrogen availability has often been thought to limit plantproductivity, especially in forest trees. In recent decades, theinput of atmospheric nitrogen to forest ecosystems has in-creased dramatically (Skeffington 1990). Nitrogen depositionsin the form of NOx, NH3, NO3

− and NH4+ reach a total of about

20 kg N ha−1 year−1 in central Europe, and may be as high as40 to 60 kg N ha−1 year−1 in some areas (van Breemen et al.

1987). Not only the amount but also the nature of the depositednitrogen may affect forest ecosystems (Nihlgård 1985, vanDijk and Roelofs 1988, Zöttl 1990). In soils with low cationavailability, high nitrogen inputs, especially in the form ofNH4

+, induce ion imbalances by either interfering with theuptake of other ions or causing a relative deficiency in treetissues as a result of increased growth rates (Huettl 1990,Weissen et al. 1990). This nutritional stress can be furtherenhanced as a result of high atmospheric acidity input leadingto soil acidification and displacement of adsorbed Mg and Caby Al ions in the cell walls of roots (Ulrich 1987). In addition,direct uptake of gaseous ammonia and nitrogen oxides maycause leaf damage as well as cation losses from the foliage (vander Eerden 1982, Nihlgård 1985).

In acidic forest soils of the temperate zone, NH4+ is the major

nitrogen source and is preferentially taken up by some coniferspecies, including Norway spruce (Picea abies (L.) Karst.)(Marschner et al. 1991, Flaig and Mohr 1992, Gezelius andNäsholm 1993). Uptake of NH4

+ by roots leads to acidificationof the soil solution causing leaching of cations from the uppersoil horizons. Because NH4

+ uptake is believed to compete withthe uptake of cations, Mg deficiencies may become morepronounced in trees growing on Mg-poor soils; several studieshave shown that increased NH4

+ nutrition causes a decrease inthe uptake of cations and a reduction in foliar concentrationsof Mg, K, and P (Ingestad 1979, Boxman and Roelofs 1988,Gijsman 1990), and opposite effects were observed by increas-ing the supply of NO3

− (van Dijk and Roelofs 1988, Mohr1994).

The yellowing symptoms associated with forest declinehave often been related to Mg deficiencies (Landmann et al.1987, Forschner and Wild 1988, Cape et al. 1990, Huettl et al.1990, Hölldampf et al. 1993, Ke and Skelly 1994). The histo-logical damage in conifer needles caused by Mg deficienciesis well documented. A lack of Mg induces abnormal accumu-lation of starch in the mesophyll cells of chlorotic older nee-dles, possibly because export of carbohydrates is impeded bythe collapse of phloem cells (Fink 1989, Forschner et al. 1989,Fink 1991). It is not known if the cellular damage induced byMg deficiency is modified by the form of nitrogen supplied.Beside indirect effects of the supplied N form on uptake andtransport of Mg, and therefore on Mg nutrition, direct impactsof the applied NO3

−/NH4+-ratio might also interact with Mg

Histology of magnesium-deficient Norway spruce needles influencedby nitrogen source

LAURENCE PUECH and BEATE MEHNE-JAKOBS

Institute of Forest Botany and Tree Physiology, Albert-Ludwigs-University, Bertoldstrasse 17, D-79085 Freiburg, Germany

Received July 6, 1995

Tree Physiology 17, 301--310© 1997 Heron Publishing----Victoria, Canada

deficiency. For example, high NH4+ supply has been reported to

alter leaf chloroplasts of cultivated plants, inducing a suppres-sion of starch synthesis (Puritch and Barker 1967, Matsumotoet al. 1969, Mehrer and Mohr 1989). The aim of our investiga-tion, therefore, was to examine the influence of differentNO3

−/NH4+-ratios on the Mg, chlorophyll and starch concentra-

tions and the histology of needles of young Mg-deficientNorway spruce trees.

Material and methods

Plant material and study design

In October 1991, 100 six-year-old, non-mycorrhizal Norwayspruce trees (Picea abies) derived from one clone(No. 1213/113, provenance Rothenkirchen 84011, Franken-wald) were each maintained in sand culture in a 70-liter pot ina special outdoor facility. Each pot was individually suppliedwith nutrients by means of a continuously circulating solutionthat was refilled continuously and completely replaced every3 days. The composition of the nutrient solution, which wasdesigned to achieve optimum growth according to Ingestad’sprinciples (Ingestad 1962, 1979), is presented in Table 1.Initially, the plants were grown with a sufficient (Treatment I)or a limiting Mg supply to induce Mg deficiency (Treat-ment II). In spring 1992, the nitrogen source was varied in asubset of trees from Treatment II to achieve a NO3

−- or NH4+-

dominated nutrition (Treatments III and IV) (see Table 1).However, the total nitrogen concentration of 5 mM was notchanged. Increases in NO3

− and NH4+ concentrations were asso-

ciated with increases in the K+ and SO42− concentrations, re-

spectively, of the nutrient solutions (Table 1). During each3-day period of circulation through the plant pots, the pH ofthe nutrient solutions decreased from an initial value of 6.0 (5.7in Treatment IV) to 4.1--3.8 in the NH4

+-dominated nutrientsolutions (Treatments I, II and IV). In the NO3

−-dominatedtreatment (Treatment III), the pH remained relatively stable at

pH 6.0. The plants were subjected to the experimental regimesfor one growing season.

Light microscopy

At the end of September 1992, 10 current-year and 10 one-year-old sun-exposed needles were removed from shoots of thethird whorl of trees randomly selected from each treatment.Samples were collected at midday (1100--1300 h) on a sunnyday. Three approximately 2-mm-long segments were cut fromthe middle of each needle and immediately fixed in coldglutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for one day.The samples were then dehydrated in an ascending series ofacetone concentrations and embedded in methacryl resin(S. Fink, Albert-Ludwig-Universität, Freiburg, FRG, unpub-lished method). Semithin sections (3 µm) of the embeddedsegments were cut with a diamond knife on a Leica-2065-mi-crotome (Leica Instruments GmbH, Nuβloch, FRG).

Histological observations and a differential survey of carbo-hydrates, phenolics and lipid-like compounds were performedon the sections. The sections were stained for polysaccharidescontaining vicinal glycol groups with periodic acid and Schiffsreagent (PAS) as described by Gerlach (1977) and mounted inEukitt (Hans Thoma, Freiburg, FRG). The autofluorescentproperties of lignin, phenolics and lipids under excitation at395--440 nm were used as the counterstain. For this purpose,the sections were observed on a Zeiss-Axiophot microscopewith epi-fluorescence (Carl Zeiss, Oberkochen, FRG) usingthe filters BP 395--440 (excitation filter), FT 460 (beamsplit-ter) and LP 470 (barrier filter). Photographs were taken withan integrated camera on 100 ASA black-and-white film.

Lignin was stained with phloroglucin in HCl (von Aufseβ1973). Lipids were stained with Nile blue (Jensen 1962) andsuberin and cutin were stained with Sudan IV (Jensen 1962).Tannins were stained with ferric chloride (Jensen 1962) andproteins were stained with Coomassie blue R250 in Clarke’ssolution (Cawood et al. 1978).

Determination of starch, chlorophyll and nutrient elements

At the end of September 1992, current-year and one-year-oldshoots were harvested around noon from randomly selectedtrees (n = 6 per treatment). Needles were removed, immedi-ately frozen in liquid nitrogen and stored at −80 °C. The frozenneedle tissue was homogenized with a microdismembrator(Braun, Melsungen, FRG). The resulting powder was freeze-dried at --25 °C and stored under vacuum at −20 °C.

For the determination of foliar starch concentrations, 2- to6-mg aliquots of needle homogenate were extracted for 1 hwith 0.5 ml of 0.5 M NaOH in Eppendorf vials (Eppendorf-Netheler-Hinz GmbH, Hamburg, FRG) at ambient tempera-ture (Dekker and Richards 1971). To remove free glucose, theextract was incubated at 95 °C for 3 min, adjusted to pH 4.6with 0.5 M acetic acid, and then centrifuged at 10,000 g for5 min (Einig and Hampp 1990). Twenty- to 60-ml aliquots ofthe supernatant were used for enzymatic determination ofstarch according to Outlaw and Manchester (1979). The pro-cedure was optimized to obtain a 90% or higher recovery of an

Table 1. Mineral nutrient composition of the four treatment solutions(µM). The balance between anions and cations was made with H+.

Treatment

Ion I II III IV

Mg2+ 203 41 41 41NO3

− 2170 2170 3240 169NH4

+ 2860 2860 1740 4767SO4

2− 460 460 460 1920K+ 1500 1500 3690 1500PO4

3− 1370 1370 1370 1370Ca2+ 275 275 275 275Na+ -- 322 322 --Fe3+ 12 12 12 12Mn2+ 4.70 4.70 4.70 4.70Cu2+ 0.32 0.32 0.32 0.32Zn2+ 0.70 0.70 0.70 0.70BO3

3− 2.40 2.40 2.40 2.40MoO 4

2− 0.07 0.07 0.07 0.07

302 PUECH AND MEHNE-JAKOBS

TREE PHYSIOLOGY VOLUME 17, 1997

added internal standard (maize starch, Sigma Chemical Co.,St. Louis, MO).

Chlorophyll concentrations were determined by spectro-photometry after needle tissue homogenization and extractionin 80% acetone (Ziegler and Egle 1965), and calculated ac-cording to Lichtenthaler (1987). Magnesium and K concentra-tions in needles were analyzed after dry ashing and digestionwith HNO3/HCl by the AAS/AES flame technique (PerkinElmer 4000, Perkin-Elmer & Co GmbH, Überlinger, FRG).The N and S concentrations were determined with a CNSanalyzer (Carlo Erba Instruments, Rodano, Italy). To evaluatethe data, means and standard errors (SE) were calculatedseparately for each needle age class and treatment. Analysis ofvariance (one-way ANOVA) and the Tukey test were used toanalyze differences among the treatments within each needleage class.

Results

Needle element and chlorophyll concentrations

The influence of nitrogen sources on Mg deficiency was ana-lyzed by varying the NO3

−/NH4+-ratio of the nutrient solution

(0.76 = Treatment II, 1.86 = Treatment III, and 0.035 = Treat-ment IV, Table 1). The nutrient solution used in the Mg-suffi-cient treatment contained a fivefold higher concentration ofMg and a NO3

−/NH4+-ratio of 0.76 (Treatment I). After one

growing season, Mg concentrations in current-year and one-year-old needles of Mg-limited trees differed significantlydepending on whether NO3

− or NH4+ was the major nitrogen

source (Figure 1a). Current-year needles from trees suppliedwith NO3

− as the major N source (Treatment III) exhibited two-to threefold higher Mg concentrations than current-year need-les of trees supplied with NH4

+ as the major N source (Treat-ment IV) (Figure 1a). The differences in foliar Mgconcentrations between trees grown in the Mg-limited Treat-ments III and IV cannot be explained by dilution effects causedby different growth rates of the needles, because both needleweight and Mg concentration were higher in needles grown inthe NO3

−-dominated nutrient treatment (Treatment III) than theNH4

+-dominated treatment (Treatment 1V) (Table 2 and Fig-ure 1a). Chlorophyll concentrations of current-year needleswere influenced similarly by the treatments (Figure 1b). Com-pared with current-year needles, there was a similar but smallereffect of nitrogen source on Mg concentrations of one-year-oldneedles (Figure 1a). In contrast, reductions in chlorophyllconcentrations of one-year-old needles were only significantin the NH4

+-dominated nutrient treatment (Treatment IV, Fig-ure 1b).

Visible symptoms of yellowing occurred only in current-year needles of trees in the Mg-limiting treatments. The symp-toms were most pronounced in trees in the NH4

+-dominatedtreatment (Treatment IV), followed by trees in the treatmentwith a balanced NO3

−/NH4+-ratio (Treatment II). Only slight

yellowing was observed in current-year needles of trees in theNO3

−-dominated treatment (Treatment III). In accordance withearlier findings, the Mg concentrations, calculated as % of

Figure 1. Magnesium (a) and chlorophyll concentrations (b) in cur-rent-year and one-year-old needles of Norway spruce grown withsufficient (Treatment I) or limiting Mg supply (Treatments II--IV).Ratios of NO3

−/NH4+ were: 0.76 (Treatments I and II), 1.86 (Treat-

ment III) and 0.035 (Treatment IV). Bars indicate means (± SE, n = 6).Different letters indicate significant differences between treatments.

Table 2. Fresh weight of 100 needles (gfw), N, K and S concentrations(mg g−1 dry weight), and Mg concentrations in % of the N concentra-tion in current-year needles of Norway spruce grown with a sufficient(Treatment I) or a limited Mg supply (Treatments II, III and IV).Ratios of NO3

−/NH4+ in the nutrient solutions were: 0.76 (Treatments I

and II), 1.86 (Treatment III) and 0.035 (Treatment IV) (n = 6, SE isgiven in brackets). Different letters within rows indicate significantdifferences between treatments.

Treatment

I II III IV

gfw 0.74 (0.05) a 0.75 (0.09) a 0.89 (0.10) a 0.74 (0.07) a

N 32 (0.7) a 28 (0.9) ab 27 (1.1) b 28 (1.6) ab

S 1.7 (0.07) a 1.4 (0.12) a 1.3 (0.14) a 1.4 (0.15) a

K 10.3 (0.6) b 11.7 (0.8) b 21.2 (1.1) a 6.9 (0.2) c

Mg in 4.0 (0.21) a 1.1 (0.17) c 1.8 (0.26) b 0.6 (0.03) c

% of N

NEEDLE HISTOLOGY INFLUENCED BY MG DEFICIENCY AND N SOURCE 303

TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com

nitrogen concentration, were slightly below the physiologicalthreshold value of 2% for Norway spruce (Mehne-Jakobs1996) in the NO3

−-dominated treatment, and well below thethreshold value in the NH4

+-dominated treatments (TreatmentsII and IV; Table 2).

Needle N and S concentrations were slightly lower in allMg-limited treatments than in the Mg-sufficient treatment(Table 2). Needle S concentrations were not influenced byvariation in the NO3

−/NH4+-ratio or by a fourfold increase in

nutrient solution SO42− concentration in the NH4

+-dominatedtreatment (Treatment IV). Needle K concentrations, however,decreased significantly with decreasing NO3

−/NH4+-ratios. In

the NO3−-dominated treatment (Treatment III), high foliar K

concentrations might reflect the increased K concentration inthis treatment (K served as the counterion for NO3

−, cf. Ta-ble 1).

Microscopy

Figures 2--4 are fluorescence micrographs of needle sectionsstained with PAS. In these micrographs, the red fluorescenceof stained polysaccharides and the nuclei appear grey, whereasthe blue-green fluorescence of lignin and the yellow fluores-cence of lipids appear white.

Vascular bundle Healthy current-year needles were charac-terized by an intact epidermis, a turgid mesophyll and an intactvascular bundle (Figure 2a). Between the xylem and phloem,cambial cells (C) are organized in two or three rows (Fig-ure 2b). Their complex content was characterized by a yellow-brown fluorescence (arrowhead) and showed a positivestaining with Sudan IV for lipids and with Coomassie blue forproteins. All sieve cells (Ph) had a wide open lumen. Youngersieve cells showed a heterogeneous content whereas the older

Figure 2. Cross sections of Mg-suffi-cient needles. (a) Current-year need-le (Magnification = 38×). (b)Vascular bundle of the same needle(165×). The cambial cells show afluorescent content (arrowhead) andthe transfusion parenchyma cellscontain starch (arrow). (c) Smallstarch grains in chloroplasts ofmesophyll cells of a current-yearneedle (338×). (d) Vascular bundle ofa one-year-old needle (188×). Someolder sieve cells are distorted (arrow-heads). (e) Starch grains in chloro-plasts of mesophyll cells ofone-year-old needle (345×). Abbre-viations: Ep = epidermis, M = meso-phyll, Bs = bundle sheath, Vb =vascular bundle, X = xylem, C =cambium, Ph = phloem, A = albumi-nous cells, Tp = transfusion paren-chyma cells, Tt = transfusiontracheids, S = starch, Ch = chloro-plasts.



sieve cells appeared empty. The albuminous cells (A) borderingthe phloem were well developed and filled with light fluoresc-ing material including large nuclei. In the one-year-old needles,aging is revealed by the crushed older sieve cells (Figure 2d:arrowhead).

The Mg-deficient treatment caused premature aging in thevascular bundle, characterized by an increased proportion ofsieve cell deformations (Figures 3b, 3d, 4c, 4e, Ph) in current-year and one-year-old needles, and by modifications of thecambium in one-year-old needles including additional cellrows, interruptions of cell rows and cell distortions (Figure 3d:arrowhead, Figure 4e). Nitrogen source had no influence onthe symptoms expressed by the cambium. In the Mg-deficienttreatments, the amount of collapsed sieve cells was increasedin needles grown in the NO3

−-dominated treatment (Treat-ment III, Figures 3b and 3d) or balanced treatment (Treat-

ment II, sections not shown), but was only slightly increasedin the NH4

+-dominated treatment (Treatment IV, Figures 4c and4e) compared with phloem cells in needles from Mg-sufficientcontrol trees. However, a proportion of the sieve cells remainedfunctional in all trees in the Mg-deficient treatments.

Mesophyll Chloroplasts of needles from the Mg-sufficienttreatment were almost devoid of starch at the end of the growingseason (Figures 2a, 2c, 2e). Only small starch grains (S) wereobserved in chloroplasts (Ch) of the mesophyll surrounding thevascular bundle and in the endodermis and transfusion paren-chyma cells (Figures 2b: arrow, 2c, 2e). The amount of starchin needles of Mg-deficient trees cultivated with either a bal-anced NO3

−/NH4+-ratio (Treatment II) or an NO3

−-dominated Nform (Treatment III) did not differ markedly from that inneedles of Mg-sufficient trees (Figures 3a, 3e; sections ofTreatment II not shown). The starch concentration of current-

Figure 3. Cross sections of need-les from Treatment III (Mg-defi-cient, NO3

−-dominated nutrition).(a) Mesophyll cells of a current-year needle (Magnification =353×). The number of starchgrains in the chloroplasts shownon the photograph is relativelyhigh for the treatment. (b) Vascu-lar bundle of the same needle(195×). Some sieve cells distor-tions were induced by the treat-ment (arrowhead). (c)One-year-old needle (30×). (d)Vascular bundle of a one-year-old needle with a higher propor-tion of distorted sieve cells(218×). Cambium with an in-creased division array and somedistorted cells (arrowhead). (e)Small starch grains in chloro-plasts of mesophyll cells of a one-year-old needle (330×).Abbreviations: Ep = epidermis,M = mesophyll, Bs = bundlesheath, Vb = vascular bundle,X = xylem, C = cambium, Ph =phloem, A = albuminous cells,Tt = transfusion tracheids, S =starch, Ch = chloroplasts.



year needles in Treatments II and III was very heterogeneous.In the NH4

+-dominated treatment (Treatment IV), there was alarge increase in current-year needles. Many large starch grainswere observed in all mesophyll cells (Figures 4a and 4b) andalso in guard cells of the stomata and in hypodermal cells(Figures 4a: arrows). Starch accumulation also occurred in thetransfusion parenchyma cells (Tp) and in some of the albumi-nous cells (A) bordering the youngest sieve cells (Figure 4c:arrow). Starch grains were smaller in one-year-old needles thanin current-year needles of Treatment IV (Figure 4d).

The microscopic observations were confirmed by biochemi-cal analysis. Compared to controls, deficient Mg supply com-

bined with a balanced NO3−/NH4

+ supply or NO3−-dominated

nutrition caused a twofold increase in starch concentration ofthe current-year needles, whereas the Mg-deficient plus NH4

+-dominated nutrient regime caused a fivefold increase in starchconcentration of current-year needles (Figure 5). In one-year-old needles, treatment-related effects were similar to those ofcurrent-year needles but less pronounced.

Few spherules were observed in mesophyll cells of current-year needles in the Mg-sufficient treatment and in the Mg-de-ficient plus NO3

−-dominated treatment (Treatments I and III).The number of spherules was greater in Mg-deficient needleswhen nitrogen sources were balanced (Treatment II, sections

Figure 4. Cross sections of needlesfrom Treatment IV (Mg-deficient,NH4

+-dominated nutrition). (a) Pe-ripheral part of a current-year need-le (Magnification = 218×). Starchaccumulated in the mesophyll aswell as in the hypodermis (arrows).Tannin spherules are present in thevacuoles. (b) Inner part of the meso-phyll (345×). (c) Slightly distortedphloem, and albuminous cells show-ing a modified content and smallstarch grains (arrow) (158×). (d)Starch grains in the mesophyll chlo-roplasts of a one-year-old needle(390×). (e) Vascular bundle of aone-year-old needle with some dis-tortions of the sieve cells (arrow-head) and cambial modifications asin Figure 3e (203×). Abbreviations:Cu = cuticle, Hyp = hypodermis,Ts = tannin spherules, Ep = epider-mis, X = xylem, C = cambium,Ph = phloem, A = albuminous cells,Tt = transfusion tracheids, Tp =transfusion parenchyma cells, Ts =tanin spherules, S = starch, Ch =chloroplasts.



not shown), and the increase was greatly enhanced when thenitrogen supply was NH4

+-dominated (Treatment IV, see Fig-ures 4a and 4b: Ts). Although all spherules exhibited the sameautofluorescence under blue-violet excitation, they stained dif-ferently depending on size. The smaller droplets present in theouter mesophyll cells of the needles grown in the NH4

+-domi-nated nutrient regime (Figure 4a) were positively stained byNile blue for lipids, Coomassie blue for proteins and ferricchloride for tannins, whereas the larger droplets in the innerpart of the mesophyll remained almost unstained and showedautofluorescence. Because the staining intensity increasedwith decreasing spherule size, it is possible that the stains werephysically adsorbed on the surface of the large drops. Nospherules were observed in one-year-old needles in any of thetreatments.

Discussion

In Norway spruce, Mg concentrations of 0.9 to 1.7 mg g−1 drymatter are considered optimum, whereas values of lessthan 0.7 mg g−1 indicate deficiency (Ingestad 1962, 1979,Bergmann 1988). Throughout the literature, Mg deficiencysymptoms in Norway spruce are described as tip-yellowing ofthe older needles, whereas the younger needles remain green(Mies and Zöttl 1985, Bergmann 1988, Kaupenjohann andZech 1989, Lange et al. 1989). This difference between need-les of different ages has been related to the translocation of Mgfrom one-year-old needles to the new flush (Weikert et al.1989). In the present study, however, we observed that Mgdeficient treatment caused chlorosis of current-year needles,whereas one-year-old needles remained green, indicating thatMg translocation into the developing shoots did not decreasethe Mg concentrations of the one-year-old needles below thedeficiency threshold value (Figure 1). Conversely, the amountof Mg supplied by root uptake and by translocation from

one-year-old needles was insufficient to prevent current-yearneedles from developing chlorosis. Previously, Mehne-Jakobs(1994) observed yellowing of current-year needles of Norwayspruce trees grown with a severely restricted Mg supply(5 µM), whereas current-year leaves of trees supplied with amoderately reduced Mg supply (41 µM) did not exhibit pig-ment loss. A difference in the availability of nitrogen likelyaccounts for the occurrence of current-year needle chlorosisunder conditions of moderately reduced Mg supply in thepresent study and its absence in the previous study (Mehne-Jakobs 1994): a higher nitrogen concentration was supplied inthe present experiment than in the earlier study (5 versus3.5 mM). There is evidence that the development of Mg defi-ciency in conifers depends on the relative availabilities of Mgand nitrogen (Ingestad 1979, Weissen et al. 1990). Previously,the physiological threshold value for Mg in needles was foundto be 2% of the nitrogen concentration (Mehne-Jakobs 1996).Magnesium concentrations in current-year needles were belowthis threshold value in all of the Mg-limited treatments (Treat-ments II, III and IV, Table 2). Concentrations of both Mg andchlorophyll were strongly influenced by nitrogen source (Fig-ures 1a and 1b). Current-year needles of trees grown withNO3

− as the predominant form of N exhibited only slightreductions in Mg and chlorophyll concentrations (Treat-ment III, Figure 1b), whereas NH4

+-dominated nutrition causedsevere pigment loss (Treatment IV, Figure 1b). Because bothchlorophyll and Mg concentrations were equally influenced bynitrogen source, the intensity of the chlorosis can be attributedto Mg deficiency rather than to toxic effects of NH4

+. Thedecrease in needle Mg concentration with increasing NH4

+

supply cannot be explained by dilution caused by increasedgrowth, because needle weight was highest in the treatmentwith the lowest NH4

+ supply (Treatment III, Table 2). Thenegative influence of the reduced NO3

−/NH4+-ratio on foliar Mg

concentration is likely to have been caused by competitionbetween NH4

+ uptake and uptake of other cations (Marschner1995). Decreased foliar Mg concentrations combined withenhanced yellowing have also been observed after ammoniumfertilization of Norway spruce stands suffering from Mg defi-ciency (Feger 1990, Hüttl and Fink 1991).

Histological investigation revealed slightly premature agingof the vascular bundle in Mg-deficient needles, which wascharacterized by an increase in sieve cell distortions, especiallywhen nutrition was NO3

−-dominated, and by modifications ofthe cambium in all Mg-deficiency treatments. Similar symp-toms of precocious aging have been observed in needles col-lected from Mg-deficient Norway spruce (Fink 1989,Forschner et al. 1989, Hannick et al. 1993). Hannick et al.(1993) noted that the abnormalities first occurred in the cam-bium, then in the phloem, and finally in the xylem. In thepresent study, the Mg-deficient needles generally exhibitedalterations of the cambium but few anomalies of the phloemcells. Collapse of sieve cells is a characteristic structural symp-tom in chlorotic older needles of Mg-deficient spruce (Para-meswaran et al. 1985, Fink 1989, Forschner et al. 1989, Fink1991). In the present experiment, the trees were exposed to adeficient Mg supply for only one growing season, and this did

Figure 5. Starch concentrations in current-year and one-year-old need-les of Norway spruce trees grown with sufficient (Treatment I) orlimiting Mg supply (Treatments II--IV) (means ± SE, n = 6). Treat-ments refer to the following NO3

−/NH4+-ratios in the nutrient solutions:

0.76 (Treatments I and II), 1.86 (Treatment III) and 0.035 (Treat-ment IV). Different letters indicate significant differences betweentreatments.



not induce true phloem collapse either in the yellowing cur-rent-year or in the green one-year-old needles. However, simi-lar treatments applied after a one-year period of Mg deficiencytreatment induced severe phloem collapse and cambial abnor-malities in one-year-old needles (Puech and Mehne-Jakobs,unpublished results). We conclude, therefore, that the minoranomalies in the vascular area of Mg-deficient needles couldrepresent an early stage of pathological alteration that precedesphloem collapse caused by prolonged Mg deficiency.

Because phloem collapse induced by Mg deficiency hasonly been observed in one-year-old and older needles, it mightbe associated with the translocation of Mg from one-year-oldneedles to the developing shoots during spring. Fink (1992)suggested that during flushing in spring, Mg is quicklymoblized from the area surrounding the phloem, leading to asudden and localized severe lack of Mg. Because Mg plays animportant role in the phloem loading process (Balke andHodges 1975, Giaquinta 1983), a severe lack of local Mgmight cause a large decrease in osmotic pressure in the sievecells, which consequently collapse. Drastic reductions inphloem loading have been observed in source leaves of beanas an early effect of Mg deficiency (Cakmak et al. 1994). In thepresent study, the one-year-old needles probably still con-tained enough Mg to compensate for any localized lack of Mgin the vascular area caused by translocation of Mg into the newflush, and therefore phloem collapse was prevented (cf. Fig-ure 1).

Another symptom apparently related to low Mg concentra-tions, was the occurrence of spherules or droplets in the meso-phyll cells of current-year needles. Similar spherules havebeen described previously and identified as vacuolar tannininclusions (Chafe and Durzan 1973, Baur and Walkinshaw1974, Chabot and Chabot 1975), and were probably condensedtannins or proanthocyanidins (Haslam 1988). Multiple stain-ing of the spherules might be explained by their associationwith a wide range of cellular compounds. Accumulation ofpolyphenols and subsequently tannins in plant cells may rep-resent a protective mechanism against herbivores (Claussenet al. 1992). In addition, such compounds have been reportedto accumulate in plants exposed to various abiotic stress fac-tors, such as air pollutants (Soikkeli 1981, Jordan et al. 1991)or mineral deficiencies (Fink 1989, Forschner et al. 1989).Based on the findings that condensed tannins could be in-volved in antioxidative processes in plants (Torel et al. 1986,Feucht et al. 1994), and that Mg deficiency may induce oxida-tive stress (Polle et al. 1994), we suggest that the accumulationof spherules in the mesophyll cells represents a defense reac-tion against oxidative stress induced by Mg deficiency.

Starch accumulated in mesophyll cells of Mg-limited trees,especially in current-year needles, even though CO2-fixationrates were reduced to less than 50% of those of the Mg-suffi-cient needles (Mehne-Jakobs, unpublished results). Increasedamounts of starch in Mg-deficient spruce needles have beenobserved in both field investigations and controlled experi-ments (Forschner et al. 1989, Fink 1991, Mehne-Jakobs 1995).Photosynthate accumulation occurs before chlorophyll con-centrations decrease (Mehne-Jakobs 1995), and is probably a

result of the reduction in phloem export of sucrose caused byMg deficiency (Cakmak et al. 1994).

Accumulations of starch and tannin spherules in needlemesophyll cells were most pronounced in the NH4

+-dominatedtreatment, which contained the lowest concentrations of Mgand chlorophyll. Because ammonium toxicity reduces starchsynthesis (Puritch and Barker 1967, Matsumoto et al. 1969,Mehrer and Mohr 1989), the increased symptoms observed inthe NH4

+-dominated treatment can be attributed to an increasedseverity of Mg deficiency, most probably induced by cationantagonism at the root level, rather than to phytotoxic effectsof ammonium.

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