Tree Physiology 17, 301--310
© 1997 Heron Publishing----Victoria, Canada
Histology of magnesium-deficient Norway spruce needles influenced
by 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
Summary Effects of magnesium deficiency and variation in
nitrate to ammonium ratio on needle histology and chlorophyll
concentration were investigated in current-year and one-yearold needles of clonal Norway spruce trees (Picea abies (L.)
Karst.). Six-year-old trees were grown for one year in sand
culture with circulating nutrient solutions containing a sufficient (0.2 mM) or a limiting (0.04 mM) concentration of Mg.
The nitrogen concentration was not varied (5 mM), but the
NO −3 /NH +4 -ratio was adjusted to 0.76 in Mg-sufficient and to
1.86, 0.76 or 0.035 in Mg-limited plants. Visible symptoms of
Mg deficiency occurred only in current-year needles, indicating adequate Mg nutrition before the experiment. Under conditions of Mg limitation, chlorophyll and Mg concentrations
were lowest in needles of trees supplied with NH +4 as the major
nitrogen source and highest in needles of trees supplied with
NO −3 as the major nitrogen source. In current-year and oneyear-old needles, starch accumulation induced by Mg deficiency was increased when NH +4 was the major nitrogen source.
The accumulation of tannin spherules in current-year needles,
which occurred in response to Mg deficiency, also increased
with decreasing NO −3 /NH +4 -ratios. Deficient Mg supply caused
premature aging in tissues of the vascular bundle, as indicated
by modifications of the cambium and increased amounts of
collapsed sieve cells. The number of collapsed sieve cells was
slightly lower in needles grown in a NH +4 -dominated nutrient
regime than in needles grown in a NO −3 -dominated nutrient
regime. We conclude that NH +4 was not directly toxic to Norway
spruce trees at the applied concentrations. However, effects of
Mg deficiency were considerably greater in an NH +4 -dominated
nutrient regime than in a NO −3 -dominated nutrient regime.
Keywords: chlorophyll, magnesium deficiency, needle histology, Picea abies, starch accumulation.
Introduction
Nitrogen availability has often been thought to limit plant
productivity, especially in forest trees. In recent decades, the
input of atmospheric nitrogen to forest ecosystems has increased dramatically (Skeffington 1990). Nitrogen depositions
in the form of NOx, NH3, NO −3 and NH +4 reach a total of about
20 kg N ha −1 year −1 in central Europe, and may be as high as
40 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 deposited
nitrogen may affect forest ecosystems (Nihlgård 1985, van
Dijk and Roelofs 1988, Zöttl 1990). In soils with low cation
availability, high nitrogen inputs, especially in the form of
NH +4 , induce ion imbalances by either interfering with the
uptake of other ions or causing a relative deficiency in tree
tissues as a result of increased growth rates (Huettl 1990,
Weissen et al. 1990). This nutritional stress can be further
enhanced as a result of high atmospheric acidity input leading
to soil acidification and displacement of adsorbed Mg and Ca
by Al ions in the cell walls of roots (Ulrich 1987). In addition,
direct uptake of gaseous ammonia and nitrogen oxides may
cause leaf damage as well as cation losses from the foliage (van
der Eerden 1982, Nihlgård 1985).
In acidic forest soils of the temperate zone, NH +4 is the major
nitrogen source and is preferentially taken up by some conifer
species, including Norway spruce (Picea abies (L.) Karst.)
(Marschner et al. 1991, Flaig and Mohr 1992, Gezelius and
Näsholm 1993). Uptake of NH +4 by roots leads to acidification
of the soil solution causing leaching of cations from the upper
soil horizons. Because NH +4 uptake is believed to compete with
the uptake of cations, Mg deficiencies may become more
pronounced in trees growing on Mg-poor soils; several studies
have shown that increased NH +4 nutrition causes a decrease in
the uptake of cations and a reduction in foliar concentrations
of Mg, K, and P (Ingestad 1979, Boxman and Roelofs 1988,
Gijsman 1990), and opposite effects were observed by increasing the supply of NO −3 (van Dijk and Roelofs 1988, Mohr
1994).
The yellowing symptoms associated with forest decline
have 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 histological damage in conifer needles caused by Mg deficiencies
is well documented. A lack of Mg induces abnormal accumulation of starch in the mesophyll cells of chlorotic older needles, possibly because export of carbohydrates is impeded by
the collapse of phloem cells (Fink 1989, Forschner et al. 1989,
Fink 1991). It is not known if the cellular damage induced by
Mg deficiency is modified by the form of nitrogen supplied.
Beside indirect effects of the supplied N form on uptake and
transport of Mg, and therefore on Mg nutrition, direct impacts
of the applied NO −3 /NH +4 -ratio might also interact with Mg
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PUECH AND MEHNE-JAKOBS
deficiency. For example, high NH +4 supply has been reported to
alter leaf chloroplasts of cultivated plants, inducing a suppression of starch synthesis (Puritch and Barker 1967, Matsumoto
et al. 1969, Mehrer and Mohr 1989). The aim of our investigation, therefore, was to examine the influence of different
NO −3 /NH +4 -ratios on the Mg, chlorophyll and starch concentrations and the histology of needles of young Mg-deficient
Norway spruce trees.
Material and methods
Plant material and study design
In October 1991, 100 six-year-old, non-mycorrhizal Norway
spruce trees (Picea abies) derived from one clone
(No. 1213/113, provenance Rothenkirchen 84011, Frankenwald) were each maintained in sand culture in a 70-liter pot in
a special outdoor facility. Each pot was individually supplied
with nutrients by means of a continuously circulating solution
that was refilled continuously and completely replaced every
3 days. The composition of the nutrient solution, which was
designed to achieve optimum growth according to Ingestad’s
principles (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 (Treatment II). In spring 1992, the nitrogen source was varied in a
subset of trees from Treatment II to achieve a NO −3 - or NH +4 dominated nutrition (Treatments III and IV) (see Table 1).
However, the total nitrogen concentration of 5 mM was not
changed. Increases in NO −3 and NH +4 concentrations were associated with increases in the K+ and SO 24− concentrations, respectively, of the nutrient solutions (Table 1). During each
3-day period of circulation through the plant pots, the pH of
the nutrient solutions decreased from an initial value of 6.0 (5.7
in Treatment IV) to 4.1--3.8 in the NH +4 -dominated nutrient
solutions (Treatments I, II and IV). In the NO −3 -dominated
treatment (Treatment III), the pH remained relatively stable at
pH 6.0. The plants were subjected to the experimental regimes
for one growing season.
Light microscopy
At the end of September 1992, 10 current-year and 10 oneyear-old sun-exposed needles were removed from shoots of the
third whorl of trees randomly selected from each treatment.
Samples were collected at midday (1100--1300 h) on a sunny
day. Three approximately 2-mm-long segments were cut from
the middle of each needle and immediately fixed in cold
glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for one day.
The samples were then dehydrated in an ascending series of
acetone concentrations and embedded in methacryl resin
(S. Fink, Albert-Ludwig-Universität, Freiburg, FRG, unpublished method). Semithin sections (3 µm) of the embedded
segments were cut with a diamond knife on a Leica-2065-microtome (Leica Instruments GmbH, Nuβloch, FRG).
Histological observations and a differential survey of carbohydrates, phenolics and lipid-like compounds were performed
on the sections. The sections were stained for polysaccharides
containing vicinal glycol groups with periodic acid and Schiffs
reagent (PAS) as described by Gerlach (1977) and mounted in
Eukitt (Hans Thoma, Freiburg, FRG). The autofluorescent
properties of lignin, phenolics and lipids under excitation at
395--440 nm were used as the counterstain. For this purpose,
the sections were observed on a Zeiss-Axiophot microscope
with epi-fluorescence (Carl Zeiss, Oberkochen, FRG) using
the filters BP 395--440 (excitation filter), FT 460 (beamsplitter) and LP 470 (barrier filter). Photographs were taken with
an integrated camera on 100 ASA black-and-white film.
Lignin was stained with phloroglucin in HCl (von Aufseβ
1973). Lipids were stained with Nile blue (Jensen 1962) and
suberin and cutin were stained with Sudan IV (Jensen 1962).
Tannins were stained with ferric chloride (Jensen 1962) and
proteins were stained with Coomassie blue R250 in Clarke’s
solution (Cawood et al. 1978).
Determination of starch, chlorophyll and nutrient elements
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
2170
2860
460
1500
1370
275
-12
4.70
0.32
0.70
2.40
0.07
41
2170
2860
460
1500
1370
275
322
12
4.70
0.32
0.70
2.40
0.07
41
3240
1740
460
3690
1370
275
322
12
4.70
0.32
0.70
2.40
0.07
41
169
4767
1920
1500
1370
275
-12
4.70
0.32
0.70
2.40
0.07
NO −3
NH +4
SO 24 −
K+
PO 34 −
Ca2+
Na+
Fe3+
Mn2+
Cu2+
Zn2+
BO 33 −
MoO 24 −
At the end of September 1992, current-year and one-year-old
shoots were harvested around noon from randomly selected
trees (n = 6 per treatment). Needles were removed, immediately frozen in liquid nitrogen and stored at − 80 °C. The frozen
needle tissue was homogenized with a microdismembrator
(Braun, Melsungen, FRG). The resulting powder was freezedried at --25 °C and stored under vacuum at − 20 °C.
For the determination of foliar starch concentrations, 2- to
6-mg aliquots of needle homogenate were extracted for 1 h
with 0.5 ml of 0.5 M NaOH in Eppendorf vials (EppendorfNetheler-Hinz GmbH, Hamburg, FRG) at ambient temperature (Dekker and Richards 1971). To remove free glucose, the
extract was incubated at 95 °C for 3 min, adjusted to pH 4.6
with 0.5 M acetic acid, and then centrifuged at 10,000 g for
5 min (Einig and Hampp 1990). Twenty- to 60-ml aliquots of
the supernatant were used for enzymatic determination of
starch according to Outlaw and Manchester (1979). The procedure was optimized to obtain a 90% or higher recovery of an
TREE PHYSIOLOGY VOLUME 17, 1997
NEEDLE HISTOLOGY INFLUENCED BY MG DEFICIENCY AND N SOURCE
303
added internal standard (maize starch, Sigma Chemical Co.,
St. Louis, MO).
Chlorophyll concentrations were determined by spectrophotometry after needle tissue homogenization and extraction
in 80% acetone (Ziegler and Egle 1965), and calculated according to Lichtenthaler (1987). Magnesium and K concentrations in needles were analyzed after dry ashing and digestion
with HNO3/HCl by the AAS/AES flame technique (Perkin
Elmer 4000, Perkin-Elmer & Co GmbH, Überlinger, FRG).
The N and S concentrations were determined with a CNS
analyzer (Carlo Erba Instruments, Rodano, Italy). To evaluate
the data, means and standard errors (SE) were calculated
separately for each needle age class and treatment. Analysis of
variance (one-way ANOVA) and the Tukey test were used to
analyze differences among the treatments within each needle
age class.
Results
Needle element and chlorophyll concentrations
The influence of nitrogen sources on Mg deficiency was analyzed by varying the NO −3 /NH +4 -ratio of the nutrient solution
(0.76 = Treatment II, 1.86 = Treatment III, and 0.035 = Treatment IV, Table 1). The nutrient solution used in the Mg-sufficient treatment contained a fivefold higher concentration of
Mg and a NO −3 /NH +4 -ratio of 0.76 (Treatment I). After one
growing season, Mg concentrations in current-year and oneyear-old needles of Mg-limited trees differed significantly
depending on whether NO −3 or NH +4 was the major nitrogen
source (Figure 1a). Current-year needles from trees supplied
with NO −3 as the major N source (Treatment III) exhibited twoto threefold higher Mg concentrations than current-year needles of trees supplied with NH +4 as the major N source (Treatment IV) (Figure 1a). The differences in foliar Mg
concentrations between trees grown in the Mg-limited Treatments III and IV cannot be explained by dilution effects caused
by different growth rates of the needles, because both needle
weight and Mg concentration were higher in needles grown in
the NO −3 -dominated nutrient treatment (Treatment III) than the
NH +4 -dominated treatment (Treatment 1V) (Table 2 and Figure 1a). Chlorophyll concentrations of current-year needles
were influenced similarly by the treatments (Figure 1b). Compared with current-year needles, there was a similar but smaller
effect of nitrogen source on Mg concentrations of one-year-old
needles (Figure 1a). In contrast, reductions in chlorophyll
concentrations of one-year-old needles were only significant
in the NH +4 -dominated nutrient treatment (Treatment IV, Figure 1b).
Visible symptoms of yellowing occurred only in currentyear needles of trees in the Mg-limiting treatments. The symptoms were most pronounced in trees in the NH +4 -dominated
treatment (Treatment IV), followed by trees in the treatment
with a balanced NO −3 /NH +4 -ratio (Treatment II). Only slight
yellowing was observed in current-year needles of trees in the
NO −3 -dominated treatment (Treatment III). In accordance with
earlier findings, the Mg concentrations, calculated as % of
Figure 1. Magnesium (a) and chlorophyll concentrations (b) in current-year and one-year-old needles of Norway spruce grown with
sufficient (Treatment I) or limiting Mg supply (Treatments II--IV).
Ratios of NO −3 /NH +4 were: 0.76 (Treatments I and II), 1.86 (Treatment 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 concentration 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 NO −3 /NH +4 in the nutrient solutions were: 0.76 (Treatments I
and II), 1.86 (Treatment III) and 0.035 (Treatment IV) (n = 6, SE is
given in brackets). Different letters within rows indicate significant
differences between treatments.
Treatment
gfw
I
II
III
0.74 (0.05) a
0.75 (0.09) a 0.89 (0.10) a
IV
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
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PUECH AND MEHNE-JAKOBS
nitrogen concentration, were slightly below the physiological
threshold value of 2% for Norway spruce (Mehne-Jakobs
1996) in the NO −3 -dominated treatment, and well below the
threshold value in the NH +4 -dominated treatments (Treatments
II and IV; Table 2).
Needle N and S concentrations were slightly lower in all
Mg-limited treatments than in the Mg-sufficient treatment
(Table 2). Needle S concentrations were not influenced by
variation in the NO −3 /NH +4 -ratio or by a fourfold increase in
nutrient solution SO 24− concentration in the NH +4 -dominated
treatment (Treatment IV). Needle K concentrations, however,
decreased significantly with decreasing NO −3 /NH +4 -ratios. In
the NO −3 -dominated treatment (Treatment III), high foliar K
concentrations might reflect the increased K concentration in
this treatment (K served as the counterion for NO −3 , cf. Table 1).
Microscopy
Figures 2--4 are fluorescence micrographs of needle sections
stained with PAS. In these micrographs, the red fluorescence
of stained polysaccharides and the nuclei appear grey, whereas
the blue-green fluorescence of lignin and the yellow fluorescence of lipids appear white.
Vascular bundle Healthy current-year needles were characterized by an intact epidermis, a turgid mesophyll and an intact
vascular bundle (Figure 2a). Between the xylem and phloem,
cambial cells (C) are organized in two or three rows (Figure 2b). Their complex content was characterized by a yellowbrown fluorescence (arrowhead) and showed a positive
staining with Sudan IV for lipids and with Coomassie blue for
proteins. All sieve cells (Ph) had a wide open lumen. Younger
sieve cells showed a heterogeneous content whereas the older
Figure 2. Cross sections of Mg-sufficient needles. (a) Current-year needle (Magnification = 38×). (b)
Vascular bundle of the same needle
(165×). The cambial cells show a
fluorescent content (arrowhead) and
the transfusion parenchyma cells
contain starch (arrow). (c) Small
starch grains in chloroplasts of
mesophyll cells of a current-year
needle (338×). (d) Vascular bundle of
a one-year-old needle (188×). Some
older sieve cells are distorted (arrowheads). (e) Starch grains in chloroplasts of mesophyll cells of
one-year-old needle (345×). Abbreviations: Ep = epidermis, M = mesophyll, Bs = bundle sheath, Vb =
vascular bundle, X = xylem, C =
cambium, Ph = phloem, A = albuminous cells, Tp = transfusion parenchyma cells, Tt = transfusion
tracheids, S = starch, Ch = chloroplasts.
TREE PHYSIOLOGY VOLUME 17, 1997
NEEDLE HISTOLOGY INFLUENCED BY MG DEFICIENCY AND N SOURCE
sieve cells appeared empty. The albuminous cells (A) bordering
the phloem were well developed and filled with light fluorescing 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 the
vascular bundle, characterized by an increased proportion of
sieve cell deformations (Figures 3b, 3d, 4c, 4e, Ph) in currentyear and one-year-old needles, and by modifications of the
cambium in one-year-old needles including additional cell
rows, interruptions of cell rows and cell distortions (Figure 3d:
arrowhead, Figure 4e). Nitrogen source had no influence on
the symptoms expressed by the cambium. In the Mg-deficient
treatments, the amount of collapsed sieve cells was increased
in needles grown in the NO −3 -dominated treatment (Treatment III, Figures 3b and 3d) or balanced treatment (Treat-
305
ment II, sections not shown), but was only slightly increased
in the NH +4 -dominated treatment (Treatment IV, Figures 4c and
4e) compared with phloem cells in needles from Mg-sufficient
control trees. However, a proportion of the sieve cells remained
functional in all trees in the Mg-deficient treatments.
Mesophyll Chloroplasts of needles from the Mg-sufficient
treatment were almost devoid of starch at the end of the growing
season (Figures 2a, 2c, 2e). Only small starch grains (S) were
observed in chloroplasts (Ch) of the mesophyll surrounding the
vascular bundle and in the endodermis and transfusion parenchyma cells (Figures 2b: arrow, 2c, 2e). The amount of starch
in needles of Mg-deficient trees cultivated with either a balanced NO −3 /NH +4 -ratio (Treatment II) or an NO −3 -dominated N
form (Treatment III) did not differ markedly from that in
needles of Mg-sufficient trees (Figures 3a, 3e; sections of
Treatment II not shown). The starch concentration of current-
Figure 3. Cross sections of needles from Treatment III (Mg-deficient, NO −3 -dominated nutrition).
(a) Mesophyll cells of a currentyear needle (Magnification =
353×). The number of starch
grains in the chloroplasts shown
on the photograph is relatively
high for the treatment. (b) Vascular bundle of the same needle
(195×). Some sieve cells distortions were induced by the treatment (arrowhead). (c)
One-year-old needle (30×). (d)
Vascular bundle of a one-yearold needle with a higher proportion of distorted sieve cells
(218×). Cambium with an increased division array and some
distorted cells (arrowhead). (e)
Small starch grains in chloroplasts of mesophyll cells of a oneyear-old needle (330×).
Abbreviations: Ep = epidermis,
M = mesophyll, Bs = bundle
sheath, Vb = vascular bundle,
X = xylem, C = cambium, Ph =
phloem, A = albuminous cells,
Tt = transfusion tracheids, S =
starch, Ch = chloroplasts.
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PUECH AND MEHNE-JAKOBS
Figure 4. Cross sections of needles
from Treatment IV (Mg-deficient,
NH +4 -dominated nutrition). (a) Peripheral part of a current-year needle (Magnification = 218×). Starch
accumulated in the mesophyll as
well as in the hypodermis (arrows).
Tannin spherules are present in the
vacuoles. (b) Inner part of the mesophyll (345×). (c) Slightly distorted
phloem, and albuminous cells showing a modified content and small
starch grains (arrow) (158×). (d)
Starch grains in the mesophyll chloroplasts of a one-year-old needle
(390×). (e) Vascular bundle of a
one-year-old needle with some distortions of the sieve cells (arrowhead) and cambial modifications as
in Figure 3e (203×). Abbreviations:
Cu = cuticle, Hyp = hypodermis,
Ts = tannin spherules, Ep = epidermis, X = xylem, C = cambium,
Ph = phloem, A = albuminous cells,
Tt = transfusion tracheids, Tp =
transfusion parenchyma cells, Ts =
tanin spherules, S = starch, Ch =
chloroplasts.
year needles in Treatments II and III was very heterogeneous.
In the NH +4 -dominated treatment (Treatment IV), there was a
large increase in current-year needles. Many large starch grains
were observed in all mesophyll cells (Figures 4a and 4b) and
also in guard cells of the stomata and in hypodermal cells
(Figures 4a: arrows). Starch accumulation also occurred in the
transfusion parenchyma cells (Tp) and in some of the albuminous cells (A) bordering the youngest sieve cells (Figure 4c:
arrow). Starch grains were smaller in one-year-old needles than
in current-year needles of Treatment IV (Figure 4d).
The microscopic observations were confirmed by biochemical analysis. Compared to controls, deficient Mg supply com-
bined with a balanced NO −3 /NH +4 supply or NO −3 -dominated
nutrition caused a twofold increase in starch concentration of
the current-year needles, whereas the Mg-deficient plus NH +4 dominated nutrient regime caused a fivefold increase in starch
concentration of current-year needles (Figure 5). In one-yearold needles, treatment-related effects were similar to those of
current-year needles but less pronounced.
Few spherules were observed in mesophyll cells of currentyear needles in the Mg-sufficient treatment and in the Mg-deficient plus NO −3 -dominated treatment (Treatments I and III).
The number of spherules was greater in Mg-deficient needles
when nitrogen sources were balanced (Treatment II, sections
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NEEDLE HISTOLOGY INFLUENCED BY MG DEFICIENCY AND N SOURCE
Figure 5. Starch concentrations in current-year and one-year-old needles of Norway spruce trees grown with sufficient (Treatment I) or
limiting Mg supply (Treatments II--IV) (means ± SE, n = 6). Treatments refer to the following NO −3 /NH +4 -ratios in the nutrient solutions:
0.76 (Treatments I and II), 1.86 (Treatment III) and 0.035 (Treatment IV). Different letters indicate significant differences between
treatments.
not shown), and the increase was greatly enhanced when the
nitrogen supply was NH +4 -dominated (Treatment IV, see Figures 4a and 4b: Ts). Although all spherules exhibited the same
autofluorescence under blue-violet excitation, they stained differently depending on size. The smaller droplets present in the
outer mesophyll cells of the needles grown in the NH +4 -dominated nutrient regime (Figure 4a) were positively stained by
Nile blue for lipids, Coomassie blue for proteins and ferric
chloride for tannins, whereas the larger droplets in the inner
part of the mesophyll remained almost unstained and showed
autofluorescence. Because the staining intensity increased
with decreasing spherule size, it is possible that the stains were
physically adsorbed on the surface of the large drops. No
spherules were observed in one-year-old needles in any of the
treatments.
Discussion
In Norway spruce, Mg concentrations of 0.9 to 1.7 mg g −1 dry
matter are considered optimum, whereas values of less
than 0.7 mg g −1 indicate deficiency (Ingestad 1962, 1979,
Bergmann 1988). Throughout the literature, Mg deficiency
symptoms in Norway spruce are described as tip-yellowing of
the older needles, whereas the younger needles remain green
(Mies and Zöttl 1985, Bergmann 1988, Kaupenjohann and
Zech 1989, Lange et al. 1989). This difference between needles of different ages has been related to the translocation of Mg
from one-year-old needles to the new flush (Weikert et al.
1989). In the present study, however, we observed that Mg
deficient treatment caused chlorosis of current-year needles,
whereas one-year-old needles remained green, indicating that
Mg translocation into the developing shoots did not decrease
the Mg concentrations of the one-year-old needles below the
deficiency threshold value (Figure 1). Conversely, the amount
of Mg supplied by root uptake and by translocation from
307
one-year-old needles was insufficient to prevent current-year
needles from developing chlorosis. Previously, Mehne-Jakobs
(1994) observed yellowing of current-year needles of Norway
spruce trees grown with a severely restricted Mg supply
(5 µM), whereas current-year leaves of trees supplied with a
moderately reduced Mg supply (41 µM) did not exhibit pigment loss. A difference in the availability of nitrogen likely
accounts for the occurrence of current-year needle chlorosis
under conditions of moderately reduced Mg supply in the
present study and its absence in the previous study (MehneJakobs 1994): a higher nitrogen concentration was supplied in
the present experiment than in the earlier study (5 versus
3.5 mM). There is evidence that the development of Mg deficiency in conifers depends on the relative availabilities of Mg
and nitrogen (Ingestad 1979, Weissen et al. 1990). Previously,
the physiological threshold value for Mg in needles was found
to be 2% of the nitrogen concentration (Mehne-Jakobs 1996).
Magnesium concentrations in current-year needles were below
this threshold value in all of the Mg-limited treatments (Treatments II, III and IV, Table 2). Concentrations of both Mg and
chlorophyll were strongly influenced by nitrogen source (Figures 1a and 1b). Current-year needles of trees grown with
NO −3 as the predominant form of N exhibited only slight
reductions in Mg and chlorophyll concentrations (Treatment III, Figure 1b), whereas NH +4 -dominated nutrition caused
severe pigment loss (Treatment IV, Figure 1b). Because both
chlorophyll and Mg concentrations were equally influenced by
nitrogen source, the intensity of the chlorosis can be attributed
to Mg deficiency rather than to toxic effects of NH +4 . The
decrease in needle Mg concentration with increasing NH +4
supply cannot be explained by dilution caused by increased
growth, because needle weight was highest in the treatment
with the lowest NH +4 supply (Treatment III, Table 2). The
negative influence of the reduced NO −3 /NH +4 -ratio on foliar Mg
concentration is likely to have been caused by competition
between NH +4 uptake and uptake of other cations (Marschner
1995). Decreased foliar Mg concentrations combined with
enhanced yellowing have also been observed after ammonium
fertilization of Norway spruce stands suffering from Mg deficiency (Feger 1990, Hüttl and Fink 1991).
Histological investigation revealed slightly premature aging
of the vascular bundle in Mg-deficient needles, which was
characterized by an increase in sieve cell distortions, especially
when nutrition was NO −3 -dominated, and by modifications of
the cambium in all Mg-deficiency treatments. Similar symptoms of precocious aging have been observed in needles collected 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 cambium, then in the phloem, and finally in the xylem. In the
present study, the Mg-deficient needles generally exhibited
alterations of the cambium but few anomalies of the phloem
cells. Collapse of sieve cells is a characteristic structural symptom in chlorotic older needles of Mg-deficient spruce (Parameswaran et al. 1985, Fink 1989, Forschner et al. 1989, Fink
1991). In the present experiment, the trees were exposed to a
deficient Mg supply for only one growing season, and this did
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308
PUECH AND MEHNE-JAKOBS
not induce true phloem collapse either in the yellowing current-year or in the green one-year-old needles. However, similar treatments applied after a one-year period of Mg deficiency
treatment induced severe phloem collapse and cambial abnormalities in one-year-old needles (Puech and Mehne-Jakobs,
unpublished results). We conclude, therefore, that the minor
anomalies in the vascular area of Mg-deficient needles could
represent an early stage of pathological alteration that precedes
phloem collapse caused by prolonged Mg deficiency.
Because phloem collapse induced by Mg deficiency has
only been observed in one-year-old and older needles, it might
be associated with the translocation of Mg from one-year-old
needles to the developing shoots during spring. Fink (1992)
suggested that during flushing in spring, Mg is quickly
moblized from the area surrounding the phloem, leading to a
sudden and localized severe lack of Mg. Because Mg plays an
important role in the phloem loading process (Balke and
Hodges 1975, Giaquinta 1983), a severe lack of local Mg
might cause a large decrease in osmotic pressure in the sieve
cells, which consequently collapse. Drastic reductions in
phloem loading have been observed in source leaves of bean
as an early effect of Mg deficiency (Cakmak et al. 1994). In the
present study, the one-year-old needles probably still contained enough Mg to compensate for any localized lack of Mg
in the vascular area caused by translocation of Mg into the new
flush, and therefore phloem collapse was prevented (cf. Figure 1).
Another symptom apparently related to low Mg concentrations, was the occurrence of spherules or droplets in the mesophyll cells of current-year needles. Similar spherules have
been described previously and identified as vacuolar tannin
inclusions (Chafe and Durzan 1973, Baur and Walkinshaw
1974, Chabot and Chabot 1975), and were probably condensed
tannins or proanthocyanidins (Haslam 1988). Multiple staining of the spherules might be explained by their association
with a wide range of cellular compounds. Accumulation of
polyphenols and subsequently tannins in plant cells may represent a protective mechanism against herbivores (Claussen
et al. 1992). In addition, such compounds have been reported
to accumulate in plants exposed to various abiotic stress factors, 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 involved in antioxidative processes in plants (Torel et al. 1986,
Feucht et al. 1994), and that Mg deficiency may induce oxidative stress (Polle et al. 1994), we suggest that the accumulation
of spherules in the mesophyll cells represents a defense reaction against oxidative stress induced by Mg deficiency.
Starch accumulated in mesophyll cells of Mg-limited trees,
especially in current-year needles, even though CO2-fixation
rates were reduced to less than 50% of those of the Mg-sufficient needles (Mehne-Jakobs, unpublished results). Increased
amounts of starch in Mg-deficient spruce needles have been
observed in both field investigations and controlled experiments (Forschner et al. 1989, Fink 1991, Mehne-Jakobs 1995).
Photosynthate accumulation occurs before chlorophyll concentrations decrease (Mehne-Jakobs 1995), and is probably a
result of the reduction in phloem export of sucrose caused by
Mg deficiency (Cakmak et al. 1994).
Accumulations of starch and tannin spherules in needle
mesophyll cells were most pronounced in the NH +4 -dominated
treatment, which contained the lowest concentrations of Mg
and chlorophyll. Because ammonium toxicity reduces starch
synthesis (Puritch and Barker 1967, Matsumoto et al. 1969,
Mehrer and Mohr 1989), the increased symptoms observed in
the NH +4 -dominated treatment can be attributed to an increased
severity of Mg deficiency, most probably induced by cation
antagonism at the root level, rather than to phytotoxic effects
of ammonium.
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