Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:
Tree Physiology 17, 677--685
© 1997 Heron Publishing----Victoria, Canada
Effects of foliar potassium concentration on morphology,
ultrastructure and polyamine concentrations of Scots pine needles
ANNE JOKELA,1 TYTTI SARJALA,2 SEPPO KAUNISTO2 and SATU HUTTUNEN1
1
University of Oulu, Department of Biology, Botany, P.O. Box 333, FIN-90571 Oulu, Finland
2
The Finnish Forest Research Institute, Parkano Research Station, FIN-39700 Parkano, Finland
Received November 27, 1996
Summary We examined the effects of three foliar potassium
concentrations (high, intermediate and low) on the morphology, ultrastructure and polyamine concentrations of currentyear and 1- and 2-year-old needles of 30-year-old Scots pine
(Pinus sylvestris L.) trees. Foliar K concentration had only a
slight effect on needle morphology. The sclerenchyma cell
walls were thinner, the xylem area was larger, and the resin
ducts were smaller in needles with a low K concentration than
in needles with a high or intermediate K concentration. In
addition, the bundle sheath cells were collapsed in needles
having a low K concentration. The secondary growth of phloem
tissue and the mesophyll area were greater in needles with a
high or intermediate K concentration than in needles with a low
K concentration, possibly indicating greater production of
photoassimilates in these trees. At the ultrastructural level,
mesophyll cells with enlarged central vacuoles and small vacuoles containing electron-dense material were common in needles having a low K concentration. Enlargement of the central
vacuole coincided with an exponential increase in putrescine
concentration in needles with a low K concentration, suggesting that the central vacuole may function as a storage site for
putrescine.
Keywords: needle morphology, Pinus sylvestris, potassium deficiency, putrescine.
Introduction
Potassium (K) is characterized by high mobility in plants;
however, K uptake by plants is highly selective and closely
coupled with metabolic activity (Marschner 1995). Potassium
has important roles in enzyme activation, osmoregulation and
carbohydrate translocation in plant cells (Lüttge and Clarkson
1989). There is evidence that cell extension is related to the K
content of leaves. Thus, leaf area, cell size and turgor are lower
in expanding leaves of bean plants suffering from K deficiency
than in expanding leaves of bean plants well-supplied with K
(Mengel and Arneke 1982). Although there have been no
studies of the morphological responses of conifer needles to K
deficiency, Holopainen and Nygren (1989) reported that K
deficiency results in specific ultrastructural changes in Scots
pine seedlings including extension of the vacuolar system,
injuries to the tonoplast structure and increased deposition of
cytoplasmic lipids. However, it is not known whether similar
ultrastructural changes also occur in K-deficient needles of
different age classes in mature Scots pine trees.
A foliar K concentration of 3.5--4.0 mg gDW−1 is indicative
of severe K deficiency in Scots pine (Pinus sylvestris L.) during the nongrowing period (Paarlahti et al. 1971). Throughout
the year, the characteristic biochemical response of Scots pine
to K deficiency is the accumulation of putrescine (Sarjala and
Kaunisto 1993). Although putrescine accumulation is considered indicative of K deficiency, it has also been shown to occur
in response to various other stresses (Flores 1991).
The present study was undertaken to determine whether K
deficiency induces morphological and ultrastructural changes
in needles of different age classes in mature Scots pine trees.
Specifically, we tested the hypotheses that (1) foliar K concentrations alter both the ultrastructure and morphology of Scots
pine needles and (2) there is a close relationship among needle
microscopic structure and foliar K and putrescine concentrations.
Materials and methods
Site description and foliar sampling
Scots pine needles were collected in August 1992 from a
fertilization experiment located at Kuru (61°55′ N, 23°44′ E)
in western Finland. The site is an ombrotrophic, low-sedge
open bog with a deep peat layer. It was fertilized in 1967 with
800 kg ha −1 of rock phosphate (P 115 kg ha −1) and 200 kg ha −1
of KCl (K 100 kg ha −1). Severe potassium deficiency symptoms were observed in the late 1980s and a potassium refertilization trial was established in 1989 with seven treatments:
unfertilized and phosphorus fertilized controls, four potassium
sources of different solubility (KCl, K2CO3, KPO3, biotite) and
a mixture of KCl and biotite (Sarjala and Kaunisto 1993). Plot
mean height ranged from 4 to 6 m.
According to earlier observations (Sarjala and Kaunisto
1993), the needles of trees in the different plots differ widely
in K and polyamine concentrations. On the basis of these
results, we selected a subset of 25 trees for foliar polyamine
and nutrient analyses, of which three sets of five trees repre-
678
JOKELA ET AL.
senting high (> 5.3 mg gDW−1), intermediate (3.5--5.0 mg
gDW−1) and low (< 3.0 mg gDW−1) needle K concentrations were
selected for microscopy studies of needle morphology and
ultrastructure in August 1992. Needles of the selected trees
sampled for the microscopy studies were generally green, but
some older needles with low K concentrations often exhibited
yellow tips. The polyamine, K, N and P concentrations of
current-year (c), 1-year-old (c + 1) and 2-year-old (c + 2)
needles were analyzed. Because needles for nutrient analyses
are usually collected during the nongrowing period, foliar
sampling for polyamine and nutrient analyses was performed
in December 1992 from the same 15 trees. The data obtained
from the nutrient analyses were used to interpret the results of
the microscopy studies. Additionally, needles were sampled in
September 1995 for measurements of needle length, thickness
and width and for potassium and putrescine analyses.
Nutrient analyses and foliar free polyamines
Needles for nutrient analyses were taken to the laboratory in
plastic bags and stored at − 20 °C until analyzed. Nutrients
were analyzed by methods routinely used at the Forest Research Institute, Parkano, Finland as described by Halonen
et al. (1983). Total N was measured in oven-dried material by
the Kjeldahl method. Dry-ashed material was used for the
determination of K by flame atomic spectrophotometry
(Varian AA-30) and for the spectrophotometric analysis of P.
Needle samples for polyamine analysis were kept in ice until
taken to the laboratory and stored at − 80 °C. Free polyamines
(putrescine, spermidine and spermine) were extracted from the
needle samples with 5% HClO4, dansylated and analyzed by
HPLC as described by Sarjala and Kaunisto (1993).
Sample preparation for microscopy studies
In August 1992, needles (c = current-year, c + 1 = 1-year-old
and c + 2 = 2-year-old needles) for the microscopy studies were
collected in test tubes containing 0.05 M sodium cacodylate
buffer (pH 7) and 1.5% glutaraldehyde + paraformaldehyde
prefixative. After prefixation, 0.5-mm-thick cross sections
were cut from the middle portion of each needle, postfixed
with OsO4 and embedded in Ladd’s Epon as described by
Reinikainen and Huttunen (1989). An ultramicrotome
(Reichert Jung ULTRACUT E, Vienna, Austria) was used to
cut semi-thin sections (1--3 µm) for the light microscopy
studies and ultra-thin sections for electron microscopy (50--70
nm). The semi-thin sections were stained with toluidine blue
and the ultra-thin sections were stained with lead citrate and
uranyl acetate.
Needle morphology
Cross sections for light microscopy were taken from the middle of needles that were green and had no visible symptoms of
nutrient deficiency. The total number of needles examined was
111, i.e., two to three needles per needle year and eight to nine
needles per tree were observed. The samples were examined
with a Nikon OPTIPHOT-2 light microscope, and morphological measurements were performed with a digital image analyzer (Microscale TM/TC, Digithurst Ltd., Royston, England)
and a video camera (Hitachi KP-C571 CCD color camera).
The needle morphological variables measured were: needle
thickness, needle width, needle area, mesophyll area per needle area (%), epidermis + hypodermis area per needle area (%),
central cylinder area per needle area (%), sclerenchyma cell
wall thickness, phloem area per needle area (%), xylem area
per needle area (%), bundle sheath cell index (radial width of
bundle sheath cell/tangential width of bundle sheath cell measured on the adaxial side of needle cross sections), resin duct
area per needle area (%, measured on the abaxial side of needle
cross sections) and resin duct number per needle area (mm −2)
(Figure 1). Phloem and xylem were observed in one of the two
vascular bundles of the needle. The resin duct area was based
on measurements of two resin ducts from the abaxial side of
the needle. The compression and shrinkage of tissues during
embedding in plastic and sectioning were assumed to be minimal and similar in all samples (Toth 1982). Injured phloem
cells in the vascular bundle were also determined. The swelling
of parenchyma cells and collapse of sieve cells were regarded
as phloem injuries (Fink 1991, Jokela et al. 1995).
In September 1995, current-year needles were collected
from the same trees that were sampled in August and December 1992, and the length, thickness and width of the needles
were measured with a digital caliper. Needles were collected
from three trees from an unfertilized control plot and from
three trees from a plot fertilized with KPO3. Measurements
were made on 50 needles (one needle per fascicle) from current-year branches. Thickness and width were measured in the
middle region of the needle and were, therefore, comparable
with the image analysis measurements of needle thickness and
width made on needle cross sections in August 1992 (Figure 1).
Observations at the ultrastructural level
Ultra-thin sections were examined by the scanning transmission electron microscope (JEM 100CX II, JEOL, Tokyo, Japan). Cell organelles and cytoplasm in mesophyll were
examined in a total of 234 cells of 37 needles. The central
vacuole and cytoplasm in transfusion parenchyma were examined in 34 cells of 12 needles. Altogether, one to two needles
per needle year, and two to six needles per tree were studied.
Statistical analysis
The morphometric data were subjected to an analysis of variance (ANOVA), Kruskall-Wallis test and t-test using the
SPSS-PC software package (Jandel Corp., San Rafael, CA). In
all statistical analyses of the microscopic measurements, a
tree-specific mean was used and four to five trees represented
each K concentration. The polyamine and nutrient concentration data were subjected to linear and nonlinear regression
analyses and variance analysis using the BMDP software
package (University of California Press, Berkeley, CA).
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF POTASSIUM ON SCOTS PINE NEEDLES
679
Figure 1. Morphological
measurements made by image
analysis on cross sections of
Scots pine needles.
Table 1. Effects of needle age and classification by foliar K concentration based on an earlier study (Sarjala and Kaunisto 1993) on concentrations
of K (mg gDW−1), P (mg gDW−1) and N (% DW) and on N/P, N/K and K/P ratios in Scots pine needles sampled from five trees per treatment in
August and December 1992. Different letters within a row indicate significant differences between foliar K concentrations (P = 0.05). Needle age:
c = current-year, c + 1 = 1-year-old, and c + 2 = 2-year-old needles.
Variable
August
K (mg gDW−1)
P (mg gDW−1)
N (% DW)
N/P
N/K
K/P
December
K (mg gDW−1)
P (mg gDW−1)
N (% DW)
N/P
N/K
K/P
Needle
age
High K
Mean ± SD
Intermediate K
Mean ± SD
Low K
Mean ± SD
P
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
5.95 ± 0.79 a
4.73 ± 0.80 a
4.35 ± 0.80 a
1.74 ± 0.35 a
1.39 ± 0.26
1.28 ± 0.23
1.17 ± 0.08 a
1.08 ± 0.06 a
1.08 ± 0.05
6.9 ± 1.2
8.0 ± 1.3
8.4 ± 1.4
2.0 ± 0.3 a
2.3 ± 0.3 a
2.7 ± 0.3 a
3.5 ± 0.8 a
3.5 ± 0.9 a
3.5 ± 0.8 a
4.12 ± 0.47 b
3.33 ± 0.68 b
2.95 ± 0.60 b
1.35 ± 0.10 b
1.17 ± 0.19
1.17 ± 0.16
1.12 ± 0.05 a
1.05 ± 0.09 a
1.07 ± 0.12
8.3 ± 0.5
9.1 ± 1.1
9.2 ± 0.9
2.7 ± 0.3 b
3.2 ± 0.5 b
3.7 ± 0.4 b
3.1 ± 0.4 a
2.9 ± 0.6 a
2.5 ± 0.4 b
2.55 ± 0.30 c
2.41 ± 0.10 c
1.86 ± 0.15 c
1.72 ± 0.19 a
1.43 ± 0.16
1.06 ± 0.24
1.35 ± 0.05 b
1.38 ± 0.13 b
1.24 ± 0.27
7.9 ± 1.0
9.7 ± 1.0
12.3 ± 4.6
5.3 ± 0.9 c
5.7 ± 0.6 c
6.8 ± 1.9 c
1.5 ± 0.2 b
1.7 ± 0.2 b
1.8 ± 0.4 c
0.000
0.000
0.000
0.037
0.145
0.305
0.000
0.000
0.299
0.071
0.100
0.118
0.000
0.000
0.003
0.000
0.002
0.002
c
c+1
c
c+1
c
c+1
c
c+1
c
c+1
c
c+1
5.99 ± 0.85 a
5.55 ± 1.02 a
1.94 ± 0.49 a
1.86 ± 0.43
1.25 ± 0.12 a
1.24 ± 0.07
6.8 ± 1.6
6.9 ± 1.5
2.1 ± 0.3 a
2.3 ± 0.4 a
3.3 ± 0.9 a
3.1 ± 0.9 a
4.44 ± 0.35 b
3.95 ± 0.40 b
1.44 ± 0.17 b
1.36 ± 0.24
1.18 ± 0.05 a
1.35 ± 0.47
8.2 ± 0.8
10.5 ± 5.3
2.7 ± 0.2 b
3.5 ± 1.7 ab
3.1 ± 0.4 a
3.0 ± 0.5 a
3.04 ± 0.36 c
3.09 ± 0.23 c
1.91 ± 0.12 a
1.70 ± 0.16
1.40 ± 0.07 b
1.42 ± 0.07
7.4 ± 0.8
8.4 ± 1.1
4.6 ± 0.5 c
4.6 ± 0.4 b
1.6 ± 0.3 b
1.8 ± 0.3 b
0.000
0.000
0.047
0.056
0.004
0.593
0.406
0.422
0.000
0.014
0.001
0.014
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
680
JOKELA ET AL.
K/P ratios indicated a K deficiency in needles of trees in the
low foliar K class.
For both the August and December samplings, K and P
concentrations decreased with increasing needle age. Both K
and P concentrations were higher in December than in August
for all needle age classes and for all foliar K classes.
Results
Nutrient concentrations
Trees classified on the basis of an earlier study (Sarjala and
Kaunisto 1993) as having high, intermediate or low foliar K
concentrations fell into the same categories on the basis of
measurements made in the present study (Table 1). In trees in
the intermediate foliar K class, most macronutrients were
present in optimal concentrations, with the exception that P
concentration and the N/K ratio were well below optimum
values (Table 1). Optimum P concentrations were present in
needles in trees of the high and low foliar K classes. Foliar N
concentrations were below the deficiency limit in trees of the
intermediate and high foliar K classes (N, N/P and N/K,
Table 1), whereas the K/P ratio was in balance. The N/K and
Needle morphology
Foliar K concentration had only a slight effect on needle
morphology. Needle thickness, width (Tables 2 and 3) and
length (Table 3) were not significantly affected by foliar K
concentration. Needles with a low K concentration had the
largest needle area, whereas needles with a high K concentration had the largest relative area of mesophyll and the smallest
central cylinder area (Table 2). The sclerenchyma cell walls
were thinnest in needles with a low K concentration (Fig-
Table 2. Light microscopic image analysis measurements of needle morphological variables. Different letters within a row indicate significant
differences between the foliar K concentration classes (P = 0.05). Needles were collected in August 1992. Needle age: c = current-year, c + 1 =
1-year-old, and c + 2 = 2-year-old needles; and n = number of trees observed.
Variable
Needle thickness
(mm)
Needle width (mm)
Needle area (mm2)
Mesophyll area (%)
Epidermis + hypodermis area (%)
Central cylinder
area (%)
Sclerenchyma cell
wall thickness (µm)
Phloem area (%)
Xylem area (%)
Bundle sheath
cell index
Resin duct area (%)
Resin duct
number mm −2
Intermediate K
P
Needle
High K
age
n
Mean ± SD
n
Mean ± SD
n
Mean ± SD
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
0.781 ± 0.033
0.801 ± 0.069
0.780 ± 0.066
1.671 ± 0.069
1.690 ± 0.104
1.647 ± 0.182
1.071 ± 0.870
1.105 ± 0.139
1.122 ± 0.205
57.59 ± 2.59
59.65 ± 2.03
58.20 ± 2.29
12.68 ± 1.59
10.95 ± 1.06
12.11 ± 1.25
29.72 ± 1.85
29.40 ± 2.16
29.69 ± 2.02
6.02 ± 0.58
5.73 ± 0.66
5.73 ± 0.71 a
0.54 ± 0.09
0.76 ± 0.03
0.90 ± 0.15
0.60 ± 0.01
0.60 ± 0.11
0.62 ± 0.09
0.57 ± 0.10
0.62 ± 0.03
0.73 ± 0.04
0.91 ± 0.17
0.76 ± 0.12
0.72 ± 0.13
9.69 ± 1.26
8.86 ± 0.83
8.23 ± 0.53
4
5
5
5
5
5
4
5
5
4
5
5
4
5
5
4
5
5
4
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
0.811 ± 0.064
0.772 ± 0.017
0.774 ± 0.026
1.705 ± 0.161
1.540 ± 0.420
1.681 ± 0.149
1.110 ± 0.153
0.976 ± 0.248
1.082 ± 0.086
56.63 ± 1.27
57.57 ± 2.71
57.96 ± 3.39
11.41 ± 0.53
10.79 ± 1.44
11.33 ± 0.57
31.96 ± 1.43
31.64 ± 2.00
30.71 ± 2.98
5.66 ± 0.91
5.12 ± 0.49
4.68 ± 0.42 b
0.60 ± 0.06
0.73 ± 0.02
0.90 ± 0.18
0.58 ± 0.05
0.69 ± 0.31
0.60 ± 0.07
0.58 ± 0.10
0.66 ± 0.08
0.76 ± 0.24
0.99 ± 0.17
1.05 ± 0.33
0.76 ± 0.12
9.04 ± 2.08
8.94 ± 4.24
7.41 ± 1.79
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
0.812 ± 0.066
0.802 ± 0.093
0.808 ± 0.104
1.766 ± 0.189
1.824 ± 0.238
1.658 ± 0.357
1.210 ± 0.268
1.203 ± 0.262
1.149 ± 0.368
57.12 ± 2.11
56.68 ± 1.58
57.63 ± 1.29
11.95 ± 1.58
11.78 ± 1.29
11.83 ± 1.32
30.93 ± 3.28
31.58 ± 2.56
30.55 ± 1.43
6.44 ± 1.22
5.61 ± 0.88
4.63 ± 0.67 b
0.62 ± 0.01
0.70 ± 0.01
0.80 ± 0.11
0.67 ± 0.01
0.66 ± 0.16
0.66 ± 0.13
0.56 ± 0.08
0.60 ± 0.04
0.72 ± 0.08
0.87 ± 0.11
0.73 ± 0.18
0.84 ± 0.28
8.19 ± 1.15
7.92 ± 0.56
7.34 ± 1.83
TREE PHYSIOLOGY VOLUME 17, 1997
Low K
0.622
0.878
0.613
0.600
0.651
0.977
0.491
0.445
0.913
0.677
0.075
0.763
0.432
0.326
0.566
0.298
0.310
0.733
0.359
0.364
0.026
0.566
0.878
0.468
0.275
0.878
0.733
0.650
0.185
0.651
0.185
0.114
0.810
0.249
0.196
0.566
EFFECTS OF POTASSIUM ON SCOTS PINE NEEDLES
Table 3. Leaf morphological variables (measured with a digital caliper) and potassium (K) and putrescine concentrations of current-year
needles collected in September 1995. Fifty needles per tree were
measured on three trees per plot.
Variable
High K
Mean ± SD
Low K
Mean ± SD
P
(t-test)
37.03 ± 9.23
42.78 ± 4.64
0.389
Needle thickness
(mm)
0.67 ± 0.02
0.65 ± 0.04
0.426
Needle width (mm)
1.46 ± 0.02
1.50 ± 0.10
0.562
5.58 ± 0.10
2.37 ± 0.33
0.002
150.26 ± 52.77
2392.74 ± 1568.10
0.131
Needle length (mm)
K mg gDW
−1
Putrescine
nmol gFW−1
ure 2a) and thickest in 2-year-old needles with a high K concentration (Figure 2b; P = 0.026, Table 2).
The phloem area was greater in 1- and 2-year-old needles
than in current-year needles, and the difference was significant
in needles with a high K (P = 0.025) or an intermediate K
concentration (P = 0.022) but not in needles with a low K
681
concentration (P = 0.098). In contrast, needle age and foliar K
concentration had few significant effects on xylem area, although xylem area was larger in needles with a low K concentration than in needles with an intermediate or a high K
concentration. When calculated in relation to the central cylinder area, the phloem and xylem areas showed the same relationship as for whole needle area (data not shown). There was
no relationship between phloem cell injury and foliar K concentration.
The bundle sheath cell index was smallest (radial to tangential width of cell was smaller) in needles with a low K concentration and was independent of needle age (Table 2). In needles
with a low K concentration, the bundle sheath cells were
collapsed (Figure 2c), whereas the bundle sheath cells were
normal in shape in needles with a high K concentration (Figure 2d). The resin duct area was smallest in current-year and
1-year-old needles with a low K concentration, and the number
of resin ducts was smallest in needles with a low K concentration and was independent of needle age.
Observations at ultrastructural level
Ultrastructural observations of the mesophyll (Table 4) revealed an enlarged central vacuole in current-year and 1- and
Figure 2. Photomicrographs of cross sections of Scots pine needles. (a) Thin sclerenchyma cell walls (S) in a cross section of a 2-year-old needle
with a low K concentration. Bar = 0.1 mm. (b) Normal sclerenchyma cell walls (S) in a cross section of a 2-year-old needle with a high K
concentration. Bar = 0.1 mm. (c) Collapsed bundle sheath cells (arrows) in a cross section of a 1-year-old needle with a low K concentration.
Bar = 0.02 mm. (d) Normal-shaped bundle sheath cells (arrows) in a cross section of a 1-year-old needle with an intermediate K concentration.
Bar = 0.02 mm. Abbreviations: m = mesophyll, p = phloem, r = resin duct, t = transfusion tissue, and x = xylem.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
682
JOKELA ET AL.
Table 4. Effect of foliar K concentration class on ultrastructure of needles collected in August 1992. Needle age: c = current-year, c + 1 = 1-year-old,
and c + 2 = 2-year-old needles.
Variable
High K
Intermediate K
Low K
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
MESOPHYLL
Central vacuole
Enlarged central vacuole
Proliferation of tonoplast
--1
+
-+
+2
+
-+
-++3
+
+
+
--
+
+
+
+
Chloroplast
Swelling of thylakoids
+
+
--
+
+
+
++
+
+
Cytoplasm
Lipid accumulations
Small vacuoles with
electron-dense material
Extensive vesiculation
+
--
++
+
++
+
+
--
+
--
+
--
+
--
++
++
++
+
--
--
+
--
--
--
--
+
+
Mitochondria
Swelling of mitochondria
--
--
+
--
--
--
--
--
+
TRANSFUSION PARENCHYMA
Central vacuole
Enlarged central vacuole
--
+
--
--
--
+
--
+
--
Cytoplasm
Lipid accumulations
--
+
--
--
--
--
--
+
+
1
2
3
-- = Not observed.
+ = Occasionally observed.
++ = Frequently observed.
Figure 3. Ultrastructure of mesophyll cells (a--c) of 1-year-old
needles with a low K concentration and of a transfusion parenchyma cell (d) of a 2-year-old
needle with a low K concentration. (a) Large central vacuole
(cv) and an intensive accumulation of small vacuoles with darkstained material in the cytoplasm
(arrow). Bar = 1 µm. (b) Same
cell as in (a) showing vesiculated appearance of the cytoplasm, arrow = a small vacuole.
Bar = 1 µm. (c) Lipid accumulations (L) adjacent to the
tonoplast, which is located between lipids and dark-stained
tannin accumulations (T). Chloroplast (c) with starch grain (s)
and an intercellular space (I).
Bar = 1 µm. (d) Transfusion parenchyma cell with many lipid
accumulations (L), cv = central
vacuole, s = starch grain in chloroplast. Bar = 2 µm. Abbreviations: c = chloroplast, m =
mitochondria, w = cell wall.
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF POTASSIUM ON SCOTS PINE NEEDLES
2-year-old needles with a low K concentration. Proliferation of
the tonoplast was slightly less frequent in needles with a low
K concentration than in needles with an intermediate or high
K concentration. The formation of small vacuoles with electron-dense material in mesophyll cells (Figure 3a) was observed in needles with a low or high K concentration, but not
in needles with an intermediate K concentration. Sometimes,
these small vacuoles gave the cell a vesiculated appearance
(extensive vesiculation in Table 4) (Figure 3b).
The swelling of chloroplast thylakoids and occurrence of
lipid accumulations in the cytoplasm were abundant and independent of foliar K concentration. Lipid accumulations were
often adjacent to the tonoplast (Figure 3c). Slight mitochondrial swelling was observed in the 2-year-old needles with a
low or high K concentration. Cytoplasmic lipid accumulations
in the transfusion parenchyma occurred slightly more frequently in needles with a low K concentration than in needles
with an intermediate or high K concentration (Figure 3d),
whereas an enlarged central vacuole in transfusion parenchyma cells was observed at all foliar K concentrations.
Free polyamine concentrations
Polyamine analysis of current-year and 1- and 2-year-old
needles of the 25 selected trees indicated a negative correlation
between putrescine and K concentrations that declined with
needle age (regression analysis: current-year needles
y = 22191 x−3.20 and r 2 = 0.622, 1-year-old needles
y = 138019 x− 5.33 and r 2 = 0.554, 2-year-old needles
y = 9154 x− 2.97 and r 2 = 0.313; x = K concentration and y = putrescine concentration). In 1992, putrescine concentrations in
needles with a low K concentration were significantly higher
Figure 4. Putrescine, spermidine, and spermine concentrations in
current-year and 1- and 2-year-old needles in August and December
1992. Statistically significant differences between foliar K concentrations are indicated by different letters.
683
than putrescine concentrations in needles with an intermediate
or high K concentration (Figure 4). Spermidine and spermine
concentrations were lower in needles with a low K concentration than in needles with an intermediate or high K concentration (Figure 4). The changes in polyamine concentration in
response to foliar K concentration were independent of needle
age and were observed in both August and December, except
for spermine concentration, which was lower in December
than in August for all needle age classes. In September 1995,
putrescine concentration in needles with a low K concentration
was higher than in needles with a high K concentration (Table 3), but the difference was not statistically significant.
Discussion
Foliar K concentration had only slight effects on needle length,
thickness and width. The higher proportion of the mesophyll
area in needles with a high K concentration may indicate that
these needles had a high photosynthetic potential, which in
turn may have affected tree growth. Potassium fertilization has
been shown to increase the volume growth and basal area of
Scots pine growing on peatlands (Kaunisto 1989). Moreover,
Baillon et al. (1988) reported that photosynthetic rates were
lower in K-deficient Norway spruce seedlings than in seedlings receiving an adequate supply of K; however, in Scots pine
seedlings, photosynthetic rate was not inhibited until foliar K
concentration was reduced to 2.4 mg gDW−1 (Nygren and Hari
1992). Neither photosynthesis nor tree growth was measured
in our study.
The thin cell walls of the sclerenchyma tissue in 2-year-old
needles with a low K concentration may indicate injury similar
to that found in needles deficient in boron, potassium or phosphorus (Raitio 1979, 1981). The development of thin sclerenchyma cell walls may be a response to nutrient imbalance
caused by an excess N supply (Jokela et al. 1995).
The enhanced formation of secondary phloem in needles
with an intermediate or high K concentration compared to
needles with a low K concentration may have been a result of
enhanced photosynthesis caused by K fertilization (Dünisch
and Bauch 1994). Furthermore, Hartt (1969) showed that K
has an important role in photoassimilate transport in the
phloem of sugar cane. The formation of secondary phloem but
not secondary xylem in Scots pine needles is in agreement with
the findings of Ewers (1982), who reported that cambium in
the vascular bundle produces secondary phloem but not secondary xylem in several conifer species including Scots pine.
The finding that the xylem area was slightly larger in current-year needles with a low K concentration than in currentyear needles with an intermediate or a high K concentration is
of interest because polyamine transport takes place in the
xylem (Bagni and Pistocchi 1991). Putrescine enhances xylogenesis of xylem cells in Helianthus tuber (Phillips et al. 1988)
and it may influence cambial activity in gymnosperms. For
example, in the stem cambial zone of Norway spruce, putrescine is the most abundant polyamine during the period of
greatest cambial activity in spring and summer (Königshofer
1991).
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
684
JOKELA ET AL.
Although phloem injuries have often been attributed to nutrient imbalance (Fink 1991, 1993, Jokela et al. 1995, 1996),
we found no effect of foliar K concentration on phloem cell
integrity. The slightly collapsed bundle sheath cells of needles
with a low K concentration may be related to nutrient stress in
general or K deficiency in particular. Collapsed bundle sheath
cells have been associated with the accumulation of secondary
substances, and modifications in the bundle sheath structure
have been found in response to fumigation with SO2 and O3
(Maier-Maercker and Koch 1992). The decrease in size and
number of resin ducts with decreasing foliar K concentration
may indicate that the terpene-based defensive mechanism is
compromised in trees suffering from K deficiency (Burr and
Clancy 1992).
In our study, foliar K concentration influenced the size of the
central vacuole of mesophyll cells but had no effect on the
occurrence of tonoplast injuries or lipid accumulation. Lipid
accumulations were frequently observed but they were independent of foliar K concentration. They were often located
adjacent to the tonoplast, and may indicate tonoplast disintegration (Zwiazek and Shay 1987) or cold hardening during late
summer (Holopainen et al. 1992). Holopainen and Nygren
(1989) observed that K deficiency causes an extension of the
vacuolar system in mesophyll cells, fragmentation of the
tonoplast and an increase in cytoplasmic lipids in needles of
Scots pine seedlings. According to Holopainen and Nygren
(1989), small vacuoles with tannin deposits are associated with
K deficiency. We observed many small vacuoles with electrondense material that gave the cytoplasm a vesiculated appearance in needles with a low K concentration. We conclude that
the vacuole system may be particularly sensitive to K deficiency, because K is important in osmoregulation (Lüttge and
Clarkson 1989).
Changes in the vacuolar system could also be explained by
changes in the polyamine concentration of K-deficient needles. We observed increased concentrations of putrescine and
decreased concentrations of spermidine and spermine in needles with a low K concentration (cf. Sarjala and Kaunisto 1993).
Flores (1991) suggested that the accumulation of putrescine,
possibly in the vacuole (Pistocchi et al. 1988), compensates for
the decrease in pH of cells of K-deficient plants.
Changes in Scots pine needle structure caused by K deficiency differed from those caused by P (injuries to mitochondrial structure) and N deficiencies (changes in the cytoplasm
and chloroplast structure) (Palomäki and Holopainen 1994,
1995). Interpretation of the symptoms of K deficiency is complicated because of the effects of foliar K concentration on the
balance of the other macronutrients. For example, needle N
was at an adequate concentration for growth in needles with a
low K concentration, whereas needles with an intermediate or
high K concentration were deficient in N (cf. Paarlahti et al.
1971, Kaunisto 1987). Differences between nutrient interactions in Scots pine trees growing in the field and those that
occur in seedlings growing under controlled environment conditions may account for the differences between our observations and those reported by Holopainen and Nygren (1989).
We conclude that foliar K concentration has a small effect
on needle morphology. Needles with a low K concentration
were characterized by thinner sclerenchyma cell walls, a
slightly larger xylem area, less secondary growth of phloem,
smaller resin ducts and more collapsed bundle sheath cells than
needles classified as containing an intermediate or high K
concentration. A large accumulation of putrescine occurred in
needles with a low K concentration and was accompanied by
the development of a vacuolar system in mesophyll cells.
Acknowledgments
This research was supported by the Kone Foundation and the Finnish
Research Program on Climate Change. Dr. Jaana Bäck and Dr. Sagar
V. Krupa are thanked for their valuable comments on the manuscript
as are Mrs. Tellervo Siltakoski, Mrs. Mervi Saaranen and M.Sc. Eija
Kukkola for technical assistance. The language of the manuscript was
revised by Mrs. Leena Kaunisto.
References
Bagni, N. and R. Pistocchi. 1991. Uptake and transport of polyamines
and inhibitors of polyamine metabolism in plants. In Biochemistry
and Physiology of Polyamines in Plants. Eds. R.D. Slocum and
H.E. Flores. CRC Press, Boca Raton, FL, pp 105--120.
Baillon, F., X. Dalschaert, S. Grassi and F. Gaiss. 1988. Spruce
photosynthesis: possibility of early damage diagnosis due to exposure to magnesium or potassium deficiency. Trees 2:173--179.
Burr, K.E. and K.M. Clancy. 1992. Douglas-fir needle anatomy in
relation to western spruce budworm (Lepidoptera: Tortricidae).
J. Econ. Entomol. 86:93--99.
Dünisch, O. and J. Bauch. 1994. Influence of mineral elements on
wood formation of old growth spruce (Picea abies (L.) Karst.).
Holzforschung 48 Suppl:5--14.
Ewers, F.W. 1982. Secondary growth in needle leaves of Pinus longaeva (bristlecone pine) and other conifers: quantitative data. Am.
J. Bot. 69:1552--1559.
Fink, S. 1991. Structural changes in conifer needles due to Mg and K
deficiency. Fert. Res. 27:23--27.
Fink, S. 1993. Microscopic criteria for the diagnosis of abiotic injuries
to conifer needles. In Forest Decline in Atlantic and Pacific Regions. Eds. R.F. Hüttl and D. Müller-Dombois. Springer-Verlag,
Berlin, pp 175--188.
Flores, H.E. 1991. Changes in polyamine metabolism in response to
abiotic stress. In Biochemistry and Physiology of Polyamines in
Plants. Eds. R.D. Slocum and H.E. Flores. CRC Press, Boca Raton,
FL, pp 213--228.
Halonen, O., H. Tulkki and J. Derome. 1983. Nutrient analysis methods. Metsäntutkimuslaitoksen tiedonantoja 121. Valtion painatuskeskus, Helsinki, 28 p.
Hartt, C.E. 1969. Effects of potassium deficiency upon translocation
of 14C in attached blades and entire plants of sugarcane. Plant
Physiol. 44:1461--1469.
Holopainen, T. and P. Nygren. 1989. Effects of potassium deficiency
and simulated acid rain, alone and in combination, on the ultrastructure of Scots pine needles. Can. J. For. Res. 19:1402--1411.
Holopainen, T., S. Anttonen, A. Wulff, V. Palomäki and L. Kärenlampi.
1992. Comparative evaluation of the effects of gaseous pollutants,
acidic deposition and mineral deficiencies: structural changes in the
cells of forest plants. Agric. Ecosyst. Environ. 42:365--398.
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF POTASSIUM ON SCOTS PINE NEEDLES
Jokela, A., J. Bäck, S. Huttunen and R. Jalkanen. 1995. Excess nitrogen fertilization and the structure of Scots pine needles. Eur. J. For.
Pathol. 25:109--124.
Jokela, A., V. Palomäki, S. Huttunen and R. Jalkanen. 1996. Effects of
root damage on the nutritional status and structure of Scots pine
needles. J. Plant Physiol. 148:317--323.
Kaunisto, S. 1987. Effect of refertilization on the development and
foliar nutrient contents of young Scots pine stands on drained mires
of different nitrogen status. Commun. Inst. For. Fenn. 140, 58 p.
Kaunisto, S. 1989. Jatkolannoituksen vaikutus puuston kasvuun
vanhalla ojitusalueella. Summary: Effect of refertilization on tree
growth in an old drainage area. Folia For. 724:1--15.
Königshofer, H. 1991. Distribution and seasonal variation of polyamines in shoot-axes of spruce (Picea abies (L.) Karst.). J. Plant
Physiol. 137:607--612.
Lüttge, U. and D.T. Clarkson. 1989. Mineral nutrition: Potassium.
Prog. Bot. 50:51--73.
Maier-Maercker, U. and W. Koch. 1992. Histological examination of
spruce needles from a long-term gas exchange experiment in pure
and polluted air in the field. Trees 6:186--194.
Marschner, H. 1995. Mineral nutrition of higher plants. Academic
Press, New York, 889 p.
Mengel, K. and W.W. Arneke. 1982. Effect of potassium on the water
potential, the osmotic potential, and cell elongation in leaves of
Phaseolus vulgaris. Physiol. Plant. 54:402--408.
Nygren, P. and P. Hari. 1992. Effect of foliar application with acid mist
on the photosynthesis of potassium-deficient Scots pine seedlings.
Silva Fenn. 26:133--144.
Paarlahti, K., A. Reinikainen and H. Veijalainen. 1971. Nutritional
diagnosis of Scots pine by needle and peat analysis. Commun. Inst.
For. Fenn. 74, 58 p.
685
Palomäki, V. and T. Holopainen. 1994. Effects of phosphorus deficiency and recovery fertilization on growth, mineral concentration,
and ultrastructure of Scots pine needles. Can. J. For. Res. 24:2459-2468.
Palomäki, V. and T. Holopainen. 1995. Effects of nitrogen deficiency
and recovery fertilization on ultrastructure, growth and mineral
concentrations of Scots pine needles. Can. J. For. Res. 25:198--207.
Phillips, R., M.C. Press, L. Bingham and G. Grimmer. 1988. Polyamines in cultured artichoke explants: effects are primarily on
xylogenesis rather than cell division. J. Exp. Bot. 39:473--480.
Pistocchi, R., F. Keller, N. Bagni and P. Matile. 1988. Transport and
subcellular localization of polyamines in carrot protoplasts and
vacuoles. Plant Physiol. 87:514--518.
Raitio, H. 1979. Growth disturbances of Scots pine caused by boron
deficiency on an afforested abandoned peatland field. Folia For.
412:1--16.
Raitio, H. 1981. Effects of macronutrient fertilization on the structure
and nutrient content of pine needles on a drained short sedge bog.
Folia For. 465:1--9.
Reinikainen, J. and S. Huttunen. 1989. The level of injury and needle
ultrastructure of acid rain-irrigated pine and spruce seedlings after
low temperature treatment. New Phytol. 112:29--39.
Sarjala, T. and S. Kaunisto. 1993. Needle polyamine concentrations
and potassium nutrition in Scots pine. Tree Physiol. 13:87--96.
Toth, R. 1982. An introduction to morphometric cytology and its
application to botanical research. Am. J. Bot. 69:1694--1706.
Zwiazek, J. and J. Shay. 1987. Fluoride- and drought-induced structural alterations of mesophyll and guard cells in cotyledons of jack
pine (Pinus banksiana). Can. J. Bot. 65:2310--2317.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
© 1997 Heron Publishing----Victoria, Canada
Effects of foliar potassium concentration on morphology,
ultrastructure and polyamine concentrations of Scots pine needles
ANNE JOKELA,1 TYTTI SARJALA,2 SEPPO KAUNISTO2 and SATU HUTTUNEN1
1
University of Oulu, Department of Biology, Botany, P.O. Box 333, FIN-90571 Oulu, Finland
2
The Finnish Forest Research Institute, Parkano Research Station, FIN-39700 Parkano, Finland
Received November 27, 1996
Summary We examined the effects of three foliar potassium
concentrations (high, intermediate and low) on the morphology, ultrastructure and polyamine concentrations of currentyear and 1- and 2-year-old needles of 30-year-old Scots pine
(Pinus sylvestris L.) trees. Foliar K concentration had only a
slight effect on needle morphology. The sclerenchyma cell
walls were thinner, the xylem area was larger, and the resin
ducts were smaller in needles with a low K concentration than
in needles with a high or intermediate K concentration. In
addition, the bundle sheath cells were collapsed in needles
having a low K concentration. The secondary growth of phloem
tissue and the mesophyll area were greater in needles with a
high or intermediate K concentration than in needles with a low
K concentration, possibly indicating greater production of
photoassimilates in these trees. At the ultrastructural level,
mesophyll cells with enlarged central vacuoles and small vacuoles containing electron-dense material were common in needles having a low K concentration. Enlargement of the central
vacuole coincided with an exponential increase in putrescine
concentration in needles with a low K concentration, suggesting that the central vacuole may function as a storage site for
putrescine.
Keywords: needle morphology, Pinus sylvestris, potassium deficiency, putrescine.
Introduction
Potassium (K) is characterized by high mobility in plants;
however, K uptake by plants is highly selective and closely
coupled with metabolic activity (Marschner 1995). Potassium
has important roles in enzyme activation, osmoregulation and
carbohydrate translocation in plant cells (Lüttge and Clarkson
1989). There is evidence that cell extension is related to the K
content of leaves. Thus, leaf area, cell size and turgor are lower
in expanding leaves of bean plants suffering from K deficiency
than in expanding leaves of bean plants well-supplied with K
(Mengel and Arneke 1982). Although there have been no
studies of the morphological responses of conifer needles to K
deficiency, Holopainen and Nygren (1989) reported that K
deficiency results in specific ultrastructural changes in Scots
pine seedlings including extension of the vacuolar system,
injuries to the tonoplast structure and increased deposition of
cytoplasmic lipids. However, it is not known whether similar
ultrastructural changes also occur in K-deficient needles of
different age classes in mature Scots pine trees.
A foliar K concentration of 3.5--4.0 mg gDW−1 is indicative
of severe K deficiency in Scots pine (Pinus sylvestris L.) during the nongrowing period (Paarlahti et al. 1971). Throughout
the year, the characteristic biochemical response of Scots pine
to K deficiency is the accumulation of putrescine (Sarjala and
Kaunisto 1993). Although putrescine accumulation is considered indicative of K deficiency, it has also been shown to occur
in response to various other stresses (Flores 1991).
The present study was undertaken to determine whether K
deficiency induces morphological and ultrastructural changes
in needles of different age classes in mature Scots pine trees.
Specifically, we tested the hypotheses that (1) foliar K concentrations alter both the ultrastructure and morphology of Scots
pine needles and (2) there is a close relationship among needle
microscopic structure and foliar K and putrescine concentrations.
Materials and methods
Site description and foliar sampling
Scots pine needles were collected in August 1992 from a
fertilization experiment located at Kuru (61°55′ N, 23°44′ E)
in western Finland. The site is an ombrotrophic, low-sedge
open bog with a deep peat layer. It was fertilized in 1967 with
800 kg ha −1 of rock phosphate (P 115 kg ha −1) and 200 kg ha −1
of KCl (K 100 kg ha −1). Severe potassium deficiency symptoms were observed in the late 1980s and a potassium refertilization trial was established in 1989 with seven treatments:
unfertilized and phosphorus fertilized controls, four potassium
sources of different solubility (KCl, K2CO3, KPO3, biotite) and
a mixture of KCl and biotite (Sarjala and Kaunisto 1993). Plot
mean height ranged from 4 to 6 m.
According to earlier observations (Sarjala and Kaunisto
1993), the needles of trees in the different plots differ widely
in K and polyamine concentrations. On the basis of these
results, we selected a subset of 25 trees for foliar polyamine
and nutrient analyses, of which three sets of five trees repre-
678
JOKELA ET AL.
senting high (> 5.3 mg gDW−1), intermediate (3.5--5.0 mg
gDW−1) and low (< 3.0 mg gDW−1) needle K concentrations were
selected for microscopy studies of needle morphology and
ultrastructure in August 1992. Needles of the selected trees
sampled for the microscopy studies were generally green, but
some older needles with low K concentrations often exhibited
yellow tips. The polyamine, K, N and P concentrations of
current-year (c), 1-year-old (c + 1) and 2-year-old (c + 2)
needles were analyzed. Because needles for nutrient analyses
are usually collected during the nongrowing period, foliar
sampling for polyamine and nutrient analyses was performed
in December 1992 from the same 15 trees. The data obtained
from the nutrient analyses were used to interpret the results of
the microscopy studies. Additionally, needles were sampled in
September 1995 for measurements of needle length, thickness
and width and for potassium and putrescine analyses.
Nutrient analyses and foliar free polyamines
Needles for nutrient analyses were taken to the laboratory in
plastic bags and stored at − 20 °C until analyzed. Nutrients
were analyzed by methods routinely used at the Forest Research Institute, Parkano, Finland as described by Halonen
et al. (1983). Total N was measured in oven-dried material by
the Kjeldahl method. Dry-ashed material was used for the
determination of K by flame atomic spectrophotometry
(Varian AA-30) and for the spectrophotometric analysis of P.
Needle samples for polyamine analysis were kept in ice until
taken to the laboratory and stored at − 80 °C. Free polyamines
(putrescine, spermidine and spermine) were extracted from the
needle samples with 5% HClO4, dansylated and analyzed by
HPLC as described by Sarjala and Kaunisto (1993).
Sample preparation for microscopy studies
In August 1992, needles (c = current-year, c + 1 = 1-year-old
and c + 2 = 2-year-old needles) for the microscopy studies were
collected in test tubes containing 0.05 M sodium cacodylate
buffer (pH 7) and 1.5% glutaraldehyde + paraformaldehyde
prefixative. After prefixation, 0.5-mm-thick cross sections
were cut from the middle portion of each needle, postfixed
with OsO4 and embedded in Ladd’s Epon as described by
Reinikainen and Huttunen (1989). An ultramicrotome
(Reichert Jung ULTRACUT E, Vienna, Austria) was used to
cut semi-thin sections (1--3 µm) for the light microscopy
studies and ultra-thin sections for electron microscopy (50--70
nm). The semi-thin sections were stained with toluidine blue
and the ultra-thin sections were stained with lead citrate and
uranyl acetate.
Needle morphology
Cross sections for light microscopy were taken from the middle of needles that were green and had no visible symptoms of
nutrient deficiency. The total number of needles examined was
111, i.e., two to three needles per needle year and eight to nine
needles per tree were observed. The samples were examined
with a Nikon OPTIPHOT-2 light microscope, and morphological measurements were performed with a digital image analyzer (Microscale TM/TC, Digithurst Ltd., Royston, England)
and a video camera (Hitachi KP-C571 CCD color camera).
The needle morphological variables measured were: needle
thickness, needle width, needle area, mesophyll area per needle area (%), epidermis + hypodermis area per needle area (%),
central cylinder area per needle area (%), sclerenchyma cell
wall thickness, phloem area per needle area (%), xylem area
per needle area (%), bundle sheath cell index (radial width of
bundle sheath cell/tangential width of bundle sheath cell measured on the adaxial side of needle cross sections), resin duct
area per needle area (%, measured on the abaxial side of needle
cross sections) and resin duct number per needle area (mm −2)
(Figure 1). Phloem and xylem were observed in one of the two
vascular bundles of the needle. The resin duct area was based
on measurements of two resin ducts from the abaxial side of
the needle. The compression and shrinkage of tissues during
embedding in plastic and sectioning were assumed to be minimal and similar in all samples (Toth 1982). Injured phloem
cells in the vascular bundle were also determined. The swelling
of parenchyma cells and collapse of sieve cells were regarded
as phloem injuries (Fink 1991, Jokela et al. 1995).
In September 1995, current-year needles were collected
from the same trees that were sampled in August and December 1992, and the length, thickness and width of the needles
were measured with a digital caliper. Needles were collected
from three trees from an unfertilized control plot and from
three trees from a plot fertilized with KPO3. Measurements
were made on 50 needles (one needle per fascicle) from current-year branches. Thickness and width were measured in the
middle region of the needle and were, therefore, comparable
with the image analysis measurements of needle thickness and
width made on needle cross sections in August 1992 (Figure 1).
Observations at the ultrastructural level
Ultra-thin sections were examined by the scanning transmission electron microscope (JEM 100CX II, JEOL, Tokyo, Japan). Cell organelles and cytoplasm in mesophyll were
examined in a total of 234 cells of 37 needles. The central
vacuole and cytoplasm in transfusion parenchyma were examined in 34 cells of 12 needles. Altogether, one to two needles
per needle year, and two to six needles per tree were studied.
Statistical analysis
The morphometric data were subjected to an analysis of variance (ANOVA), Kruskall-Wallis test and t-test using the
SPSS-PC software package (Jandel Corp., San Rafael, CA). In
all statistical analyses of the microscopic measurements, a
tree-specific mean was used and four to five trees represented
each K concentration. The polyamine and nutrient concentration data were subjected to linear and nonlinear regression
analyses and variance analysis using the BMDP software
package (University of California Press, Berkeley, CA).
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF POTASSIUM ON SCOTS PINE NEEDLES
679
Figure 1. Morphological
measurements made by image
analysis on cross sections of
Scots pine needles.
Table 1. Effects of needle age and classification by foliar K concentration based on an earlier study (Sarjala and Kaunisto 1993) on concentrations
of K (mg gDW−1), P (mg gDW−1) and N (% DW) and on N/P, N/K and K/P ratios in Scots pine needles sampled from five trees per treatment in
August and December 1992. Different letters within a row indicate significant differences between foliar K concentrations (P = 0.05). Needle age:
c = current-year, c + 1 = 1-year-old, and c + 2 = 2-year-old needles.
Variable
August
K (mg gDW−1)
P (mg gDW−1)
N (% DW)
N/P
N/K
K/P
December
K (mg gDW−1)
P (mg gDW−1)
N (% DW)
N/P
N/K
K/P
Needle
age
High K
Mean ± SD
Intermediate K
Mean ± SD
Low K
Mean ± SD
P
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
5.95 ± 0.79 a
4.73 ± 0.80 a
4.35 ± 0.80 a
1.74 ± 0.35 a
1.39 ± 0.26
1.28 ± 0.23
1.17 ± 0.08 a
1.08 ± 0.06 a
1.08 ± 0.05
6.9 ± 1.2
8.0 ± 1.3
8.4 ± 1.4
2.0 ± 0.3 a
2.3 ± 0.3 a
2.7 ± 0.3 a
3.5 ± 0.8 a
3.5 ± 0.9 a
3.5 ± 0.8 a
4.12 ± 0.47 b
3.33 ± 0.68 b
2.95 ± 0.60 b
1.35 ± 0.10 b
1.17 ± 0.19
1.17 ± 0.16
1.12 ± 0.05 a
1.05 ± 0.09 a
1.07 ± 0.12
8.3 ± 0.5
9.1 ± 1.1
9.2 ± 0.9
2.7 ± 0.3 b
3.2 ± 0.5 b
3.7 ± 0.4 b
3.1 ± 0.4 a
2.9 ± 0.6 a
2.5 ± 0.4 b
2.55 ± 0.30 c
2.41 ± 0.10 c
1.86 ± 0.15 c
1.72 ± 0.19 a
1.43 ± 0.16
1.06 ± 0.24
1.35 ± 0.05 b
1.38 ± 0.13 b
1.24 ± 0.27
7.9 ± 1.0
9.7 ± 1.0
12.3 ± 4.6
5.3 ± 0.9 c
5.7 ± 0.6 c
6.8 ± 1.9 c
1.5 ± 0.2 b
1.7 ± 0.2 b
1.8 ± 0.4 c
0.000
0.000
0.000
0.037
0.145
0.305
0.000
0.000
0.299
0.071
0.100
0.118
0.000
0.000
0.003
0.000
0.002
0.002
c
c+1
c
c+1
c
c+1
c
c+1
c
c+1
c
c+1
5.99 ± 0.85 a
5.55 ± 1.02 a
1.94 ± 0.49 a
1.86 ± 0.43
1.25 ± 0.12 a
1.24 ± 0.07
6.8 ± 1.6
6.9 ± 1.5
2.1 ± 0.3 a
2.3 ± 0.4 a
3.3 ± 0.9 a
3.1 ± 0.9 a
4.44 ± 0.35 b
3.95 ± 0.40 b
1.44 ± 0.17 b
1.36 ± 0.24
1.18 ± 0.05 a
1.35 ± 0.47
8.2 ± 0.8
10.5 ± 5.3
2.7 ± 0.2 b
3.5 ± 1.7 ab
3.1 ± 0.4 a
3.0 ± 0.5 a
3.04 ± 0.36 c
3.09 ± 0.23 c
1.91 ± 0.12 a
1.70 ± 0.16
1.40 ± 0.07 b
1.42 ± 0.07
7.4 ± 0.8
8.4 ± 1.1
4.6 ± 0.5 c
4.6 ± 0.4 b
1.6 ± 0.3 b
1.8 ± 0.3 b
0.000
0.000
0.047
0.056
0.004
0.593
0.406
0.422
0.000
0.014
0.001
0.014
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
680
JOKELA ET AL.
K/P ratios indicated a K deficiency in needles of trees in the
low foliar K class.
For both the August and December samplings, K and P
concentrations decreased with increasing needle age. Both K
and P concentrations were higher in December than in August
for all needle age classes and for all foliar K classes.
Results
Nutrient concentrations
Trees classified on the basis of an earlier study (Sarjala and
Kaunisto 1993) as having high, intermediate or low foliar K
concentrations fell into the same categories on the basis of
measurements made in the present study (Table 1). In trees in
the intermediate foliar K class, most macronutrients were
present in optimal concentrations, with the exception that P
concentration and the N/K ratio were well below optimum
values (Table 1). Optimum P concentrations were present in
needles in trees of the high and low foliar K classes. Foliar N
concentrations were below the deficiency limit in trees of the
intermediate and high foliar K classes (N, N/P and N/K,
Table 1), whereas the K/P ratio was in balance. The N/K and
Needle morphology
Foliar K concentration had only a slight effect on needle
morphology. Needle thickness, width (Tables 2 and 3) and
length (Table 3) were not significantly affected by foliar K
concentration. Needles with a low K concentration had the
largest needle area, whereas needles with a high K concentration had the largest relative area of mesophyll and the smallest
central cylinder area (Table 2). The sclerenchyma cell walls
were thinnest in needles with a low K concentration (Fig-
Table 2. Light microscopic image analysis measurements of needle morphological variables. Different letters within a row indicate significant
differences between the foliar K concentration classes (P = 0.05). Needles were collected in August 1992. Needle age: c = current-year, c + 1 =
1-year-old, and c + 2 = 2-year-old needles; and n = number of trees observed.
Variable
Needle thickness
(mm)
Needle width (mm)
Needle area (mm2)
Mesophyll area (%)
Epidermis + hypodermis area (%)
Central cylinder
area (%)
Sclerenchyma cell
wall thickness (µm)
Phloem area (%)
Xylem area (%)
Bundle sheath
cell index
Resin duct area (%)
Resin duct
number mm −2
Intermediate K
P
Needle
High K
age
n
Mean ± SD
n
Mean ± SD
n
Mean ± SD
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
0.781 ± 0.033
0.801 ± 0.069
0.780 ± 0.066
1.671 ± 0.069
1.690 ± 0.104
1.647 ± 0.182
1.071 ± 0.870
1.105 ± 0.139
1.122 ± 0.205
57.59 ± 2.59
59.65 ± 2.03
58.20 ± 2.29
12.68 ± 1.59
10.95 ± 1.06
12.11 ± 1.25
29.72 ± 1.85
29.40 ± 2.16
29.69 ± 2.02
6.02 ± 0.58
5.73 ± 0.66
5.73 ± 0.71 a
0.54 ± 0.09
0.76 ± 0.03
0.90 ± 0.15
0.60 ± 0.01
0.60 ± 0.11
0.62 ± 0.09
0.57 ± 0.10
0.62 ± 0.03
0.73 ± 0.04
0.91 ± 0.17
0.76 ± 0.12
0.72 ± 0.13
9.69 ± 1.26
8.86 ± 0.83
8.23 ± 0.53
4
5
5
5
5
5
4
5
5
4
5
5
4
5
5
4
5
5
4
5
5
5
5
5
5
5
5
4
5
5
5
5
5
5
5
5
0.811 ± 0.064
0.772 ± 0.017
0.774 ± 0.026
1.705 ± 0.161
1.540 ± 0.420
1.681 ± 0.149
1.110 ± 0.153
0.976 ± 0.248
1.082 ± 0.086
56.63 ± 1.27
57.57 ± 2.71
57.96 ± 3.39
11.41 ± 0.53
10.79 ± 1.44
11.33 ± 0.57
31.96 ± 1.43
31.64 ± 2.00
30.71 ± 2.98
5.66 ± 0.91
5.12 ± 0.49
4.68 ± 0.42 b
0.60 ± 0.06
0.73 ± 0.02
0.90 ± 0.18
0.58 ± 0.05
0.69 ± 0.31
0.60 ± 0.07
0.58 ± 0.10
0.66 ± 0.08
0.76 ± 0.24
0.99 ± 0.17
1.05 ± 0.33
0.76 ± 0.12
9.04 ± 2.08
8.94 ± 4.24
7.41 ± 1.79
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
0.812 ± 0.066
0.802 ± 0.093
0.808 ± 0.104
1.766 ± 0.189
1.824 ± 0.238
1.658 ± 0.357
1.210 ± 0.268
1.203 ± 0.262
1.149 ± 0.368
57.12 ± 2.11
56.68 ± 1.58
57.63 ± 1.29
11.95 ± 1.58
11.78 ± 1.29
11.83 ± 1.32
30.93 ± 3.28
31.58 ± 2.56
30.55 ± 1.43
6.44 ± 1.22
5.61 ± 0.88
4.63 ± 0.67 b
0.62 ± 0.01
0.70 ± 0.01
0.80 ± 0.11
0.67 ± 0.01
0.66 ± 0.16
0.66 ± 0.13
0.56 ± 0.08
0.60 ± 0.04
0.72 ± 0.08
0.87 ± 0.11
0.73 ± 0.18
0.84 ± 0.28
8.19 ± 1.15
7.92 ± 0.56
7.34 ± 1.83
TREE PHYSIOLOGY VOLUME 17, 1997
Low K
0.622
0.878
0.613
0.600
0.651
0.977
0.491
0.445
0.913
0.677
0.075
0.763
0.432
0.326
0.566
0.298
0.310
0.733
0.359
0.364
0.026
0.566
0.878
0.468
0.275
0.878
0.733
0.650
0.185
0.651
0.185
0.114
0.810
0.249
0.196
0.566
EFFECTS OF POTASSIUM ON SCOTS PINE NEEDLES
Table 3. Leaf morphological variables (measured with a digital caliper) and potassium (K) and putrescine concentrations of current-year
needles collected in September 1995. Fifty needles per tree were
measured on three trees per plot.
Variable
High K
Mean ± SD
Low K
Mean ± SD
P
(t-test)
37.03 ± 9.23
42.78 ± 4.64
0.389
Needle thickness
(mm)
0.67 ± 0.02
0.65 ± 0.04
0.426
Needle width (mm)
1.46 ± 0.02
1.50 ± 0.10
0.562
5.58 ± 0.10
2.37 ± 0.33
0.002
150.26 ± 52.77
2392.74 ± 1568.10
0.131
Needle length (mm)
K mg gDW
−1
Putrescine
nmol gFW−1
ure 2a) and thickest in 2-year-old needles with a high K concentration (Figure 2b; P = 0.026, Table 2).
The phloem area was greater in 1- and 2-year-old needles
than in current-year needles, and the difference was significant
in needles with a high K (P = 0.025) or an intermediate K
concentration (P = 0.022) but not in needles with a low K
681
concentration (P = 0.098). In contrast, needle age and foliar K
concentration had few significant effects on xylem area, although xylem area was larger in needles with a low K concentration than in needles with an intermediate or a high K
concentration. When calculated in relation to the central cylinder area, the phloem and xylem areas showed the same relationship as for whole needle area (data not shown). There was
no relationship between phloem cell injury and foliar K concentration.
The bundle sheath cell index was smallest (radial to tangential width of cell was smaller) in needles with a low K concentration and was independent of needle age (Table 2). In needles
with a low K concentration, the bundle sheath cells were
collapsed (Figure 2c), whereas the bundle sheath cells were
normal in shape in needles with a high K concentration (Figure 2d). The resin duct area was smallest in current-year and
1-year-old needles with a low K concentration, and the number
of resin ducts was smallest in needles with a low K concentration and was independent of needle age.
Observations at ultrastructural level
Ultrastructural observations of the mesophyll (Table 4) revealed an enlarged central vacuole in current-year and 1- and
Figure 2. Photomicrographs of cross sections of Scots pine needles. (a) Thin sclerenchyma cell walls (S) in a cross section of a 2-year-old needle
with a low K concentration. Bar = 0.1 mm. (b) Normal sclerenchyma cell walls (S) in a cross section of a 2-year-old needle with a high K
concentration. Bar = 0.1 mm. (c) Collapsed bundle sheath cells (arrows) in a cross section of a 1-year-old needle with a low K concentration.
Bar = 0.02 mm. (d) Normal-shaped bundle sheath cells (arrows) in a cross section of a 1-year-old needle with an intermediate K concentration.
Bar = 0.02 mm. Abbreviations: m = mesophyll, p = phloem, r = resin duct, t = transfusion tissue, and x = xylem.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
682
JOKELA ET AL.
Table 4. Effect of foliar K concentration class on ultrastructure of needles collected in August 1992. Needle age: c = current-year, c + 1 = 1-year-old,
and c + 2 = 2-year-old needles.
Variable
High K
Intermediate K
Low K
c
c+1
c+2
c
c+1
c+2
c
c+1
c+2
MESOPHYLL
Central vacuole
Enlarged central vacuole
Proliferation of tonoplast
--1
+
-+
+2
+
-+
-++3
+
+
+
--
+
+
+
+
Chloroplast
Swelling of thylakoids
+
+
--
+
+
+
++
+
+
Cytoplasm
Lipid accumulations
Small vacuoles with
electron-dense material
Extensive vesiculation
+
--
++
+
++
+
+
--
+
--
+
--
+
--
++
++
++
+
--
--
+
--
--
--
--
+
+
Mitochondria
Swelling of mitochondria
--
--
+
--
--
--
--
--
+
TRANSFUSION PARENCHYMA
Central vacuole
Enlarged central vacuole
--
+
--
--
--
+
--
+
--
Cytoplasm
Lipid accumulations
--
+
--
--
--
--
--
+
+
1
2
3
-- = Not observed.
+ = Occasionally observed.
++ = Frequently observed.
Figure 3. Ultrastructure of mesophyll cells (a--c) of 1-year-old
needles with a low K concentration and of a transfusion parenchyma cell (d) of a 2-year-old
needle with a low K concentration. (a) Large central vacuole
(cv) and an intensive accumulation of small vacuoles with darkstained material in the cytoplasm
(arrow). Bar = 1 µm. (b) Same
cell as in (a) showing vesiculated appearance of the cytoplasm, arrow = a small vacuole.
Bar = 1 µm. (c) Lipid accumulations (L) adjacent to the
tonoplast, which is located between lipids and dark-stained
tannin accumulations (T). Chloroplast (c) with starch grain (s)
and an intercellular space (I).
Bar = 1 µm. (d) Transfusion parenchyma cell with many lipid
accumulations (L), cv = central
vacuole, s = starch grain in chloroplast. Bar = 2 µm. Abbreviations: c = chloroplast, m =
mitochondria, w = cell wall.
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF POTASSIUM ON SCOTS PINE NEEDLES
2-year-old needles with a low K concentration. Proliferation of
the tonoplast was slightly less frequent in needles with a low
K concentration than in needles with an intermediate or high
K concentration. The formation of small vacuoles with electron-dense material in mesophyll cells (Figure 3a) was observed in needles with a low or high K concentration, but not
in needles with an intermediate K concentration. Sometimes,
these small vacuoles gave the cell a vesiculated appearance
(extensive vesiculation in Table 4) (Figure 3b).
The swelling of chloroplast thylakoids and occurrence of
lipid accumulations in the cytoplasm were abundant and independent of foliar K concentration. Lipid accumulations were
often adjacent to the tonoplast (Figure 3c). Slight mitochondrial swelling was observed in the 2-year-old needles with a
low or high K concentration. Cytoplasmic lipid accumulations
in the transfusion parenchyma occurred slightly more frequently in needles with a low K concentration than in needles
with an intermediate or high K concentration (Figure 3d),
whereas an enlarged central vacuole in transfusion parenchyma cells was observed at all foliar K concentrations.
Free polyamine concentrations
Polyamine analysis of current-year and 1- and 2-year-old
needles of the 25 selected trees indicated a negative correlation
between putrescine and K concentrations that declined with
needle age (regression analysis: current-year needles
y = 22191 x−3.20 and r 2 = 0.622, 1-year-old needles
y = 138019 x− 5.33 and r 2 = 0.554, 2-year-old needles
y = 9154 x− 2.97 and r 2 = 0.313; x = K concentration and y = putrescine concentration). In 1992, putrescine concentrations in
needles with a low K concentration were significantly higher
Figure 4. Putrescine, spermidine, and spermine concentrations in
current-year and 1- and 2-year-old needles in August and December
1992. Statistically significant differences between foliar K concentrations are indicated by different letters.
683
than putrescine concentrations in needles with an intermediate
or high K concentration (Figure 4). Spermidine and spermine
concentrations were lower in needles with a low K concentration than in needles with an intermediate or high K concentration (Figure 4). The changes in polyamine concentration in
response to foliar K concentration were independent of needle
age and were observed in both August and December, except
for spermine concentration, which was lower in December
than in August for all needle age classes. In September 1995,
putrescine concentration in needles with a low K concentration
was higher than in needles with a high K concentration (Table 3), but the difference was not statistically significant.
Discussion
Foliar K concentration had only slight effects on needle length,
thickness and width. The higher proportion of the mesophyll
area in needles with a high K concentration may indicate that
these needles had a high photosynthetic potential, which in
turn may have affected tree growth. Potassium fertilization has
been shown to increase the volume growth and basal area of
Scots pine growing on peatlands (Kaunisto 1989). Moreover,
Baillon et al. (1988) reported that photosynthetic rates were
lower in K-deficient Norway spruce seedlings than in seedlings receiving an adequate supply of K; however, in Scots pine
seedlings, photosynthetic rate was not inhibited until foliar K
concentration was reduced to 2.4 mg gDW−1 (Nygren and Hari
1992). Neither photosynthesis nor tree growth was measured
in our study.
The thin cell walls of the sclerenchyma tissue in 2-year-old
needles with a low K concentration may indicate injury similar
to that found in needles deficient in boron, potassium or phosphorus (Raitio 1979, 1981). The development of thin sclerenchyma cell walls may be a response to nutrient imbalance
caused by an excess N supply (Jokela et al. 1995).
The enhanced formation of secondary phloem in needles
with an intermediate or high K concentration compared to
needles with a low K concentration may have been a result of
enhanced photosynthesis caused by K fertilization (Dünisch
and Bauch 1994). Furthermore, Hartt (1969) showed that K
has an important role in photoassimilate transport in the
phloem of sugar cane. The formation of secondary phloem but
not secondary xylem in Scots pine needles is in agreement with
the findings of Ewers (1982), who reported that cambium in
the vascular bundle produces secondary phloem but not secondary xylem in several conifer species including Scots pine.
The finding that the xylem area was slightly larger in current-year needles with a low K concentration than in currentyear needles with an intermediate or a high K concentration is
of interest because polyamine transport takes place in the
xylem (Bagni and Pistocchi 1991). Putrescine enhances xylogenesis of xylem cells in Helianthus tuber (Phillips et al. 1988)
and it may influence cambial activity in gymnosperms. For
example, in the stem cambial zone of Norway spruce, putrescine is the most abundant polyamine during the period of
greatest cambial activity in spring and summer (Königshofer
1991).
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
684
JOKELA ET AL.
Although phloem injuries have often been attributed to nutrient imbalance (Fink 1991, 1993, Jokela et al. 1995, 1996),
we found no effect of foliar K concentration on phloem cell
integrity. The slightly collapsed bundle sheath cells of needles
with a low K concentration may be related to nutrient stress in
general or K deficiency in particular. Collapsed bundle sheath
cells have been associated with the accumulation of secondary
substances, and modifications in the bundle sheath structure
have been found in response to fumigation with SO2 and O3
(Maier-Maercker and Koch 1992). The decrease in size and
number of resin ducts with decreasing foliar K concentration
may indicate that the terpene-based defensive mechanism is
compromised in trees suffering from K deficiency (Burr and
Clancy 1992).
In our study, foliar K concentration influenced the size of the
central vacuole of mesophyll cells but had no effect on the
occurrence of tonoplast injuries or lipid accumulation. Lipid
accumulations were frequently observed but they were independent of foliar K concentration. They were often located
adjacent to the tonoplast, and may indicate tonoplast disintegration (Zwiazek and Shay 1987) or cold hardening during late
summer (Holopainen et al. 1992). Holopainen and Nygren
(1989) observed that K deficiency causes an extension of the
vacuolar system in mesophyll cells, fragmentation of the
tonoplast and an increase in cytoplasmic lipids in needles of
Scots pine seedlings. According to Holopainen and Nygren
(1989), small vacuoles with tannin deposits are associated with
K deficiency. We observed many small vacuoles with electrondense material that gave the cytoplasm a vesiculated appearance in needles with a low K concentration. We conclude that
the vacuole system may be particularly sensitive to K deficiency, because K is important in osmoregulation (Lüttge and
Clarkson 1989).
Changes in the vacuolar system could also be explained by
changes in the polyamine concentration of K-deficient needles. We observed increased concentrations of putrescine and
decreased concentrations of spermidine and spermine in needles with a low K concentration (cf. Sarjala and Kaunisto 1993).
Flores (1991) suggested that the accumulation of putrescine,
possibly in the vacuole (Pistocchi et al. 1988), compensates for
the decrease in pH of cells of K-deficient plants.
Changes in Scots pine needle structure caused by K deficiency differed from those caused by P (injuries to mitochondrial structure) and N deficiencies (changes in the cytoplasm
and chloroplast structure) (Palomäki and Holopainen 1994,
1995). Interpretation of the symptoms of K deficiency is complicated because of the effects of foliar K concentration on the
balance of the other macronutrients. For example, needle N
was at an adequate concentration for growth in needles with a
low K concentration, whereas needles with an intermediate or
high K concentration were deficient in N (cf. Paarlahti et al.
1971, Kaunisto 1987). Differences between nutrient interactions in Scots pine trees growing in the field and those that
occur in seedlings growing under controlled environment conditions may account for the differences between our observations and those reported by Holopainen and Nygren (1989).
We conclude that foliar K concentration has a small effect
on needle morphology. Needles with a low K concentration
were characterized by thinner sclerenchyma cell walls, a
slightly larger xylem area, less secondary growth of phloem,
smaller resin ducts and more collapsed bundle sheath cells than
needles classified as containing an intermediate or high K
concentration. A large accumulation of putrescine occurred in
needles with a low K concentration and was accompanied by
the development of a vacuolar system in mesophyll cells.
Acknowledgments
This research was supported by the Kone Foundation and the Finnish
Research Program on Climate Change. Dr. Jaana Bäck and Dr. Sagar
V. Krupa are thanked for their valuable comments on the manuscript
as are Mrs. Tellervo Siltakoski, Mrs. Mervi Saaranen and M.Sc. Eija
Kukkola for technical assistance. The language of the manuscript was
revised by Mrs. Leena Kaunisto.
References
Bagni, N. and R. Pistocchi. 1991. Uptake and transport of polyamines
and inhibitors of polyamine metabolism in plants. In Biochemistry
and Physiology of Polyamines in Plants. Eds. R.D. Slocum and
H.E. Flores. CRC Press, Boca Raton, FL, pp 105--120.
Baillon, F., X. Dalschaert, S. Grassi and F. Gaiss. 1988. Spruce
photosynthesis: possibility of early damage diagnosis due to exposure to magnesium or potassium deficiency. Trees 2:173--179.
Burr, K.E. and K.M. Clancy. 1992. Douglas-fir needle anatomy in
relation to western spruce budworm (Lepidoptera: Tortricidae).
J. Econ. Entomol. 86:93--99.
Dünisch, O. and J. Bauch. 1994. Influence of mineral elements on
wood formation of old growth spruce (Picea abies (L.) Karst.).
Holzforschung 48 Suppl:5--14.
Ewers, F.W. 1982. Secondary growth in needle leaves of Pinus longaeva (bristlecone pine) and other conifers: quantitative data. Am.
J. Bot. 69:1552--1559.
Fink, S. 1991. Structural changes in conifer needles due to Mg and K
deficiency. Fert. Res. 27:23--27.
Fink, S. 1993. Microscopic criteria for the diagnosis of abiotic injuries
to conifer needles. In Forest Decline in Atlantic and Pacific Regions. Eds. R.F. Hüttl and D. Müller-Dombois. Springer-Verlag,
Berlin, pp 175--188.
Flores, H.E. 1991. Changes in polyamine metabolism in response to
abiotic stress. In Biochemistry and Physiology of Polyamines in
Plants. Eds. R.D. Slocum and H.E. Flores. CRC Press, Boca Raton,
FL, pp 213--228.
Halonen, O., H. Tulkki and J. Derome. 1983. Nutrient analysis methods. Metsäntutkimuslaitoksen tiedonantoja 121. Valtion painatuskeskus, Helsinki, 28 p.
Hartt, C.E. 1969. Effects of potassium deficiency upon translocation
of 14C in attached blades and entire plants of sugarcane. Plant
Physiol. 44:1461--1469.
Holopainen, T. and P. Nygren. 1989. Effects of potassium deficiency
and simulated acid rain, alone and in combination, on the ultrastructure of Scots pine needles. Can. J. For. Res. 19:1402--1411.
Holopainen, T., S. Anttonen, A. Wulff, V. Palomäki and L. Kärenlampi.
1992. Comparative evaluation of the effects of gaseous pollutants,
acidic deposition and mineral deficiencies: structural changes in the
cells of forest plants. Agric. Ecosyst. Environ. 42:365--398.
TREE PHYSIOLOGY VOLUME 17, 1997
EFFECTS OF POTASSIUM ON SCOTS PINE NEEDLES
Jokela, A., J. Bäck, S. Huttunen and R. Jalkanen. 1995. Excess nitrogen fertilization and the structure of Scots pine needles. Eur. J. For.
Pathol. 25:109--124.
Jokela, A., V. Palomäki, S. Huttunen and R. Jalkanen. 1996. Effects of
root damage on the nutritional status and structure of Scots pine
needles. J. Plant Physiol. 148:317--323.
Kaunisto, S. 1987. Effect of refertilization on the development and
foliar nutrient contents of young Scots pine stands on drained mires
of different nitrogen status. Commun. Inst. For. Fenn. 140, 58 p.
Kaunisto, S. 1989. Jatkolannoituksen vaikutus puuston kasvuun
vanhalla ojitusalueella. Summary: Effect of refertilization on tree
growth in an old drainage area. Folia For. 724:1--15.
Königshofer, H. 1991. Distribution and seasonal variation of polyamines in shoot-axes of spruce (Picea abies (L.) Karst.). J. Plant
Physiol. 137:607--612.
Lüttge, U. and D.T. Clarkson. 1989. Mineral nutrition: Potassium.
Prog. Bot. 50:51--73.
Maier-Maercker, U. and W. Koch. 1992. Histological examination of
spruce needles from a long-term gas exchange experiment in pure
and polluted air in the field. Trees 6:186--194.
Marschner, H. 1995. Mineral nutrition of higher plants. Academic
Press, New York, 889 p.
Mengel, K. and W.W. Arneke. 1982. Effect of potassium on the water
potential, the osmotic potential, and cell elongation in leaves of
Phaseolus vulgaris. Physiol. Plant. 54:402--408.
Nygren, P. and P. Hari. 1992. Effect of foliar application with acid mist
on the photosynthesis of potassium-deficient Scots pine seedlings.
Silva Fenn. 26:133--144.
Paarlahti, K., A. Reinikainen and H. Veijalainen. 1971. Nutritional
diagnosis of Scots pine by needle and peat analysis. Commun. Inst.
For. Fenn. 74, 58 p.
685
Palomäki, V. and T. Holopainen. 1994. Effects of phosphorus deficiency and recovery fertilization on growth, mineral concentration,
and ultrastructure of Scots pine needles. Can. J. For. Res. 24:2459-2468.
Palomäki, V. and T. Holopainen. 1995. Effects of nitrogen deficiency
and recovery fertilization on ultrastructure, growth and mineral
concentrations of Scots pine needles. Can. J. For. Res. 25:198--207.
Phillips, R., M.C. Press, L. Bingham and G. Grimmer. 1988. Polyamines in cultured artichoke explants: effects are primarily on
xylogenesis rather than cell division. J. Exp. Bot. 39:473--480.
Pistocchi, R., F. Keller, N. Bagni and P. Matile. 1988. Transport and
subcellular localization of polyamines in carrot protoplasts and
vacuoles. Plant Physiol. 87:514--518.
Raitio, H. 1979. Growth disturbances of Scots pine caused by boron
deficiency on an afforested abandoned peatland field. Folia For.
412:1--16.
Raitio, H. 1981. Effects of macronutrient fertilization on the structure
and nutrient content of pine needles on a drained short sedge bog.
Folia For. 465:1--9.
Reinikainen, J. and S. Huttunen. 1989. The level of injury and needle
ultrastructure of acid rain-irrigated pine and spruce seedlings after
low temperature treatment. New Phytol. 112:29--39.
Sarjala, T. and S. Kaunisto. 1993. Needle polyamine concentrations
and potassium nutrition in Scots pine. Tree Physiol. 13:87--96.
Toth, R. 1982. An introduction to morphometric cytology and its
application to botanical research. Am. J. Bot. 69:1694--1706.
Zwiazek, J. and J. Shay. 1987. Fluoride- and drought-induced structural alterations of mesophyll and guard cells in cotyledons of jack
pine (Pinus banksiana). Can. J. Bot. 65:2310--2317.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com