Trees (1995) 9: 134- 142

�9 Springer-Verlag 1995

Aluminium causes nutrient imbalance and structural changes in the needles of Scots pine without inducing clear root injuries

Sari Janhunen, Virpi Palomiiki, Toini Holopainen

Ecological Laboratory, Department of Environmental Sciences, University of Kuopio. P.O.Box 1627, FIN-70211 Kuopio, Finland

Received: 27 January 1994/Accepted: 16 May 1994

Abstract. The effects of aluminium chloride (AICI3) treatments (50 and 150 mg/l) on 3-year-old Scots pine (Pinus sylvestris L.) seedlings were studied in a sand culture during 2 growing periods in an open field experi- ment. Even by the end of the first growing period, a decline was observed in the concentrations of Ca, Mg and P within the needles, and of Ca and Mg in the roots. After the second growing period, increased N and K concentrations were observed in the needles of Al-treated seedlings. Both the needles and roots of Al-treated seedlings showed, after the second growing period, a decline in growth and increased concentrations of AI as the amount of AICI3 in the nutrient solution increased. Al-induced changes in needle structure were found to be symptomatic of a nutrient imbalance, particularly of Mg and P. Al-stress did not result in any observable changes in root anatomy or in the number of mycorrhizas. Scots pine proved to be rather resistant to Al-stress, indicating that direct Al-injuries are not likely in the field, though Al-stress may be a contributing factor in the formation of nutrient imbalances.

Key words: Pinus sylvestris L. - Aluminium - Nutrients - Mycorrhiza - Ultrastructure

Introduction

Many physiological disorders caused by aluminium in different plants are well established in the literature. One of the most obvious symptoms is reduction in root growth (Taylor 1988; Rengel 1992). Common shoot responses to increased levels of AI include decreased shoot biomass, and reduced stem and leaf production, as well as delayed budbreak and leaf expansion (Cronan et al. 1989). How- ever, tissue mineral concentration may be more sensitive to AI than are growth indices (Cronan et al. 1989), since many

Correspondence to: S. Janhunen

studies indicate early decreases in concentrations of diva- lent cations in various plant species (Ca and Mg) after A1 treatment (Roy et al. 1988; Godbold 1991; Rengel 1992). However, changes in other tissue mineral concentrations are not as pronounced. N and K concentrations may either increase or decrease (Roy et al. 1988), and P concentrations have also shown both increased (Entry et al. 1987; Vogelei and Rothe 1988) and decreased (Foy 1974) levels in response to AI treatment.

The symptoms of AI toxicity in tree roots are well known, including reduced elongation and branching, a generalized thickening of the roots, and necrosis of cells near the root meristems (Cronan et al. 1989). Reports on the role of mycorrhizas in the response of trees to AI treatment have often been contradictory, since the role may vary with respect to different ectomycorrhizal fungi (Thompson and Medve 1984). Cumming and Weinstein (1990a-c) and Wilkins and Hodson (1989) reported that mycorrhizal infection in Pinus rigida and Picea abies prevented AI toxicity; however, Jentschke et al. (1991) did not find beneficial effects of mycorrhizas in preventing AI toxicity in spruce [Picea abies (L.) Karst.l seedlings.

Some studies have also focused on the cellular changes in trees under Al-stress. In red spruce (Picea rubens Sarg.) roots, AI can induce premature vacuolation, accumulation of phenolic-like material, loss of cells from peripheral cell layers, formation of intercellular spaces, increased disrup- tion of cellular membranes, degeneration of the cytoplasm (McQuattie and Schier 1990) and callose formation in the root tips of Picea abies (Jorns et al. 1991 ). Correspondingly, examination of the Pinus rigida roots (McQuattie and Schier 1992) showed that aluminum caused deterioration and increased vacuolation. At higher AI levels (50 mg AI/I), intracellular fungal hyphae and bacteria were also observed. However, in a study of the Scots pine (Pinus sylvestris L.), Metzler and Oberwinkler (1987) found very few meriste- matic abortions in the root tips at low pH (3.0). Structural changes related to air pollution in conifer needles have also been extensively studied (Sutinen 1987; Fink 1988, 1989; Holopainen et al. 1992), but little attention has until recently focused on needle structure under Al-stress. McQuattie and

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S c h i e r (1993) f o u n d a d v a n c e d ce l lu la r d i s i n t e g r a t i o n in the s te la r and i n n e r m e s o p h y l l t i s sues o f Pinus rigida need les . T h e r e f o r e , the a im of th is s t udy w as to d e t e r m i n e h o w the e x p e c t e d n u t r i e n t i m b a l a n c e s w o u l d a f fec t s h o o t d e v e l o p - m e n t and n e e d l e u l t r as t ruc tu re . A n o t h e r i m p o r t a n t a i m was to s tudy w h e t h e r the s t ruc tura l c h a n g e s c a u s e d by k n o w n A1 s t ress in the roo t s and m y c o r r h i z a s o f Pinus sylvestris are s imi l a r to those p r e v i o u s l y f o u n d in the f ine roots o f o t h e r c o n i f e r o u s t ree spec ies .

Materials and methods

Plant material and experimental treatments

Three-year-old nursery grown Scots pine (Pinus sylvestris L.) seed- lings (total number 55) originated from central Finland, were planted on 10 May 1990 in 7.5-1 plastic pots containing quartz sand. Seedlings were grown in an open field at the botanical garden of the University of Kuopio (62 ~ 53'N, 27 ~ 37'E) during 2 growing periods in 1990 and 1991. The surface of the field was coated with a black plastic sheet and the pots were placed on grates laying on the plastic to prevent contact with the soil. The growth medium was protected from rain by plastic covers placed over the pots.

Seedlings were divided into four study groups: normal and acid controls and two Al-treatments (A1-50 and Al-150). Each treatment was watered with an optimum nutrient solution planned for Scots pine (Ingestad 1979) containing constant levels of N-P-K-Ca-Mg/ 1 0 0 - 1 4 - 4 0 - 6 - 6 (50 ppm N). The pH of the normal control treatment was maintained at about 4.9 and that of the acid-control and Al-treatments were adjusted between 3.5 and 3.8. Aluminium, in the form of A1C13, was added to the nutrient solution at concentrations of 50 mg AN and 150 mg A1/1 when the Ca/AI molar ratios were 0.041 and 0.014, respectively. Seedlings were watered (0.15 1 nutrition solution/each time) according to weather conditions in order to keep the sand culture moist from 28 May to 11 October 1990 (amount of nitrogen added 122 kg/ha) and from 7 May to 30 August 1991 (amount of nitrogen added 101 kg/ha). During the first growing period the seedlings needed more watering with nutrient solution (69 times in the first and 50 times in the second growing period), since the weather conditions were drier than during the second summer. At the end of the first growing period (23 October 1990), five seedlings from each treatment were sampled, with the remainder of the seedlings being sampled on 3 September 1991. The latter series of seedlings were overwintered in the open field covered by excised spruce branches and snow.

Visual observations and growth parameters

and counted after 10-15 min staining in Ponceau S (Daughtridge et al. 1986), and the total number of mycorrhizas and short roots per 100 cm were determined. In addition to mycorrhizas, all abnormally swollen and branched long root tips and the total number of long root tips were counted. All observations were made from the whole sample of about 200 cm consisting of 1 - 8 cm long root pieces collected randomly throughout the root system. Roots were then oven dried at +85 ~ C, the corresponding dry weights were measured, and the roots were processed for chemical analyses.

Ultrastructure. For the ultrastructural study the samples of needles from the middle part of the leading shoot were collected three times: 15 October 1990, 5 August 1991 and 2 September 1991. Five to ten current and previous-year needles representing different seedlings were collected from each treatment. Needles were placed in a 2% glutaraldehyde fixative in 0.1 M (0.05 M) phosphate buffer (pH 7; Soikkeli 1980). A 1-mm piece of the lower part of the apical third of each needle was cut under a fixative drop. The samples from the mycorrhizal rootlets were collected and placed in a 2.5% glutaralde- hyde fixative from several different seedlings of each treatment before the roots were frozen.

The samples of needles and rootlets were prefixed in 2% and 2.5% glutaraldehyde, respectively, postfixed in buffered 1% OsO4 solution, dehydrated in a graded ethanol series, infiltrated and finally embedded in Ladd's LX 112 resin (Soikkeli 1980;" Holopainen and Heinonen- Tanski 1993). Semitbin sections (about 1 - 2 gm), cross sections of needles and longitudinal sections of short roots, were prepared for light microscopy (Zeiss, Standard microscope 16) and stained with toluidine blue. Thin sections from needles and roots were cut with a diamond knife for transmission electron microscopy (JEOL 1200 S) and stained on grids with uranyl acetate and lead citrate. Replicate samples from both current and previous-year needles (n = 5 - 6 ) and from roots (n = 4) representing each treatment were examined. In addition to ultrastruc- tural observations the length of chloroplasts and starch grains and number of plastoglobules in the cross section of chloroplasts were measured from pictures taken at a magnification of 10000 from randomly selected mesophyll cells. At the end of the experiment the number of thylakoids in granum stacks were counted and the ratio of chloroplast length to width was determined.

Chemical analyses

For analyses of R Ca, Mg and K from roots and needles (0.1 g samples) sulphuric acid-hydrogen peroxide digestion (Allen 1989) was used, and for nitrogen analyses Kjeldahl digestion (Allen 1989) was used. Calcium, magnesium and potassium were determined by AAS (Perkin-Elmer 460) and phosphorus by the molybdenum blue method (Allen 1989). The aluminium content of the roots and needles were analysed at the Finnish Forest Research Institute at Vantaa by plasma- emission spectrophotometry (ICP) from ashed and HCl-dissolved material.

Shoots. The external condition of the seedlings, especially the colour of the needles and germination of the buds, were assessed during the 1991 growing period.

The total shoot length, current-year leading shoot length and, in 1991, the previous-year leading shoot length were measured. The number of current- and previous-year needles in the leading shoot were counted and the average length of the current-year needles (12 per shoot) were measured. The shoots were oven dried at +60 ~ C, the corresponding dry weights of the present needle generations and the total shoot weight were measured, and samples were prepared for chemical analyses.

Roots. The roots were thoroughly cleaned with tap water on sieves and thereafter stored at - 2 4 ~ C until analy sis. Root morphology, the colour of the roots, the percentage of dead roots (%), the branching density and curling of roots and abnormalities at the surface were assessed as the roots were thawing. The mycorrhizal short root tips were identified

Statistical methods

The data were subjected to 1-way ANOVA and the treatment means were compared using Tukey's Multiple Range Test (SPSS-PC pro- grams).

Results

Visual observations and growth parameters

A t the b e g i n n i n g o f the s e c o n d s u m m e r f l u s h i n g o f the b u d s d id no t d i f fe r s ign i f i can t ly b e t w e e n t r e a tmen t s , w i th all s eed l ings s ta r t ing to g row no rma l ly . H o w e v e r , d u r i n g the

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Table 1. Current year and whole leader shoot lengths, root/shoot ratios (dry weight), needle densities in current and previous year shoots and mean needle lengths in current year shoots in the growing period 1991. Means + SD (n = 4-11)

Shoot length Shoot length Root/shoot ratio Needle density Needle density Mean needle current whole current previous length

Control 18.1 _+3.7b~ 45.6_+ 8.lab 0.51 • 11.0+ 1.2a 24.4+4.9b 4.9• Acid-control 19.3 • 3.6b 54.5 + 6.4b 0.55 + 0.07a 14.1 _+ 2.6a 19.8 + 2.6ab 3.8 + 0,5a

AI-50 15.4 + 5.0b 43.8 + 10.6b 0.44 + 0.07a 12.8 + 3.0a 17.1 • 3.2a 3.9 _+ 0.5a A1-150 9.4 • 4.3a 32.3 + 8.1 a 0.43 _ 0.09a 20,6 • 6.3b 23.5 ___ 6.6b 3.1 + 0.9a F 6.996 7.316 2.615 7.804 4.083 12.954 P 0.002 0.001 0.076 0.001 0.017 0,000

a Means followed by the same letter are not significantly different (P <0.05) according to Tukey's Multiple Range test

Table 2. Nutrient and aluminium concentrations (means + SD, n = 4 - 5 ) in current and previous year needles and roots after the growing period 1990 (rag/g)

N K P Ca Mg A1

Current year needles Control 16.05 + 1.92aa 8.96 _+ 1.85a 1.69 + 0.14b 2.04 ___ 0.46b 1.14 + 0.14c 0.17 + 0.06a Acid-control 17.48 • 1.08a 8.49 + 1.10a 1.67 _ 0.18b 1.78 + 0.47ab 0.88 • 0.10b 0.22 + 0.05a A1-50 15.13 + 2.43a 8.91 + 0.57a 1.09 + 0.05a 1.16 ___ 0.61 a 0.57 • 0.15a 0.30 • 0.12ab Al-150 14.85 +0.51a 8.48+ 1.24a 1.15 • 1.12+0.17a 0.58 +0.09a 0.43 + 0.09b F 1.903 0.210 10.398 5.085 24.898 8.443 P 0.176 0.888 0.001 0.012 0.000 0.001

Previous year needles Control 13.53 + 1.24ab 7.35 + 0.78a 1.49 + 0.08b 4.23 + 0.52a 1.36 + 0.20b 0.23 + 0.05a Acid-control 15.62 + 0.60b 7.70 + 1.28a 1.46 + 0.1 lb 4.14 + 0.94a 1.02 ___ 0.21 a 0.25 + 0.04a AI-50 13.53 +2.44ab 7.83 +0.56a 1.05+0.14a 3.84+ 1.12a 0.95 +0.22a 0.38 +0.13ab A1-150 11.35 + 0.93a 8.16 + 0.82a 0.99 + 0.29a 3.76 ___ 0.45a 0.99 + 0.03a 0.45 + 0.1 lb F 6.752 0.689 11.336 0.404 5.406 7.227 P 0.005 0.572 0.000 0.753 0.009 0.003

Roots Control 14.29 + 1.52a 5.97 + 1.02a 1.63 + 0.30a 2.84 + 0.67b 1.17 _ 0.18b 1.96 ___ 0.65a Acid-control 14.31 + 1.74a 6.28 + 0.65a 1.50 + 0.16a 2.69 + 0.65b 1.12 + 0.12b 2.14 + 0.84a A]-50 13.50 + 3.24a 5.55 + 0.87a 1.17 + 0.16a 1.33 + 0.07a 0.74 • 0.09a 3.49 + 0.45b Al-150 13.45• 1.32a 6.67• 1.79a 1.24 +0.39a 1 . 3 2 + _ _ 0 . 1 2 a 0.76• 3.91 • F 0.259 0.787 2.688 16.066 12.644 11.902 P 0.854 0.520 0.087 0.000 0.000 0.000

a Means followed by the same letter are not significantly different (P <0.05) according to Tukey's Multiple Range Test

35

3O

25

o~ 2O . m

O

J= 15

-,3 l o

I~J= CONTROL ~7~= ACID-CONTROL

[~= A I - 5 0

~ 1 = A1-150

NEW OLD NEEDLES NEEDLES

ab b

r s

z F / ~J

WHOLE SHOOTS

b

b

/ A

ROOTS

Fig. 1. Dry weights of current (new) and previous year (old) needles, whole shoots and roots after the second growing period

s e c o n d g r o w i n g p e r i o d a b o u t h a l f o f the seed l ings in the A I - 1 5 0 t r e a t m e n t g roup h a d b e c o m e y e l l o w - g r e e n .

A l u m i n i u m d e c r e a s e d the g r o w t h o f the seed l ings , as d e m o n s t r a t e d by the l eng ths and dry we igh t s o f the s h o o t m e a s u r e d af te r the s e c o n d g r o w i n g per iod. T h e d e c r e a s e in the l eng th o f the c u r r e n t - y e a r l ead ing shoo t and o f the w h o l e shoo t was g rea tes t in the seed l ings r e c e i v i n g A l - 1 5 0 c o m p a r e d to the o the r t r e a t m e n t s (Tab le 1). M o r e o v e r , the d e c r e a s e in the dry w e i g h t o f c u r r e n t - y e a r need le s was s ign i f i can t ly g rea te r for A l - 1 5 0 t han for the n o r m a l con t ro l t r e a t m e n t g roup (Fig. 1); howeve r , n o d i f f e r ences in the p r e v i o u s - y e a r need le s we re f o u n d b e t w e e n the t r e a t m e n t g roups (Fig. 1). R o o t dry w e i g h t was s ign i f i can t ly l o w e r in the A l - 1 5 0 t r e a t m e n t g roup t han in b o t h con t ro l g roups (Fig. 1), t h o u g h no s ign i f i can t d i f f e r ences (Table 1) we re o b s e r v e d in the r o o t / s h o o t rat io.

In all t r e a t m e n t s wi th l o w e r pH, wi th or w i t h o u t a l u m i n i u m , the need l e s we re c lea r ly shor t e r t han in the n o r m a l con t ro l t r e a t m e n t at the end o f the e x p e r i m e n t (Tab le 1). T h e dens i ty o f the need l e s ( n u m b e r o f n e e d l e s / shoo t l eng th ) in the c u r r e n t - y e a r l ead ing shoots o f the A l - 1 5 0 t r e a t m e n t g roup was s ign i f i can t ly h i g h e r t han in

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Table 3. Nutrient and aluminium concentrations (mean _ SD, n = 4 -5) in current and previous year needles and roots after the growing period 1991 (mg/g)

N K P Ca Mg A1

Current year needles Control 11.63 ,,, 1.89a~ 6.82 _ 1.09a 0.99 _ 0.18ab 1.73 ___ 0.12b 0.92 _ 0.12b 0. t 3 +__ 0.03a Acid-control 10.98 _ 2.68a 7.34 • 0.56a 1.25 • 0.16b 1.45 ___ 0.18b 0.99 ___ 0.09b 0.22 ,,, 0.02a A1-50 17.35 ,,, 2.49b 9.98 • 0.78b 0.72 -t- 0.15a 0.46 _ 0.19a 0.37 ___ 0.07a 0.28 -t- 0.08a Al-150 14.35 • 9.57_ 1.17b 0.77 • 0.25 • 0.27 +0.07a 0.48 +0.20b F 4.696 9.916 10.734 112.603 81.489 9.015 P 0.017 0.00i 0.000 0.000 0.000 0.001

Previous year needles Control 10.62 ,,, 3.33a 5.09 ___ 0.86a 0.71 ___ O. 19ab 3.37 ___ 0.80b 0.78 _ 0.25b 0.16 _+ 0.04a Acid-control 11.38_ 2.77ab 6.38_ 1.28a 1.04+0.19b 3.20• 0.86_0.10b 0.29• A1-50 20.22 • 6.04c 4.88 • 1.29a 0.51 _ 0.04a 1.58 ___ 0.48a 0.39 _ 0.13a 0.39 • 0.10a AI-t50 19.00_ 4.64bc 4.90___ 1.18a 0.62 +__ 0.33a 1.23 • 0.32_0.10a 0.79_ 0.29b F 6.534 1.914 5.662 20.572 15.232 12.261 P 0.004 0.168 0.008 0.000 0.000 0.000

Roots Control 10.17 _+ 3.03a 5.43 ___ 0.75a 1.20 • 0.15a 2.34 ,,, 0.50b 1.14 + 0.14b 2.90 • 0.39a Acid-control 9.53 • 0.4 la 5.67 • 1.00a 1.26 + 0.20a 2.12 _ 0.14b 1.22 + 0.16b 3.32 • 0.53a A1-50 12.51 + 2.39a 6.54 ___ 1.23a 1.08 _ 0.26a 1.32 _+ 0.15a 0.57 • 0.13a 5.17 -+- 0,73b Al-150 10.94_ 2.11a 6.12• 1.18• 0.79• 0.42_0.19a 6.16• F 1.695 1.217 0.483 23.191 29.495 31.490 P 0.208 0.336 0.699 0.000 0.000 0.000

a Means followed by the same letter are not significantly different (P <0.05) according to Tukey's Multiple Range Test

Table 4. Lengths of chloroplasts and starch grains, number of plastoglobuli per chloroplast cross-section measured from EM-photo- graphs after the first growing period, 1990 (means _ SD)

Length of Length of Number of chloroplast starch plastoglobuli/ (Bm) (Bm) chloroplast

cross-section

Current year needles Control 4.55 • 1.33ab a 0.95 • 21.4_ 14.1b Acid-control 5.12_0.59b 1.36• 25.7--- 10.6b A1-50 4.13 _ 0.97a 1.01 • 20.4 ___ 10.9b Al-150 4.47___ 0.88ab 1.09__+ 0.98a 11.9+ 5.2a F 3.556 1.173 6.008 P 0.017 0.323 0.001

Previous year needles Control 4.76--- 1.07b 0.46• 28.9+ 12.2b Acid-control 4.49 + 1.24ab 0.29 _ 0.54ab 16.0 • 8.6a A1-50 4.22_ 0.96ab 0.60_0.69b 21.8,,, l l .9a Al-150 4.01 _0.86a 0.16• 16.0+ 8.7a F 3.374 5.729 10.208 P 0.021 0.001 0.000

a Means followed by the same letter are not significantly different (P <0.05) according to Tukey's Multiple Range Test

the o ther seedl ings. Moreove r , the need le dens i ty o f the p rev ious -yea r lead ing shoot was h igh not only in the A l -150 group but also in the normal controls , wi th only the A1-50 group showing a l ower density.

Nutrient concentrations

(Tables 2, 3). In the roots, a decrease in m a g n e s i u m and ca l c ium was observed , whereas the phosphorus concent ra - t ions showed no s ignif icant d i f fe rences be tween groups (Tables 2, 3).

A l u m i n i u m t rea tment dec reased the n i t rogen concent ra - t ion o f the p rev ious -yea r need les in 1990, but increased the concent ra t ions o f both the current and the p rev ious -yea r needles in 1991 (Tables 2, 3). The on ly s igni f icant differ- ence in the po tass ium concent ra t ions was obse rved in the second year w h e n both a l u m i n i u m t rea tments s igni f icant ly inc reased the po ta s s ium concen t ra t ion in the cur ren t -year need les (Tables 2, 3).

The a l u m i n i u m concent ra t ions o f both roots and need les inc reased wi th increas ing exposure level . This increase was mos t d iscernib le in the roots o f both study years, where A1 concent ra t ions were about t0 t imes h igher than in the needles (Tables 2, 3).

Root symptoms and mycorrhizas

The pe rcen tage o f l iv ing mycor rh i za l root tips ranged in 1990 f rom about 49% to 75% and in 1991 f rom 88% to 95% of the total short roots in the di f ferent t r ea tment groups. Never the less , a l u m i n i u m t rea tment did not s ignif- icant ly affect the quant i ty o f mycor rh i za l or total short roots (data not shown). The n u m b e r o f swol len and branched root tips was genera l ly l ow and did not di f fer s igni f icant ly be tween the t rea tment groups.

A l u m i n i u m t rea tment dec reased the phosphorus , m a g n e - s ium and ca l c ium concent ra t ions s igni f icant ly (excep t for the decrease in Ca concen t ra t ion in 1990 o f p r ev ious -yea r needles) in both need le genera t ions o f both study years

Microscopic observations

Needles. The only change in Al - t rea ted seed l ings obse rved at l ight mic roscop ica l l eve l was a sl ight swel l ing o f p h l o e m

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Figs. 2 - 3 . Light micrographs of vascular tissue of Scots pine needles. Bars = 100 ~m. Fig. 2. Control needle showing intact phloem (p). Fig. 3, Needle exposed to A1 (Al-150). A swollen phloem cell (asterisk) is observable adjacent to normal phloem (p) tissue. Figs. 4 - 7 . Transmission electron micrographs of Scots pine needles. Bars" = 1 ~m. Fig, 4. The structure of current-year needle mesophyll cell (acid-control). s = starch grain, m = mitochondrion.

Fig, 5. Chloroplasts (c) of a current year needle in Al-treated (Al-150) seedling. Note light staining plastoglobuli (arrows). Fig, 6. Decreased number of thylakoids (arrows) in granum stacks in the current year needles of Al-150, s = starch grain. Fig. 7. Cavities (arrows) and lipid (l) inside a chloroplast of a Al-treated previous year needle (A1-50), s = starch grain. Note also swelling of mitochondria (m)

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Table 5. Lengths of chloroplast and starch grains, number of plastoglobuli per chloroplast cross-section, numbers of thylakoids per granum and ratio of chloroplast length to width measured from EM-photographs after the second growing period, 1991 (means • SD)

Length of chloroplast Length of starch Number of plastoglobuli/ Number of Ratio of chloro- (gm) (gm) chloroplast cross-section thylakoids/granum plast length/width

Current year needles Control 5.09 ___ 0.81 a a 2.72 • 1.15ab 8.9 + 3.8a 7.0 _ 4.4b 2.32 ___ 0.59ab Acid-control 4.96 • 0.90a 3.26 • 1.18b 10.1 • 6.1 ab 5.8 • 3.6a 2.02 • 0.46a A1-50 4.75 • 0.62a 2.47 • 1.09ab 14.3 ___ 6.5c 6.0 • 3.2a 2.07 • 0.45ab A1-150 5.00 • 1.03a 2.13 • 1.60a 14.2 • 6.7bc 5.2 • 2.8a 2.46 • 0.89b F 0.848 5.028 6.507 11.746 3.202 P 0.470 0.002 0.000 0.000 0.026

Previous year needles Control 4.52 • 1.14a 1.63 • 1.14a 19.0 ___ 8.5a 8.9 • 6.4b 2.64 • 0.72b Acid-control 5.38 • 0.90b 2.24 • 1.23a 19.4 • 6.6a 7.5 • 5.0a 2.76 • 0.65b A1-50 4.84 • 1.00ab 1.93 • 1.28a 22.7 • 13.9a 6.9 • 5.0a 2.02 • 0.48a A1-150 4.59 • 1.14a 1.64 • 1.61 a 20.5 ___ 10.0a 6.9 • 4.9a 2.69 ___ 0.63b F 4.065 1.655 0.860 7.389 8.352 P 0.009 O. 179 0.464 0.000 0.000

a Means followed by the same letter are not significantly different (P < 0.05) according to Tukey's Multiple Range Test

cells (Fig. 3). This phenomenon was also seen in some controls (Fig. 2), but the degree of swelling in the A1-150 group was greater than that in the other treatment groups.

Both the seedlings in the normal control and the acid control treatment groups showed an intact ultrastrncture of the mesophyll cells (Fig. 4) throughout the experiment. No consistent trend that could be related to Al-treatments was observed in the chloroplast length or in length/width ratio (Tables 4, 5). A significant decrease in starch grain size was observed in the oldest needles in 1990 and in the youngest needle generations of the following year (Tables 4, 5). Exposure to a higher level of aluminium caused a decrease in the number of plastoglobules after the first growing period, but after the second growing period the number significantly increased in the current-year needles (Tables 4, 5, Fig. 5). Lowered pH and aluminium treatments significantly decreased the number of thylakoids in granum stacks as compared to the normal control group (Table 5, Fig. 6).

Some chloroplasts showed cavities between the thyla- koids, which in some cases also contained lipids (Fig. 7). These cavities were observed after the second growing period and appeared mainly in those groups receiving aluminium treatments.

Swelling of the mitochondria was observed in almost all treatment groups (Fig. 7). The central vacuole was found to have an irregular edge in groups not receiving aluminium treatment, consisting of a rough tannin layer and small vesicles. The amount of cytoplasmic lipid bodies increased slightly in the current-year needles of Al-treated seedlings towards the end of the experiment.

Mycorrhizal roots. Both light and electron microscopic observations revealed that the majority (about 75%) of the sampled brown dichotomous mycorrhizas were well developed ectomycorrhizas, but ectendomycorrhizas were also frequently found. The only symptom of mycorrhizas at the ultrastructural level after 2 growing periods was a slight increase in vacuolation in the fungal cells of Al-treated (Al-150) seedlings compared to those receiving the normal control treatment.

Discussion

Growth responses

Variable responses with respect to shoot and root growth in different species have been described earlier in the litera- ture. Schier (1985) found no significant differences in the terminal shoot growth of red spruce (Pieea rubens Sarg.) seedlings at A1 concentrations up to 200 mg Aid. Nosko et al. (1988) observed a decreased root to shoot ratio in white spruce [Picea glauca (Moench) Voss] seedlings, indicating root growth reduction and biomass allocation to the shoots. Some reduction in the growth of Scots pine occurred at the level of 50 mg A1/1 after one growing period (Arovaara and Ilvesniemi 1990). However, in the present study significant reductions in dry weight were found only after the second year of treatment at a higher A1 level of 150 mg A1/1. In this study, no significant effect of A1 on the root/shoot ratio was observed, indicating that growth reduction occurred simultaneously both in the shoots and roots. In the Scots pine, aluminium seemed to not only reduce the needle length but also reduced the leading shoot length resulting in greater needle density.

Nutrient concentrations

These results support the findings previously described by Foy 1974, Roy et al. 1988 and Taylor 1988 showing that calcium and magnesium concentrations decreased both in the roots and needles. This decrease was already observable after the first growing period supporting the observation that nutrient uptake is affected before a decline in growth is detected (Cronan et al. 1989) and may actually be the reason for diminished growth. Nutritional changes between the first and the second year are possibly due to continued exposure, since exposure treatments were the same in the both years. A decrease in calcium and magnesium concen- trations started from an aluminium concentration of 50 mg A1/1 which agrees with the observations of Arovaara and Ilvesniemi (1990) concerning Scots pine.

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Calcium concentrations in the needles were sufficient or far above the deficiency limits (range of optimum) reported by Ingestad (1962). The lowest Ca concentration (below the deficiency limit) was measured after the second year in the current-year needles of the A1-150 group, apparently due to the disturbed uptake and immobile nature of calcium (Bukovac and Wittwer 1957; Helmisaari 1990). Magne- sium concentrations in the needles were near or below the deficiency limits (Ingestad 1962) in both aluminium treat- ments, but after the second year low levels were also found in the controls. After the first year, the control treatments produced calcium and magnesium levels very similar to those found for Scots pine needles in natural forest stands, but after the second year the levels were lower than those in forest stands (M~ilk6nen et al. 1990).

Our study shows that the P concentrations of needles were reduced in both years beginning from a treatment concentration of 50 mg A1/1, though root concentrations did not appear to be significantly affected. Needle P concentra- tions were below deficiency limits (Ingestad 1962) for the A1 treatment groups, with low concentrations being also found after the second year in the control treatment groups. The P concentrations in the needles of the control treatment group were quite similar or lower than in the needles of pines growing in forest stands (M~ilkrnen et al. 1990; Raitio 1990). One reason for the declined P concentration in needles might be the co-precipitation of A1 with phosphate in the apoplast causing the formation of aluminium phos- phate in the root or mycorrhiza tissues (Foy et al. 1978; Vare 1990). Another explanation for such reduced P concentrations has been proposed by Clarkson (1965) who reported altered biochemical pathways associated with P uptake in A1 stress.

Nitrogen and potassium concentrations in the current- year needles were found to increase in both A1 treatment groups. This increase could be due to the decline in growth of Al-treated seedlings leading to an increase in elemental concentrations. Therefore, the yellow-green colour ob- served in the needles of Al-treated seedlings may be due to a magnesium rather than nitrogen deficiency. Grransson and Eldhuset (1991) reported increased N and K uptake rate per unit growth rate for Al-exposed pine which might also be the case in our study. In general, the N and K concentrations in needles were usually slightly below the deficiency limits presented by Ingestad (1962). M~ilkrnen et al. (1990) and Raitio (1990) reported lower K concen- trations in natural stands of Scots pines than in our experiment, but the N levels were very similar.

In this study, aluminium concentrations in the needles increased with increasing level of exposure to A1, though all treatments except Al-150 seemed to be within the normal range compared to levels measured in forest grown pines (Raitio 1990; Vilkka et al. 1990). A corre- sponding accumulation of A1 in needles has earlier been observed for Scots pine (Arovaara and Ilvesniemi 1990) as well as in other plant species (Foy et al. 1978). Aluminium concentrations in the roots were as great as 10-20 times higher than those in the needles and increased with time of exposure. In control treatment group, this might be related to an increase in H § production over the course of the study due to e.g. nitrification (Olsthoorn et al. 1991), which in

turn could increase the amount of soluble A1 in the growth medium.

Although the present study did not examine the local- ization of AI in the roots, earlier studies have reported that A1 can accumulate in the cell walls of the epidermis and cortex (Schaedle et al. 1989; Hodson and Wilkins 1991). The endodermis has been considered a barrier to the apoplastic movement of ions in the root; however, the increase in shoot A1 content found in this study shows that the endodermis can not restrict all A1 movement. The formation of aluminium polyphosphate granules in the ectomycorrhizal hyphae (V~re 1990) may thus also in- crease the root A1 concentration together with the phos- phorus concentration.

Root symptoms and mycorrhizas

This study indicates that the roots were strongly mycor- rhizal and that the A1 concentrations used did not have any significant effect on the mycorrhizal numbers. Earlier studies have shown that ectomycorrhizal fungi do not prevent A1 from reaching the root cortex and displacing Mg and Ca in Picea abies seedlings (Jentschke et al. 1991). However, several authors (Wiikins and Hodson 1989; Cumming and Weinstein 1990a-c) have reported that rhizospheric fungi may reduce the effects of aluminium. In this study, it is possible that mycorrhiza formation may have reduced the toxicity of A1 to Scots pine, since no typical injuries were seen in the root systems at the macroscopical level and any changes in ultrastructure found were minimal. Furthermore, it seems that the nutrient imbalances in this study may have resulted from direct inhibition of uptake rather than from changes in root morphology or mycorrhiza number.

Needle structure

Our results seem to indicate a clear relationship between changes in needle structure and nutrient levels. Also McQuattie and Schier (1993) found this type of relation in the needles of Pinus rigida seedlings where foliar concentrations of P, Ca and Mg decreased as A1 concentra- tions increased and changes in the structure of chloroplasts appeared leading to localized cell collapse at higher A1 concentration (50 mg AI/1). In this study, such drastic effects were not found and the needle tissues and cell organelles retained their integrity throughout the experi- ments. Altogether the ultrastructural symptoms were rela- tively slight and only limited physiological consequences can be expected.

At the ultrastructural level the most interesting results came from morphometric measurements. The length of chloroplasts seemed to be dependent on the nitrogen concentration measured from the needles: the more nitro- gen the longer the chloroplasts. This finding is in agree- ment with the results of Fink (1989) and Holopainen et al. (1992) who described a reduction in the size of spruce and pine chloroplasts at nitrogen deficiency. The length of

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starch grains in chloroplasts found in the summertime were greater in Al-treated seedlings, but in both the autumn of 1990 and 1991 the length o f starch grains showed a greater decline in Al-150 than in control seedlings. This phenom- enon may be related to delayed photosynthate translocation due to retarded phloem function caused by Mg or Ca deficiency (Fink 1989) during the active photosynthesiz- ing period in summer. The presence of swollen phloem cells, a symptom of Mg deficiency (Fink 1989; Holopainen et al. 1992) support the presence of Mg deficiency in the Al-treated seedlings, and coincides well with the nutrient data and observations on starch accumulation. These findings agree also with the results of McQuattie and Schier (1993) who suggested the same reason for starch accumulation in Pinus rigida under Al-stress.

By the end of this study, there was an obvious increase in the number of plastogtobules and a decrease in the number o f thylakoids that may have consenquences on the photosynthesizing capacity of the needles. Such changes have earlier been associated to several nutrient (Mg, P, N, K) deficiencies (Fink 1989; Holopainen et al. 1992). However, in the present study, these symptoms seem to reflect primarily Mg and P deficiencies. The presence of mitochondrial swelling probably indicates a P deficiency (Holopainen et al. 1992). This symptom was to some extent seen in all treatment groups, which fits well with generally low P concentrations in the needles.

A greater number of cavities inside the chloroplast have earlier been connected to a K deficiency (Holopainen and Nygren 1989). In contrast, the cavities observed in this study were mainly found in those Al-treatment groups where the needle K concentrations were seen to be increased. On the other hand, the rough tannin layer and small vesicles close to the tonoplast, which have earlier been related to a K-deficiency (Holopainen and Nygren 1989), were found to be most abundant in the control treatment groups, where in the present study K concentra- tions were also found to be lower.

Fink (1989) demonstrated that severe Ca deficiency may lead to phloem collapse and accumulation of tannins in the vacuoles, as well as separation of thylakoids and accumula- tion of starch in Norway spruce needles. Though a moder- ate calcium deficiency was also found to develop in our experiment, such drastic changes were not seen, probably because the Scots pine, a typical calcifuge species, is well adapted to low soil pH and a low Ca supply, which are often related to increased levels of A1 (Rengel 1992) in soil water.

Scots pine tolerance to AI

Schaedle et al. (1989) classified Scots pine as a resistant species, with toxic A1 concentrations rising to 3 0 0 0 - 5 0 0 0 gM (about 8 1 - 1 3 5 mg A1/1) before a decline in growth could be detected. In our study, the toxic A1 concentration in the optimum nutrient solution was to some extent lower (1850 gM) after the second growing period. Arovaara and Ilvesniemi (1990) reported 50 mg A1/1 (1850 ~ / ) to be the limit for optimum nutrient treat- ments, but at lowered nutrient level the growth reduction

limit was 1 0 - 2 0 mg Aid ( 3 7 0 - 7 4 0 ~14). This is only slightly higher than the levels measured in Finnish soil solutions which were 1 - 5 mg Aid ( 3 7 - 1 8 5 /aM; Ilves- niemi 1991). According to Ilvesniemi (1991), A1 is more harmful to Scots pine when the ratio of N to other nutrients drops far below the optimum.

Our study shows that Scots pine seedling roots do not develop specific structural symptoms even under rather severe Al-stress. However, Al-stress does cause nutrient imbalance in the needles of Scots pine, as revealed by diminished Mg, Ca and P levels and corresponding ultra- structural symptoms. Under field conditions, direct alumi- nium-induced injuries to Scots pine are not likely, though Al-stress may be a contributing factor in the formation of nutrient imbalances.

Acknowledgements. This work was financially supported by the Academy of Finland. We are grateful to Dr. Helvi Heinonen-Tanski and Dr. Jarmo Holopainen for critically reading the manuscript, and to Mrs. Mirja Korhonen, Mrs. Raija Pitk~nen, Mr. Jarkko Utriainen and Mr. Raimo Pesonen for their technical assistance, and finally to Ken Pennington for the linguistical revision of the manuscript.

References

Allen SE (1989) Chemical analysis of ecological materials. Black- well's, Oxford

Arovaara H, Ilvesniemi H (1990) The effects of soluble inorganic aluminium and nutrient imbalances on Pinus sylvestris and Picea abies seedlings. In: Kauppi P, Kentt/imies K, Anttila P (eds) Acidification in Finland. Springer, Berlin Heidelberg New York, pp 715-733

Bukovac MJ, Wittwer SH (1957) Absorption and mobility of foliar applied nutrients. Plant Physiol 32:428-435

Clarkson DT (1965) The effect of aluminium and some other trivalent metal cations on cell division in the root apices of Allium cepa. Ann Bot 29:309-315

Cronan CS, April R, Bartlett RJ, Bloom PR, Driscoll CT, Gherini SA, Henderson GS, Joslin JD, Kelly JM, Newton RM, Parnell RA, Patterson HH, Raynal DJ, Schaedle M, Schofield CL, Sucoff El, Tepper HB, Thornton FC (1989) Aluminium toxicity in forests exposed to acidic deposition: the ALBIOS results. Water Air Soil Pollut 48: 181-192

Cumming JR, Weinstein LH (1990a) Aluminium-mycorrhizal interac- tions in the physiology of pitch pine seedlings. Plant Soil 125: 7-18

Cumming JR, Weinstein LH (1990b) Utilization of ALPO4 as a phosphorus source by ectomycorrhizal Pinus rigida Mill. seed- lings. New Phytol 116: 99-106

Cumming JR, Weinstein LH (1990c) Nitrogen source effects on A1 toxicity in nonmycorrhizal and mycorrhizal pitch pine (Pinus rigida) seedlings. 1. Growth and nutrition. Can J Bot 68: 2644-2652

Daughtridge AT, Boese SR, Pallardy SG, Garrett HE (1986) A rapid staining technique for assessment of ectomycorrhizal infection of oak roots. Can J Bot 64:1101-1103

Entry JA, Cromack K Jr, Stafford S, Castellano MA (1987) The effect of pH and aluminum concentration on ectomycorrhizal formation in Abies balsamea. Can J For Res 17:865-871

Fink S (1988) Histological and cytological changes caused by air pollutants and other abiotic factors. In: Schulte-Hostede S, Darrall N, Blank LW, Wellburn AR (eds) Air pollution and plant metab- olism. Elsevier, London, pp 36-54

Fink S (1989) Pathological anatomy of conifer needles subjected to gasenous air pollutants or mineral deficiencies. Aquilo Ser Bot 27: 1-6

142

Foy CD (1974) Effects of aluminum on plant growth. In: Carson EW (ed) The plant root and its environment. University Press of Virginia, Charlottesville, pp 601-642

Foy CD, Chaney RL, White MC (1978) The physiology of metal toxicity in plants. Annu Rev Plant Physiol 29 :511-566

Godbold DL (1991) Aluminium decreases root growth and calcium and magnesium uptake in Picea abies seedlings. In: Wright RJ, Baligar VC, Murrmann RP (eds) Plant-soil interactions at low pH. Kluwer, Dordrecht, pp 747-753

Grransson A, Eldhuset TD (1991) Effects of aluminium on growth and nutrient uptake of small Picea abies and Pinus sylvestris plants. Trees 5 : 1 3 6 - 1 4 2

Helmisaari H-S (1990) Temporal variation in nutrient concentrations of Pinus sylvestris needles. Scand J For Res 5: 177-193

Hodson M J, Wilkins DA (1991 ) Localization of aluminium in the roots of Norway spruce [Picea abies (L.) Karst.] inoculated with Paxillus involutus Fr. New Phytol 118:273-278

Holopainen T, Heinonen-Tanski H (1993) Effects of different nitrogen sources on the growth of Scots pine seedlings and the ultrastructure and development of their mycorrhizae. Can J For Res 23: 362-372

Holopainen T, Nygren P (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, Anttonen S, Wulff A, Palom~iki V, Karenlampi L (1992) Comparative evaluation of the effects of gaseous pollutants, acidic deposition and mineral deficiencies: structural changes in the cells of forest plants. Agric Ecosyst Env 42 :365-398

Ilvesniemi H (1991) Studies on the effects of acid deposition on soil chemical characteristics and seedling growth. PhD Thesis. Uni- versity of Helsinki, Department of Silviculture

Ingestad T (1962) Macro element nutrition of pine, spruce, and birch seedlings in nutrient solutions. Statens Skogsforskningsinstitut 51 :7

Ingestad T (1979) Mineral nutrient requirements of Pinus silvestris and Picea abies seedlings. Physiol Plant 4 5 : 3 7 3 - 3 8 0

Jentschke G, Schlegel H, Godbold DL (1991) The effect of aluminium on uptake and distribution of magnesium and calcium in roots of mycorrhizal Norway spruce seedlings. Physiol Plant 82 :266-270

Jorns AC, Hecht-Buchholz C, Wissemeier AH (1991) Aluminium- induced callose formation in root tips of Norway spruce [Picea abies (L.) Karst.]. Z Pflanzenernaehr Bodenk 154:349-353

M~ilk~nen E, Derome J, Kukkola M (1990) Effects of nitrogen inputs on forest ecosystems estimation based on long-term fertilization experiments. In: Kauppi P, Kentt~imies K, Anttila P (eds) Acid- ification in Finland. Springer, Berlin Heidelberg New York, pp 325-347

McQuattie CJ, Schier GA (1990) Response of red spruce seedlings to aluminium toxicity in nutrient solution: alternations in root anatomy. Can J For Res 20: 1001-1011

McQuattie CJ, Schier GA (1992) Effect of ozone and aluminum on pitch pine (Pinus rigida) seedlings: anatomy of mycorrhizae. Can J For Res 22 :1901-1916

McQuattie CJ, Schier GA (1993) Effect of ozone and aluminum on pitch pine (Pinus rigida) seedlings: needle ultrastructure. Can J For Res 23: 1375-1387

Metzler B, Oberwinkler F (1987) The in-vitro-mycorrhization of Pinus sylvestris L. and its dependence on the pH-value. Eur J For Path 17:385-397

Nosko P, Brassad P, Kramer JR, Kershaw KA (1988) The effect of aluminum on seed germination and early seedling establishment, growth and respiration of white spruce (Picea glauca). Can J Bot 66 :2305-2310

Olsthoorn AFM, Keltjens WG, Van Baren B, Hopman MCG (1991) Influence of ammonium on fine root development and rhizosphere pH of Douglas-fir seedlings in sand. Plant Soil 133:75-81

Raitio H (1990) The foliar chemical composition of young pines (Pinus sylvestris L.) with or without decline. In: Kauppi P, Kentt~imies K, Anttila P (eds) Acidification in Finland. Springer, Berlin Heidelberg New York, pp 699-713

Rengel Z (1992) Role of calcium in aluminium toxicity. New Phytol 121:499-513

Roy AK, Sharma A, Talukder G (1988) Some aspects of aluminum toxicity in plants. Bot Rev 54: 145-178

Schaedle M, Thornton FC, Raynal DJ, Tepper HB (1989) Response of tree seedlings to aluminum. Tree Physiol 5 : 3 3 7 - 3 5 6

Schier GA (1985) Response of red spruce and balsam fir seedlings to aluminum toxicity in nutrient solutions. Can J For Res 15 :29-33

Soikkeli S (1980) Ultrastructure of the mesophyll in Scots pine and Norway spruce: seasonal variation and molarity of the fixative buffer. Protoplasma 103:241-252

Sutinen S (1987) Cytology of Norway spruce needles. II. Changes in yellowing spruces from the Taunus Mountains, West Germany. Eur J For Pathol 17 :74 -85

Taylor GJ (1988) The physiology of aluminum phytotoxicity. In: Sigel H, Sigel A (eds) Metal ions in biological systems, vol. 24. Aluminum and its role in biology. Dekker, New York, pp 123-163

Thompson GW, Medve RJ (1984) Effects of aluminum and manganese on the growth of ectomycorrhizal fungi. Appl Env Microbiol 48: 556-560

V~ire H (1990) Aluminium polyphosphate in the ectomycorrhizal fungus Suillus variegatus (Fr.) O. Kunze as revealed by energy dispersive spectrometry. New Phytol 116:663-668

Vilkka L, Aula I, Nuorteva P (1990) Comparison of the levels of some metals in roots and needles of Pinus sylvestris in urban and rural environments at two times in the growing season. Ann Bot Fenn 2 7 : 5 3 - 5 7

Vogelei A, Rothe GM (1988) Die Wirkung von S~iure und Aluminium- ionen auf den Nahrelementgehalt und den histologischen Zustand nichtmykorrhizierter Fichtenwurzeln [Picea abies (L.) Karst.]. Forstw Cbl 107:348-357

Wilkins DA, Hodson MJ (1989) The effects of aluminium and Paxillus involutus Fr. on the growth of Norway spruce [Picea abies (L.) Karst.]. New Phytol 113:225-232