Annals of Botany 77 : 1–10, 1996

Fine Structure of Acid Mist Treated Sitka Spruce Needles :

Open-top Chamber and Field Experiments

ANU WULFF*, ALAN CROSSLEY† and LUCY J. SHEPPARD†

*Department of Ecology and En�ironmental Science, Uni�ersity of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland

and † Institute of Terrestrial Ecology, Edinburgh Research Station, Penicuik, Midlothian EH26 0QB, UK

Received: 27 April 1995 Accepted: 28 July 1995

Sitka spruce grafts (clones DF and 141) grown in open-top chambers (OTC) and ‘mature ’, 6–8 m tall Sitka spruce(clone DF) grown in the field were exposed to acid mist containing an equimolar ion mixture of H

#SO

%and NH

%NO

$at pH 2±5. Mist was applied 4 times a week (4¬1 mm) in the OTC experiment and twice a week (2¬2 mm) on averagein the field experiment, between May and Oct. 1991. Samples for light and electron microscopy were collected in Nov.and Jan. following acid mist treatment. Acid mist significantly decreased the amount of calcium deposited in the outerepidermal cell walls, the reduction being most pronounced in the OTCs. Ultrastructurally, acid mist caused asignificant increase in chloroplast and grana width. Other symptoms associated with acid mist included swelling ofchloroplast thylakoids, chloroplast protrusions, cytoplasm vacuolization, increase in large lipid accumulations andsickle-shaped chloroplast thylakoids. In the OTCs, acid mist hastened the acquisition of frost hardening in bothclones. In the field, the control trees exhibited more frost injury than the acid mist treated trees suggesting, again, thatacid mist had either hastened or enhanced the stage of frost hardiness of treated trees. In general, acid mist inducedchanges were more pronounced in the OTCs than in the field. # 1996 Annals of Botany Company

Key words : Picea sitchensis, Sitka spruce, acid mist, fine structure, calcium oxalate, frost hardening.

INTRODUCTION

Interest in the deposition and effects of cloud and mistcontaining pollutants increased rapidly in the 1980s becauseof decline in forest health at high altitude. For example, thehealth of red spruce (Picea rubens Sarg.) in eastern NorthAmerica, and that of silver fir (Abies alba) and Norwayspruce (Picea abies L. Karst.) in Europe has been found tobe impaired at high altitude sites (Schulze, Lange and Oren,1989; Johnson, 1992). These high altitude forests arefrequently ‘ in-cloud’ which may result in substantial iondeposition to the canopy surface since the concentration ofthe major pollutant ions is two to ten times greater incloudwater than in rain (Fowler et al., 1988).

Acid mist has been observed to disturb conifer physiologyin several ways. Fowler et al. (1989), Cape et al. (1991),Eamus and Murray (1993) and Sheppard, Leith and Cape(1994) have shown in open-top chamber experiments thatfrost sensitivity in autumn increases in red, Norway andSitka spruce seedlings following acid mist treatment atpH 2±5. Changes in frost hardiness of only a few degreesCelsius are important since they can significantly increasethe risk of frost damage (Sheppard, Smith and Cannell,1989). Exposure to acidic mist has also been observed tocause direct foliar injury, e.g. on red spruce seedlings treatedat pH 3 (Jacobson et al., 1990) or 3±5 (Leith et al., 1989), andNorway spruce at pH less than 2±75 (Leisen and Marschner,1990). By contrast, Sitka spruce seedlings have shown novisible injury at pH 3 (Percy and Baker, 1988) and were only

* Corresponding author.

injured at pH 2±5 when exposed frequently to acid mist(Sheppard et al., 1994). Repeated wet}dry periods havegenerally been shown to increase the detrimental effects ofacid mist (Sheppard, Cape and Leith, 1993). The risk offoliar injury, growth changes and increased frost sensitivityincrease when concentration ratios of sulphate to nitrate inthe mist are high (Jacobson et al., 1990; Cape et al., 1991).

Ultrastructural studies have corroborated these physio-logical effects, identifying areas of damaged tissue ororganelles. Cellular collapse at pH 2±6 and decreasedmesophyll air space and starch with increasing acidity havebeen observed in yellow poplar (mist was applied for 4 h d−"

for 8 weeks) (Crang and McQuattie, 1986). These are typicalsymptoms of acute damage induced by acid rain in broad-leaved species (Evans and Curry, 1979). An increase in thelarge cytoplasmic lipid areas, cytoplasmic vesiculation, poordifferentiation of cell organelles and tonoplast and plasma-lemma irregularities were found in the mesophyll cells ofScots pine and Norway spruce in long-term field experimentswith acid rain at pH 3 (Ba$ ck, Neuvonen and Huttunen,1994). Increases in large lipid accumulations in the cyto-plasm of conifer needles have also been described in otheracid rain experiments (Holopainen and Nygren, 1989;Saastamoinen and Holopainen, 1989), and in papers fromareas with elevated SO

#or NO

xemissions (Anttonen, 1992).

In Scots pine, protrusions and irregular crook shapedchloroplasts have been observed following acid rain treat-ment at pH 3 (Holopainen and Nygren, 1989).

Ultrastructural observations can also provide a tool forthe assessment of winter hardening since there are severalfeatures which typify a ‘winter appearance’, for example,

0305-7364}96}010001­10 $12.00}0 # 1996 Annals of Botany Company

2 Wulff et al.—Acid Mist Treated Sitka Spruce Needles

reticulate appearance of cytoplasm, chloroplasts (sometimesirregularly shaped) which have migrated away from cellwalls and gathered together, and the absence of starchgrains (Soikkeli, 1978; Reinikainen and Huttunen, 1989;Fincher, 1992).

Acid mist has been observed to change the compositionand structure of surface waxes of spruce needles (Percy andBaker, 1990) which could result in enhanced loss of mineralsby leaching. Though acid mist and acid rain experiments onconifers have shown enhanced leaching of foliar cations,especially K+, Ca#+ and Mg#+, no reductions in overall leafcontent of leached cations have usually been detectable(Mitterhuber, Pfanz and Kaiser, 1989). This is supposedlydue to enhanced root uptake. However, reduction of boundcalcium in the outer epidermal cell walls has been observedin Norway spruce needles following acid mist treatment(Fink, 1991b). The loss of calcium in outer epidermal cellwalls can be used as an indicator of acid mist impacts on theneedle epidermis though calcium crystals there representphysiologically inactive calcium. Calcium is usually boundas calcium oxalate in plants, sometimes as calcium carbonateor pectins (Fink, 1991a) and in gymnosperms has mainlybeen found in cell walls of secondary phloem, and epidermalcells and extracellularly on the outside of the walls ofmesophyll cells which face the intercellular spaces (Fink,1991a).

In the present study our specific aims were to answer thefollowing questions: (a) What kinds of ultrastructuralchanges does acid mist cause in Sitka spruce needles and dothey relate to increased frost sensitivity? (b) Does acid misttreatment result in similar changes in the OTCs and in thefield experiment? (c) Does acid mist reduce the amount ofcalcium in outer epidermal cell walls in Sitka spruce?

MATERIALS AND METHODS

Acid mist treatments

Octagonal open-top chambers (OTC) glazed with 3 mmhorticultural glass, floor area 7±0 m# and height 2±3 m, wereused to study the effects of acid mist on 1 m high grafts ofSitka spruce [Picea sitchensis (Bong.) Carr.]. This graftedmaterial was derived from physiologically mature trees, atleast 30 years old, enabling an examination of the responseof mature foliage to acid mist under identical conditions tothose used in earlier studies with seedlings (Sheppard, Capeand Leith, 1993). In this study two clones (DF and 141),provided by the UK Forestry Commission, were used.Clone DF originated from a tree showing severe frostinjury, (provenance Queen Charlotte Island) growing inprovenance trial at Wark forest, Northumberland, UK.Clone 141, provenance unknown, was selected in 1964 fromGlenbranter Forest, Argyll, UK. Chambers were fitted withpolyethylene ceilings to exclude rainfall and watered via thebase of the pots using capillary matting (Fowler et al., 1989).Three replicate chambers were used for each treatment. Thegrafts were exposed to mist (containing an equimolar ionmixture of H

#SO

%and NH

%NO

$at pH 2±5 and 5±0) at a

frequency of 4¬1 mm applications on consecutive dayseach week between 14 May and 11 Nov. 1991. Droplets,

with a mass mean diameter of approx. 90 µm, were appliedvia a disc spinning at approx. 5000 rpm. Treatmentsprovided 3±5 kg H, 48 kg N and 55 kg S ha−" and 0±07 kg H,! 1 kg N and S ha−" for pH 2±5 and 5 treatments, re-spectively. This represents a dose three times higher thanthat measured in the Scottish uplands.

In the field experiment at Glencorse (3°13« W, 55°51« N,186 m above sea level, near Edinburgh, Scotland) blocks offour trees, which were 6–8 m tall and at least 19 years old,growing on a rich agricultural soil, were surrounded by aframework which supported roller blinds. Blinds werepulled down to isolate blocks during mist application, andraised between treatment periods to minimize chambereffects. The chosen site, of 144 trees from a single clone(‘DF’, taken as cuttings from a 10-year-old parent tree in1972), was on a slight slope which had induced differentialgrowth so the trees were stratified into five height classes. Asimilar mixture of H

#SO

%and NH

%NO

$was used as in the

OTCs and the spraying regime between May and Oct. 1991aimed at an average of 2¬2 mm per week. Droplet diameterwas approximately 60 µm. A second treatment with theaddition of 30 mg l−" sodaglass spheres (% 20 µm) wasapplied to simulate anthropogenic particulates in cloud.The chamber treatments were arranged as pairs, for eachheight class, as ‘acid mist ’ with or without particles (4­4trees). The control trees (eight per height class) were selectedas individual trees outside chambers near the acid misttreated trees. Treatments provided 3 kg H, 47 kg N and51 kg S ha−" for each acid mist chamber in 1991.

Trees in the OTCs experienced frost (minimum ®6 °C)on six occasions before sampling for microscopy (Sheppardet al., 1994). Temperature data for the field site is notavailable but since the distance between the OTC and fieldsite is only approx. 700 m, temperatures can be expected tobe rather similar at both sites. Because of the absence ofprotecting walls, the daily minimum temperatures mighthave been colder at the field site than in the OTCs.

Microscopy

At the OTC site, current year needle samples from theclones DF and 141 were collected on 24 Nov. 1991. Onegreen needle from the upper third of each tree from each ofthe three chambers (five trees per chamber) was removedand fixed. Finally 15 needles per treatment and per clonewere examined using a JEOL 1200 transmission electronmicroscope resulting in 208 micrographs from which 259chloroplasts were observed. In the field experiment, twocontrasting tree height classes were chosen, the shortest(height class 1) and the tallest (height class 5). Upwardfacing, green current year needles were collected from thesouth-eastern branches at a height about 1±6 m, from themid-section of the shoot. On 12 Nov. 1991, six needles pertree were collected for microscopy. On 28 Jan. 1992, onlyone needle from the same trees was sampled (from each offour acid mist, four acid mist­particles and eight controltrees per height class). From the autumn samples, threeneedles per tree were examined microscopically resulting in155 electron micrographs, from which 216 mesophyll cells(217 chloroplasts) were observed. One hundred and twenty

Wulff et al.—Acid Mist Treated Sitka Spruce Needles 3

six micrographs were taken from the Jan. samples, fromwhich 134 mesophyll cells (150 chloroplasts) were examined,three to six per tree (mean 4±4), 69 from the height class 1and 65 from the height class 5.

Needle pieces (0±5–1 mm in length) from the middle of theneedles were fixed in 2±5% Karnovsky (12 Nov. 1991)(Karnovsky, 1965) or glutaraldehyde (24 Nov. 1991 and 28Jan. 1992) fixative in 0±1 phosphate buffer for 18 h,postfixed with 1% OsO

%for 6 h and dehydrated in graded

acetone series (50, 70, 95 and 100%, each 2¬20 min).Needle pieces in Eppendorf tubes (caps on to exclude anymoisture) were gradually infiltrated with Spurr’s resin(Spurr, 1969) (2 h in 3:1, 2 h in 1:1 and overnight in 1:3acetone}resin solution, 3 d in pure resin) in a rotator,embedded in LKB 2208–156 molds and polymerized for atleast 48 h (70 °C). For electron microscopy ultra-thinsections, cut on a Reichert-Jung Ultracut E ultramicrotome,were stained with vacuum-packed stabilized solutions ofuranyl acetate (UltroStain I) and lead citrate (UltroStainII). A JEOL JEM 1200 Ex transmission electron microscopeoperating at 80 kVwas used for ultrastructural observations.

Glyoxal-bis-(2-hydroxyanil) (GBHA) stain was used tovisualize distribution of bound calcium in epidermal cellwalls using light microscopy (Pearse, 1985). The GBHA (asa 4% solution in absolute ethanol) was applied to the1–2 µm sections on gelatin covered slides as a mixedsolution containing nine parts GBHA and two parts 2

NaOH. After 10 min staining the sections were dehydratedin 70%, 96% and absolute ethanol, dried at roomtemperature and mounted in Permount. By this methodbound calcium could be seen as red deposits. As the redcolour fades gradually, the observations were completedwithin 24 h. Each epidermal cell (150–190 epidermal cellsper needle) in the OTC samples were classified visually inone of the three different classes (" 80, ! 80 and 0%) basedon the calcium coverage of outer epidermal cell wall. Fieldsamples, which were processed later, were examined in moredetail, and were classified to four different classes (100," 50, ! 50 and 0%).

Statistics

Differences in the calcium distribution classes between thetreatments were analysed with a χ# test based on theweighted number of examined epidermal cells. Analysis ofvariance (Manova procedure) (SPSS Inc., Chicago, Illinois)was used to test for effects of mist on continuousultrastructural parameters. In the OTC experiment thechamber mean (n¯ 3) was the unit of replication. In theGlencorse field experiment, there were no significantdifferences between acid mist with or without particles, soresults were combined for statistical analysis so that fourtree blocks were used as a replicate unit, resulting in tworeplicates per height class.

RESULTS

Light microscopy

There were no specific differences related to treatment inneedle structure seen by light microscopy for either the OTC

or field experiment. In both experiments, the epidermal,mesophyll and endodermal cells were intact and no changesresulting from acid mist treatment were detected in theintercellular space. The hypodermis was consistently com-posed of one to three cell layers. Vascular tissue was normalexcept in the tissue from a few of the field grown trees wherecollapse of phloem could occassionally be observed.

Bound calcium was mainly restricted to the phloem andouter epidermal cell walls. Sometimes a thin layer ofcalcium was also observed in the inner epidermal cell wall.No intracellular calcium oxalate deposition was observed,but some calcium deposits were occasionally seen in theintercellular space. Acid mist treatment significantly de-creased the amount of calcium in the outer epidermal cellwalls (Tables 1 and 2). This reduction in calcium was alwaysgreatest in the stomatal areas (in hypodermal subsidiarycells) or in otherwise indented areas, and lowest at theneedle edges. Abaxial sides of the needles were moreaffected by acid mist than adaxial sides. The reduction incalcium deposition became more severe with time at theOTC site. In Oct., reduction (data not shown) in calciumdeposition was 7% increasing to 25% in late Nov.Generally, the reduction in calcium deposition followingacid mist treatment was less marked in the field than in theOTCs.

Transmission electron microscopy

Mesophyll cells of control needles were usually charac-terized by intact cell structures. The chloroplasts were lensshaped, the stromawas light and the amount of plastoglobulismall. No major irregularities in plasmalemma or tonoplast

T 1. Distribution (%) of calcium content of the outerepidermal cell walls in the current year Sitka spruce needles.OTC-site, 24 No�. 1991, clones DF and 141. Each epidermalcell was classified to one of the three different classes (" 80%,! 80% or 0%) based on the calcium co�erage (seen as reddeposits) of outer epidermal cell wall. Adaxial (upper) andabaxial (lower) sides of needles were examined separately.Values represent means of 9 (clone 141) to 12 (clone DF) trees(SD in parentheses). Different letters below the columnsindicate significant difference in the distribution of the classesbetween the treatments within each side, χ# test (based on the

number of examined cells), P! 0±000

Adaxial side Abaxial side

Class Controls Acid mist Controls Acid mist

Clone DF" 80% 28 (13) 17 (9) 57 (16) 32 (16)! 80% 35 (8) 28 (10) 30 (15) 45 (12)

0% 37 (17) 55 (16) 13 (3) 23 (10)a b a b

Clone 141" 80% 37 (16) 27 (9) 47 (19) 22 (11)! 80% 33 (9) 41 (6) 40 (16) 56 (12)

0% 30 (17) 32 (7) 13 (5) 22 (11)a b a b

4 Wulff et al.—Acid Mist Treated Sitka Spruce Needles

T 2. Distribution (%) of calcium content of the outerepidermal cell walls in the current year Sitka spruce needles atGlencorse, 12 No�. 1991, in height classes 1 and 5. Eachepidermal cell was classified to one of the four different classes(100%, " 50%, ! 50% or 0%) based on the calciumco�erage (seen as red deposits) of outer epidermal cell wall.Adaxial (upper) and abaxial (lower) sides of needles wereexamined separately. Values represent means of 21–24 needles(SD in parantheses). Different letters below the columnsindicate significant difference between the classes and treat-ments within each side, χ# test (based on the number of

examined cells), P! 0±05

Adaxial side Abaxial side

Class Controls Acid mist Controls Acid mist

Height class 1100% 42 (13) 44 (15) 43 (15) 43 (15)

" 50% 27 (8) 28 (10) 41 (11) 38 (8)! 50% 20 (8) 19 (9) 12 (9) 15 (12)

0% 11 (7) 9 (7) 4 (5) 4 (5)a a a a

Height class 5100% 49 (16) 47 (13) 49 (18) 44 (16)

" 50% 25 (8) 28 (9) 36 (10) 38 (10)! 50% 20 (9) 19 (8) 11 (8) 14 (9)

0% 5 (6) 6 (5) 3 (6) 4 (5)a b a b

P¯ 0±048 P¯ 0±001

were detected. Mitochondria were intact in all the samples.Curling of plasmalemma in the mesophyll cells wasoccassionally seen in both the control and treated trees.Chloroplast length tended to decrease following acid misttreatment but the decrease was significant only in wintersamples from the short trees in the field trial. On the otherhand, acid mist significantly increased the chloroplast width(Tables 3 and 4). Maximum thylakoid grana thickness wasalso significantly increased by acid mist treatment, in clone141 in the OTCs (Table 3) and in the tall trees in the field,clone DF (Table 4). Thylakoid swelling was substantiallyincreased in acid mist treated trees in the OTCs (Figs 1 and2) and also slightly in the field treated trees (Tables 5 and 6).

In the OTCs, treatment with acid mist speeded up theonset of hardening in both clones (Table 5) as evident from,for example, the reticulate appearance of the cytoplasm,which occurred 50% more frequently in acid mist-treated

T 3. Results of micrograph measurements, OTC site, 24 No� 1991. Values are means of 15 trees (SD in parentheses).(Analysis of �ariance, Mano�a procedure, n¯ 3)

Clone DF Clone 141

Controls Acid mist P Controls Acid mist P

Length of the chloroplasts, µm 6±3 (1±2) 6±1 (1±3) 0±35 5±9 (1±1) 5±7 (0±8) 0±16Width of the chloroplasts, µm 2±3 (0±5) 2±6 (0±6) 0±24 2±3 (0±7) 2±8 (0±7) 0±048Maximum thickness of grana, µm 0±3 (0±2) 0±2 (0±1) 0±14 0±4 (0±2) 0±6 (0±3) 0±09

trees of clone DF, and the reduced amount of starch inclone 141. Both controls and acid mist treated trees of clone141 were ultrastructurally more winter-hardy than trees ofclone DF, more than 90% of trees in clone 141 showingwinter-type appearance of cytoplasm (Fig. 3). Peripheralvesicles in the chloroplasts (Fig. 3) occurred more frequentlyin clone 141 than in clone DF, and also more in the acid misttreated trees than in the controls in clone DF. In both clonescytoplasmic vacuoles and small lipid bodies were 10–20%more frequent in acid mist treated than in control trees.Large lipid bodies were generally rare in the present study,and were most frequent (in 7% of trees) in acid mist treatedtrees of clone DF in the OTCs.

In the field, irregularities in chloroplast shape in clone DFin Jan. were not concentrated in the sides adjacent to cellwall as protrusions as in the OTCs (Figs 2 and 3) insteadchloroplasts assumed a lobate or amoeboid-like appearance(Fig. 4) in both height classes. In the field exposure, lipid-like bodies were frequently present, atypically inside thechloroplasts (Figs 5 and 6). These lipid-like bodies wereirregularly shaped and larger than plastoglobuli but stainedin a similar way to plastoglobuli, and sometimes resembledagglomerations of large plastoglobuli. The presence of lipid-like bodies inside the chloroplast was twice as frequent inthe tallest trees as in the shortest trees, and substantiallymore pronounced in control than treated trees.

Between 30 and 80% of cells from the field werecharacterized by a disintegrated cytoplasm, seen as a verysparse cytoplasm, i.e. decrease of ER and ribosomes, whichcould not be classified as either ‘summer-type’ or ‘winter-type’. Sparse cytoplasm occurred, usually simultaneously,with dark cytoplasmic bodies (Fig. 6), and was mostpronounced at the lowest needle nitrogen concentrations(! 1±4% d. wt). In the tall trees, sparse cytoplasm wasalways linked with atypical lipid-like bodies inside thechloroplasts, but this was not always the case in the shorttrees, implying that the perturbation was greater in the talltrees.

Small starch grains were still present in some Nov.samples (0–25%). Also in mid-winter small starch grainswere occasionally present (0–50%), especially in the controltrees, suggesting that they were less hardy in Jan. than theacid mist treated trees. In Jan., all trees from all heightclasses and treatments had some abnormal crooked orsickle-shaped thylakoids (Fig. 4). This symptom was threetimes as frequent in acid mist treated trees as in the controls.(Table 6).

Wulff et al.—Acid Mist Treated Sitka Spruce Needles 5

T 4. Results of micrograph measurements in Glencorse, 12 No�. 1991 and 28 Jan. 1992. Values are means of eight trees(s.d. in parentheses)

Height class 1 Height class 5

Controls Acid mist P Controls Acid mist P

12 Nov. 1991Length of the chloroplast, µm 6±4 (0±5) 6±2 (0±8) 0±65 6±4 (0±7) 6±2 (0±4) 0±72Width of the chloroplast, µm 2±8 (0±5) 3±3 (0±4) 0±097 2±9 (0±4) 2±7 (0±4) 0±29Maximum thickness 0±36 (0±06) 0±33 (0±07) 0±38 0±34 (0±1) 0±39 (0±1) 0±67of grana, µm

28 Jan. 1992Length of the chloroplast, µm 7±0 (0±4) 6±5 (0±7) 0±041 6±8 (0±5) 6±9 (0±9) 0±92Width of the chloroplast, µm 2±9 (0±5) 3±3 (0±3) 0±003 2±8 (0±3) 3±7 (0±2) 0±039Maximum thickness 0±23 (0±1) 0±21 (0±07) 0±59 0±26 (0±05) 0±32 (0±06) 0±005of grana, µm

T 5. Results of classified parameters of Sitka spruce needle ultrastructure in OTCs, 24 No�. 1991. Values are percentagesof trees ha�ing class 0 (symptom absent) or 1 (symptom present) as a dominating score. Each treatment group contains 15 trees

Clone DF Clone 141

Controls Acid mist Controls Acid mist

Class 0 Class 1 Class 0 Class 1 Class 0 Class 1 Class 0 Class 1

Shape of the chloroplast 93 7 50 50 60 40 14 86(0¯ ellipsoid, 1¯ irregular)

Starch grains 80 20 71 29 73 27 86 14Swelling of thylakoids 87 13 36 64 73 27 43 57Peripheral vesicles in the chloroplast 67 33 29 71 7 93 14 86Lipid bodies inside the chloroplast 100 0 93 7 100 0 100 0Small cytoplasmic lipid bodies 40 60 29 71 33 67 14 86Appearance of cytoplasm 67 23 21 79 0 100 7 93(0¯ summer-type, 1¯winter-type)

Cytoplasmic vacuoles 47 53 29 71 27 73 7 93

T 6. Results of classified parameters of Sitka spruce needle ultrastructure in Glencorse, 12 No�. 1991 and 28 Jan. 1992.Values are percentages of trees ha�ing class 0 (symptom absent) or 1 (symptom present) as a dominating score. Each treatment

group contains 8 trees

Height class 1 Height class 5

Controls Acid mist Controls acid mist

Class 0 Class 1 Class 0 Class 1 Class 0 Class 1 Class 0 Class 1

12 Nov. 1991Shape of the chloroplast 25 75 0 100 13 87 63 37(0¯ ellipsoid, 1¯ irregular)

Starch grains 100 0 88 12 25 75 75 25Sickle-type thylakoids 100 0 100 0 100 0 100 0Lipid bodies inside the chloroplast 63 37 88 12 25 75 75 25Small cytoplasmic lipid bodies 0 100 0 100 0 100 38 62Cytoplasmic vacuoles 13 87 13 87 0 100 13 87

28 Jan. 1992Shape of the chloroplast 25 75 0 100 25 75 0 100(0¯ ellipsoid, 1¯ irregular)

Starch grains 50 50 100 0 75 25 88 12Swelling of thylakoids 88 12 75 25 100 0 88 12Sickle-type thylakoids 75 25 25 75 88 12 25 75Lipid bodies inside the chloroplast 75 25 88 12 50 50 63 37Small cytoplasmic lipid bodies 0 100 0 100 0 100 0 100Cytoplasmic vacuoles 25 75 25 75 25 75 13 87

6 Wulff et al.—Acid Mist Treated Sitka Spruce Needles

F 1–6. Electron micrographs from green current year Sitka spruce needles after acid mist treatment in open-top chambers (OTC) or in theGlencorse field experiment. Fig. 1. Control needle, clone DF, OTC. Note the intact chloroplast and dense cytoplasm. ¬13500. Fig. 2. Acid misttreated needle, clone DF, OTC. Note the swelling of thylakoids and protrusions (arrows) adjacent to the cell wall. ¬15000. Fig. 3. Acid misttreated needle, clone 141, OTC. Note the reticulate cytoplasm (*) and protrusions, increased height of grana and peripheral vesicles (arrows) inthe chloroplast. ¬11500. Fig. 4. Control needle, January, Glencorse. Note the irregularly shaped chloroplast and sickle-shaped thylakoids

Wulff et al.—Acid Mist Treated Sitka Spruce Needles 7

DISCUSSION

In the present study a significant reduction in the amount ofcalcium in the outer epidermal cell walls was found for Sitkaspruce in both the OTC and field experiment. Fink (1991b,1993) has observed similar acid mist-induced decreases incalcium in Norway spruce needles. Though foliar calciumreduction was not significant in whole leaves in our study(Sheppard et al., 1994) the observed loss of calcium from theouter epidermal cell walls might indicate the presence ofacid mist-induced leaching processes at least in the epi-dermis. Huttunen, Turunen and Reinikainen (1991) foundCaSO

%-crystallites on needle surfaces after simulated acid

rain treatment and regarded these as an indicator ofnutrient leaching. In Norway spruce, Tenberge (1992)detected the sudden appearance of calcium crystals in outerepidermal cell walls in late Jun. This happened concurrentlywith a reduction in permeability of the cuticular membraneto water. He postulated that calcium which is excreted withthe transpiration stream may crystallize beneath thecuticular layer. A reduction in the amount of precipitatedcalcium in the outer epidermal cell walls following acid mist-treatment suggests an increased permeability of the cuticularmembrane and therefore enhanced water loss via cuticulartranspiration. One factor contributing to epidermal calciumleaching might have been acid mist-induced increasedweathering and wettability of needle surface (Percy andBaker, 1988; Turunen and Huttunen, 1991). The morepronounced reduction of calcium in the indented areas andon the abaxial side, observed in the present study, may beassociated with the longer contact time.

Deposition of calcium oxalate crystals in the epidermisrepresents the excess, physiologically inactive, calcium andthus a reduction will not directly impair plant physiology.However, since calcium has essential structural function inthe strengthening of the cell walls (Marschner, 1986), theabsence of calcium in the epidermis may indicate formationof ‘channels ’ which could promote leaching of other ions. Itis also possible that the absence of calcium oxalate, especiallyin stomatal area, may affect stomatal functioning sincecalcium oxalate has been hypothesized to regulate theapoplastic free calcium which in turn in high concentrationsinhibits the stomatal opening (Ruiz and Mansfield, 1994).The presence of roofs in the OTCs exacerbates the inimicaleffect of acid mist, since dilution or the washing effect of rainis excluded. This was evident from the OTC experimentwhere the reduction in epidermal calcium was morepronounced than in the field experiment.

No additional acid mist-induced effects were observed bylight microscopy. In contrast to broad-leaved species, wheremesophyll cell disruption and decreased intercellular spacehave been observed (Evans and Curry, 1979; Crang andMcQuattie, 1986), mesophyll cells of Sitka spruce wereintact and no changes in intercellular space volume weredetected. Collapsed epidermal cells and malformed stomata

(arrows). ¬11500. Fig. 5. Acid mist treated needle, January, Glencorse. Note the lipid-like bodies (*) inside the chloroplast, dark bodies in thecytoplasm (arrows) and vacuolization of cytoplasm. ¬14000. Fig. 6. Acid mist treated needle, January, Glencorse. Note the sparse disintegratingcytoplasm (*). Dark stained bodies in the cytoplasm are in close contact with vacuoles (arrow). ¬11500. C, Chloroplast ; CW, cell wall ; CV,

central vacuole ; G, granum; M, mitochondrion; PG, plastoglobuli ; S, starch; T, tannin; V, vacuole.

were also absent in our study, though these symptoms havebeen detected in Norway spruce seedlings following acidmist exposure (pH 2±5) (Fink, 1993). The absence of thesesymptoms may be due to sampling only green, healthy-looking needles for microscopy.

Acid mist thickened grana, previously associated withhigh nitrogen inputs (Soikkeli and Ka$ renlampi, 1984; Ba$ ckand Huttunen, 1992) and increased chloroplast width,earlier observed in Norway spruce needles treated with pH 3solution (composed of nitric acid and sulphuric acid in theratio 1:2) (Ba$ ck and Huttunen, 1992). Both N and S areessential plant nutrients (Marschner, 1986) and an increasein supply of these elements due to acid deposition appearsto have initially positive effects on chloroplasts. The changeto more rounded chloroplasts has earlier been described inassociation with SO

#exposure (Ka$ renlampi and Houpis,

1986) so it is possible that the observed increase inchloroplast width (resulting in a more rounded shape) mightbe related to a surplus of sulphur.

Irregularities in chloroplast shape in the OTCs resembledthe reported protrusions of chloroplasts in Scots pine afteracid rain exposure (Holopainen and Nygren, 1989). Similarprotrusions have also been observed in conjunction withcombined acid mist and NO

#exposure in yellow poplar

(Crang and McQuattie, 1986). This type of irregularity inchloroplast shape might be a response to increased acidityon the cell surface which would result in partial withdrawalof the plastids further away from the cell wall.

Chloroplast thylakoids usually form a network, parallelto the long diameter of the chloroplast, which consist ofsingle nonappressed stroma thylakoids and appressed granathylakoids (Schnepf, 1980). In our study, abnormal sickle-shaped thylakoids existed in Jan. samples from the field,being three times as frequent in acid mist treated trees as inthe controls. In the autumn samples there were no suchsymptoms, suggesting this feature to be associated withwinter conditions after acid mist exposure. To our knowl-edge such thylakoids have not been previously reported.The irregular, amoeboid shape of the chloroplasts, as well asperipheral vesicles in the chloroplasts, appears to beassociated with winter hardening, in Sitka spruce also, sincethese symptoms appeared in parallel with winter-typecytoplasm and reduction of starch accumulation. Peripheralchloroplast vesicles have been suggested to indicate thestorage of membrane material and be representative ofadaptation to cold (Ba$ ck, Huttunen and Kristen, 1993). Theamoeboid shape of chloroplasts has previously beendescribed in conjunction with winter hardening in Scotspine and Norway spruce (Ba$ ck and Huttunen, 1992).

In most studies acid mist has been found to reduce frostresistance or delay winter hardening (Fowler et al., 1989;Cape et al., 1991; Sheppard et al., 1993) though beneficialeffects of adequate N on frost hardiness have also beenestablished (DeHayes, Ingle and Waite, 1989; Klein, PerkinsMyers, 1989; Caporn, Risager and Lee, 1994). In our study,

8 Wulff et al.—Acid Mist Treated Sitka Spruce Needles

acid mist hastened the winter hardening process. This hasimportant implications for frost hardiness measurements,since, as shown in another part of this experiment, the effectof acid mist may be masked because control trees are lesshardy than treated trees resulting in freezing treatmentsthemselves overriding the treatment effect (Wulff, Leith andSheppard, 1994). This would also explain the results, forexample in Cape et al.’s (1991) study, where there was nosignificant difference in frost hardiness in early stages offrost hardening but was apparent when seedlings wereappreciably frost hardened. The importance of geneticcomposition in determining the timing of acquiring differentdegrees of hardiness is well known (Cannell and Sheppard,1982) and was reconfirmed in our study. Acid mist-inducedhastening of frost hardiness in the OTCs could be seenparticularly clearly in the less hardy clone DF.

In the field, the cytoplasm from a large proportion ofmesophyll cells appeared disintegrated and contained darkbodies. This is thought to represent frost injury, since theclone in question is known to be frost sensitive and it hasbeen shown that cytoplasm disintegration and accumulationof dark material in it are early ultrastructural signs of frostinjury (Holopainen and Holopainen, 1988; Reinikainen andHuttunen, 1989). The dark bodies in our study werefrequently associated with vacuoles suggesting a proteinousorigin (Vigil et al., 1985). Since the lowest foliar nitrogencontents were related to this sparse, disintegrating cytoplasmit is possible that the increased N inputs from acid mist hadhelped to augment reserves of proteins which, in turn,facilitate and acceleratemembrane repair processes. Interest-ingly, control needles had more of this cell type and morestarch, indicating that control trees were also less hardythan acid mist treated trees in the field.

The positive effects of acid mist on frost hardening may berelated to physiological maturity of the trees. Sheppard etal. (1994) found within this same experiment that maturegrafts showed a smaller reduction in hardiness, measured aselectrolyte leakage, at an equivalent dose than that foundpreviously with Sitka spruce seedlings. Sheppard et al.(1994) sugggested this to be due to less enhanced foliarsulphate concentrations in mature trees as compared toseedlings. Morphological differences between seedlings andmature Sitka spruce trees (Steele, Coutts and Yeoman, 1989)could also contribute to the higher susceptibility of seedlings.The higher specific leaf area and the smaller weight of thecuticular waxes in young trees (Steele et al., 1989) mayexacerbate acid mist-induced responses in seedlings.

Foliar potassium (K) concentrations were relatively smallin the field site, especially in the short trees where the meanK% was 0±78³0±12 (d.wt) (Sheppard, unpubl. res.). Atendency toward K deficiency could explain the phloemcollapse observed in the field with the light microscope(Fink, 1991; Holopainen et al., 1992). Potassium, as themajor cytoplasmic cation, is known to be an important ionin osmoregulation, and plants receiving an inadequatesupply of K are often more suspectible to frost damage(Marschner, 1986). In conjunction with K deficiency anoccurrence of lipid-like bodies inside the chloroplasts hasbeen reported (Holopainen and Nygren, 1989). The greaternumbers of chloroplasts containing lipid-like bodies in the

control trees compared with the acid mist treated trees may,thus, reflect a slightly higher suspectibility to frost injury inthe control trees. Similarly shaped material to the lipid-likebodies inside the chloroplasts, though more dark-stained,have been reported in injured Scots pine and Norway sprucecells after a low temperature treatment as well (Reinikainenand Huttunen, 1989) suggesting that these bodies areassociated with frost.

Interestingly, the less irregular shape of the chloroplaststogether with less numerous cytoplasmic lipid bodies wouldsuggest an acid mist-induced delay in hardening processes inthe tall trees of Glencorse field experiment in the autumn.This would support those earlier studies where delayed orreduced frost hardiness due to acid deposition has beenproposed (e.g. Friedland et al., 1984; Fowler et al., 1989;Cape et al., 1991). The most significant increase in thethickness of grana in these same trees suggests that the inputof extra N was diverted more to stimulate photosyntheticprocesses (cf. Eamus and Murray, 1993) than to frosthardening processes in tall trees. However, the morepronounced presence of disintegrating cytoplasm, starchand lipid-like bodies inside the chloroplasts in the untreatedtall trees suggest an opposite effect to frost hardening andmakes it difficult to assess acid mist-induced effects in thetall trees in autumn.

The differences in acid mist-induced responses betweenthe OTCs and the Glencorse field experiment may be due tothe mist application procedure. More frequent misting andexclusion of rain intensified the effect of acid mist in theOTCs. This could explain the substantially higher frequencyof thylakoid swelling in the OTCs compared to the fieldsamples. Acid deposition induced increases in large lipidareas and cytoplasm vacuolization (Ba$ ck and Huttunen,1992, Ba$ ck et al., 1994) were also only present in the OTCs.Vacuolization of the cytoplasm has been observed followingSO

#exposure in a 3 years’ open-field study with Scots pine

and Norway spruce, too, where it was connected todetoxification of excess sulphate or low pH (Wulff andKa$ renlampi, 1995). Furthermore, comparison of field andOTC results was complicated because of a large proportionof existing frost injury symptoms in both control andtreated trees in the field.

In conclusion, acid mist induced ultrastructural symptomsin Sitka spruce were relatively few and resembled thoseobserved with other conifer species, except for the sickle-shaped thylakoids in winter samples which represented anew symptom associated with acid mist. Calcium reductionin the outer epidermal cell walls following acid misttreatment was significant, indicating the presence of acidmist-induced leaching processes in the epidermis. Winterhardening processes were clearly affected by the acid mist.In the Glencorse field experiment, sparse, disintegratingcytoplasm, most obviously due to a frost injury, was relatedto the lowest foliar nitrogen concentrations. Lipid-likebodies inside the chloroplasts were also associated withfrost injury in our study. The more pronounced effects in theOTCs highlight the importance of high frequency of mistingsand wet}dry cycles.

Wulff et al.—Acid Mist Treated Sitka Spruce Needles 9

ACKNOWLEDGEMENTS

This study was financed by the Academy of Finland. Wethank Ms Sari Sihvonen and the staff at ITE, EdinburghResearch Station, for their valuable technical assistance.

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