J. Phytopathology 122, 1—12 (1988)© 1988 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0931-1785

Institut fiir Allgemeine Botanik der Johannes Gutenberg-UniversitdtSaarstr. 21, D-6500 Mainz, Federal Republic of Germany

Electron Microscopic Studies of Spruce Needlesin Connection with the Occurrence of Novel Forest Decline

I. Investigations of the Mesophyll

GEORG JUNG and ALOYSIUS WILD

Authors' address: GEORC JUNG and ALOYSIUS WILD, Institut fiir Allgemeine Botanik der JohannesGutenberg-Universitat, Saarstr. 21, D-6500 Mainz, Federal Republic of Germany.

With 12 figures

Received January 29, 1987; accepted July 22, 1987

Abstract

Needles of four spruce trees showing different degrees of novel kinds of forest decline wereinvestigated by electron microscopy. Green needles appearing at least superficially still intact wereselected for the present investigation. Most of the mesophyll appeared to be undamaged. However,groups of atypical mesophyll cells were found close to the endodermis or the hypodermis. Thechloroplasts of the apparently damaged cells were particularly affected. Changes in the matrix of thechloroplasts, i.e. increased affinity to osmium, occurrence of extensive nests of plastoglobuli, as wellas damage to the membranes, i.e. lesions in the envelope and abnormal thylakoid membranes, wereobserved. Signs of decomposition of other cellular structures including mitochondria were alsodetectable. There appeared to be a close correlation between the degree of damage at the whole treelevel and the degree of damage occurring at the cellular level. It is concluded that particularly the lipidsand the proteins of the membranes are affected by anthropogenic air pollutants and natural stressors.The altered membrane structure may for instance cause abnormal osmotic conditions for the cellularcompartments and may impair transport processes and thus lead to loss of function not only of thecells but also of the whole needle.

Zusammenfassung

Elektronenmikroskopische Untersuchungen von Fichtennadeinim Zusammenhang mit dem Auftreten neuartiger Waldschaden

Im Zusammenhang mit dem Auftreten neuartiger Waldschaden wurden Nadeln von vierunterschiedlich stark geschadigten Fichten elektronenmikroskopisch untersucht. Es handelte sichausschliefilich um grvine, makroskopisch noch unversehrt aussehende Nadeln von Seitentrieben ersterOrdnung, die dem siebten Quid entstammten. In den untersuchten Schnitten war nie das gesamte

*) Reprint requests to Prof. Dr. A. WiLD

U.S. Copyright Clearance Center Code Statement: 0931-1785/88/2201-0001$02.50/0

2 JUNG and WILD

Mesophyll geschikligt, sondern immer nur einzelne Zellgruppen nahe der Endodermis oder unterhalbder Hypodermis. Das Ausmafi der Schaden, nach dem die Einzelbaume ausgewahlt worden waren,spiegelte sich in etwa auf dem Niveau der Nadeln wider. Besonders differenziert und charakteristischerwiesen sich die Schadigungen der Chloroplasten. Hier zeigten sich sowohl Veranderungen derMatrix wie die Zunahme der Osmiophilie oder umfangreiche Nester von Plastoglobuli, als auch derMembranen, d. h. Lasionen des Envelope sowie Schwellungen, Verzerrungen und Kontrastzu- oder-abnahme der Thylakoide. Parallel dazu waren Zerfallserscheinungen der Mitochondrien und andererZellstrukturen zu beobachten. Daraus wird geschlossen, dafi durch die Einwirkung anthropogenerLuftschadstoffe und natiirlicher Stressoren insbesondere die Lipide und Proteine der Zellmembranengeschadigt werden, was unter anderem auch zu veranderten osmotischen Verhaltnissen in denZellkompartimenten und zu Storungen von Transportprozessen fiihrt. Die Schadigung der Zellmem-branen kann schliefilich zur Einbufie der Funktionalitat der Zellen selbst und der ganzen Nadelfiihren.

For several years, forest decline has been occurring over large areas inEurope. According to the present state of research, this disease appears to becomplex, i.e. the causes are considered to be very diverse and one stressor alonerarely plays the major role. Today, the influence of climatic, edaphic andmicrobial aspects is undisputed at limited locations and times. Nevertheless,anthropogenic air pollution is likely to be responsible for the exceptional intensityof damage and for the spreading of forest injury over large regions (MOHR 1984,ULRICH 1984, ELSTNER et al. 1985).

In the past, the experimental approach was to treat plants under controlledconditions with the major pollutants. It could be repeatedly demonstrated thateven the tiniest destructions which cannot be discerned macroscopically or withthe light microscope can be diagnosed in the electron microscope. A further fieldof research was opened up for the phytopathologist when plants from the biotopedisplaying symptoms caused by unknown factors were investigated and theresults compared with those obtained from plants grown under standardizedconditions. The aim was to determine whether the plants growing in naturalhabitats show symptoms similar to those obtained under laboratory conditions.

GoDZiK and KNABE (1973) have already investigated various pine speciesfrom the Ruhr area and from Upper Silesia. In material taken from polluted areasthey observed alterations of the chloroplasts. They concluded that ultrastructuralphenomena can be used to demonstrate the action of air pollutants on conifers. Inrecent years, this concept was further developed by SOIKKELI (1978) and SOIKKELIand TuoviNEN (1979). They carried out extensive investigations on needle mate-rial from Picea abies and they were able to confirm that certain damage types arecaused by pollution.

The aim of our study was to monitor over one vegetation period thealterations to the needles of Picea abies from an area with significant tree damage,and to interprete the damage types. This study is part of a project involving notonly cytomorphological but also physiological and biochemical aspects, e.g.analysis of photosynthesis and the photosynthetic apparatus, of nitrogen metabo-lism, plant nutrients, water balance, and of phytohormones (BODE et al. 1985,WILD and BODE 1986, BENNER and WILD 1986, WILD 1987a, b, WILD 1988).

A special problem is that edaphic, climatic and other environmental factorsoften vary to such an extent that exact information on the conditions prior to the

Electron Microscopic Studies of Spruce Needles 3

investigation is not available, particularly not for perennial plants. Nevertheless,we consider it essential to compare electron microscopic data with the occurrenceof new kinds of tree damage. Only then will we be able to determine the exactcause for the disease.

Material and Methods

Material. Needles of spruce (Picea abies [L.] Karst.) were taken from a stock of trees, about 80years old, growing on a southwestern slope in the forests of Konigstein (FRG) in the Taunusmountains (Forest District Glashiitten, division 43/44). The plantation is located in a mountain areaabout 665—757 m above sea level. The soil was acid brown earth on micaceous sandstone and"taunus" quarzite basis.

Four trees were selected. The characteristics are noted in Table 1. The scale of symptoms rangedfrom green-looking trees with little loss of needles to heavily injured trees with yellow needles andintensive loss of needles.

Table 1Characteristics of the selected trees

Tree Ratio of yellow needles Foliage presentto entire mass of needles in %

A < 1/3 90—100B < 1/3 30— 50C > 1/3 90—100D > 1/3 30— 50

The samples were taken exclusively from the middle of lateral twigs grown at boughs of theseventh whorl. Care was taken that samples were collected from similar positions of the trees at eachsampling date. Only green needles of the 1984 generation were considered. Sampling took place at thefollowing dates: 20 April, 1 July, and 7 October 1985.

Methods. Immediately after the branch was taken, the needles were cut off and put into asolution of 1 % glutaraldehyde and 1 % formaldehyde in phosphate buffer (0.02 M, pH 7.4). Thematerial was then transported in a cold bag at about 4—7 °C to the laboratory.

Small segments of 1—2 mm length were cut (immersed in fixation solution) with a razor bladefrom the middle of needles within 4—^5 hours. The sections were transferred to a fresh solution of thesame fixative, vacuum infiltrated and then kept at least one hour at room temperature.

After that the concentration of the fixation solution was raised from 2 % / 2 % glutaraldehyde/formaldehyde to 3 % / 3 % and then to 4 % / 4 % at about one hour intervals.

Post-fixation Was in 2 % osmium tetroxide in 0.05 M phosphate buffer, pH 7.4, for two hours.The samples were then washed in buffer and dehydrated in a graded acetone series and embedded inSpurr's epoxy resin (SPURR 1969). Ultra-thin sections of 60—90 nm were cut on a LKB III ultra-microtome with a diamond knife, stretched with xylene and put on grids. They were first stained with0.5 % uranyl acetate and then with 2 % lead citrate. The sections were examined in a Zeiss EM 9Aand a Hitachi H 600 electron microscope.

Results*»

Only green needles were investigated in this study to identify early markersof damage. Initially it was necessary to estabhsh the appearance of normal intactcells in relatively healthy material. Tree A (Table 1) met these requirements.

1*

4 JUNG and WILD

Mesophyll cells deviating from this state were characterized as damage types andthey were compared with the apparently normaly type. Special attention was paidto the chloroplasts, because they showed distinct alterations. The samples takenat different seasons were treated separately. Clear differences were observedbetween the spring material and the summer/autumn material.

The spring collection

Normal type (Fig. 1). The central vacuole occupies most of the space of themesophyll cells. It contains polyhydroxyphenols (tannins). The tannins coat thetonoplast as small droplets or are distributed as larger droplets in the cell sap.Occasionally, small vacuoles are found in the parietal cytoplasm. The nucleuseither is suspended in the middle of the cell by cytoplasmic strands or it is locatedin the parietal cytoplasm. It mostly shows an irregular outline and coarsegranulation and contains one to several nucleoli.

The cytoplasm is finely granulated, interspersed by little endoplasmicreticulum, and contains a large number of ribosomes and mitochondria. Highlyosmiophilic droplets are found occasionally. The main mass of the protoplasm ismade up by the chloroplasts. The lens-shaped chloroplasts contain a large centralstarch grain occounting for the major proportion of their volume. The matrixshows moderate granulation and several small nests of piastoglobuli. Distinctgrana are visible. The grana thylakoids are interconnected by a few stromathylakoids.

Occasional pits occur in the cell walls. In the cytoplasm bordering the pitmembrane, smooth endoplasmic reticulum structures are found in large numbers;these communicate both with the endoplasmic reticulum and with the plas-modesmata. The plasmalemma shows an irregular outline.

The following abnormalities were found:Damage type 1 (Figs. 2 and 3). The cytoplasm appears to be shrunk.

Electron-translucent vacuoles are fringed by osmiophilic material. The tonoplastsare no longer discernible as such. The matrix of the plastids is highly opaque, withflocculant granulation which may become entirely black. The thylakoids appearto be packed close together and can be barely discriminated or stand out as brightlines against a dark background. The thylakoids are considerably lessosmiophilic, but there is a dramatic increase of osmiophilia in the matrix (Fig. 3).Occasionally, normal plastids may be found between the altered plastids (Fig. 3).Thylakoid swelling does not occur in spring. The plastoglobuli have not signifi-cantly increased.

Damage type 2 (Fig. 4). Here, the cytoplasm lacks the typical features. Onlyvacuoles with slightly osmiophilic borders and fine electron-dense precipitate canstill be distinguished. Mitochodria, ribosomes and endoplasmic reticulum arebarely discernible. The tonoplast is intact. The chloroplasts are highly swollen,but the envelope remains intact. The thylakoids are located centrally or packedtogether at the periphery. They have very intense contrast and are more or lessswollen, in some cases with transition to vesiculation. The spacing of the loculivaries with the degree of swelling. Together with the starch grain, the thylakoid

Electron Microscopic Studies of Spruce Needles

r , ^ r:vc

Figs. 1—4. ultrathin sections of needles collected in springFig. 1. normal type

Fig. 2. damage type 1Fig. 3. damage type 1 (thylakoids 1>)

Fig. 4. damage type 2 (electron translucent spaces •= )

6 JUNG and WILD

system makes up just half of the plastids. Some vesicles are seen close to theenvelope. These vesicles may possibly be invaginations of the envelope. In somecases they may invaginate deeply into the plastids. The chloroplasts showthylakoid-free regions which arise peripherally; these contain dense clumpymaterial. The regions include electron-translucent spaces containing finely dis-persed precipitates.

Damage type 3 (Fig. 5). The cytoplasm is only a vesicular osmiophilic mass.The mitochondria degenerate to vesicles. The chloroplasts are swollen and insome cases even burst. Some matrix is still visible, but it is restricted to islets. Thethylakoids are swollen with formation of vesicles. Only remnants of grana arerecognizable. The tonoplast is still intact and the vacuole is free of tannins.

Special damage type of tree B (Fig. 6). A variant damage type was found inone of the trees (tree B). The plastids displays in some cases drastically increasednumbers of plastoglobuli. Often more than half of the plastid volume is filled byplastoglobuli. Damage types 1 and 2 were completely absent in this tree.

The summer and autumn collections

Normal type. The cytoplasm appears less densely granulated than in spring.Ribosomes are only scarce and the smooth endoplasmic reticulum is lessdeveloped. However, the mitochondria and plastids are still numerous. Insummer, the chloroplasts often have more than 10 layers of thylakoids pergranum. A large proportion of the plastid volume is occupied by the enormousstarch granule. The number of plastoglobuli is strongly reduced. The concentra-tion of tannins appears to be substantially reduced. All the other structures of thecell are organized as in spring.

The numbering of the damage types will be the same as for the springcollection. The differences in the ultrastructure will be listed in the following:

Damage type 1 (Figs. 7, 8, 9). The damage type occurs in a similar way tospring. The mitochondria do not have well organized tubuli. They appear to becoarsely granulated and are interspersed with membrane residues. The outline ofthe tonoplast is indicated by a thick layer of dense black agglomerations (prob-ably tannins). In the vacuole, there is a diffuse precipitate. Only chloroplasts arecomparatively little affected. Nevertheless, the thylakoids are more distinct thanin spring. Plastoglobuli occur in small nests. In addition, there are electron-translucent holes in the matrix. The holes appear to rise by invagination of theinner envelope membrane (Fig. 9) and by swelling of thylakoids. The plastidenvelope shows evaginations towards the plasmalemma. Vesicles close to theplasmalemma are filled by masses of electron-dense material.

Damage type 2 is absent in summer and autumn.Damage type 3 (Fig. 10). This type is almost identical to that in the spring

collection. However, there are large lipid droplets in the cytoplasm. The chloro-plast envelope often displays large protrusions.

Damage type 4 (Fig. 11). This type occurs in summer but mainly in autumn.It showed peculiar plastid deviants. Here, the thylakoids appear to be disarrangedand undulated. The stacking of the grana thylakoids is also lost.

Electron Microscopic Studies of Spruce Needles

)>

, . . ^ • - , . . . > • • • : • - - - -

• 1

. - . . • > • , . - — " -

1 • -

• )

i>

, - • / • • • \

\ . .

. ' . • ' • ' - .

_i : :

Figs. 5—8. spring collection (Figs. 5, 6), summer and autumn collections (Figs. 7, 8)Fig. 5. damage type 3 (formation of residual grana • )

Fig. 6. special damage type of tree BFig. 7. damage type 1

Fig. 8. damage type 1 (invaginations of the envelope • ; electron translucent hole D)

JUNG and WILD

Figs. 9—12. summer and autumn collectionsFig. 9. damage type 1 (electron translucent holes in the matrix; invagination —

Fig. 10. damage type 3 (protrusions • )Fig. 11. damage type 4 (droplets • ; round-shaped vesicles 1>)

Fig. 12. special damage type of tree B

- • )

Electron Microscopic Studies of Spruce Needles 9

The cytoplasm displays further aberrations. Zones are disclosed in which thecytoplasm is not granulated but filled with dense collections of droplets. Thereare also zones of dark, round-shaped vesicles appearing empty. The mitochondriatend to swell and to obliterate.

Special damage type of tree B (Fig. 12). A special damage type of tree B wasagain observed. This time there are less plastoglobuli. However, granum stacksare markedly reduced in size. Electron transparent holes occur.

Discussion

It should be stressed that only certain groups of atypical mesophyll cellswere found, namely close to the endodermis or the hypodermis. Most of themesophyll appeared to be undamaged. There seemed to be a close correlationbetween the degree of damage at the whole tree level (Table 1) and the degree ofdamage occurring at the cellular level. It remains to be seen whether cells are ableto repair the damage described.

It would be speculation to place the damage types into a time sequence. Theyshould all be discussed seperately as long as intermediate types are not found.

The dark matrix of the plastids of the damage type 1 was observed in plantstreated with ozone (THOMSON et al 1966 b, 1974, ATHANASIOUS 1980), or withnitrogen oxides (THOMSON and SWANSON 1972), PAN (THOMSON et al 1966 a,THOMSON and SWANSON 1972), or with SO2 (FISCHER et al 1973, MLODZIANOWSKI

and BiALOBOK 1977, MALHOTEA 1976). The authors discuss that a dehydration ofthe plastids probably caused by osmotic with-drawal of water is responsible forthe increase in contrast. It is assumed that the agents such as ozone mainly affectthe cell membranes, particularly the membrane proteins (TINGEY and TAYLOR

1982, MuDD 1982). The amino acids with sulfhydryl groups (methionine, cys-teine, cystine) and the aromatic amino acids (tryptophane, histidine, tyrosine) arefor instance sensitive towards ozonolysis (MUDD 1982).

Furthermore, activated oxygen species (singlet oxygen and oxygen freeradicals) occur preferentially in the chloroplasts because of the continuousproduction of oxygen in the presence of photo-activated pigment molecules andof reduced redox components with highly negative redox potential. This processis promoted by various stress factors including anthropogenic air pollutants. Thehighly reactive oxygen species can lead to destructive oxidation of cellularcomponents and can cause disintegration of the cell membranes. Of particularimportance are peroxidations of unsaturated fatty acids of the membrane lipids(ELSTNER et al 1985, BEKINA and GUSEINOVA 1986, DODGE and GILHAM 1986,

WILD 1987a, b).MICHAEL et al. (1982) assume that changes in the membrane permeability

after SO2 fumigation are responsible for the raised frost sensitivity of spruce.BECKERSON and HOFSTRA (1980) found no altered membrane conductivities afteradministration of low doses of SO2 (0.15 ppm) over longer periods (1—5 days) inwhite beans, soya and in radish. However, they measured greatly raised mem-brane conductivities after ozone application to the same species.

10 JUNG and WILD

It is important in this context that the envelope is frequently folded, but onlyon the chloroplast side towards the plasmalemma. These folds apparently give riseto vesicles which are filled with osmiophilic material. They show little resem-blance to the massive invagination reported by COLQHOUN et al. (1975) andATHANASIOUS (1980) in senescent Raphanus sativus plants. However, the foldsresemble the structures described by GODZIK and SASSEN (1974) for SOa-exposedPhaseolus plants. They observed constrictions of vesicles to the inside of thechloroplasts. SWANSON et al. (1973) and THOMSON et al. (1974) observed a similarphenomenon of folding and accumulation of osmiophilic material between theenvelope and the plasmalemma in Phaseolus plants treated with ozone. It is notknown, why this process only occurs in this particular region of the plastids.

It is interesting to note that in cells of the damage type 1 occasionally intactchloroplasts occur in the vicinity of damaged chloroplasts (Fig. 3). Actually thecell shown in Fig. 3 shows striking similarity to cells found by GODZIK andKNABE (1973) and by SOIKKELI and TUOVINEN (1979) in SO2-exposed plants grownin natural habitats.

Furthermore, damage type 2 cells are also found by SOIKKELI and TUOVINEN

(1979). They describe the thylakoid-free zones in the peripheral stroma, theswellings of the thylakoid system and the vesicles close to the envelope. Thissupports the view that anthropogenic air pollutants can cause destructions of themembrane systems, mainly in the plastids. The peripheral membrane-free spacesshown in Fig. 4 were also described by FISCHER et al. (1973) and MIYAKE et al.(1984) in SO2-treated material.

In damage type 2 it is obvious that the contrastibility of all membranes hasincreased, similarly to what has been reported by PELL and WEISSBERGER (1976) inozone-treated Glycine plants.

There is strong evidence that swelling of the thylakoids in general andspecifically in our case are a basic stress response. This symptom was observedafter treatment with ozone (ATHANASIOUS 1980, MIYAKE et al. 1984), NOx(WELLBURN et al. 1972), and SO2 (WELLBURN et al. 1972, MALHOTRA 1976), insenescent plants (HURKMAN 1979, HUDAK 1981, TAGEEVA et al. 1981), and underwater stress conditions (FELLOWS and BOYER 1976).

In our case, not only the thylakoids but also the plastids were swollen. Thisindicates that swelling was caused by major changes of osmotic conditions.Membrane alterations including transport processes may be responsible.

Gells similar to damage type 3 were observed by SOIKKELI (1978) after frostdamage. MICHAEL et al. (1982) report that SO2-exposed spruces react to lowtemperatures with a major fall in the net rate of photosynthesis. Especially inwinter when ground frost, low humidity, high irradiation, and high levels of SO2caused by the inversion weather coincide this damage may be strongest.

The damage type 4 which occurs in summer but mainly in autumn is open todiscussion at the present state. However, the extremely high numbers of plasto-globuli in the special damage type of tree B are an indication for light stressconditions (WILD 1987a, 1988).

Electron Microscopic Studies of Spruce Needles 11

The electron microscopic study is also supported by physiological data ofour group obtained from material taken at the same time and from the samebranches. It was shown that the more severely damaged trees (G and D) have amuch lower chlorophyll content than less severely diseased trees. The Mg "content of needles collected from these trees is also significantly lower (WILD andBODE 1986, WILD 1987b). It was suspected that the cause for the two symptomswas a severe destruction of the photosynthetic membranes. Our present studyconfirms that membrane damages occur in apparently healthy green needles.

In conclusion, we now have strong evidence that novel forest decline iscaused at least by a contribution of pollutants of the atmosphere. It appears as ifthe biomembranes are the early point of attack inside the cells. Especially thechloroplasts react either by swelling or shrinkage, indicating a disturbed osmoticbalance and an impairment of transport processes of the cell compartments.

These investigations were supported by the Federal Environmental Office (UmweltbundesamtBerlin) and the Commission of the European Communities (Brussels). We are grateful to Dr. G.ElSENBElS and Dr. K. HONOMICHL for providing the facihties to perform the electron microscopicstudies.

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