J. Cell Sci. 75, 195-205 (1985) 195Printed in Great Britain © The Company of Biologists Limited 1985

CHANGES OCCURRING IN CHLOROPLASTS OF

PHASEOLUS FOLLOWING INFECTION BY

SCLEROTINIA: A CYTOCHEMICAL STUDY

V. N. TARIQ AND P. JEFFRIESBiological Laboratory, University of Kent, Canterbury, Kent CTZ 7NJ, U.K

SUMMARY

Dramatic changes occur in the infrastructure of chloroplasts within host tissues during theinfection oiPhaseolus by the plant pathogenic fungus Sclerotinia. Electron-opaque deposits developinitially in the peripheral region of the chloroplast stroma, in cells in advance of the hyphal front. Asinfection continues, the amount and intensity of deposition increases and spreads throughout thechloroplast. The deposits possess a high degree of structural integrity in the form of a crystallinesquare lattice with 10 nm periodicity. Enzyme digestion studies have been used to show that thedeposits are proteinaceous. Similar deposits are also induced by treatment with oxalic and citricacids, but not by the potassium salts of these acids buffered at pH7-2. It is suggested that a pHchange within the chloroplasts, resulting from oxalic acid secretion by the fungus, is responsible forinduction of protein deposition. The likelihood that the deposits are ribulose, 1,5-bisphosphatecarboxylase-oxygenase is discussed.

INTRODUCTION

There are several reports of the appearance of crystals or spherulites (orderedaggregates) of proteinaceous material (e.g. see Gunning, Steer & Cochrane, 1968) inthe stroma of healthy plastids. Similar observations have been recorded for plastidsthat have undergone severe physiological stress (e.g. see De Greef & Verbelen, 1973;Wrischer, 1973). Gunning (1965) used the term 'stromacentre' to describe a fibrillarspherulite formed in the plastids of healthyAvena leaves. In a later study (Gunningeial. 1968), staining reactions suggested the absence of nucleic acid from thestromacentre and that the latter was probably proteinaceous. Electron micrographsinterpreted as illustrating the disaggregation of the stromacentre into free particles,suggested that the resulting units were indistinguishable from the finely particulatebackground material of the stroma, believed to be the CCVfixing enzyme ribulose1,5-bisphosphate carboxylase-oxygenase (RuDP-carboxylase; EC 4.1.1.39). Thisenzyme (also known as 'Fraction 1' protein) is a major protein in the stroma ofplastids.

Gunning et al. (1968) also showed that fibrillar material, similar to thestromacentre of Avena leaves, became apparent in the stroma of plastids in portionsof Phaseolus vulgaris L. leaves subjected to high-speed centrifugation, or in leavespartially dehydrated as a result of induced plasmolysis or wilting. Similar observations

Key words: chloroplast ultrastructure, Sclerotinia, oxalic acid, plant pathogenesis, proteindigestion.

196 V. -N. Tariq and P. Jeffries

of the crystallization of components of the stroma in plasmolysed leaf cells have beenreported by Wrischer (1973) and Shumway, Weier & Stocking (1967), whileThompson, Dugger & Palmer (1965, 1966) showed that material from the stroma ofPhaseolus plastids could be precipitated in semi-crystalline arrays following thetreatment of leaf tissue with air pollutants, such as peroxyacetyl nitrate or ozone.

Many reports on the existence of crystalline, proteinaceous inclusions in plant cellsor organelles have concentrated on the natural occurrence of this phenomenon inhealthy plant tissue, or the induction of depositions as a result of physiological stressby a variety of mechanical or chemical treatments. During an investigation of theinfection of Phaseolus tissue by the phytopathogenic ascomycete fungus Sclerotinia,it was discovered that chloroplasts of the host underwent dramatic changes in theirultrastructural appearance (Tariq, 1984). Electron-opaque deposits, variable in formbut often exhibiting a high degree of structural organization, appeared in the stroma ofthe chloroplasts. The change in appearance of these organelles was greatest in thosecells close to the penetration site of the pathogen.

This paper provides additional information on the structure and chemical nature ofthese deposits, obtained by electron microscopy and ultrastructural cytochemistry.

MATERIALS AND METHODS

Plant materialSeeds of Phaseolus vulgaris, cultivars The Prince, Laura and Groffy, and P. cocdneus L. cv.

Achievement (seed source: Elsoms Seeds Ltd, U.K.) were sown in John Innes no. 3 compost (4seeds/14cm diam. pot). The beans were glasshouse-grown under natural light conditions and aminimum temperature of 12°C.

Fungal materialIsolate SS4074 of Sclerotinta sclerotiorum Lib. de Bary (obtained from infected Chrysanthemum

spp.) and isolate SM03 of S. minor Jagger (from Dr Van Etten, Canada) were maintained at 25 °C, inthe dark, on 3-9% (w/v) Difco potato dextrose agar (PDA), containing 18 p.p.m. penicillin and50 p.p.m. streptomycin.

Inoculation of plant materialFully expanded primary leaves of 2- to 3-week old bean seedlings were excised and the cut ends of

their petioles wrapped in tissue saturated with sterile distilled water. Leaf tissue was inoculated withthe pathogen using agar plugs (5 mm diam.) cut from the advancing margin of a 3-day-old colony ofthe fungus on PDA, and inverted on the adaxial surface of the leaf (two inocula/half leaf).Inoculated material was incubated in plastic sandwich boxes, lined with saturated water-absorbentpaper, at 20 °C, under fluorescent lights (3500 lux), or in the dark at 25 °C. Following incubation for10— 23 h, samples of leaf tissue bearing infection sites were fixed in 0-5% (w/v) parafor-maldehyde—1 % (w/v) glutaraldehyde in 0-1 M-sodium cacodylate-HCl buffer, pH 7-2, for 90 minbefore preparation for electron microscopy.

Effect of oxalic and citric acids on chloroplastsThe adaxial surface of primary leaves, excised from 2-week-old bean seedlings, was evenly

abraded using a dense suspension of purified Kieselguhr (BDH Chemicals Ltd, U.K.) in sterile

Chlomplast changes during infection 197

distilled water. The leaves were then rinsed three times in sterile distilled water. Discs (10 mmdiam.) of leaf tissue were cut and placed in one of the following solutions (two discs/solution): (1)0-1 M, 0-01 M or 0-001 M-oxalic acid (pH 1 -9, 2-5 and 3-2, respectively); (2) 0-1 M or 0-01 M-citric acid(pH2-4 and 2-8, respectively); (3) 0-01 M or 0001 M-oxalic acid-KOH (pH7-2); (4) 0-01 M or0-001 M-citric acid-KOH (pH7-2); (5) sterile distilled water (pH7-2). The leaf discs wereincubated in their respective solutions for 3 h at room temperature (~ 21 °C), after which time theywere removed and rinsed briefly in sterile distilled water. Samples of leaf tissue (approx. 2 mm )were cut from the centre of each disc and fixed in paraformaldehyde-glutaraldehyde in 0-1M-cacodylate buffer (pH 7-2) for 1 h before their preparation for transmission electron microscopy.

Electron microscopyLeaf tissue was prepared for electron microscopy using the procedure previously described (Tariq

& Jeffries, 1984).

Protein digestionUltrathin serial sections (~ 90 nm) of embedded leaf tissue were mounted on hexagonal 300 mesh

copper or nickel grids, coated with Formvar. After drying overnight the grids were submerged in10% (v/v) hydrogen peroxide for 20min at 37 °C (Thorsch & Esau, 1983). After a brief wash indistilled water the experimental sections were treated at 37 °C for 7-30 h with one of the following:0-2% (w/v) aqueous protease, pH7-3 (Protease type xiv, Sigma Chemical Co. ,U.K.) ;0-5% (w/v)pepsin (Sigma Chemical Co., U.K. P-7000) in 0-1 M-HC1; or 0-3 % (w/v) aqueous trypsin, pH 8-0(Sigma Chemical Co., T-0134). Control grids, bearing sections cut serially to those used in thedigestion tests, were treated simultaneously using water in place of the enzyme. After incubation thegrids were washed briefly in distilled water and allowed to dry. Sections were stained using uranylacetate (2%, w/v) and lead citrate (Reynolds, 1963) and examined in a Philips 410 transmissionelectron microscope.

OBSERVATIONS

Infected host tissueAs reported previously (Tariq, 1984; Tariq & Jeffries, 1984) the infection of

healthy Phaseolus leaf tissue, occurring 8 to 10 h after inoculation, was preceded bythe formation of appressoria on the host cuticle. The cuticle was then breached by apenetration peg, which, upon entry into the upper epidermal wall of the host,developed into a subcuticular vesicle. Twelve hours after inoculation subcuticularhyphae were observed radiating out from the point of penetration, whilst infectionhyphae, growing inter- and intracellularly, extended into the ground tissues (i.e.palisade and mesophyll) and reached the lower epidermis. The vascular tissues withinthe leaf exhibited few signs of cellular disorganization at this stage during the infectionprocess.

One of the most dramatic changes occurring during the colonization of host tissuetook place within the chloroplasts. Chloroplasts in advance of the mycelial front of theinfection hyphae, or in the immediate vicinity of inter- and intracellular hyphae of thepathogen, possessed electron-opaque deposits within the stroma (Fig. 1). Theintensity of this deposition decreased in chloroplasts farther away from the pen-etration point, while chloroplasts in regions of healthy host tissue appeared normal

198 V.-N. Tariq and P. Jeffries

Figs 1-3. For legends see p. 200

Chloroplast changes during infection 199

Figs 4—10. For legends see p. 200

200 V.-N. Tariq and P. Jeffries

and apparently unaffected (Fig. 2). The electron-opaque deposits developed initiallyat the edge of the chloroplasts, in close proximity to the double membrane forming theboundary of these organelles (Fig. 1), but eventually spread throughout the stroma.Fig. 3 illustrates an early stage during formation of the deposits and reveals that theyinitially appeared as a crystalline square lattice of periodicity approximately 10 nm.However, as the size and intensity of the deposits increased, their appearance becamemore amorphous. Variations in the size and ultrastructural appearance of the depositsmay be a reflection of: (1) variations in the plane of sectioning; and (2) the orientationof the deposits with respect to the electron beam. When the crystal was orientated sothat the electron beam was parallel to one of the crystallographic planes of the depositthe image appeared as a series of parallel striations. The distance between striationswas approximately 10 nm (Fig. 4). Tilting the specimen with respect to the electronbeam caused the orientation of the striations to change through 90°, whilst thedistance between the striations remained the same (Fig. 6). When the electron beam

Fig. 1. Two adjacent cells within the mesophyll of a primary leaf of P. vulgaris (cv.Laura), 20h after inoculation with isolate SS4074. Chloroplasts possess electron-opaquedeposits in early (filled arrow) and late (open arrow) stages of formation. X17 250.

Fig. 2. A healthy chloroplast within the mesophyll of a leaf of P. coccineus (cv.Achievement), lOh after inoculation with isolate SS4O74. X24960.

Fig. 3. Early stage of deposit formation (arrows) in the stroma of a chloroplast of thepalisade layer in P. vulgaris (cv. Prince), 10h after inoculation with isolate SS4074.X41600.

Figs 4—6. Deposits in stroma exhibiting square crystalline lattice. From mesophyll cell ofP. vulgaris (cv. Groffy), 23 h after inoculation with isolate SM03. X73 100. Fig. 4. Tiltangle, - 9 ° ; Fig. 5. Tilt angle, 0°; Fig. 6. Tilt angle, +12°.

Fig. 7. Higher magnification of deposit from mesophyll cell of P. vulgaris (cv. Groffy),23 h after inoculation with isolate SM03. X116 550.

Fig. 8. Extensive, amorphous electron-opaque deposits in chloroplasts of a mesophyll cellof P. vulgaris (cv. The Prince), 12 h after inoculation with isolate SS4074. X9890.

Fig. 9. Electron-opaque deposits in chloroplasts of the palisade layer of an uninfected leafof P. vulgaris (cv. The Prince) following incubation in 0-001 M-oxalic acid (pH 3-2).X7826.Fig. 10. Higher magnification of Fig. 9 showing fibrillar (•) or lattice (arrow) structuredependent on orientation of deposits with respect to plane of sectioning. X 34 400.

Figs 11-20. Results of protein digestion study.Fig. 11. Extensive deposition within chloroplast of a cell of P. vulgaris (cv. The

Prince), 12h after inoculation with isolate SS4074. X12470.Fig. 12. Serial section to that shown in Fig. 11 following incubation in pepsin for 15 h.

X12 470.Fig. 13. Chloroplasts from a mesophyll cell of P. vulgaris (cv. The Prince) 12 h after

inoculation with isolate SS4074. X7826.Fig. 14. Serial section to that shown in Fig. 13, following incubation in pepsin for 25 h.

Note the complete digestion of the electron-dense deposits. X7826.Fig. 15. Hypha (arrow) of SS4074 within mesophyll cell of P. vulgaris (cv. The Prince)

12 h after inoculation. The adjacent chloroplasts exhibit extensive electron-opaquedeposition. X3698.

Fig. 16. Serial section to that shown inFig. 15 folio wing incubation in protease f or 15 h.The deposits have been digested. X3698.

Chloroplast changes during infection 201

Figs 11-16

202 V.-N. Tariq and P. Jeffries

19

Fig. 17. Chloroplast from uninfected region of the mesophyll of P. vulgaris (cv. ThePrince) following incubation in water for 25 h. X29 240.

Fig. 18. Chloroplast from uninfected region of the mesophyll of P. vulgaris (cv. ThePrince) following incubation in pepsin for 25 h. There has been little change inultrastructural appearance, when compared with the control tissue shown in Fig. 17.X24940.Fig. 19. Chloroplast from leaf of P. vulgaris (cv. The Prince) following incubation in0-01 M-oxalic acid, pH 2-5. Note the presence of electron-opaque deposits. X9890.

Fig. 20. Serial section to that shown in Fig. 19, following incubation in protease for 25 h.The electron-opaque deposits have been digested. X9890.

was simultaneously parallel to both crystallographic planes the internal structure ofthe deposit appeared as a square lattice (Figs 5, 7). Deposits orientated randomly withrespect to the electron beam appeared amorphous (Figs 1,8).

Chloroplast changes during infection 203

Effect of oxalic and citric acids

The treatment of healthy leaf tissue with oxalic acid (1-100 mM) (Figs 9, 10) orcitric acid (10-100 mM) resulted in the appearance of electron-opaque deposits withinthe stroma of the chloroplasts. Ultrastructurally, these deposits appeared very similarto those formed in leaf tissue as a result of infection by Sclerotinia. The chloroplasts incontrol tissue, incubated in distilled water, remained healthy in appearance.Incubation of leaf tissue in solutions of the potassium salts of these acids, at pH 7-2,did not result in the formation of deposits and the chloroplasts appeared unaffected bythis treatment, suggesting it was the pH change, resulting in an acidic environmentwithin the tissue, that was responsible for the deposition of crystalline material withinthe stroma of chloroplasts.

Protein digestion

The size and ultrastructural appearance of the chloroplast deposits suggested thatthey were proteinaceous and a protein digestion study was carried out to corroboratethis observation. The results are illustrated in Figs 11-20. Figs 11 and 12 representserially cut sections of the same chloroplasts. The chloroplasts in Fig. 11, incubated indistilled water, possess a high degree of electron-dense deposition. Fig. 12 illustratesthe complete digestion of these deposits during incubation for 25 h in pepsin. Similarresults were obtained when tissue was treated with pepsin for 7 or 15 h (Figs 13, 14)and with protease for 7—25 h (Figs 15, 16). Treatment with trypsin resulted in onlypartial digestion of deposits. Healthy chloroplasts within the same tissue remainedunaffected by each proteolytic treatment (Figs 17, 18). The deposits induced byoxalic and citric acids were similarly digested by proteolytic action (Figs 19, 20).

DISCUSSION

Electron-opaque proteinaceous deposits were formed in the chloroplasts ofPhaseolus leaf tissue, as a result of infection of the latter by Sclerotinia species.Comparison of our results with published reports has revealed considerable similaritybetween the deposits illustrated here and those of Gunning et al. (1968) and Wrischer(1973). These previous reports have suggested that the deposits were RuDP-carboxylase (Fraction 1 protein), a protein of approximately 560X103 molecularweight and pH optimum 7-2-8-9 (Kirk & Tilney-Bassett, 1978). The stroma containsnumerous other proteins as well as the enzymes involved in the CC^-fixation cycle ofphotosynthesis. However, at least half of the water-soluble protein of the chloroplastis RuDP-carboxylase (Kirk & Tilney-Bassett, 1978). RuDP-carboxylase is therefore aprotein in the right location and in sufficient quantity to form the electron-opaquedeposit described here, although confirmation of this would require the isolation anddetailed biochemical characterization of the deposits. Haselkorn, Fernindez-Mordn,Kieras & Van Bruggen (1965) undertook a combined electron microscopic andbiochemical investigation of Fraction 1 protein from Chinese cabbage leaves. Theirattempts to crystallize the isolated protein from ammonium sulphate solutions

204 V.-N. Tariq and P. Jeffries

produced fibrous precipitates, which when aggregated appeared to be linear. Theexamination of negatively stained particles of Fraction 1 protein revealed that thelatter consisted of a cube, approximately 12 nm along each edge, containing 24subunits. There is thus some structural similarity between this aggregate and thedeposits induced in Phaseolus by Sclerotinia. Our observation of only partialdigestion of deposits by trypsin may reflect the fact that trypsin is a more specificproteolytic enzyme, catalysing the hydrolysis of peptide bonds whose carbonylfunction is donated by a lysine or an argenine residue (Lehninger, 1978).

In summary, our results indicate that a major stromal protein within chloroplasts ofPhaseolus tissue, undergoes precipitation as a consequence of the colonization of thistissue by Sclerotinia. We believe that this protein may be RuDP-carboxylase, and thatits deposition is the result of physiological stress brought about by the decrease in pH,caused by the secretion of oxalic acid by these fungi. Sclerotinia species are relativelyunspecialized necrotrophic pathogens, similar in many respects to Botrytis, yet thedeposition of crystalline material has not previously been observed in any othernecrotrophic infection. However, Sclerotinia differs from most other necrotrophs inthe secretion of relatively large amounts of oxalic acid during the colonization of hosttissues (Marciano, Di Lenna & Magro, 1983; Noyes & Hancock, 1981) and this mayexplain these differences in ultrastructural changes during pathogenesis. As a specificexample, Maxwell & Lumsden (1970) detected up to 48-3 mg of oxalic acid per gramdry weight of infected bean hypocotyl tissue. The role of oxalic acid duringpathogenesis remains uncertain, but several workers (Bateman & Beer, 1965;Marciano et al. 1983; Maxwell & Lumsden, 1970) have suggested that it actssynergistically with the cell wall-degrading enzymes secreted by the pathogen, bothby chelating cations inhibitory to the action of these enzymes, and by reducing the pHof the host tissue, providing a favourable acid environment for their action. Thetreatment of healthy host tissue with oxalic and citric acids resulted in deposits withinthe chloroplasts similar to those formed after infection, whilst treatment of tissue withthe potassium salts of these acids, at pH7-2, had no significant effect upon theirstructural organization. This suggests that precipitation of this electron-opaquematerial within the chloroplasts may be due to the dramatic decrease in pH, from pH 6to pH 4 (Marcianoetal. 1983; Maxwell & Lumsden, 1970; Noyes & Hancock, 1981),occurring during pathogenesis by Sclerotinia. A few other fungi, such as Sclerotiumrolfsii Sacc. (Bateman & Beer, 1965) andEndothiaparasitica (Havir & Anagnostakis,1983), also produce oxalic acid but no ultrastructural observations of thesehost-pathogen interactions have been published.

We thank the University of Kent, for the award of a studentship to the senior author (V.-N. T.),and Dr Keith Gull for helpful discussions.

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(Received 22 August 1984 -Accepted 5 November 1984)