Plant Physiol. (1986) 82, 1145-11470032-0889/86/82/1145/03/$01.00/0

Communication

Restriction Enzyme Analysis of Tomato Chloroplast andChromoplast DNA1

Received for publication November 6, 1985 and in revised form May 13, 1986

CHRISTINA M. HUNT, Ross C. HARDISON, AND CHARLES D. BOYER*Department ofHorticulture (C.M.H., C.D.B.) and Molecular and Cell Biology (R.C.H.), The PennsylvaniaState University, University Park, Pennsylvania 16802

ABSTRACT

Plastid DNA was isolated from the chloroplasts of tomato (Lycoper-sicon esculentum var Traveler 76) leaves and the chromoplasts of ripetomato fruit. Comparisons of the two DNAs were made by restrictionendonuclease analysis using PvuII, HpaI, and BglI. No differences inthe electrophoretic banding patterns of the restricted plastid DNAs weredetected, indicating that no major rearrangements, losses, or gains ofplastid DNA accompany the transition from chloroplast to chromoplast.

Central to the ripening process in tomato fruit is the transitionof chloroplasts in the green fruit to chromoplasts in the ripe fruit.In this process the morphology and biochemical activities of theplastids are profoundly altered. The granal thylakoids character-istic of chloroplasts degenerate and carotenoid inclusions form(5, 13). Chl is lost and photosynthetic activities cease, whereassynthesis of carotenoid pigments increases dramatically (12).

Chloroplasts contain DNA which encodes proteins functionalin photosynthesis and protein and RNA components of theplastid protein synthesis apparatus (2, 17). Other plastids alsocontain DNA, sometimes in quantities greater than chloroplastsof the same plant (15). However, the role of plastid DNA in thefunction of these organelles is not known.

This study addresses the question of whether changes in theplastid DNA are involved in the transition from chloroplast tochromoplast. Plastid DNAs were isolated from the leaves andfrom the red-ripe fruit of tomato. These DNAs were digestedwith restriction enzymes PvuII, HpaI, and Bgl I, and the electro-phoretic banding patterns of the fragments were compared. Anyrearrangements or other changes in plastid DNA that occurduring the plastid transition would alter the pattern of restrictionendonuclease cleavage sites and be detected as a change in sizeof restriction fragments. In previous work, Iwatsuki et al. (6)found no differences between tomato chloroplast and chromo-plast DNAs digested with BamHI and EcoRI. However, theircomparison does not include all the fragments produced by thesetwo enzymes. This study compares all the fragments from theDNAs digested with PvuII, HpaI, and Bgl I, whose cleavage siteshave been mapped (11), allowing the plastid genome to beexamined more thoroughly.

'Contribution No. 84. Department of Horticulture, The PennsylvaniaState University. Authorized for publication as Paper No. 7287 in thejournal series of the Pennsylvania Agricultural Experiment Station.

MATERIALS AND METHODS

Isolation of Organelles. Tomato (Lycopersicon esculentum varTraveler 76) chloroplasts were isolated according to Palmer (8)from leaves (250 g) of mature greenhouse plants maintained inthe dark for 48 h to eliminate starch granules.

Chromoplasts were isolated from the pericarp tissue (700 g) ofred-ripe tomato fruit according to Camara et al. (1) with thefollowing modifications. The discontinuous gradients consistedof 17, 6, and 6 ml of 1.45, 1.05, and 0.84 M sucrose solutions,respectively. The red chromoplast band was collected and diluted4-fold with wash buffer (0.4 M sorbitol, 50 mM Tris-HCl [pH8.0], and 25 mM EDTA), and centrifuged at 20,000g for 15 minat 40C.

Mitochondria were isolated from leaves oftomato plants about25 cm tall according to Fontarnau and Hernandez-Yago (4) withthe following changes. BufferA was modified to 0.35 M mannitol,50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.1% BSA (w/v), 2 mMa-ketoglutarate, and 15 mM 2-mercaptoethanol. Immediatelybefore the addition of MgCl2, the crude mitochondrial suspen-sion was allowed to warm to room temperature, then rechilled.Under these conditions, contaminating chloroplasts lyse, whilemitochondria, in the presence of the respiratory substratea-ketoglutarate, are stable (3). After treatment with DNase andwashing, DNA was extracted from the crude mitochondrialpellet.

Extraction of DNA. DNA was isolated from each of theorganelle preparations by the method of Palmer (8). The DNAswere extracted with CsCl-saturated isopropanol to remove ethid-ium bromide, dialyzed against 10 mM Tris-HCl (pH 8.0) and 0.1mM EDTA, and extracted with phenol precipitated by ethanolfrom 2 mm ammonium acetate.

Analysis with Restriction Endonucleases. The organelle DNAswere digested with the restriction enzymes PvuII, HpaI, andBgl I (New England Biolabs) according to the supplier's instruc-tions in 100 to 200 ,d total volume, followed by ethanol precip-itation. Fragments were separated by electrophoresis on a hori-zontal 0.5% agarose gel and visualized by staining with ethidiumbromide. The gel size was 27.5. x 17.5 x 0.5 cm and electropho-resis was for 15 to 20 h at 15 to 25 mamp in Tris-borate buffer(0.089 M Tris-borate, 0.089 M boric acid, and 2 mm EDTA).

Electron Microscopy. Samples from gradient fractions werefixed with glutaraldehyde, dehydrated, and embedded in epoxyresin. Plastid sections were stained 20 min with 3% uranyl acetatein 15% methanol and 1 min with lead citrate. Several sectionsfrom representative organelle preparations were examined.

RESULTS AND DISCUSSION

To ensure that the transition from chloroplast to chromoplastwas complete in the fruit used for chromoplast DNA isolation,

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Plant Physiol. Vol. 82, 1986

FIG. 1. Cross-section of an anomalousform from a chromoplast preparation.This structure has mitochondrial-likebodies (M) within an organelle that dis-plays characteristic features of chloro-plasts: osmophilic globules (0), crystallinethyaloid membranes (CT), and lack ofgrana (x27,500).

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thin sections of the chromoplast fraction were examined in theelectron microscope. Organelles with typical chromoplast char-acteristics were observed, i.e. osmophilic globules, crystallinethylakoid membranes, and lack ofgrana (5, 13). No chloroplastswere seen. Also present were anomalous structures with typicalchromoplast characteristics but containing inclusions which ap-pear to be mitochondria (Fig. 1). Camara et al. (1) report 'agglu-

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FIG. 2. Restriction enzyme analysis oftomato organelle DNAs. Chloroplast (Ct),chromoplast (Cm), and mitochondrial(Mi) DNAs were digested with PvuII,HpaI, and BgJ I restriction endonucleasesand fractionated by electrophoresis on0.5% agarose gels. For BglI digests twolanes were overloaded to show the smallerfiagments. Migration of size marker frag-ments are indicated by numbers (kb) inthe left margin.

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11

tination of heterogeneous membranes' occurred during the iso-lation of Capsicum annuum chromoplasts and show an electronmicrograph of a membrane-bound body containing structureswhich resemble mitochondria. Schmitt and Herrmann (14) andLarsson et al. (7) have noted the presence of multiorganellecomplexes containing mitochondria in isolated chloroplast prep-arations. Similar membrane fusion products containing mito-

1146 HUNT ET AL.

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TOMATO CHROMOPLAST DNA

chondria may also have formed in tomato fruit homogenates,giving rise to anomalous forms (Fig. 1) and correlating with thepresence of mitochondrial DNA in some chromoplast DNApreparations (Fig. 2).A comparison ofthe restriction endonuclease cleavage patterns

of chloroplast and chromoplast DNA showed that no majorrearrangements occurred during the transition from chloroplastto chromoplast. Figure 2 shows the electrophoretic patterns ofchloroplast, chromoplast, and mitochondrial DNAs digestedwith PvuII, HpaI, and BgJI. The same bands are present inchromoplast DNA as in chloroplast DNA for all three restrictionenzymes. Additional mainly weaker bands are present in varyingquantities in some preparations of the chromoplast DNA (com-pare lanes 2 and 3) and in heavily loaded chloroplast DNA (lane10). These bands are identical to the major bands obtained whenmitochondrial DNA is digested with the same enzymes. Withthe additional chromoplast bands identified as contaminatingmitochondrial DNA, the results demonstrate the identity of theelectrophoretic banding patterns for the three restriction enzymedigests compared. PvuII, HpaI, and BgJI sites for tomato chlo-roplast DNA have been mapped by Palmer and Zamir (10). Thesizes of the bands obtained in this study correspond to themapped fragment sizes and all mapped fragments are shown onthe comparison gels. The distribution of PvuII, HpaI, and BgJIsites allows the entire plastid genome to be examined in the sizerange of fragments that are well-resolved on these agarose gels(1-30 kb).

In some DNA preparations, the plastid bands were not presentin the expected stoichiometry. For example, in lane 3 the chro-moplast 14.1 kb PvuII fragment is much weaker than the 15.1kb fragment, while they are present at the expected equal inten-sities in the chromoplast DNA in lane 2 and the chloroplastDNA in lane 1. Thus these differences in band intensity do notreflect differences between chloroplast and chromoplast DNA,but rather they reflect variation from one DNA preparation toanother. The observed anomalies in band intensity could notresult from plastid-specific differences in 'flipping' of the singlecopy regions due to recombination between the inverted repeats.None of the restriction fragments examined spans an entire copyof an inverted repeat, a requirement for detecting flipping (9).A HpaI restriction site in tomato plastid DNA not shown on

the maps of Palmer and Zamir (10) or Phillips (1 1) was detected.Under conditions where all other HpaI sites were completelycut, a 3.5 kb fragment was observed in both chloroplast andchromoplast DNAs. This fragment was digested into the expected3.0 kb fragment (and presumably a 0.5 kb fragment) by theaddition of more HpaI restriction endonuclease. The newlydetected HpaI site is located approximately 0.5 kb on one sideof the 3.0 kb HpaI fragment. The exact position of this site isbeing determined.

Several other studies have compared restriction digest patternsof plastid DNAs from different plastid types. Thompson (16)compared the electrophoretic EcoRI banding patterns of DNAfrom isolated chloroplasts and chromoplasts of daffodil and

found them to be identical. Scott et al. (15) compared theelectrophoretic banding patterns of plastid DNA which werepresent in total DNA isolated from different tissues of potatoand digested with BamHI and PstI. No differences were detected,although all the bands produced by these two enzymes could notbe discerned. Iwatsuki et al. (6) compared many ofthe fragmentsof tomato chloroplast and chromoplast DNAs digested withBamHI and EcoRI and found no differences. The results ofthese studies and the one reported here comparing the completeplastid genomes of tomato chloroplasts and chromoplasts indi-cate that no major rearrangements, losses, or gains, of plastidDNA accompany the transformation between plastid types. Anyrearrangements that do not alter the size of the DNA restrictionfragments (such as inversions) would have to be restricted toregions between the PvuII, HpaI, and Bgl I sites. From the mapof Palmer and Zamir (10), it can be seen that the largest suchregion is about 20 kb. Therefore, it seems unlikely that large-scale DNA rearrangements play a role in plastid switching.

Acknowledgments-The authors thank Dr. K-C. Liu for assistance with theelectron microscopy of plastids and mitochondria and C. Stratton and Dr. J.Roscher of Campbell Institute for Research and Technology for providing tomatofruit.

LITERATURE CITED

1. CAMARA B, F BARDAT, 0 DOGBO, J BRANGEON, R MONEGER 1983 Terpenoidmetabolism in plastids. Isolation and biochemical characteristics of Capsi-cum annuum chromoplasts. Plant Physiol 73: 94-99

2. ELLIS RJ 1981 Chloroplast proteins: synthesis, transport, and assembly. AnnuRev Plant Physiol 32: 111-137

3. FERGUSON IB, MS REID, RJ RoMANI 1985 Effects of low temperature andrespiratory inhibitors on calcium flux in plant mitochondria. Plant Physiol77: 877-880

4. FONTARNAU A, J HERNANDEZ-YAGO 1982 Characterization of mitochondrialDNA in Citrus. Plant Physiol 70: 1678-1682

5. HARRIS WM, AR SPURR 1969 Chromoplasts of tomato fruits. II. The redtomato. Am J Bot 56: 380-389

6. IWATSUKI N, A HIRAI, T ASAHI 1985 A comparison oftomato fruit chloroplastand chromoplast DNAs as analyzed with restriction endonucleases. PlantCell Physiol 26: 599-601

7. LARSSON C, C COLLIN, P-A ALBERTSSON 1971 Characterization of three classesof chloroplasts obtained by counter-current distribution. Biochim BiophysActa 245: 425-438

8. PALMER ID 1982 Physical and gene mapping of chloroplast DNA for Atriplextriangularis and Cucumis sativa. Nucleic Acids Res 10: 1593-1605.

9. PALMER JD 1983 Chloroplast DNA exists in two orientations. Nature 301: 9210. PALMER JD, D ZAMIR lq82 Chloroplast DNA evolution and phylogenetic

relationships in Lycopersicon. Proc Natl Acad Sci USA 79: 5006-50101 1. PHILLIPS A 1985 Restriction map and clone bank oftomato plastid DNA. Curr

Genet 10: 147-15212. RAYMUNDO LC, CO CHICHESTER, KL SIMPSON 1976 Light-dependent carote-

noid synthesis in the tomato fruit. J Agric Food Chem 24: 59-6413. Rosso SW 1968 The ultrastructure ofchloroplast development in red tomatoes.

J Ultrastruct Res 25: 307-32214. SCHMITT JM, RG HERRMANN 1977 Fractionation of cell organelles in silica sol

gradients. In DM Prescott, ed, Methods in Cell Biology, Vol 15, AcademicPress, New York, pp 177-200

15. SCOTT NS, MI TYMMs, JV POSSINGHAM 1984 Plastid-DNA levels in thedifferent tissues of potato. Planta 161: 12-19

16. THOMPSON JA 1980 Apparent identity of chromoplast and chloroplast DNAin the daffodil. Narcissus pseudonarcissus. Z Naturforsch 35C: 1101-1103

17. WHITFELD PR, W BoTTOMLEY 1983 Organization and structure of chloroplastgenes. Annu Rev Plant Physiol 34: 279-3 10

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