Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/7111506

ExpressionofChloroplastandMitochondrialGenesduringMicrosporogenesisinMaize

ARTICLEinPLANTPHYSIOLOGY·JULY1992

ImpactFactor:6.84·DOI:10.1104/pp.99.2.396·Source:PubMed

CITATIONS

29

READS

13

5AUTHORS,INCLUDING:

FrançoiseMonéger

EcolenormalesupérieuredeLyon

29PUBLICATIONS1,233CITATIONS

SEEPROFILE

RégisMache

UniversityJosephFourier-Grenoble1

120PUBLICATIONS3,407CITATIONS

SEEPROFILE

Availablefrom:RégisMache

Retrievedon:04February2016

Plant Physiol. (1992) 99, 396-4000032-0889/92/99/0396/05/$01 .00/0

Received for publication April 26, 1991Accepted December 8, 1991

Expression of Chloroplast and Mitochondrial Genes duringMicrosporogenesis in Maize1

Fran9oise Moneger, Paul Mandaron, Marie-Fran9oise Niogret, Georges Freyssinet, and Regis Mache*Laboratoire de Biologie Moleculaire V6getale, Unit6 1178 du Centre National de /a Recherche Scientifique,Universit6 Joseph Fourier, BP 53, 38041 Grenoble Cedex, France (F.M., P.M., M.-F.N., R.M.); and Biologie

Moleculaire et Cellulaire Vegetale, Rhone-Poulenc Agrochimie, BP 9163, 69263 Lyon Cedex 09, France (G.F.)

ABSTRACT

Mitochondrial and plastid gene expression has been examinedduring maize (Zea mays) microsporogenesis. Accumulation oftranscripts was found for three mitochondrial genes studied (cob,atp6, and atp9) at the mid-term of pollen development. In contrast,these mitochondrial transcripts were undetectable in mature pol-len. Southern and DNA gel blot experiments showed that the copynumber of mitochondrial genes was amplified in microspores atstages preceding the accumulation of these transcripts. Plastidtranscripts of the photosynthetic psbA and rbcL genes could notbe detected after the two mitoses, whereas precursors of the16S rRNA are detected at low levels.

Cytoplasmic maternal inheritance and CMS2 are two bio-logical traits showing the importance of organelles duringmicrosporogenesis. In the case of maize, genetic and cytolog-ical evidence has been given for the lack of paternal inherit-ance of plastid and mitochondrial DNA (2, 4, and referencestherein). Different authors have tried to explain the mecha-nisms implied in maternal inheritance (5) and different hy-potheses have been brought forward. One of these hypothesessuggests (7) destruction of the paternal plastid genome duringmicrosporogenesis in barley to account for maternal inherit-ance. Knowledge about the persistence and integrity of organ-elle DNA during pollen formation would therefore help toevaluate this hypothesis. Recently, pollen DNA from Medi-cago sativa or Antirrhinum majus, known to transmit organ-elle genes biparentally and maternally, respectively, wasprobed with a plastid-specific DNA fragment (5). It was shownthat the presence or absence of plastid DNA correlated withthe type of inheritance. It would be interesting to knowwhether this result might be generalized and valid in otherplant species.

In the case of CMS, blocks occur in critical steps in thedevelopment of the male flower. CMS has been extensivelystudied in maize and it is now accepted that the CMS phe-notype is correlated with recombination events in the mito-

This work was supported by Rh6ne Poulenc Agrochimie. M.-F.N. is financially supported by a grant from l'Institut de la RechercheAgronomique.

2 Abbreviations: CMS, cytoplasmic male sterility; PM, premature;M, mature; S, starch-containing microspores; V, vacuolated micro-spores; bp, base pairs; kb, kilobase pairs.

chondrial genome leading to the production of variant poly-peptides (at least in the case of CMS-T), which are proposedto affect development of the male flower. Lee and Warmke(14) have shown by EM studies that the number of mitochon-dria increases in anthers of both normal and T-sterile lines ofmaize. This increase takes place both in the sporogenous cellsand in the tapetum of anthers. The authors suggested that therapid multiplication of mitochondria could induce a condi-tion of stress to which the male sterile lines of maize responddifferently than the normal lines. This hypothesis emphasizesthe importance of genetic events that occur in mitochondriaduring the normal development of pollen.

Following meiosis in the male flower of maize, each mi-crospore develops individually. After vacuole formation inthe unicellular microspore, two successive haploid mitosesoccur, giving rise to a large vegetative cell and two spermcells. Starch is accumulated in the amyloplasts contained inthe vegetative cell until maturation of the pollen. A methodhas been developed in our laboratory to isolate microsporesat different steps of their development without contaminationby diploid cells from anther tissues ( 17).We present here the study of the expression of some chlo-

roplast and mitochondrial genes in isolated microspores atdifferent steps of their development. The results show thatgene transcripts from mitochondria are present at highersteady-state levels at mid-term of the microsporogenesis com-pared with leaves. Following the two mitoses in microspores,plastid transcripts, with the exception of a very low amountof 16S rRNA, are no longer detectable.

MATERIALS AND METHODS

Growth Conditions for Maize

Seeds of two fertile lines of maize (Zea mays), MO 17 andRP 704, were provided by Rh6ne Poulenc Agrochimie. Plantswere grown in a greenhouse under defined conditions: light,50,000 lux; temperature, 22°C in the day (15 h) and 20°C inthe night (9 h); RH approximately 60%. Plants producedpollen after 2 months.

Preparation of Microspores

The timing of differentiation of the microspores follows agradient along the tassel. To minimize heterogeneity beforethe preparation of microspores, tassels were separated in threegroups (17): (a) young tassels that are still embedded in the

396

ORGANELLE GENE EXPRESSION DURING MICROSPOROGENESIS

leaves and contain microspores with a vacuole (V) and (b)mid-tassels that are clearly visible and contain microsporesaccumulating starch (S1, S2). The third group containedmature tassels with dehiscent anthers. These contained thepremature microspores full of starch (PM) and completelymature microspores (M). Spikelets collected from tassels ofthe same group were quickly ground in water with a Polytron.The homogenate was fractionated by successive filtrationthrough 120- and 50-,um mesh size metallic sieves. For youngtassels, a 50-,um mesh size sieve was needed to collect the V-stage microspores. For mid-tassels a 30% Percoll cushion wasused to separate the V-stage microspores from the S micro-spores that pelleted through the cushion. The mature tasselswere shaken to collect mature pollen grains released fromdehiscent anthers. The remaining flowers, still closed, con-tained PM-stage microspores that were nearly mature com-pared with the S2-stage microspores. After filtration, micro-spores were collected and sedimented. These preparationswere free of other tissues and debris. Viability of M micro-spores was checked by incubation in germination mediumaccording to Cook and Walden (3). Material was either usedimmediately or pelleted, frozen in liquid nitrogen, and storedat -700C.

Nucleic Acids Preparation from Microspores

Microspores were ground in a liquid nitrogen-cooled Spex6700 Freezer Mill (Bioblock, Strasbourg, France). The extrac-tion of nucleic acids from the resulting powder was performedin lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2%SDS, 2% sodium sarcosinate, and 10 mm EDTA) and depro-teinized using phenol/chloroform according to ref. 18. Puri-fication of DNA was achieved by CsCl centrifugation. Diffi-culties were encountered in estimating the amount of DNAto be used for hybridization due to the presence of contami-nating polysaccharides in CsCl-purified microspore DNA. Toovercome this problem, the initial CsCl-ethidium bromidecentrifugation was followed by a centrifugation in CsCl-bis-benzimide, which separated DNA from the starch (16). In thecase of DNA extracted from M pollen, polysaccharides werestill present even after this treatment. The DNA obtained wasfound to be of sufficient purity for hybridization with differentprobes.To prepare RNA, total nucleic acid extracted from micros-

pores was precipitated with cold ethanol, pelleted, and resus-pended in water. High mol wt RNA was prepared by differ-ential precipitation from LiCl as described (18) and used forfurther analysis.

Chloroplast DNA Preparation

Chloroplasts were purified on a continuous 20 to 60%sucrose gradient and DNA was isolated from green leaves ofmaize plants following conventional procedures and followedby centrifugation in CsCl gradients.

Southern, Northern, and DNA Gel Blot Experiments

Total DNA was restricted using BamH 1 and EcoR 1 (Boeh-ringer) restriction endonucleases, electrophoretically sepa-

rated and transferred to nylon membrane using standardprocedures. "DNA gel blot" experiments were done in thesame manner as Southern experiments but the DNA was notrestricted with any enzyme prior to loading. Ten or 15 Ag oftotal cellular RNA were size fractionated by electrophoresisin a 1.2% agarose/5 mM hydroxymethyl mercury gel, stainedwith ethidium bromide, and transferred to nylon membraneby capillary blotting in 10 x SSC. Chloroplast probes werefrom spinach. Nuclear and mitochondrial probes were frommaize. The following DNA fragments were used as probes:the 750 bp XbaI-PstI intragenic fragment of the psbA gene(26); the 550 bp SaIl-EcoRV fragment containing the rpsl9gene (27); the 1200 bp EcoRI-PstI intragenic fragment of therbcL gene (30); the 1200 bp EcoRI-BamHI intragenic frag-ment of the rrn 16S gene (1); the 1200 bp EcoRI-HindIIIintragenic fragment of the atp6 gene (8); the 2200 bp XbaIfragment containing the atp9 gene (9); the intragenic 680 bpHindIII-EcoRI fragment of the cob gene(6); the 1000 bpEcoRI intragenic fragment ofthe rbcS gene. Labeling of insertswas done by the random hexamer priming method to aspecific activity ranging from 1 to 5 x 109 cpm/,ug. Prehy-bridization and hybridization conditions were at 42°C in 50%formamide, 1 M NaCl, 1% SDS, and 0.2 mg/mL salmonDNA. Samples (108 cpm) of each probe was used for hybrid-ization. Washes were performed 10 min at room temperaturein 2 x SSC, 1 h in 2 x SSC, 1% SDS at 65C, and 1 h atroom temperature in 0.1 x SSC. RNA molecular size markers(0.3-7.4 kb) from Boehringer were used in the northernexperiment with the rrn 16S probe; the 5.3 and the 1.6 kbRNA fragments of the marker are revealed with the labeledprobe. Experiments were made from two to four times,independently.

RESULTS

Expression of Chloroplast and Mitochondrial Genes inMicrospores

Homogeneous populations of microspores were used fromthe following stages defined in "Materials and Methods": V,SI, S2, PM, and M. To obtain experimental data on plastidgenes during microsporogenesis, northern-type experimentswere carried out using total RNA extracted from microsporesat these stages and from leaves with cpDNA probes containingintragenic fragments of psbA, rbcL, and rrn 16S genes (Fig.1). As can be seen after ethidium bromide staining, theamount of RNA for samples of mature stages (PM and M) islower than for the young stages, although the same amountof RNA, as estimated by UV absorbance, has been loaded onthe gel. We observe that many additional bands are presentin young microspores (V and S 1-2), which tend to disappearin older stages. These bands seem to reflect the in vivosituation instead of a degradation during the extraction proc-ess because an identical electrophoretic pattern was obtainedwith RNA extracted from fresh whole anthers immediatelydropped into liquid nitrogen after harvesting (not shown).The origin of these bands remains unknown but seems to bespecific for young microspores. A very small amount ofpsbAtranscript, but no rbcL transcript, was detected in vacuolatedmicrospores (V stage) at the beginning of microsporogenesis.

397

Plant Physiol. Vol. 99, 1992

Figure 1. Hybridization of the chloroplast psbA,rbcL, and rrn 16S gene probes to RNA isolatedfrom microspores at different stages of devel-opment (V, Si-2, PM, M) and from leaves (L).Ten micrograms of RNA were size separated onhydroxymercury gel. Ethidium bromide fluores-cence of RNAs is shown on the left side. Probeswere labeled as indicated in "Materials and Meth-ods." Exposure time was of 24 h (psbA), 96 h(rbcL), 10 h (rrn 16S with microspore RNA), or6 h (rrn 16S with leaf RNA). Size of transcriptsare indicated in kb.

V SI2 PM M L V S12 PMM iL V SI.. PM M

*25S

418S416 5

qw

psbA rbc

At later stages, no transcripts from the two photosyntheticgenes could be detected even after prolonged exposure time.The results obtained with the rrn 16S probe reveal the

presence ofa small amount ofan RNA fragment with a highersize than the mature 16S rRNA. The size of the fragmentcorresponds approximately to that ofthe precursor 16S rRNA(about 1.9 kb). This putative pre-16S RNA is present at allstages of microspore development but possibly in a decreasingamount. The presence of the same precursor 16S rRNAspecies was also detected in mature pollen in another inde-pendent experiment (not shown).

Similar experiments were performed using mitochondrialprobes prepared from cob, atp6, and atp9 genes (Fig. 2). Thepresence of abundant transcripts from these genes is revealedin S2 microspores. In contrast, no transcripts could be de-tected at a significant level in M pollen. In leaves, the atp9and cob transcripts are clearly detected, whereas the atp6transcripts were only detected after a longer exposure time ofthe autoradiograph.

Analysis of Plastid DNA Contained in Microspores

Southern experiments using chloroplast gene probes werecarried out to see whether the absence of transcripts could be

S2 M L S. M L S. M L

* _

....* f::

cob atp9

L-.'p6

atp6

Figure 2. Hybridization of the mitochondrial cob, atp6, and atp9probes to RNA isolated from S2 microspores, from pollen at dehis-cence (stage M) and from leaves (L). Fifteen micrograms of RNA weresize separated on an agarose gel containing hydroxymercury. Ethid-ium bromide fluorescence of RNA is shown on the left side . Autora-diographs were obtained after 8 h (atp6 and cob probes) or 1 h (atp9)of exposure. Transcripts indicated with a star are more clearly de-tected in leaf RNA after a longer time of exposure of the autoradi-ograph but are not detected in M pollen RNA.

related to a disappearance of the plastid genome. The psbA,rbcL, and rpsl 9 genes have been located in specific restrictedfragments of maize chloroplast DNA by other authors (13).The psbA gene is in the 4.6-kb BamHI-8 fragment, the rbcLgene in the 4.35-kb BamHI-9 fragment, and the rpsl9 genein the 5.3-kb BamHI-6 and 4.6-kb BamHI-8 fragments or inthe 2.2-kb EcoRI-m fragment. In our experiments, thesecharacteristic fragments were detected in leafDNA as well asin the microspore DNA (Fig. 3). Plastid DNA is also presentin M pollen as shown by the identification of the BamHIrestriction fragment bearing the psbA gene. The three chlo-roplast gene probes reveal additional bands that are detectedafter a longer exposure in microspore DNA but not in leafDNA: a 12- and a 5.6- kb BamHI fragment containingsequences homologous to the rbcL gene, two EcoRI fragments(of 4 and 1.8 kb, respectively) and one 10.5-kb BamHIfragment revealed by the rpsl9 probe, and a 6.7-kb BamHIfragment revealed by the psbA probe. By their sizes, thesecannot be explained as incomplete digestion bands. Theycould be of mitochondrial origin because it is well established

psbA rbcL rps 19L S2 M L U2 L 82 L 82

_ 4.6 4

:~~~~~~~~~~~~~~~~~~~~' 2:

B3amHll BamHl BamHl F-'-oRl

L 52 L 5

I .6.8.s64

Figure 3. Hybridization of organelle gene probes to restricted DNAfragments from leaves (L), microspores (S2), and mature pollen (M).Total DNA was restricted by BamHl or EcoRI. Sizes of restrictionfragments are given in kb. Probes used are indicated at the bottomof each autoradiogram. The size of additional bands observed onoverexposed autoradiograms is also indicated. The amount of DNAloaded on the gel is: L and S2, 0.3 Ag; M, 1 /tg.

MONEGER ET AL.398

ORGANELLE GENE EXPRESSION DURING MICROSPOROGENESIS

that some cpDNA have been transferred to the mitochondrialgenome (15, 23-25).

Analysis of Mitochondrial DNA Contained in Microspores

An amplification of the mtDNA in microspores relative toleaves is observed by Southern-type hybridizations performedwith mitochondrial probes (Fig. 3). Total DNA from leavesor from microspores at the stage of starch accumulation (S2)was digested with BamHI and hybridized with mitochondrialatp6 and cob probes. Ethidium bromide fluorescence con-firmed that complete digestion was achieved (not shown). Thetwo genes are much more abundant in S2 microspores thanin leaves. Identical restriction fragments are revealed withatp6 and cob probes in leaf and microspore DNA. The 6.8-kb atp6 and 13-kb cob fragments correspond to the copy ofthe gene located on the "master circle" molecule according toFauron and Havlik (11). The other fragments likely corre-spond to copies of the gene located on submolecules of themtDNA. Two fragments of 1.95 and 1.8 kb bearing the cobgene appear specifically in microspore DNA. They are notdetected in leaf DNA even after a longer time of exposure.They might arise from amplification of submolecules of themitochondrial genome or from recombination events occur-ring during microsporogenesis.A second series ofexperiments were undertaken to estimate

the relative amount of mitochondrial genes during develop-ment of microspore. The same samples of unrestricted DNAextracted from microspores at different stages ofdevelopmentwere purified by electrophoresis and after blotting were hy-bridized with a nuclear probe (rbcS) and two different mito-chondrial genes (cob and atp6). The results (Fig. 4) clearlyshow that mitochondrial genes are present in all the stages ofdevelopment of the microspores, even in the M pollen grainwhere no expression ofthe three genes examined was detected(Fig. 2). The amount of mitochondrial DNA reached a max-imum at a time of microspore development between stages Vand S.

V Si S2 M L

.... .

cobFigure 4. Hybnidization of nuclear (rbcS) and mitochondrial (cob andatp6) gene probes to total DNA isolated from microspores at severalstages of development or from leaves. DNA was purified by electro-phoresis before blotting (see "DNA gel blot" experiments in "Materialsand Methods"). The same samples of DNA were used for the threehybridization experiments. Ten micrograms of DNA were depositedin each slot. Blots were hybridized with random-primed labeledprobes (rbcS, 1t9X 16 iCpM;cob, 5 x 1 m6cpm; atp6, 15 x 1e6CpM).X-ray films were exposed for 48 h (rbcS and cob) or 96 h (atp6).Stages of microspore development are given at the top of the figure.

DISCUSSION

Our results indicate an increase of the steady-state level ofmitochondrial transcripts in microspores at mid-term of de-velopment. The accumulation of mitochondrial transcriptsmight result from a gene dosage effect because it correspondsto the amplification of specific mitochondrial genes. We can-not exclude that, alternatively, activation of the transcriptionof the mitochondrial genes or stabilization of the mitochon-drial transcripts occurs in mid-term microspores.Very little is known about mitochondrial gene expression

during pollen development in comparison with nuclear geneexpression (19). It has been found that the accumulation ofcytosolic transcripts occurs during two separate phases ofmicrospore development. First, the "early" genes becomeactivated after meiosis, and second, the "late" genes becomeactive after microspore mitosis. The mRNAs of the latter setof genes increase in abundance up to maturity (19). Theobserved pattern of mitochondrial gene expression duringmicrosporogenesis appears to be distinct from either the earlyor late nuclear gene expression pattern. Mitochondrial tran-script abundance is highest in the middle stages of pollendevelopment but decreases significantly toward maturity. Wecan speculate that the increase of mitochondrial transcripts isrelated to an increase in mitochondrial proteins and possiblyto a higher respiration rate.An electron microscopic observation of microspores during

development has allowed Lee and Warmke (14) to detect anincrease in the number of mitochondria as well as a decreasein size of individual mitochondria at postmeiotic stages. Thisobservation has not been correlated with gene expression. Ourresults show an increase in mitochondrial gene copy numberrelative to the copy number of a nuclear gene and we suggestthat this increase is related to the larger number of mitochon-dria observed (14). Mitochondrial division, DNA replication,and gene transcription might occur concomitantly during themid-phase of microspore development.

Amplification of the mitochondrial genome might haveimportant consequences in the context of CMS. Following asuggestion ofWarmke and Lee (28), we speculate that genomeamplification is at the origin of the abortion of mitochondriain anthers of the male sterile lines of maize. The products ofchimeric genes occurring in cytoplasmic sterile lines (10, 22,29) might result in the abortion of anther cells in overexpres-sion of mitochondrial genes accompanying amplification ofpart of the mitochondrial genome, which does not take placein other tissues of the plant.The psbA and the rbcL genes have been used as represent-

ative plastid-encoded genes. Very low numbers of transcriptsof the psbA gene have been detected in juvenile microsporesonly. These transcripts might be remnants of premeiotictranscription. Our results indicate that the two photosyntheticgenes are not transcribed at an appreciable level during mi-crosporogenesis, or that a rapid degradation of the RNAproducts occurs. This result might be extended to the expres-sion of other plastid-encoded photosynthetic genes in micros-pores. A comparable situation occurs in chromoplasts thatresult from the redifferentiation of chloroplasts in tissues,such as those of the tomato fruit during ripening. In the latter,the transcript levels for photosynthetic genes decrease to low

399

Plant Physiol. Vol. 99, 1992

or nondetectable levels in parallel with the differentiation ofchloroplasts into chromoplasts (12, 21).We assume that the rRNA species detected in microspores

corresponds to the unprocessed form of 16S rRNA. As freeRNA molecules appear to be degraded in plastids, any existingpre- 16S rRNA molecules are probably found in complexeswith ribosomal proteins. The detection of pre- 16S rRNAmight suggest that the synthesis of plastid ribosomes, andhence translation, in microspores is inhibited, or proceeds atan extremely low level. A similar situation might occur intomato chromoplasts where transcripts of the plastid genecoding for the ribosomal protein S4 has been undetectable(12) and where the level of 16S rRNA decreases duringchromoplast differentiation (20).

Results concerning the plastid genome in microspores showthat no rearrangement or deletion ofthe few chloroplast genesthat have been tested occurs in microspores during theirdevelopment or at maturity. Our results are in contrast withthose obtained by Corriveau et al. (5). These authors couldnot detect plastid DNA in M pollen of A. majus, a species,like maize, known to transmit plastids maternally. We suggestthat plastidDNA is present in the vegetative cells ofthe pollenonly and not in sperm cells. The absence of plastid in spermcells would explain the strict maternal inheritance previouslyobserved (2, 4).

ACKNOWLEDGMENTS

We thank Prof. C.S. Levings III and Prof. C. Leaver for theirauthorization to use mitochondrial probes originated from theirlaboratory. We are grateful to F. Vedel and M. Lebrun for the gift ofclones, and to H. Pesey for her assistance.

LITERATURE CITED

1. Briat JF, Dron M, Loiseaux S, Mache R (1982) Structure andtranscription of the spinach chloroplast operon rDNA leaderregion. Nucleic Acids Res 10: 6865-6878

2. Conde M-F, Pring D-R, Levings C-S III (1979) Maternal inher-itance oforganelle DNA's in Zea mays-Zea perennis reciprocalcross. J Hered 70: 2-4

3. Cook FS, Walden DB (1965) The male gametophyte of Zea maysL. II. In vitro germination. Can J Bot 43: 779-786

4. Corriveau JL, Coleman AW (1988) Rapid screening method todetect potential biparental inheritance of plastid DNA andresults for over 200 angiosperm species. Am J Bot 75:1433-1458

5. Corriveau JL, Goff LJ, Coleman AW (1990) Plastid DNA is notdetectable in the male gametes and pollen tubes of an angio-sperm (Antirrhinum majus) that is maternal for plastid inher-itance. Curr Genet 17: 439-444

6. Dawson AJ, Jones VP, Leaver CJ (1984) The apocytochrome bgene in maize mitochondria does not contain introns and ispreceded by a potential ribosome binding site. EMBO J 3:2107-2113

7. Day A, Ellis THN (1984) Chloroplast DNA deletions associatedwith wheat plants regenerated from pollen: possible basis formaternal inheritance of chloroplasts. Cell 39: 359-368

8. Dewey RE, Levings CS III, Timothy DH (1985) Nucleotidesequence of ATPase subunit 6 gene of maize mitochondria.Plant Physiol 79: 914-919

9. Dewey RE, Schuster AM, Levings CS III, Timothy DH (1985)Nucleotide sequence of Fo-ATPase proteolipid (subunit 9)gene of maize mitochondria. Proc Natl Acad Sci USA 82:1015-1019

10. Dewey RE, Levings CS III, Timothy DH (1986) Novel recom-binations in the maize mitochondrial genome produce aunique transcriptional unit in the Texas male-sterile cytoplasm.Cell 44: 439-449

11. Fauron CMR, Havlik M (1988) The BamHl, Xhol, SmaIrestriction enzyme maps of the normal maize mitochondrialgenome genotype B37. Nucleic Acids Res 16: 10395-10396

12. Kobayashi H, Ngernprasirtsiri J, Akazawa T (1990) Transcrip-tional regulation and DNA methylation in plastids duringtransitional conversion ofchloroplasts to chromoplasts. EMBOJ 9: 307-313

13. Larrinua IM, Muskavitch KMT, Gubbins EJ, Bogorad L (1983)A detailed restriction endonuclease site map of the Zea maysplastid genome. Plant Mol Biol 2: 129-140

14. Lee SLJ, Warmke HE (1979) Organelle size and number infertile and T-cytoplasmic male-sterile corn. Am J Bot 66:141-148

15. Lonsdale DM, Hodge TP, Howe CJ, Stern DB (1983) Maizemitochondrial DNA contains a sequence homologous to theribulose-1,5-bisphosphate carboxylase large subunit gene ofchloroplast DNA. Cell 34: 1007-1014

16. Macherel D, Kobayashi H, Akazawa T (1985) Amyloplast nu-cleoids in sycamore cells and presence in amyloplast DNA ofhomologous sequences to chloroplast genes. Biochem BiophysRes Commun 133: 140-146

17. Mandaron P, Niogret MF, Mache R, Moneger F (1990) In vitroprotein synthesis in isolated microspores ofZea mays at severalstages of development. Theor Appl Genet 79: 134-138

18. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, NY

19. Mascarenhas JP (1990) Gene activity during pollen develop-ment. Annu Rev Plant Physiol Plant Mol Biol 41: 317-338

20. Piechulla B, Chonoles Imlay KR, Gruissem W (1985) Plastidgene expression during fruit ripening in tomato. Plant MolBiol 5: 373-384

21. Piechulla B, Pichersky E, Cashmore AR, Gruissem W (1986)Expression of nuclear and plastid genes for photosynthesis-specific genes proteins during tomato fruit development andripening. Plant Mol Biol 7: 367-376

22. Rottmann WH, Brears T, Hodge TP, Lonsdale DM (1987) Amitochondrial gene is lost via homologous recombination dur-ing reversion of CMS T maize to fertility. EMBO J 6:1541-1546

23. Sederoff RR, Ronald P, Bedinger P, Rivin C, Walbot V, BlandM, Levings CS III (1986) Maize mitochondrial plasmid SIsequences share homology with chloroplast gene psbA. Ge-netics 113: 469-472

24. Stern DB, Lonsdale DM (1982) Mitochondrial and chloroplastgenomes of maize have a 12-kilobase DNA sequence in com-mon. Nature 299: 698-702

25. Stern DB, Palmer JD (1984) Extensive and widespread homol-ogies between mitochondrial DNA and chloroplast DNA inplants. Proc Natl Acad Sci USA 81: 1946-1950

26. Thomas F, Zeng GQ, Mache R, Briat JF (1988) Transcriptionstudy of the genes encoded in the region of the junctionbetween the large single copy and the inverted repeat A ofspinach chloroplast DNA. Plant Mol Biol 10: 447-457

27. Thomas F, Massenet 0, Dorne AM, Briat JF, Mache R (1988)Expression of the rp123, rpl2 and rpsl9 genes in spinachchloroplasts. Nucleic Acids Res 16: 2461-2472

28. Warmke HE, Lee SLJ (1978) Pollen abortion in T cytoplasmicmale-sterile corn (Zea mays): a suggested mechanism. Science200: 561-563

29. Young EG, Hanson MR (1987) A fused mitochondrial geneassociated with cytoplasmic male sterility is developmentallyregulated. Cell 50: 41-49

30. Zurawski G, Perrot B, Bottomley W, Whitfeld PR (1981) Thestructure of the gene for the large subunit of ribulose-1,5-bisphosphate-carboxylase from spinach chloroplast DNA. Nu-cleic Acids Res 9: 3251-3269

400 MONEGER ET AL.