Copyright 0 1990 by the Genetics Society of America

Identification of a Maize Nuclear Which Influences the Size and Number of cox2 Transcripts in Mitochondria of Perennial Teosintes

Pam Cooper, Ed Butler’, and Kathleen J. Newton Division of Biological Sciences, Universityof Missouri, Columbia Missouri 6521 1 Manuscript received February 20, 1990 Accepted for publication June 21, 1990

ABSTRACT The involvement of nuclear in mitochondrial was investigated by identifying alterations in mitochondrial gene expression that occur when teosinte are introduced into certain maize inbred nuclear backgrounds. The cytoplasms from the teosintes Zea perennis, Zea diploperennis, and Zea luxurians were introduced into the maize A619 or W23 lines by recurrent backcrossing. Northern analysis revealed that the Z. perennis and Z. diploperennis mitochondrial cox2 transcript patterns were dependent upon the maize nuclear genotype. In a W23 nuclear background, these teosinte mitochondria have two major transcripts of 1.9 and 1.7 kb, whereas in an A619 background, they have three major transcripts of 1.9, 1.5 and 1.3 kb. No effect of nuclear background on cox2 transcripts was observed for plants possessing Z. luxurians . All teosinte-maize combinations possess larger, minor cox2 transcripts of 3.9, 3.3 and 3.0 kb; nuclear background has no effect on these transcripts. Immunoblot analysis showed a threefold reduction of the COX11 polypeptide in Z. perennis-A619 combinations compared to Z. perennis-W23 combinations. All the major and minor transcripts possess both cox2 exons. The cox2 intron is missing from all the major transcripts and is present only in the 3.9- and 3.0-kb minor transcripts. The 1.7- and 1.3-kb transcripts are missing untranslated regions 3’ to the cox2 gene; therefore at least some of the size heterogeneity is due to differential termination or downstream processing. Genetic analyses indicate that a single nuclear gene is responsible for the observed differences in the major cox2 transcripts, and that A6 19 carries the dominant allele. This gene, designated Mct, is specific for ~0x2,as no transcript size differences were observed for the other two mitochondrial cox genes.

EVERAL and mitochondrial enzyme crossing, the nuclear becomesessentially that S complexes consist of subunits encoded by both of the maize inbred, while the maternally inherited the cytoplasmic and nuclear . Thus proper cytoplasmicgenomes remain those of theoriginal function requires a compatible interaction teosinte. When cytoplasm from the perennial teosinte between gene products from two distinct genomes Zea perennis is present with the nuclear genotype of that occurs when those genomes have evolved under A619 or of other Oh43-derived maize inbreds, the the same selective pressures. Nuclear cytoplasmic in- resulting plants aremale sterile (GRACENand GROCAN compatibility may be found within a species, or as a 1974; LAUCHNAN and GABAY-LAUGHNAN 1983). Out- result of intergeneric or interspecific hybridization, crossesof these 2. perennis-maize combinationsto withits outwardmanifestation being a develop- other maize lines restored fertility. Zea diploperennis mentally aberrant phenotype (GRUN 1976). in an A6 19 backgroundalso results in partial or full Cytoplasmicmale sterility (CMS) is a frequently male sterility (J. KERMICLE,personal communication; encounteredphenotype in plantsthat is generally K. J. NEWTON,unpublished observations). consideredto result from disturbances in nuclear- A second trait attributed to an incompatibility be- mitochondrialinteractions (DUVICK 1965; HANSON tweenteosinte cytoplasmic andmaize nuclear ge- and CONDE 1985).CMS plants fail to shed functional nomes is an abnormal kernellplant phenotype desig- pollen, and the traitis maternally inherited. CMS can nated “teosinte cytoplasm associated miniature” (tcm) arise following interspecific crosses between maizeand (ALLEN,EMENHISER and KERMICLE1989). This trait certainteosintes (wildrelatives of maize). Teosinte was originally identified in 2. perennis plants whose cytoplasmcan be introducedinto a maizenuclear nuclear genome had been replaced by that of maize background by using the maize line as the male parent inbred W23 (KERMICLEand LONNQUIST1973). Ker- in a program of recurrent backcrossingto the teosinte- nels are much smaller but structurallysimilar to nor- derived female line. After several generationsof back- malkernels. They germinate more slowly and give

’ Present address: Arimna Cancer Center, University of Arizona, Tuscon rise to short-statured, pale-green, yet fertile plants. Arimna 85724. Normal kernel and plant development becan restored

Geneticb 126 46 1-46? (October, 1990) 462 P. Cooper, E. Butler and K. J. Newton in these teosinte-maize combinations by outcrosses to as no qualitative transcript differences were identified maizelines, suchas A619, that possess dominant for the other two mitochondrialcox genes. nuclear rectifiers (Rcml, Rcm2) of the kernel defect (ALLEN,EMENHISER and KERMICLE1989). Teosintes MATERIALS AND METHODS more closely relatedto maize, i.e., those classified along with maize in Zea Section Zea (DOEBLEYand Teosinte andmaize stocks: The nuclear genomes of ILTIS1980) are not tcm susceptible(ALLEN, EMEN- three teosinte species, 2. perennis (Collins and Kempton), 2. diploperennis (Guzman, collection 777), and 2. luxurians HISER and KERMICLE1989). Only combinations of the (Guatemala, Wilkes, collection 51 186) were replaced with more distantly related teosintes found in Zea Section that of maize by successively backcrossing withinbred lines Luxurianteswith W23 exhibitthe tcm phenotype A619 or W23. The initial five backcrosses weremade at the (ALLEN, EMENHISERand KERMICLE1989). University of Wisconsinby JERRY KERMICLE,and backcross- Although a number of nuclear genes that regulate ing was continued at the University of Missouri. Analysis of plant materials was begun after at least six generations of mitochondrial gene expression have been identified backcrossing; data presented here are from materials back- in yeast (TZAGOLOFFand MYERS1986), with the ex- crossed at least nine generations to the respectivemaize ception of nuclearfertility restorers (HANSONand inbred lines. Youngear shoots were used for thepreparation CONDE 1985), few such genes have been described in of mitochondria. The ear shoots were harvested from field- plants. We have begun to identify molecular altera- or greenhouse-grown material when the silks had just emerged from the surrounding husk leaves. tions that result from incompatible combinations of Preparation of mitochondrial RNA: A crude mitochon- teosinte cytoplasmic genomes and maize nuclear ge- drial pellet was prepared from homogenate of 20-50 g of nomesas a means of studyinghow nuclear genes surface-sterilized ear shoots as described by STERNand regulatemitochondrial gene expression in higher NEWTON (1986). The pellet was resuspended in a wash buffer consisting of 0.35 M sorbitol, 50 mM Tris-HCI (pH plants. We have used lines accessions derived from of 8) and 20 mM EDTA. The sample was layered onto a 20/ three species of teosintes in Section Luxuriantes: the 35/47/60% (w/v) sucrose step gradient and centrifuged in perennials 2. perennis and Z. diploperennis, and the a Beckman SW 41 rotorat 154,000 X g for 1hr. The annual Zea luxurians. The nuclear genomes of these purified mitochondria were removed from the35/47% teosintes were replaced with those of maize inbreds interface and slowly diluted threefold with wash buffer. The mitochondria were collected by centrifugation at 10,000 X W23 or A619 by serialbackcrossing. Recently we gand washed once with wash buffer. For scoring individuals, reported an effect of maize nuclear background2. on single ear shoots were homogenized and the mitochondria luxurians mitochondrialprotein synthesis (COOPER collected by differential centrifugation. They were washed and NEWTON 1989). 2. luxurians mitochondria from by a second round of centrifugation at 2000 and 10,000 X g, but were not further purified. All manipulations of mi- the A619 line synthesize a distinctive 22-kD polypep- tochondria were carried out at4". RNA was extracted from tide, whereas those fromthe W23 linedo not. Genetic the mitochondria in the presence of 1 mM aurintricarboxylic analyses indicated that synthesisof this mitochondrial acid (ATA) following the procedureof STERN andNEWTON polypeptide is controlled by a single nuclear gene. (1986). RNA was precipitated twice in 2 M LiCI, and solu- Here we reportthat maizenuclear background bilized in 25 mM Tris-HC1 (pH 8), 50 pM ATA, aliquotted, and frozen on dry ice. RNA samples were stored at -80". influences the number andsize of transcripts from the Electrophoresisand hybridization analysis of RNA: geneencoding subunit 2 ofcytochrome c oxidase Total mitochondrial RNA (0.5 pg per lane) was electropho- (~0x2)in 2. perennis and 2. diploperennis mitochondria. resed through 1.2% agarose-formaldehyde gels (MANIATIS, Three major transcripts are present in mitochondria FRITSCHand SAMBROOK1982). After staining with ethidium bromide the RNAs were blotted onto uncharged nylon from plants possessing an A619 nuclear background, filters (MSI, 0.45 pm) by wicking the gels with 10 X SSC. whereas two major transcripts are found in mitochon- RNA was UV crosslinked to the filters, and the filters baked dria from plants possessing a W23 background. Nu- for 2 hr at80". Filters were prehybridized overnight at 42" clear background has no influence on cox2 transcript in 50% deionized formamide, 25 mM Pipes-NaOH (pH 64, pattern in lines carrying 2. luxurians cytoplasm. The 5 mM EDTA, 10 X Denhardt's solution, 0.75 M NaCI, 0.1 % sodiumdodecyl sulfate (SDS) and 100 pg/ml sonicated, major transcripts all contain both exons of the gene denatured salmon sperm DNA (STERNand NEWTON1986). and lack the intron, indicating that they are processed, Hybridization with DNA probes was carried out under the mature mRNAs. Two of the major transcripts were same conditions for 16 h. Following hybridization, filters missing 3' untranslated regions ofthe gene; thus some were washed in 2 X SSC, 0.5% SDS followed by two changes of 1 X SSC, 0.5% SDS at 65" prior to autoradiography of the size heterogeneity is the result of differential using Kodak XRP-5 film and Dupont Cronex intensifying termination or downstream processing. Segregation screens. Estimation of RNA sizewas based on molecular analysis showed that a single nuclear gene is respon- size standards run alongside the samples (BRL RNA Lad- sible for determining transcript pattern. The A619 der). Cloned restriction fragments containing the genes for line carries the dominant allele responsible for the subunits I (coxl, ISAAC,JONES and LEAVER 1985), I1 (~0x2, Fox and LEAVER1981), or 111 (~0x3,HIESEL et al. 1987) of production of three transcripts, whereas W23 carries mitochondrial cytochrome c oxidase were labeled by ran- the recessive allele responsible for the production of dom priming (Pharmacia Oligolabelling Kit) according to two transcripts. This nuclear geneis specific for cox2, the manufacturer's instructions. Restriction fragments Maize Gene Affects Teosinte cox,? RNA 463 which containrd the intron ;Ind exons of the cox2 gene were A identifiedfrom ;I restriction map basedon the published nucleotidcz sequence (Fox and LEAVER1981). The appro- 123456 priate fi-;Ignwlts were elutrtl fromgels and purified by 3.9b st;lntl;lrd ~)rotncols (MANIAIXS, FRITSCH and SAMRROOK 3.3, 3.0 * 1982). Immunoblot analysis of mitochondrial : Mito- cllontlria were purified Iron1 sucrose gradients as above. then resuspended in it small volume of RNA wash buffer, illltl the cancentr;ttion of mitochondrial was cleter- minrd using the nwthod of I'EIERSON (1 977). The mito- chontlria were solubilized in sample buffer (LAEMMLI1970) ;und thc proteins separ;Ited in SDS gels with a linear poly- B ;Icryl;mlide grxtient of 12-1 8%.Equivalent anlounts of total 123456 mitocl1ondri;lI protein (20 pg) were loaded onto each gel lane ;\longside molecularlveight markers (Bio-RadLow). 1)rrplic;tte s;mples were loaded oneach half of the gel. lollowing electrophoresis, one-half of the gel was stained with Coonlassie blue R-250, and the other half WIS used for immunohlot ;lnalysis. I'roteins were electropl~oretically transferred from the gel to 0.2 prn nit~-ocellullose(Schleicher and Schuell, Keene SH)using ;I Bio-Rad Transblot cell operated at .50 V for .5 hr. Prior to tr;msfer, gels were equilibrated in transfer lx1ffc.r consisting of 12.5 nnf Tris, 96 111x1 glycine, 0.1 % SDS and 20% JleOH. Following transfer. the filter was ShiIken overnight in 20 n1M Tris-HCI (pH 7..5), 150 InM XACI and 3% fittty acid-free USA to block nonspecific pro- tein binding sites. Reaction of the filter with antibodies and probe detection using alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgC;), 5-bromo-4-chloro-3- indolyl phosphate and nitro blue tetrazolium was essentially ;IS previously described (DARR.SOMMERVILLE and ARNTZEN 1986). The filter was incubated 16 hr with ;I 1:24 dilution of an affinity-purified I-abbit anti-COX11 IgG (NIVISONand HANSON 1989). Because the antigen used to produce the antibody was a synthetic peptide conjugated to keyhole linlpet hemocyanin (KLH), 0.17 mg/mI KLH was included in the probe solution. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Boehringer-~.lannI~ein~,Indianapolis Indi- ana) was used as :I I: 1000 dilution of the stock solution. Following developnent, the filter was photographed using ennis-A6 19, Z. diploperennkA6 19), three major tran- Polaroid Type 53 film, and the negative was scanned using scripts of 1.9, 1.5 and 1.3 kb hybridized with the cox2 an LKB CTltroScan XL Laser Densitometer to quantitate probe (Figure1 A, lanes 2 and 4). Southern blot analy- the antibody signal. The filter was then stained for proteins sis indicatedthat the changes in transcriptpattern with 0.1 % India ink. were notthe result of mitochondrial genome re- arrangements or of froma duplicate cox2 RESULTS gene. Teosinte mitochondrialDNAs from Z. perennis- Influence of nuclear background on transcript W23, Z. perennis-A619, Z. diploperennis-W25, and Z. patterns for subunits I, I1 and I11 of cytochrome c diploperennis-A6 19 were digested with 13 different oxidase: Northern blot analysis of the teosinte mito- restrictionenzymes, and the Southern blotsof the chondrial RNAs was carried out using cloned restric- digests probed with the cloned fragment containing tion fragments containing the genesfor subunits I, I1 the cox2 gene. For every restriction enzyme used, the and I11 ofcytochrome c oxidase as hybridization numberof hybridizingrestriction fragments de- probes (Figure 1). When the cloned cox2 gene (Fox pended upon the mitochondrial source, but did not and IXAVER 1981) was usedas a probe,both the depend on the nuclear background; thatis, for a given nunlber andsize of hybridizing transcripts variedas a teosinte cytoplasm, the same hybridization patternwas function of nuclear background for the Z. perennis observedirrespective of the maize nuclear back- and Z. diplopermnis cytoplasms. When these teosinte ground. In all but one case, only a single hybridizing mitochondria were in a maize inbred W25 nuclear fragment was observed. The presence of a restriction background (Z. perennis-W25, Z. diploperennis-W23), site within the region delineated by the cloned probe two major 1.9- and 1.7-kb transcripts hybridized with accountsfor the instance in which morethan one theprobe (Figure IA, lanes 1 and 3). Whenthe hybridizingband was observed(data not shown). nuclear background was maize inbred A6 19 (Z. per- Therefore all the cox2 mRNAs were transcribed from 464 P. Cooper, E. Butler and I(. J.Newton -0 1 kb

EXON 1 EXON 2 5' 3' N-A N-A I H-M 03 kb 03 kb T T M E 0.7 kb 0.7 hb

A B C D

123456 1 23 4 56123456 12 345 6 -3.9, -3.3- - -- ~ 43.0.

WP

OI'IIIC cos2 II';III\( ript\ arv in kilotx~ses.

;I single copy of the gene, and the transcript differ- probes on Northern blots (Figure 2).When fragments ences did not arise due to some major rearrangement that contained either the first or second exon of the in the mitochondrial genome. Maize nuclear back- cox2 gene were used as probes (Figure 2, A and C), ground apparently had no effect on the cox2 transcript the transcript patterns were found to be identical to patterns for Z. luxurians, because two transcripts of those generated when the full length clone was used 1.9 and 1 .7 kb were detectable with the probe in both as a probe (Figure IA). If the blots were probed with nuclear backgrounds. Minor cox2 transcripts of 3.9, a fragment from the intron of the gene, none of the 3.3 and 3.0 kb were present in all the teosinte-maize major transcripts in any of the teosinte-maize combi- combinations. nations hybridized to the probe, although the larger Transcript patterns forcoxl and cox3 in the teosinte- 3.9- and 3.0-kb minor transcripts did hybridize to the maize comhinations were less complex than the cox2 intron probe (Figure 2B). transcriptpatterns, and maize nuclearbackground When a 700-bp region flanking the 3' end of the had no qualitative effect on those patterns.Two major cox2 gene was used as a probe, the minor transcripts transcripts of 2.4 and 2.1 kb hvbridized with the coxl hvbridized in all the teosinte-maize combinations (Fig- probe in every case (Figure 1 B). Teosinte mitochon- ure 2D). The 1.9- and 1.5-kb major transcripts also dria in the "23 background possess slightly higher hybridized with the probe. However, the 1 .3-kb tran- levels of the 2.4-kb transcript than teosinte mitochon- script detectable with the full length probe failed to dria in the A6 19 background. A major 1.2-kb and a hybridize with the 3' probe (Figure 2D, lanes 2 and less abundant 1.8-kb transcript were detected using 4). Also, the 1.7-kb transcript detectable with the full cox3 as the hybridization probe in all the teosinte- length probe hybridized only weakly with the 3' flank- maize combinations (Figure 1C). Nuclear background ing region in Z. perennis-W23, 2. diploperennis-PV23 seems to have some effect on the relative abundance and Z. luxurians in both maize nuclear backgrounds of the mqjor cox3 transcript. Teosinte mitochondria (Figure 2D, lanes 1, 3, 5 and 6, respectively). in the "23 backgroundcontain slightly reduced Effect of nuclear background on COXII protein amounts of the 1.2-kb transcript compared to mito- levels: To determine what effect nuclear background chondria in the A619 background (Figure IC). had on the teosinte COXII protein itself, total mito- The major teosinte cox2 transcripts are processed chondrialproteins were extracted from 2. perennis andcontain both exons: To characterizethe cox2 and Z. luxurians in both the W23 and A619 nuclear transcript patterns obtained for the two different nu- backgrounds, and subjected to SDS-polyacrylamide clear backgrounds,restriction fragments containing gel electrophoresisand immunoblotting. Equivalent different portions of the cox2-containing pZmEl clone amounts of total mitchondrialprotein were loaded (FOX and LEAVER1981) were used as hybridization onto each lane of the gel. The proteins were blotted Gene Affects TeosinteAffectsMaize Gene cox2 RNA 46.5

I..IG~~RE4.-Scgrcg;ttion ;m;llyhis 01' ros-7 tt;lnscript 1);11ter11hI)\ Sortlwrl1 blot ll\l~ridimtion.\fitorhontlri;~l KS'h \\';IS estt;lctcd from intlivitlu;tl car shoots. clrrtro~~horcsctl.hlottctl. ;tnd prol>cd wit11 rlw full Icngth ros-7 gene. L.;lncs: I,K. prrmniz cvtopl:~s~~~.\V23 1~;tckgrountl (Z. purrnni,r-\V23): 2, I:, hvt)ritl Z. prrrnnis-\V93 X A6 19: 3- IO. I);~c-kcross(Z. prrmnis-\V23 X ;if;19) X IVY:%: 1 1-22 I:?, %. perenni.+\V2:3 X ;\ti 19 sclf-~~olli~~;~tctl, Siwsmajor of I~;III- scripts ill kilolxlscs ;IW illtlicatctl ;II lcli. script" pattern (i.e., 1.9 and 1.7 kb cox2 transcripts as in Figure 1 A, lane I), were crossed by A61 9. Z. perennis-A6 19 plants, which exhibit the "three-tran- script" pattern (ie., 1.9-, 1.5- and 1.3-kb transcripts as in Figure 1 A, lane 2) were crossed by W23. The F1 hybrids Z. perennis-M"L3 X A619 (Figure 4, lane 2) and Z.perennis-A619 x W23 (not shown) both exhib- ited the three-transcript pattern upon Northern analy- sis using the cox2 clone as probe.Thus, the two- I:IC:I,RE :<.-IIIIIIIIIIIO~)~OI ;tnaI\sih of 111c(:OS11 polvpcl)titlc in transcript pattern is recessive to the three-transcript ~~~ito(-l~o~~(lri~~from ~I;IIII\ posscs\ing tcosintc cvtopl;lslrls in nl;lix pattern. IVY:\ or :\(;I 9 IIIIC~~:I~Ix~ckgrou~~tls. \litocllontlri;~l protrins werr An F? generation was produced by self-pollinating srp;~ratctI011 12-1 8% liw;tr ~)ol~~c~.vl~~~~~itlcgels containing SIIS. Z. perennis"'23 X A619 plants. Z. perennis-U'23 X I'rot(im w~cI)lottctI tonitorcllulow ;1m1 rl~cI)lots \vcrc illcul);ltctl A619 plants were also backcrossed by maize inbred \\it11 ;lllti-(:OXll lxlhit lg(;. I%ou~ltl;tntil)otlv W;IS tlc~rcrtr~lusing "23 plants. Seeds from these crosses were planted an aIL;tlinc ~)l~(~~~)l~;~t;~sc-(.o~~,j~~g;~tc(lsc.ro~~tlar\;~ntil)otlv. I~ncs: I. %. prrenni.~cvtopl;~s~~~. IV23 I~;~rkgrou~~d(Z.prrrnnis-\\'2:$): 2. Z. and mitochondrial RNA was extracted from individ- prwnnis-;\619: 3. %. prro,nis-\V2:<. sill of pl;mr ill hnc 1 : 4. %. uals. Transcriptpatterns were assessed by probing prrrnnis-;\619. sib 01- 1hnt in I;IW 2: 5. %. /us~crions-\VL'J: 6. Z. Northern blots of the mitochondrial RNA with the /lcsf~rio~f.~-.~fil~~.l'ositiol1s of' n1olcrlll;tl. III;IS~ nl;lrkers sllow1 in cox2 clone. A representative blot is shown in Figure kilothlrons 011 the right. 4A. lanes 5-22. The segregation of transcript patterns for F? and backcross generations are summarized in to nitrocellulose and reacted with the COXII antibody Table 1. If a sirlgle nuclear gene is responsible for the (~IvIsONand HANSON1989). Staining the blot with transcript differences, then a 3:l segregation in favor India ink following antibody treatment confirmed that of the three-transcript pattern is expected in the Fn the transfer of proteins from the gel to the nitrocel- generation, and a 1: 1 segregation is expected in the lulose was uniform both within and between lanes (not backcross generation. Outof 3.5 F? individuals scored, shown). The COX11 antibody bound to a protein of 25 possessed the three-transcript pattern, and 10 pos- molecular mass = 34 kD (Figure 3). Three-times more sessed the two-transcript pattern. Chi-squared analysis COX11 polypeptide was present in 2. perennis mito- of the 25:10 ratio showed that it did not deviate chondria in ;I "23 background (Figure 3, lanes 1 and significantly from 3:1 (Table 1, analysis 1). For the 3) than in an A6 19 background (Figure 5, lanes 2 and backcross, 3 of 8 individuals scored possessed the 4). More COXII protein was present in 2. luxurians three-transcript pattern. The probability of observing mitochondria as compared to 2. perennis, but there a 3:s ratio by chance when a 1:l ratio was expected was no significant difference in the level of the protein was found to be 0.73 (Table 1, analysis 2). Thus at as a function of nuclear background for Z. luxurians the conventional 5% level of significance, these data mitochondria (Figure 3, lanes 5 and 6). support the hypothesis that a single nuclear gene is A single nuclear gene determines cox2 transcript responsible for the differences in cox2 transcript size pattern: ~l%enumber of nuclear genesresponsible for and number. the effect 011 the cox2 transcript pattern in Z. perennis DIS<:USSION was determined by genetic analysis. Plants possessing %. perennis cytoplasm were used for these studies. Z. Differential efffects on mitochondrial cox2 gene perennis-W23 plants, which exhibit the "two-tran- expression are observed when Z. perennis or Z. diplo- 466 P. Cooper, E. Butler and K. J. Newton

TABLE 1

Segregation of Z. perennis cox 2 transcript patterns in Fsand backcross generations

IndividualsIndividuals Expectedtwo with three with Analysis Cross ratio n transcriptstranscripts XP p (2)

1 2. perennis-W23 X A619 X 3:1 35 25 10 0.08 0.80 > P > 0.70 2 2. perennis-(W23 X A619) X W23 1:l 8 3 5 0.73 Analysis 1: Fr sample from two sibling ears grown during two field seasons. d.f. = 1, two-sided x‘ calculated using Yate’s continuity correction. Analysis 2: backcross from a single ear. Two-sided binomial exact test. perennis cytoplasms are in association with certain (NEWTONand COE 1989). However, the three-tran- maize nuclear backgrounds. When the cytoplasm is in script pattern is present in the fertile F1 hybrids as a W23 background, two major cox2 transcripts of 1.9 well as in the sterile Z. perennis- or Z. diploperennis- and 1.7 kb are present, whereas in an A6 19 back- A6 19 plants. Thus there is no direct correlation be- ground the 1.7-kbtranscript is replaced by two smaller tween the expression of CMS and the altered cox2 transcripts of 1.5 and 1.3 kb. Although plant mito- transcripts. chondria possess genes encoding subunits I, I1 and I11 Segregation analyses of the cox2 transcript patterns of the cytochrome c oxidase complex, only cox2 tran- in 2. perennis-maize populations support the hypoth- script number is influenced by nuclear background in esis that a single nuclear gene is responsible for the the teosinte-maize combinations. Alteringnuclear observeddifferences in the major cox2 transcripts. background had no major qualitative effects on cox1 The inbred A6 19line carries the dominant allele of or cox3 transcripts. The cox2 transcript patterns for Z. this gene, which we designate as Mct (for modifier of luxurians mitochondria were not affected by maize cox2 transcripts). Plants possessing Z. perennis cyto- nuclear background; the 1.9- and 1.7-kb transcripts plasm in an A619 nuclear background also exhibit a were present in mitochondria from plants possessing threefoldreduction in accumulated levels of the either the W23 or the A619 nuclear background. COX11 polypeptide relative to the plants carrying the Z. perennis, tetraploida perennial teosinte, is same cytoplasms in a W23 background. These data thought to have derived from the diploid perennis Z. imply a reduced stability or translational efficiency of diploperennis (ILTIS et al. 1979). Thusthe similar the smaller transcripts. Alternatively, the resulting effects of nuclear background on the cox2 transcripts polypeptides may be less stable. patterns for thetwo species is not surprising. Teosinte How might the Mct gene alter cox2 transcript size? mitochondria in their own nuclear background pos- Transcript size heterogeneity can be the result of sess a two-transcript pattern (P. COOPER,unpublished multiple transcriptioninitiation sites, multiple tran- results); therefore the three-transcript pattern, seen scription terminationsites, and/or post-transcriptional when perennialteosinte mitochondria are present processing. In maize mitochondria, the transcript size with A6 19nuclear genes, represents an abnormal heterogeneity of the protein coding genes atp9, cox3 situation. and cob results from multiple sites of transcription Although miniature kernelsand CMS are two other initiation (MULLIGAN,LAU and WALBOT1988; MUL- phenotypic consequences of placing, Z. perennis, Z. LIGAN, MALONEYand WALBOT1988). Because all of diploperennis and 2. luxurians cytoplasms in associa- the teosintemajor cox2 transcripts lack the intron, tion with maize nuclear genes, there appears to beno they represent processed forms of the mRNA. Given direct causal relationship between cox2 transcript pat- that the coding region for the Z. diploperennis cox2 tern and either of these two developmental abnor- gene is virtually identical to that of maize (GWYNNet malities. Miniature kernels result when these teosinte al. 1987), and that the minimum length for a spliced cytoplasms are in association with a W23 background, mRNA would be 825 bp (FOX and LEAVER1981), and normal kernel size can be recovered by outcross- then a substantial portion of each of the teosinte cox2 ing to A6 19(ALLEN, EMENHISERand KERMICLE transcripts consists of 5’ and 3’ untranslated regions. 1989). We have found no linkage betweenaltered The presence of multipletranscripts inall of the cox2 transcript patterns and kernel/plant size. In an teosinte-maize combinations is in part due to hetero- FB populationsegregating 3:l forlarge and small geneity in the 3’ untranslated region, as judged by kernels, cox2 transcript patterns segregated independ- the lack of hybridization of the 3‘ noncoding region ently from kernel size (P. COOPER,unpublished re- of the pZmEl clone to the 1.7- and 1.3-kb transcripts. sults). Plants possessing 2. perennis or Z. diploperennis This is in contrast to maize atp9, cob and cox3 tran- cytoplasm in an A6 19 nuclear background exhibit scripts in maize (MULLIGAN,LAU and WALBOT1988; CMS but fertility is restored by outcrossing to W23 MULLIGAN, MALONEYand WALBOT1988) and atpA M a ize Gene Affects TeosinteAffects Gene Maize cox2 RNA 467 and cox2 transcripts in Oenethera (SCHUSTERet al. GWYNN,B., R. E. DEWEY,R. R. SEDEROFF,D. H. TIMOTHYand C. 1986), in which no 3' heterogenity has been identi- S. LEVINGS111, 1987 Sequence of the 18s-5s ribosomal gene region and the cytochrome oxidase I1 gene from mtDNA of fied. Althoughwe have not yet analyzed the full extent Zea diploperennis. Theor. Appl. Genet. 74 781-788. of 5' heterogenity in the teosinte cox2 transcripts, we HANSON,M. R., and F. CONDE,1985 Function and variation of can deduce from our data that the 1.7-kb transcript cytoplasmic genomes: lessons from cytoplasmic-nuclear inter- found in plants possessing a W23 nuclear background actions affection male fertility in plants. Int. Rev. Cytol. 94: arises from an initiation or processing event approxi- 2 13-267. HIESEL,R., W. SCHOBEL,W. SCHUSTERand A. BRENNICKE,1987 mately 400 bp further upstream from a similar event The cytochrome oxidase subunit I and subunit 111 genes of that gives rise to the 1.3-kb transcript found in plants Oenethera mitochondria are transcribed from identical pro- possessing an A61 9 nuclear background. The tran- moter sequences. EMBO J. 6: 29-34. scripts are similar in their sequence content in that ILTIS, H. H., J. F. DOEBLEY,R. GUZMAN andB. PAZY, 1979 Zea they possess both exons(825 bases) but have no intron dzploperennis (Graminae): a new teosinte from Mexico. Science 203: 186-188. and little or no 3' untranslated regions (Figure 2). ISAAC, P. G., V. P. JONES and C. J. LEAVER,1985 The maize Precise mapping of the 5' and 3' ends of the teosinte cytochronle c oxidase subunit I gene: sequence, expression, cox2 transcripts by S1 nuclease and primer extension and rearrangement in cytoplasmic male sterile plants. EMBO analyses will determine whether theseconclusions are J. 4: 1617-1623. true. KERMICLE,J. L., and J. H. LONNQUIST,1973 Nucleo-cytoplasmic interaction in the determination of a defective seed trait. Maize We wish to acknowledgeJERRY KERMICLEfor generously provid- Genet. Coop. Newslet. 47: 209-2 11. ing us with the teosinte-maize stocks. We are grateful to CHRIS LAEMMLI,U. K., 1970 Cleavage of structural proteins during the LEAVERfor providing us with the maize cox1 and cox2 clones, and assembly of the head of bacteriophage T4. Nature 227: 680- to AXEL BRENNICKE forthe Oenethera cox3 clone. We also thank 685. HELENNIVISON and MAUREENHANSON for the gift of the COX11 LAUCHNAN,J. R., and S. GABAY-LAUGHNAN,1983 Cytoplasmic antibody, and KARENCONE forcritically reading the manuscript. male sterility in maize. Annu. Rev. Genet. 17: 27-48. Support was provided by the National Science Foundation (DCB MANIATIS,T., E. F. FRITSCHand J. SAMBROOK, 1982Molecular 885827), the Missouri Center for Advanced Technology, and a Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory, McKnight Foundation Individual Investigator Award. Cold Spring Harbor, N.Y. MULLIGAN,R. M., G. T. LAUand V. WALBOT,1988 Numerous transcription intitiation sites exist for the maize mitochondrial LITERATURECITED genes for subunit 9 of the ATP synthase and subunit 3 of ALLENJ. O., G. K. EMENHISERand J. L. KERMICLE, 1989 cytochrome oxidase. Proc. Natl. Acad.Sci. USA 85: 7998- Miniature kernel and plant: interaction between teosinte cyto- 8002. plasmic genomes and maize nuclear genomes. Maydica 34 MULLIGAN,R. M.,A. P. MALONEYand V. WALBOT,1988 RNA 277-290. processing and multiple transcription initiation sites result in COOPER,P., and K. J. NEWTON, 1989 Maize nuclear background transcript size heterogeneity in maize mitochondria. Mol. Gen. regulates the synthesis of a 22-kDa polypeptide in Zea luxurians Genet. 211: 373-380. mitochondria. Proc. Natl. Acad. Sci. USA 86 7423-7426. NEWTON,K. J., and E. H. COE,JR., 1989 Multiple ferility restorer DARR,S. C., S. C. SOMERVILLEand C. J. ARNTZEN,1986 genes for EP (2.p.) cytoplasm. Maize Genet. Coop. Newslet. Monoclonal antibodies to the light-harvesting chlorophyll a/b 63: 68-69. protein complex of photosystem 11. J. Cell Biol. 103: 733-740. NIVISON,H. T., and M. R. HANSON,1989 Identification of a DOEBLEY,J., and H. H. ILTIS, 1980 Taxonomy of Zea. I. Subge- mitochondrial protein associated with cytoplasmic malesterility neric classification with key to taxa. Am J. Bot. 67: 982-993. in petunia. Plant Cell 1: 1121-1130. DUVICK,D. N., 1965 Cytoplasmic pollen sterility in corn. Adv. PETERSON,G. L., 1977 Simplification of the protein assay of Genet. 13: 1-56. Lowry et al. which is generally more applicable. Anal. Biochem. Fox, T. D., and C. J. LEAVER,1981 The Zea mays mitochondrial 83: 346-356. gene coding cytochrome oxidase subunit I1 has an intervening SCHUSTER,W., R. HIESEL,P. C. ISAAC,C. J. LEAVERand A. sequence and does not contain TGA codons. Cell 26: 3 15- BRENNICKE,1986 Transcript termini of messenger RNAs in 323. higher plant mitochondria. Nucleic Acids Res. 14 5943-5954. GRACEN,V. E., and C. 0. GROGAN,1974 Diversity and suitability STERN,D. B., and K. J. NEWTON, 1986 Isolation of plant mito- for hybrid production of different sources of cytoplasmic male chondrial RNA. Methods Enzymol. 118: 488-496. sterility in maize. Agron J. 66: 654-657. TZAGOLOFF,A., and A. M. MYERS, 1986 Genetics of mitochon- GRUN,P., 1976 Cytoplasmic Genetics and Evolution. Columbia Uni- drial biogenesis. Annu. Rev. Biochem. 55: 249-285. versity Press, New York. Communicating editor: M. R. HANSON