Copyright 0 1990 by the Genetics Society of America Identification of a Maize Nuclear Gene 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 genes in mitochondrial gene expression was investigated by identifying alterations in mitochondrial gene expression that occur when teosinte cytoplasms 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 cytoplasm. 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 chloroplast and mitochondrial enzyme crossing, the nuclear genome becomesessentially that S complexes consist of subunits encoded by both of the maize inbred, while the maternally inherited the cytoplasmic and nuclear genomes. Thus proper cytoplasmicgenomes remain those of theoriginal organelle 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- mal kernels. 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 respective maize 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