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Prior research has shown that the home learning environment (HLE) is critical in the development of spatial skills and that various parental beliefs influence the HLE. However, a comprehensive analysis of the impact of different parental beliefs on the spatial HLE remains lacking, leaving unanswered questions about which specific parental beliefs are most influential and whether inducing a growth mindset can enhance the spatial HLE. To address these gaps, we conducted an online study with parents of 3- to 5-year-olds. We found that parents' growth mindset about their children's ability strongly predicted the spatial HLE after controlling for parents' motivational beliefs about their children, beliefs about their own ability, children's age, children's gender, and family SES. Further, reading an article about growth mindset led parents to choose more challenging spatial learning activities for their children. These findings highlight the critical role of parents' growth mindset in the spatial HLE. Crucially, these findings demonstrate that general growth mindset messages without specific suggestions for parental practices can influence parental behavior intentions. Further, these effects were also observed in the control domain of literacy, underscoring the broad relevance of the growth mindset in the HLE.

Background: Mechanisms contributing to tissue remodeling of the infarcted heart following cell-based therapy remain elusive. While cell-based interventions have the potential to influence the cardiac healing process, there is little direct evidence of preservation of functional myocardium., Aim: The aim of the study was to investigate tissue remodeling in the infarcted heart following human embryonic stem cell-derived endothelial cell product (hESC-ECP) therapy., Materials and Methods: Following coronary artery ligation (CAL) to induce cardiac ischemia, we investigated infarct size at 1 day post-injection in media-injected controls (CALM, n = 11), hESC-ECP-injected mice (CALC, n = 10), and dead hESC-ECP-injected mice (CALD, n = 6); echocardiography-based functional outcomes 14 days post-injection in experimental (CALM, n = 13; CALC, n = 17) and SHAM surgical mice ( n = 4); and mature infarct size (CALM and CALC, both n = 6). We investigated ligand-receptor interactions (LRIs) in hESC-ECP cell populations, incorporating a publicly available C57BL/6J mouse cardiomyocyte-free scRNAseq dataset with naive, 1 day, and 3 days post-CAL hearts., Results: Human embryonic stem cell-derived endothelial cell product injection reduces the infarct area (CALM: 54.5 ± 5.0%, CALC: 21.3 ± 4.9%), and end-diastolic (CALM: 87.8 ± 8.9 uL, CALC: 63.3 ± 2.7 uL) and end-systolic ventricular volume (CALM: 56.4 ± 9.3 uL, CALC: 33.7 ± 2.6 uL). LRI analyses indicate an alternative immunomodulatory effect mediated via viable hESC-ECP-resident signaling., Conclusion: Delivery of the live hESC-ECP following CAL modulates the wound healing response during acute pathological remodeling, reducing infarct area, and preserving functional myocardium in this relatively acute model. Potential intrinsic myocardial cellular/hESC-ECP interactions indicate that discreet immunomodulation could provide novel therapeutic avenues to improve cardiac outcomes following myocardial infarction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Spiroski, McCracken, Thomson, Magalhaes-Pinto, Lalwani, Newton, Miller, Bénézech, Hadoke, Brittan, Mountford, Beqqali, Gray and Baker.)

Fraction knowledge and algebraic skill are closely linked. Algebra is a gatekeeper for advanced courses (Booth & Newton, 2012; Brown & Quinn, 2007). This study uses the person-centered approach of latent profile analysis to examine individual differences in middle schoolers' (N = 350) algebra performance at the end of the year (EOY). The relative importance of a range of fraction skills is considered in predicting the likelihood of displaying a particular profile of EOY algebra knowledge, measured by feature knowledge, equation encoding, equation solving, and word-problem skills. Notably, fraction number line estimation did not predict algebra performance on any of the four measures. Fraction arithmetic at the beginning of the year is most predictive of displaying the highest performing EOY algebra profile., (© 2021 The Authors. Child Development © 2021 Society for Research in Child Development.)

The large mitochondrial genomes of angiosperms are unusually dynamic because of recombination activities involving repeated sequences. These activities generate subgenomic forms and extensive genomic variation even within the same species. Such changes in genome structure are responsible for the rapid evolution of plant mitochondrial DNA and for the variants associated with cytoplasmic male sterility and abnormal growth phenotypes. Nuclear genes modulate these processes, and over the past decade, several of these genes have been identified. They are involved mainly in pathways of DNA repair by homologous recombination and mismatch repair, which appear to be essential for the faithful replication of the mitogenome. Mutations leading to the loss of any of these activities release error-prone repair pathways, resulting in increased ectopic recombination, genome instability, and heteroplasmy. We review the present state of knowledge of the genes and pathways underlying mitochondrial genome stability.

Maize hybrids exhibiting heterosis (hybrid vigor) were generated from inbred parents with increasing genetic distance. B73 was used as the common female parent in crosses with N192 (low heterosis), MO17 (high-heterosis 1), and NC350 (high-heterosis 2). Total and mitochondria-enriched proteomes were analyzed from ear shoots of field-grown hybrids and their inbred parents. GeLCMS (1D SDS-PAGE fractionation, trypsin digestion, LTQ Orbitrap nano-RP-LC MS/MS) was used to analyze proteins, and spectral counting was used for quantitation. In total, 3,568 proteins were identified and quantified in hybrids including 2,489 in the mitochondria-enriched fraction and 2,162 in the total protein fraction. Sixty-one proteins were differentially abundant (p < 0.05) in one or both of the high-heterosis hybrids compared with the low-heterosis hybrid. For the total proteome, eight of these showed similar trends in abundance in both of the higher-heterosis hybrids. Nine proteins showed this heterosis-correlated pattern in the mitochondrial proteome, including a mitochondria-associated target of rapamycin (TOR) protein. Although differentially abundant proteins belong to various pathways, protein, and RNA metabolism, and stress responsive proteins were the major classes changed in response to increasing heterosis.

The transfer of mitochondrial DNA (mtDNA) into nuclear genomes is a regularly occurring process that has been observed in many species. Few studies, however, have focused on the variation of nuclear-mtDNA sequences (NUMTs) within a species. This study examined mtDNA insertions within chromosomes of a diverse set of Zea mays ssp. mays (maize) inbred lines by the use of fluorescence in situ hybridization. A relatively large NUMT on the long arm of chromosome 9 (9L) was identified at approximately the same position in four inbred lines (B73, M825, HP301, and Oh7B). Further examination of the similarly positioned 9L NUMT in two lines, B73 and M825, indicated that the large size of these sites is due to the presence of a majority of the mitochondrial genome; however, only portions of this NUMT (~252 kb total) were found in the publically available B73 nuclear sequence for chromosome 9. Fiber-fluorescence in situ hybridization analysis estimated the size of the B73 9L NUMT to be ~1.8 Mb and revealed that the NUMT is methylated. Two regions of mtDNA (2.4 kb and 3.3 kb) within the 9L NUMT are not present in the B73 mitochondrial NB genome; however, these 2.4-kb and 3.3-kb segments are present in other Zea mitochondrial genomes, including that of Zea mays ssp. parviglumis, a progenitor of domesticated maize., (Copyright © 2015 Lough et al.)

Knowledge of fractions is thought to be crucial for success with algebra, but empirical evidence supporting this conjecture is just beginning to emerge. In the current study, Algebra 1 students completed magnitude estimation tasks on three scales (0-1 [fractions], 0-1,000,000, and 0-62,571) just before beginning their unit on equation solving. Results indicated that fraction magnitude knowledge, and not whole number knowledge, was especially related to students' pretest knowledge of equation solving and encoding of equation features. Pretest fraction knowledge was also predictive of students' improvement in equation solving and equation encoding skills. Students' placement of unit fractions (e.g., those with a numerator of 1) was not especially useful for predicting algebra performance and learning in this population. Placement of non-unit fractions was more predictive, suggesting that proportional reasoning skills might be an important link between fraction knowledge and learning algebra., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The phenomenon of hybrid vigor (heterosis) has long been harnessed by plant breeders to improve world food production. However, the changes that are essential for heterotic responses and the mechanisms responsible for heterosis remain undefined. Large increases in biomass and yield in high-heterosis hybrids suggest that alterations in bioenergetic processes may contribute to heterosis. Progeny from crosses between various inbred lines vary in the extent of vigor observed. Field-grown maize F₁ hybrids that consistently exhibited either low or high heterosis across a variety of environments were examined for changes in proteins that may be correlated with increased plant vigor and yield. Unpollinated ears at the time of flowering (ear shoots) were selected for the studies because they are metabolically active, rich in mitochondria, and the sizes of the ears are diagnostic of yield heterosis. Total protein and mitochondrial proteomes were compared among low- and higher-heterosis hybrids. Two-dimensional difference gel electrophoresis was used to identify allelic and/or isoform differences linked to heterosis. Identification of differentially regulated spots by mass spectrometry revealed proteins involved in stress responses as well as primary carbon and protein metabolism. Many of these proteins were identified in multiple spots, but analysis of their abundances by label-free mass spectrometry suggested that most of the expression differences were due to isoform variation rather than overall protein amount. Thus, our proteomics studies suggest that expression of specific alleles and/or post-translational modification of specific proteins correlate with higher levels of heterosis., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Cytoplasmic male sterility (CMS) in plants is usually associated with the expression of specific chimeric regions within rearranged mitochondrial genomes. Maize CMS-S plants express high amounts of a 1.6-kb mitochondrial RNA during microspore maturation, which is associated with the observed pollen abortion. This transcript carries two chimeric open reading frames, orf355 and orf77, both unique to CMS-S. CMS-S mitochondria also contain free linear DNA plasmids bearing terminal inverted repeats (TIRs). These TIRs recombine with TIR-homologous sequences that precede orf355/orf77 within the main mitochondrial genome to produce linear ends. Transcription of the 1.6-kb RNA is initiated from a promoter within the TIRs only when they are at linear ends. Reversions of CMS-S to fertility occur in certain nuclear backgrounds and are usually associated with loss of the S plasmids and/or the sterility-associated region. We describe an unusual set of independently recovered revertants from a single maternal lineage that retain both the S plasmids and an intact orf355/orf77 region but which do not produce the 1.6-kb RNA. A 7.3-kb inversion resulting from illegitimate recombination between 14-bp microrepeats has separated the genomic TIR sequences from the CMS-associated region. Although RNAs containing orf355/orf77 can still be detected in the revertants, they are not highly expressed during pollen development and they are no longer initiated from the TIR promoter at a protein-stabilized linear end. They appear instead to be co-transcribed with cytochrome oxidase subunit 2. The 7.3-kb inversion was not detected in CMS-S or in other fertile revertants. Therefore, this inversion appears to be a de novo mutation that has continued to sort out within a single maternal lineage, giving rise to fertile progeny in successive generations.

Organellar DNA transfer into the nucleus and incorporation into chromosomes are continuing processes. We have examined chloroplast DNA insertions in maize chromosomes using fluorescence in situ hybridization (FISH). Probes to detect the nuclear-plastid sequences (NUPTs) were generated using 14 overlapping fragments of the chloroplast genome. Using current FISH methods, detection of NUPTs on mitotic metaphase chromosomes requires relatively large insertions (>3 kb) with strong sequence similarity to chloroplast DNA. In the B73 inbred line, hybridization of each fragment separately resolved a total of 49 NUPT sites; fewer sites (30) were detectable when all the probes were combined. The combined set of probes was hybridized to 10 genetically useful maize inbred lines and identified 19-30 NUPTs in each line. In all but two of the lines, a prominent NUPT was present on the long arm of chromosome 5. Collectively, the number of NUPTs exceeds the number of nuclear-mitochondrial (NUMT) sites identified within the same set of inbred lines. A majority of the NUPTs are found at sites that are different from the NUMTs. Like NUMTs, the positions of the NUPTs vary greatly among the lines, suggesting that the transfers are recent as well as frequent. Thus, insertions of large segments of chloroplast and mitochondrial DNA are components of the dynamic fraction of maize nuclear genomes., (Copyright 2010 S. Karger AG, Basel.)

S-type cytoplasmic male sterility (CMS-S) in maize is associated with high levels of a 1.6-kb RNA in mitochondria. This RNA contains two chimeric open reading frames (ORFs), orf355 and orf77. The previously described nuclear restorer-of-fertility allele Rf3 causes the processing of all transcripts that contain these chimeric ORFs. The Lancaster Surecrop-derived inbred line A619 carries a restorer that is distinct from Rf3 in that it selectively reduces only the CMS-S-specific 1.6-kb RNA. We have found that 10 additional Lancaster lines carry a single restoring allele traceable to either of two inbred lines, C103 and Oh40B. The C103 and Oh40B restorers are allelic to each other, but not to Rf3. Thus, this restoring allele, designated Rf9, represents a second naturally occurring CMS-S restorer in maize. Rf9 is a less effective restorer of fertility than is Rf3; its expression is influenced by both inbred nuclear background and temperature. Rf9 acts to reduce the amounts of orf355/orf77-containing linear mitochondrial subgenomes, which are generated by recombination of circular subgenomes with CMS-S-specific linear plasmids. The 1.6-kb RNA, which is transcribed only from linear ends, is correspondingly reduced.

Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.

Flowering plants harbor the largest mitochondrial genomes reported so far. At present, the nucleotide sequences of 15 mitochondrial genomes from seven angiosperm species are available, making detailed comparative analysis feasible. The gene content is variable among the species, but the most striking feature is the fluidity of intergenic regions, where species-specific sequences predominate. Additionally, angiosperm mitochondrial genomes, even within a species, show a remarkable amount of rearrangement. We also review mitochondrial mutants in angiosperms from a genomic viewpoint, and discuss how they have arisen. The involvement of nuclear genes in mitochondrial genome stability and organization is currently being revealed through the analysis of mutants.

We have sequenced five distinct mitochondrial genomes in maize: two fertile cytotypes (NA and the previously reported NB) and three cytoplasmic-male-sterile cytotypes (CMS-C, CMS-S, and CMS-T). Their genome sizes range from 535,825 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-120 kb) account for most of the size increases. Plastid DNA accounts for 2.3-4.6% of each mitochondrial genome. The genomes share a minimum set of 51 genes for 33 conserved proteins, three ribosomal RNAs, and 15 transfer RNAs. Numbers of duplicate genes and plastid-derived tRNAs vary among cytotypes. A high level of sequence conservation exists both within and outside of genes (1.65-7.04 substitutions/10 kb in pairwise comparisons). However, sequence losses and gains are common: integrated plastid and plasmid sequences, as well as noncoding "native" mitochondrial sequences, can be lost with no phenotypic consequence. The organization of the different maize mitochondrial genomes varies dramatically; even between the two fertile cytotypes, there are 16 rearrangements. Comparing the finished shotgun sequences of multiple mitochondrial genomes from the same species suggests which genes and open reading frames are potentially functional, including which chimeric ORFs are candidate genes for cytoplasmic male sterility. This method identified the known CMS-associated ORFs in CMS-S and CMS-T, but not in CMS-C.

The P2 line of maize (Zea mays) is characterized by mitochondrial genome destabilization, initiated by recessive nuclear mutations. These alleles alter copy number control of mitochondrial subgenomes and disrupt normal transfer of mitochondrial genomic components to progeny, resulting in differences in mitochondrial DNA profiles among sibling plants and between parents and progeny. The mitochondrial DNA changes are often associated with variably defective phenotypes, reflecting depletion of essential mitochondrial genes. The P2 nuclear genotype can be considered a natural mutagenesis system for maize mitochondria. It dramatically accelerates mitochondrial genomic divergence by increasing low copy-number subgenomes, by rapidly amplifying aberrant recombination products, and by causing the random loss of normal components of the mitochondrial genomes.

Interorganellar signaling interactions are poorly understood. The maize non-chromosomal stripe (NCS) mutants provide models to study the requirement of mitochondrial function for chloroplast biogenesis and photosynthesis. Previous work suggested that the NCS6 mitochondrial mutation, a cytochrome oxidase subunit 2 (cox2) deletion, is associated with a malfunction of Photosystem I (PSI) in defective chloroplasts of mutant leaf sectors (Gu et al., 1993). We have now quantified the reductions of photosynthetic rates and PSI activity in the NCS6 defective stripes. Major reductions of the plastid-coded PsaC and nucleus-coded PsaD and PsaE PSI subunits and of their corresponding mRNAs are seen in mutant sectors; however, although the psaA/B mRNA is greatly reduced, levels of PsaA and PsaB (the core proteins of PSI) are only slightly decreased. Levels of the PsaL subunit and its mRNA appear to be unchanged. Tested subunits of other thylakoid membrane complexes--PSII, Cyt b6/f, and ATP synthase, have minor (or no) reductions within mutant sectors. The results suggest that specific signaling pathways sense the dysfunction of the mitochondrial electron transport chain and respond to down-regulate particular PSI mRNAs, leading to decreased PSI accumulation in the chloroplast. The reductions of both nucleus and plastid encoded components indicate that complex interorganellar signaling pathways may be involved.

The NB mitochondrial genome found in most fertile varieties of commercial maize (Zea mays subsp. mays) was sequenced. The 569,630-bp genome maps as a circle containing 58 identified genes encoding 33 known proteins, 3 ribosomal RNAs, and 21 tRNAs that recognize 14 amino acids. Among the 22 group II introns identified, 7 are trans-spliced. There are 121 open reading frames (ORFs) of at least 300 bp, only 3 of which exist in the mitochondrial genome of rice (Oryza sativa). In total, the identified mitochondrial genes, pseudogenes, ORFs, and cis-spliced introns extend over 127,555 bp (22.39%) of the genome. Integrated plastid DNA accounts for an additional 25,281 bp (4.44%) of the mitochondrial DNA, and phylogenetic analyses raise the possibility that copy correction with DNA from the plastid is an ongoing process. Although the genome contains six pairs of large repeats that cover 17.35% of the genome, small repeats (20-500 bp) account for only 5.59%, and transposable element sequences are extremely rare. MultiPip alignments show that maize mitochondrial DNA has little sequence similarity with other plant mitochondrial genomes, including that of rice, outside of the known functional genes. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly three-fourths of the maize NB mitochondrial genome is still of unknown origin and function.

The consequences of mitochondrial dysfunction are not limited to the development of oxidative stress or initiation of apoptosis but can result in the establishment of stress tolerance. Using maize mitochondrial mutants, we show that permanent mitochondrial deficiencies trigger novel Ca(2+)-independent signaling pathways, leading to constitutive expression of genes for molecular chaperones, heat shock proteins (HSPs) of different classes. The signaling to activate hsp genes appears to originate from a reduced mitochondrial transmembrane potential. Upon depolarization of mitochondrial membranes in transient assays, gene induction for mitochondrial HSPs occurred more rapidly than that for cytosolic HSPs. We also demonstrate that in the nematode Caenorhabditis elegans transcription of hsp genes can be induced by RNA interference of nuclear respiratory genes. In both organisms, activation of hsp genes in response to mitochondrial impairment is distinct from their responses to heat shock and is not associated with oxidative stress. Thus, mitochondria-to-nucleus signaling to express a hsp gene network is apparently a widespread retrograde mechanism to facilitate cell defense and survival.

We have examined the expression of three alternative oxidase (aox) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.

Each mitochondrion possesses a genome that encodes some of its own components. The nucleus encodes most of the mitochondrial proteins, including the polymerases and factors that regulate the expression of mitochondrial genes. Little is known about the number or location of these nuclear factors. B-A translocations were used to create dosage series for 14 different chromosome arms in maize plants with normal cytoplasm. The presence of one or more regulatory factors on a chromosome arm was indicated when variation of its dosage resulted in the alteration in the amount of a mitochondrial transcript. We used quantitative Northern analysis to assay the transcript levels of three mitochondrially encoded components of the cytochrome c oxidase complex (cox1, cox2, and cox3). Data for a nuclearly encoded component (cox5b) and for two mitochondrial genes that are unrelated to cytochrome c oxidase, ATP synthase alpha-subunit and 18S rRNA, were also determined. Two tissues, embryo and endosperm, were compared and most effects were found to be tissue specific. Significantly, the array of dosage effects upon mitochondrial genes was similar to what had been previously found for nuclear genes. These results support the concept that although mitochondrial genes are prokaryotic in origin, their regulation has been extensively integrated into the eukaryotic cell.

Cytoplasmic male sterility (CMS) and the abnormal-growth, nochromosomal stripe (NCS) phenotypes are cytoplasmically determined traits in plants. Mitochondrial DNA rearrangements involving short repeats appear to be responsible for the production of CMS reversions to fertility and NCS mutations. NCS4, a new mutant of maize CMS-S, exhibits both abnormal growth and male fertility. This mutant is unique because both mutations occurred within the same plant. Free S1 and S2 episomes normally found in CMS-S mitochondria have been lost from NCS4 plants. An S2 sequence has recombined aberrantly with a ribosomal protein coding region, rps3/rpl16. One end of the S2 sequence and the 5' end of the rps3/rpl16 transcription unit are absent from the NCS4 recombinant genome. Loss of mitochondrial ribosomal protein function is lethal; therefore, NCS4 plants are heteroplasmic for the rps3 deletion. Loss of S sequences from CMS-S mitochondria is not lethal and plants regain pollen function. Thus, although NCS4 plants have very abnormal plant phenotypes, they are male-fertile.

We have characterized two promoters of the cytochrome oxidase subunit 2 (cox2) gene in Zea perennis mitochondria present in maize lines. Initiation at a site 907 bases upstream of the start codon results in the major approximately 1900 nt cox2 transcript. A sequence just upstream of this site conforms to the consensus described for maize mitochondrial promoters and its transcription is correctly initiated in a maize mitochondrial in vitro transcription extract. A second transcription initiation site (-347) is used only when the dominant allele of a nuclear gene, Mct, is present and its use results in an additional, shorter major transcript. Sequences flanking the Mct-dependent transcription initiation site, which we have termed the conditional promoter of cox2 (cpc), do not fit the maize mitochondrial promoter consensus and do not function in the maize in vitro transcription extract. The cpc region does not hybridize with mitochondrial, chloroplast or nuclear DNAs from most maize or teosinte lines. However, the cpc sequence is found in the same position upstream of the cox2 gene in Zea diploperennis mtDNA and it has striking similarity to the previously reported 'ORF of unknown origin' fused to the ATPase subunit 6 gene in maize CMS-C mitochondria. cpc appears to represent a new type of mitochondrial promoter. Further analysis of both conditional and constitutive promoters should help us to better understand the control of transcription in plant mitochondria.

The maize NCS6 mitochondrial mutation is a partial deletion of the cytochrome oxidase subunit 2 gene (cox2) that survives heteroplasmically in the plant. Mutant mitochondria segregate from normal mitochondria during somatic development giving rise to defective sectors on the plants, including areas of kernel abortion on the ears. Embryos from NCS6 kernels can be rescued by tissue culture. Slowly growing Type II callus derived from one of these embryos has been shown by PCR analysis to be homoplasmic for the mutation, carrying only the defective mitochondrial cox2 gene. Most of the rescued embryos were heteroplasmic for normal and mutant genes and heteroplasmy was maintained in the callus cultures. However, when suspension cultures were initiated from heteroplasmic calli, normal cells were shown to have a selective advantage. When the homoplasmic cox2 mutant callus cultures were placed on regeneration medium, plantlets did not regenerate. Heteroplasmic calli were capable of regeneration under the same conditions. These studies suggest that the functioning of mitochondrial cytochrome oxidase is not essential for growth as callus, but is required for the differentiation and development of plants.

The molecular basis of the maternally inherited, heteroplasmic NCS2 mutant of maize was investigated. Analysis of the NCS2 mtDNA showed that it closely resembles the progenitor cmsT mitochondrial genome, except that the mutant genome contains a fused nad4-nad7 gene and is deleted for the small fourth exon of nad4. The rearrangement has occurred at a 16-bp repeat present in the third intron of the nad4 gene and in the second intron of the nad7 gene. Transcripts containing exon 4 of the nad4 gene are greatly reduced in mtRNA preparations from heteroplasmic NCS2 plants; larger transcripts are associated with the first three nad4 exons. Identical 5' ends of the nad4 transcripts have been mapped 396 and 247 bp upstream of the start codon in mtRNAs from both NCS2 and related non-NCS plants. The putative transcription termination signal of nad4 is deleted in mutant DNA, resulting in the production of the unique longer transcripts. The complex transcript pattern associated with nad7 is also altered in the mutant. Both nad4 and nad7 encode subunits of complex I (NADH dehydrogenase) of the mitochondrial electron transfer chain. Oxygen uptake experiments show that the functioning of complex I is specifically reduced in mitochondria isolated from NCS2 mutant plants.

Two linear DNA plasmids resembling the R1 and R2 plasmids that are present in the mitochondria of several South American strains of maize were found in mitochondria from a single source of Zea luxurians collected by L. Mazoti. The Mazoti mtDNA is closely related to mtDNAs of other Z. luxurians, but mitochondria derived from the other Z. luxurians sources lack the plasmids. The larger plasmid from Mazoti mitochondria, M1, was cloned and large portions of it were sequenced. Restriction mapping and sequence comparisons showed that approximately 4.9 kb is similar to the S1 plasmid of maize and an additional 2.6 kb is related to R1 sequences integrated into the main mitochondrial genome of N cytoplasm. Therefore, the M1 plasmid appears to be very similar to the R1 plasmid. The inverted repeats at the ends of the M1 plasmid are not identical. The left end IR is similar to the S-TIRs found at the termini of the S plasmids. The right end IR more closely resembles the integrated R1 sequences, including the "variant" region of the TIR. Whereas the variant region contains 13 bp in the S-TIRs and 15 bp in an integrated version of R1, it is 16 bp long in M1. The region of M1 that has no homology to the S1 plasmid is expressed at very low levels in Mazoti and RU cytoplasms, but at much higher levels in CMS-S mitochondria, where part of it is present in the main mitochondrial genome.

The nonchromosomal stripe (NCS6) mutation of maize is a partial deletion of the mitochondrial cytochrome oxidase subunit 2 (Cox2) gene. The Cox2 deletion and a narrow yellow striping phenotype are inherited together in a maternal fashion. The striped plants are heteroplasmic for mutant and normal Cox2 genes. Only the mutant Cox2 gene is detected within the yellow stripes, whereas both normal and mutant forms of the gene are present in the green sectors of the NCS6 plants. In the green leaves of nonstriped relatives, only the normal Cox2 gene is found. Both the structure and functioning of the chloroplasts in the yellow leaf sectors of NCS6 plants are altered. The pleiotropic effects of the NCS6 mutation suggest that mitochondrial function is required for the development of photosynthetically competent chloroplasts.

The nonchromosomal stripe 2 (NCS2) mutant of maize (Zea mays L.) has a DNA rearrangement in the mitochondrial genome that segregates with the abnormal growth phenotype. Yet, the NCS2 characteristic phenotype includes striped sectors of pale-green tissue on the leaves. This suggests a chloroplast abnormality. To characterize the chloroplasts present in the mutant sectors, we examined the chloroplast structure by electron microscopy, chloroplast function by radiolabeled carbon dioxide fixation and fluorescence induction kinetics, and thylakoid protein composition by polyacrylamide gel electrophoresis. The data from these analyses suggest abnormal or prematurely arrested chloroplast development. Deleterious effects of the NCS2 mutant mitochondria upon the cells of the leaf include structural and functional alterations in the both the bundle sheath and mesophyll chloroplasts.

A deletion eliminating part of a transcribed region of mitochondrial DNA (mtDNA) has been found in the maize nonchromosomal stripe 3 (NCS3) mutant. This results in the specific loss of a set of three mitochondrial RNAs consisting, in normal plants, of a 4.9 kb transcript, its 1.8 kb intron and the resulting processed mRNA of approximately 2.9 kb. In the NCS3 mitochondrial genome the DNA encoding the putative promoter and 5' end of the affected RNAs is missing. This transcribed region of normal maize mtDNA has been sequenced and the intron splice junction has been determined. The 2.9 kb processed mRNA carries two overlapping open reading frames (ORFs) with predicted amino acid sequences that show similarity to two Escherichia coli ribosomal proteins, S3 (rps3) and L16 (rpl16). The presence of severe stunting and striping in NCS3 plants correlates absolutely with the molecular changes described here. This fact and the impaired ability for mitochondrial protein synthesis by NCS3 plants indicate that one or both of these reading frames are translated to functional ribosomal proteins in normal maize mitochondria.

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 COXII polypeptide in Z. perennis-A619 combinations compared to Z. perennis-W23 combinations. All the major and minor transcripts posses 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 A619 carries the dominant allele.(ABSTRACT TRUNCATED AT 250 WORDS)

The nonchromosomal stripe (NCS) mutations of maize are a set of maternally inherited mutations that result in reduced plant growth and lowered reproductive capacity. NCS6 mutants are characterized by stunted growth, yellow stripes on leaves, and sectors of aborted kernels on ears. The heteroplasmic NCS6 striped plants carry both partially deleted and normal versions of the mitochondrial cytochrome oxidase subunit 2 (cox2) gene and have low levels of cox2 transcripts when compared with nonstriped control plants. Other tested mitochondrial genes and their transcripts appear to be unaltered in NCS6 plants. The level of COXII protein is also specifically reduced in the striped plants relative to that of control plants. In the NCS6 mutant mitochondrial genome, a portion of the first exon of the cox2 gene is deleted along with the 5' flanking region. The formation of the defective cox2 gene appears to have involved a recombination event between 31-bp repeats.

We describe a new maternally inherited maize mutation, nonchromosomal stripe 5 (NCS5), that adversely affects plant growth and yield. Mutant plants are characterized by reduced height, defective yellow striping on leaves, and aborted kernels on ears. NCS5 striped plants carry both normal and partially deleted versions of the mitochondrial cytochrome oxidase subunit 2 gene and exhibit greatly reduced levels of cox2 transcripts when compared with nonstriped control plants. Other mitochondrial genes and their mRNAs are not affected. Thus, the defective plant phenotype is correlated with a reduction in the number of functional cytochrome oxidase subunit 2 genes. The NCS5 mutant mitochondrial genome appears to have arisen by amplification of a rare homologous recombination product.

Inbred line Ky21 carries nuclear genes which restore fertility to all three cytoplasmic male sterile (cms) types of maize: T, C and S. By substituting the Ky21 cytoplasm into a nuclear background lacking all of the nuclear restorer genes, we have demonstrated that Ky21 contains a fertile (normal) cytoplasm. Gel electrophoresis of mitochondrial DNA from Ky21 demonstrated an approximately 2.1 kb plasmid and no evidence for a 2.35 kb plasmid found in many normal cytoplasms of North American lines of maize. A 2.1 kb plasmid had been reported to be diagnostic for the T-type cms. However, the restriction endonuclease digestion pattern of Ky21 mtDNA more closely resembled that of normal lines than T-cms. Furthermore, mitochondria of Ky21 plants did not synthesize a 13 kilodalton polypeptide, which has only been found to be synthesized by T-type mitochondria. From these molecular criteria, as well as from the genetic analysis, we conclude that the mitochondria of the Ky21 universal restorer line are normal. In having a shorter form of a linear mtDNA plasmid, Ky21 resembles cytoplasms found in Mexican races of maize.

The mitochondrial MDH isozymes in the scutellum of the mature maize (Zea mays L.) kernel are encoded by three independently inherited nuclear genes. Mdh1 is located on chromosome 8, close to the breakpoint (8L.35) of a waxy-marked reciprocal translocation between chromosomes 8 and 9. Mdh2 is located in the distal region of the long arm of chromosome 6. Mdh3 is on the long arm of chromosome 3, approximately 2.6 map units from sh2. A modifier of the mitochondrial MDH isozymes (Mmm) maps approximately 27.5 units proximal to Adh1 in the central portion of the long arm of chromosome 1. Independently assorting duplicate genes code for the soluble MDH isozymes. Mdh4 is located in the same region of chromosome 1 as Mmm, approximately 29 map units proximal to Adh1. Mdh5 maps approximately 20 units distal to a2 in the short arm of chromosome 5.--Intergenic and interallelic heterodimer formation occurs among gene products that occupy the same subcellular compartment. MDH isozymes were purified and analyzed by native-SDS two-dimensional polyacrylamide gel electrophoresis. The proposed mitochondrial MDH intergenic heterodimer bands were found to be composed of two subunits, which differ in their migrations on SDS gels; whereas, genetically defined homodimers contained only one type of subunit.--This evidence is discussed in terms of two genetic models proposed for the maize mitochondrial MDH isozymes.

Genetically defined dosage series of chromosome arms 1L, 3L, 4S, 5L, 7L, 9S, 10L and combinations of 1L-3L, collectively spanning approximately one-third of the maize genome, were examined for alterations in the expression of total protein profiles in scutellar tissue. The major effects found were negative correlations of specific proteins with the dosage of particular regions in a manner similar to that previously described for enzyme activity levels (Birchler 1979). Chromosome arms 1L, 4S and 5L produced the most severe negative effects, with 3L and 7L exhibiting this phenomenon to a lesser degree. Positive correlations of certain proteins were observed with the dosage of the 1L, 3L, 5L and 7L regions. The structural locus of one of the major scutellar proteins (PRO) is present in the long arm of chromosome 1 (Schwartz 1979), but exhibits compensation in a dosage series involving whole-arm comparisons. Multiple factors in 1L affect the level of the protein. The compound TB-1La-3L4759-3 (1L 0.20-0.39) has a slight negative effect on PRO, while TB-1La-3Le (1L 0.20-0.58) and TB-1La-3L5267 (1L 0.20-0.72) have a more pronounced negative influence. The level of this protein is not altered by the dosage of 3L. These observations suggest that compensation is brought about by the cancellation of a positive structural gene dosage effect by the negative inverse effect. Other regions of the genome that contribute to the control of PRO levels are 4S and 5L. Total protein profiles were also compared in haploid, diploid and tetraploid maize as a comparison to the aneuploid series. Most proteins exhibit structural-gene-dosage effects through the ploidy series, but others show a positive effect greater than expected from varying the structural genes. Still others are negatively affected by ploidy changes. In general, the ploidy alterations are not as great as predicted from the cumulative action of the aneuploid effects. The bearing of these observations on the biochemical basis of aneuploid syndromes is discussed.

We have investigated the inheritance of the mitochondrial DNA (mtDNA) restriction endonuclease digestion patterns of maize inbred line B37N in individual plants and pooled siblings in lineages derived from five separate plants in the third generation following successive self-pollinations. The restriction fragment patterns of the different mtDNA samples were compared after digestion with five endonucleases. No differences were visible in the mobilities of the 199 fragments scored per sample. Hybridization analysis with two different cloned mtDNA probes, one of which contains homologies to a portion of the S2 plasmid characteristic of cms-S maize, failed to reveal cryptic variation. The apparent rate of genomic change in maize mtDNA from inbred plants appears to be very slow, compared with the faster rates of change seen in maize tissue cultures and with the documented rapid rate of inter- and intraspecific variation for mammalian mtDNA.

When cytoplasm of the teosinte Zea luxurians is introduced into certain maize inbred nuclear backgrounds, the pattern of protein synthesis in the teosinte mitochondria is altered. Teosinte mitochondria purified from plants possessing a maize A619 nuclear background (Z.l.-A619 plants) synthesize a novel 22-kDa polypeptide that is associated exclusively with the membrane fraction of the organelle. Mitochondria from plants possessing a W23 nuclear background do not synthesize this protein. The F(1) hybrids Z.l.-A619 x W23 and Z.l.-W23 x A619 do not synthesize the protein. However, synthesis of the polypeptide was observed in 14 of 21 individual progeny from the backcross of the F(1) hybrid Z.l.-A619 x W23 to the pollen parent A619. These data suggest that a single nuclear gene controls the synthesis of the 22-kDa protein in mitochondria, with the recessive allele of the gene allowing expression of the polypeptide. Mitochondria from the F(1) hybrid Z.l.-A619 x Mo17 synthesize the 22-kDa protein, whereas mitochondria from Z.l.-A619 x B73 do not. Data from these outcrosses demonstrate that other maize lines also possess nuclear genes capable of regulating the synthesis of the 22-kDa Zea luxurians mitochondrial protein.

The genetic and molecular analyses of higher plant mitochondria can be facilitated by studying maternally-inherited mutations, such as the nonchromosomal stripe (NCS) mutants of maize, that have deleterious effects on plant growth. We have previously demonstrated a correlation between specific alterations in mitochondrial DNA and the expression of NCS phenotypes. In the present studies, the effects of the NCS2 mutation on mitochondrial gene expression are evaluated. Proteins synthesized by mitochondria isolated from NCS2 mutants and from related plants with normal growth have been compared. NCS2 mitochondria synthesize much reduced amounts of a single polypeptide. Probes corresponding to the mitochondrial DNA region altered in NCS2 hybridize to an aberrant set of transcripts in NCS2 mitochondria. Transcripts homologous to several previously characterized plant mitochondrial genes are similar in NCS2 and related non-mutant mitochondria.

The genetic analysis of higher plant mitochondria has been limited by a scarcity of identified mutations with known progenitors. Correspondingly, few molecular studies have been directed at types of plant mitochondrial variation other than cytoplasmic male sterility. The maternally inherited nonchromosomal stripe (NCS) mutants of maize have profound deleterious effects on plant growth and yield. We report specific alterations in mitochondrial DNA (mtDNA) for two independent, phenotypically distinct NCS mutants. NCS2 plants have a distinctive 21-kilobase Xho I mtDNA band and very reduced amounts of DNA in an 8-kilobase band that is present in the progenitor. NCS3 plants have a distinctive 20-kilobase Xho I band and a reduction in a 16-kilobase band. Our studies confirm that the affected organelle in NCS plants is the mitochondrion. Because NCS-type plants appear with a certain frequency in a particular line (WF9), this line is a potential source of additional mutations for functional and molecular analyses of maize mitochondrial genes.

We have examined mitochondrial DNA (mtDNA) sequence conservation, transcriptional patterns of mitochondrial genes, and mitochondrial translation products in four species of the plant family Cucurbitaceae, in which there is a seven-fold range in mitochondrial genome size. A set of conserved mtDNA sequences which we term "core" DNA is present in all cucurbit genomes examined. In watermelon, only those mtDNA restriction fragments which contain "core" DNA hybridize with mtRNA. Similar numbers of polypeptides are synthesized by isolated mitochondria from all species examined, but an extra set of mtDNA sequences is transcribed in the largest genome (muskmelon). Taken together, these results suggest that some mtRNA is untranslated in muskmelon. Cloned mitochondrial genes of known function, from maize, identified mitochondrial transcripts that varied in both size and number among cucurbit species.

Five independently inherited loci on five distinct chromosomes encode the mitochondrial and cytosolic isozymes of NAD-dependent malate dehydrogenase (MDH; L-malate:NAD+ oxidoreductase, EC 1.1.1.37). Multiple alleles, including electrophoretic nulls, occur for each locus. However, a single allele of normal activity at one of the three loci encoding the mitochondrial MDHs is sufficient for normal development, whereas plants with essentially no cytosolic MDH activity function normally. The requirement of a normal activity allele at one of the three structural loci encoding the mitochondrial MDHs demonstrates in plants that a commonly studied dehydrogenase enzyme is essential for normal embryogenesis.

We detected both quantitative and qualitative organ-specific differences in the total protein composition of mitochondria of maize. Labeling of isolated mitochondria from each organ demonstrated that a few protein differences are due to changes in the polypeptides synthesized by the organelle. The synthesis of developmental stage-specific mitochondrial polypeptides was found in the scutella of developing and germinating kernels. The approximately 13-kDa polypeptide synthesized by mitochondria from seedlings of the Texas (T) male-sterile cytoplasm was shown to be constitutively expressed in all organs of line B37T tested. Methomyl, an insecticide known to inhibit the growth of T sterile plants, was shown to be an effective inhibitor of protein synthesis in mitochondria from T plants.