Daryl R. Pring

Initiation and processing of atp6, T-urf13 and ORF221 transcripts from mitochondria of T cytoplasm maize

SummaryThe mitochondrial gene T-urf13 is implicated in cytoplasmic male sterility and maternal inheritance of susceptibility to disease in T cytoplasm maize. The organization of the T-urf13 region is a consequence of multiple recombinational events, one of which has duplicated a 5 kb region that is 5′ to atp6. Transcription initiation and processing sites for atp6 were identified within the 5 kb region and found to serve T-urf13 and the cotranscribed gene ORF221. Differences in atp6 transcripts from N and T cytoplasms were associated with two small insertions in the T mitochondrial genome that create a unique processing site. Fertility restoration is associated with alterations of T-urf13 transcripts and a significant reduction in the gene product, a 13 kDa polypeptide. Specifically, the abundance of major T-urf13 transcripts is slightly reduced and a novel transcript is detected in T cytoplasm plants containing the dominant nuclear restorer gene Rf1. The 5′ terminus of the restorer-specific transcript mapped internal to T-urf13 and the reduction of major transcripts in plants with Rf1 is not as marked as the reduction in the 13 kDa polypeptide. This raises the possibility that the primary action of the Rf1 gene is associated with translational control, rather than RNA processing.

Molecular analysis of genomic stability of mitochondrial DNA in tissue cultured cells of maize.

SummaryMitochondrial DNA (mtDNA) of the Black Mexican sweet line of maize isolated from tissue cultured cell suspension cultures and young seedlings was examined. Restriction fragments generated by two endonucleases were comparatively analyzed by visualization of ethidium bromide stained gels as well as by membrane hybridization with nick-translated DNA probes of plasmid-like S1 and S2 DNA. Although no major molecular alterations were seen in tissue cultured cells, the samples were clearly not identical. The variation was mainly in the stoichiometry of several restriction fragments. Hybridization analyses with S1 and S2 probes show no evidence of molecular rearrangement in this part of the genome in tissue cultured cells. Minor variations in restriction patterns could reflect alterations in frequency of circular mtDNA molecules, perhaps related to nuclear alterations during the extended period of culture.

Plasmid like DNAs associated with Mitochondria of Cytoplasmic Male-sterile Sorghum

SummaryPlasmid-like, linear DNAs were detected in preparations of mitochondrial DNA from cytoplasmic male-sterile sorghum. Designated N-1 and N-2, the DNAs exhibited molecular sizes of ca. 5,700 and 5,300 bp, respectively. The DNAs occurred in only the IS1112C entry among 24 entries examined. Electron microscopy of the DNAs indicated that the molecules were linear as isolated. Nick translation of N-1 and N-2 followed by membrane hybridization indicated substantial homology between the two DNAs, and some homology to the S-1 and S-2 maize plasmid-like DNAs. At least four additional DNA species, ranging from ca. 1,000–4,000 bp if linear, were also detected in sorghum mitochondrial DNA. The detection of plasmid-like DNAs in sorghum, with homology to the plasmid-like DNAs of maize, suggests an etiological relationship of the molecules to the inheritance and expression of cytoplasmic male sterility in sorghum, perhaps in a manner analogous to the postulated role of these elements in maize.

Polymorphism of mitochondrial DNA ‘S’ regions among normal cytoplasms of maize

Genomic variation in S1 and S2 homologous sequences, defined as the S regions, were examined in mitochondrial DNAs of 12 normal cytoplasm maize lines collected in the United States. Three genomic variants were detected among the 12 cytoplasms, eight of which were identical to the Wf9 model structure. Hybridization data with S1 and S2 DNAs and with two cosmids spanning these regions were consistent with the concept that S1 and S2 sequences are found in each normal cytoplasm. Three variations of the S1 region were established; the Wf9 structure, a second group consisting of F6, A188, and W182BN, and a third, Black Mexican. Genome structure was conserved through the S2 region in all lines examined. None of the cytoplasms included complete copies of S1; the 1400 bp repeat characteristic of S1 and S2 was absent in the S1 region of all lines. A 2.1 kb linear DNA was observed instead of a 2.3 kb DNA in F6, A188, and W182BN. Integrated copies of S1 and S2 sequences may be a constituitive characteristic of normal, male-fertile maize cytoplasms.

Physical mapping of homologous segments of mitochondrial episomes from S male-sterile maize.

Restriction endonuclease maps of two double-stranded plasmid-like DNAs, 6180 and 5175 bp each, isolated as linear molecules from the mitochondria of S-type cytoplasmic male-sterile maize were prepared. Twelve cleavage sites were mapped in each using HindIII, XhoI, EcoRI, SacI, XbaI, SalI, BamHI, and BstEII. BamHI does not cleave S-1 DNA and SalI does not cleave S-2 DNA. A 1150-bp homologous sequence in addition to the 200-bp terminal inverted repeats was terminally oriented on both DNAs by reciprocal hybridization and heteroduplex analysis.

Fertility restoration and mitochondrial nucleic acids distinguish at least five subgroups among cms-S cytoplasms of maize (Zea mays L.)

SummaryDifferences in fertility restoration and mitochondrial nucleic acids permitted division of 25 accessions of S-type male sterile cytoplasm (cms-S) of maize into five subgroups: B/D, CA, LBN, ME, and S(USDA). S cytoplasm itself (USDA cytoplasm) was surprisingly not representative of cms-S, since only two other accessions, TC and I, matched its mitochondrial DNA pattern. CA was the predominant subgroup, containing 18 of the 25 accessions. The B/D and ME subgroups were the most fertile and LBN the most sterile. The exceptional sterility of LBN cytoplasm makes it the most promising of the 25 cms-S accessions for the production of hybrid seed. The most efficient means of quantifying the fertility of the subgroups was analysis of pollen morphology in plants having cms-S cytoplasm and simultaneously being heterozygous for nuclear restorer-of-fertility (Rf) genes. This method took advantage of the gametophytic nature of cms-S restoration. The inbred NY821LERf was found to contain at least two restorer genes for cms-S. Fertility differences were correlated with mitochondrial nucleic acid variation in the LBN, ME, and S (USDA) subgroups.

A physical map of the sorghum chloroplast genome

SummaryThe chloroplast genome of the IS1112C cytoplasm of sorghum was mapped by the construction of a Bam-HI library in pUC8, and hybridization with BamHI, SalI, and PstI digests of chloroplast DNA (ctDNA) of sorghum and maize. The molecules are extensively colinear, with only one of 13 SalI fragments differing slightly from maize. Seven of 70 restriction sites differed in the two species. A total molecular size of ca. 138 kb was estimated for sorghum. The inverted repeat was not conserved between sorghum and maize, as revealed by a slightly larger BamHI 16S rDNA fragment in sorghum. Homology of a sequence adjacent to the γbcl gene and one end of the inverted repeat was detected. These homologies were also observed in maize, and suggest that the ctDNA genomes of sorghum and maize share small reiterations of sequences of the inverted repeat.

Plant Breeding: Male Sterility in Higher Plants - Fundamentals and Applications

Under normal conditions, plants undergo a life cycle that consits of an alternating vegetative sporophytic generation and a much-reduced sexual gametophytic generation. During these cycles, seeds germinate, the mature plant organs differentiate, and finally vegetative growth terminates in flower formation, which in principle leads to sepals, petals, anthers, and carpels. Male sterile mutants which cannot produce fertile pollen or functional anthers can often be observed in higher plant species. In this chapter we differentiate between nuclear and cytoplasmicnuclear male sterility (CMS); the latter is particularly useful for production of hybrid seed, which is the main application of CMS. To date, Fl hybrid varieties are produced in most agricultural and horticultural crops. The successful exploitation of heterosis requires a simple and reliable system to produce female parents and perform crosses for the production of hybrid seed. Without a CMS system, male floral organs must be removed mechanically, which is usually not economical nor practical. While nuclear male sterility is based solely on mutations which occur in nuclear genes, CMS is maternally inherited and based on changes in mitochondrial gene expression as influenced by nuclear genes. Importantly, the CMS phenotype may be corrected by nuclear fertility restoration (RF) genes. In the first section, we discuss anther and pollen development and present recent molecular data as well. In the second part, some of the most important CMS systems are presented. Finally, approaches to genetically engineering male sterility in higher plants are discussed. Genetically engineered male sterility may be applied to any crop, including those crops where CMS systems are not available or are unreliable. This area thus has a tremendous potential in plant breeding.

Transcript profiling of male-fertile and male-sterile sorghum indicates extensive alterations in gene expression during microgametogenesis

Male-sterile sorghum carrying the IS1112C cytoplasm represents an unusual example of aberrant microgametogenesis wherein microspores develop into inviable pollen that remain physically intact until anther exsertion. These inviable pollen grains do not deposit starch, yet fluoresce with the vital stain fluoroscein diacetate. We sought to elucidate the extent of differential gene expression in this subtle example of defective microgametogenesis through cDNA-AFLP transcript profiling of near-isogenic male-fertile and male-sterile plants at an early stage representing early-mid microspores to early pollen, 7–11xa0days prior to anthesis, and a late stage representing young to nearly mature pollen, spanning the terminal 96xa0h of pollen development. The transition from early to late stages is characterized by changes in abundance of nearly 33% of shared transcripts, and early- or late-specific expression of about 10% of transcripts. Male-sterile plants exhibit extensive changes in regulatory patterns characteristic of fertile plants, including premature expression of late-specific, and prolonged expression of early-specific, transcripts. Genome-wide transcriptome patterns indicate the expression of an estimated 12,000 genes in early-mid microspores, and the abundance of at least 15% of these transcripts is altered in male-sterile plants. A near-isogenic line restored to male fertility is characterized by apparent normalized expression of most of these transcripts. The development of the microgametophyte is thus characterized by dynamic programmed changes in gene expression, and the expression of male sterility compounds these changes in a complex manner.

Microspore gene expression associated with cytoplasmic male sterility and fertility restoration in sorghum

Gene expression associated with the restoration of fertility in sorghum lines carrying the IS1112C male-sterile cytoplasm was characterized through cDNA-AFLP transcript profiling and examinations of protein product abundance of selected mitochondrial genes. Microspores from near-isogenic male-sterile, fertility-restored, and normal-cytoplasm lines in two genetic backgrounds, and from four recombinant inbred lines (RILs) differing at two fertility restorer loci, were examined. We characterized seven transcript-derived fragments (TDFs) unique to a BC7F3 fertility-restored line, four of which were eliminated in a marker-selected BC11F3. Transcripts or cDNAs of the remaining three TDFs were detected in all lines examined, indicating that the unique fragments represent alleles detected by sequence polymorphisms or by enhanced transcript abundance. The TDFs were found to be derived from the non-recurrent IS1112C male parent, and certain of the TDFs may be linked to the fertility restorer alleles, consistent with analyses of the RILs. Sequences of the TDFs showed similarity with EST’s of unknown cellular function. The accumulation of mitochondrial proteins was examined in male-sterile, normal-cytoplasm, and restored BC11F3 lines. The male-sterile microspore mitochondria contained elevated levels of the nuclear-encoded alternative oxidase protein, potentially associating mitochondrial dysfunction with failed pollen development. The relative abundance of mitochondrial-encoded ATPA, ATP6 and COXII proteins, and of nuclear-encoded MnSOD, Cyt c and ALDH proteins, did not vary with cytoplasm or fertility status at the microspore stage.