Christine D. Chase

Molecular Biology and Biotechnology of Plant Organelles

The fields of organelle biology and plant biotechnology have now been inter-related for a century. Plant organelles have intrigued biologists since their defiance of Mendelian inheritance and their endosymbiont origins became apparent. The first application of organelle biotechnology was the role played by cytoplasmic male sterility in hybrid seed production, a contribution towards the “Green Revolution”. In modern times, plant organelles are again leading the way for the creation of genetically modified crops. On a global scale, 75% of GM crops are engineered for herbicide resistance and most of these herbicides target pathways that reside within plastids. Several thousand proteins are imported into chloroplasts that participate in biosynthesis of fatty acids, amino acids, pigments, nucleotides and numerous metabolic pathways including photosynthesis. Thus, from green revolution to golden rice, plant organelles have played a critical role in revolutionizing agriculture. This book details not only the basic concepts and current understanding of plant organelle genetics and molecular biology but also focuses on the synergy between basic biology and biotechnology. Forty-four authors from nine countries have contributed 24 chapters, containing 52 figures and 28 tables. Section one on organelle genomes & proteomes discusses molecular features of plastid and mitochondrial genomes, evolutionary origins, somatic and sexual inheritance, proteomics, bioinformatics and functional genomics. Section two on organelle gene expression and signalling discusses transcription, translation, RNA processing, RNA editing, introns and splicing, protein synthesis, proteolysis, import of proteins into chloroplast and mitochondria and regulation of all these processes. Section three on organelle biotechnology discusses the genetic manipulation of organelles by somatic cell genetics, the use of cytoplasmic male sterility for hybrid seed production and the exciting applications of chloroplast and nuclear genetic engineering for biotic/abiotic stress tolerance, improved fatty acid/amino acid biosynthesis, and for the production of biopharmaceuticals, biopolymers and biomaterials. 1. HISTORICAL PERSPECTIVE Plant organelles have fascinated geneticists since the rediscovery of Mendel’s laws. In 1904, Correns reported maternally inherited male sterility in savory, and similar observations were soon reported for other plant species (reviewed by Edwardson, 1956; Duvick, 1959; and Havey, chapter 23 of this volume). In 1907, Correns 2 HENRY DANIELL AND CHRISTINE D. CHASE reported maternal inheritance of mutant plastids in Mirabilis, and Bauer reported non-Mendelian, bi-parental inheritance of mutant plastids in Pelargonium (reviewed by Hagemann, 2000; Hagemann, chapter 4 of this volume; Maier and SchmitzLinneweber, chapter 5 of this volume). At the same time, the stage was being set for the first application of organelle genetics to biotechnology. In 1908, Shull and East rediscovered hybrid vigour, or heterosis, in maize, and this ultimately led to the commercial development of F1 hybrid maize (reviewed by Duvick, 2001). The production of F1 hybrid seed on a commercial scale requires large, uniform populations of male-sterile plants. For many crops, this has been achieved primarily through the use of cytoplasmically inherited male sterility (CMS) (reviewed by Duvick, 1959; Havey, chapter 23). Long before molecular approaches demonstrated that CMS resulted from gain-of-function mutations in the mitochondrial genome (reviewed by Chase and Gabay-Laughnan, chapter 22 of this volume), this organelle-encoded trait was extensively exploited in commercial agriculture. Since their discovery, mitochondrial CMS genes have served as useful reporter genes in basic studies of plant mitochondrial gene expression. (See chapter 22 for examples). Daniell et al. (chapter 16) review the first chloroplast-encoded CMS trait, engineered using the phaA gene coding for β-ketothiolase. Thus the basic science and practical applications of organelle biology have now been synergistically linked for a century. 2. EVOLUTION AND MOLECULAR BIOLOGY Questions concerning the evolution of organelles have been a key force driving studies of organelle molecular biology. The endosymbiont origin of cellular organelles was proposed as early as 1882 and this hypothesis was further developed early in the twentieth century (reviewed by Margulis, 1970). Molecular features of organelle genes and genomes (reviewed by Gray, chapter 2 of this volume) provided the confirming data for the endosymbiont hypothesis. Molecular research over the subsequent decades revealed many prokaryotic features in the modern-day plant organelles, including some aspects of organelle division, genome organization and coding content, transcription, translation, RNA processing, and protein turn-over. These features are discussed in chapters 3, 5 and 6, 8, 10, 12 and 15 of this volume, respectively. For genome maps, models or graphical representations of pathways, readers are referred to color plates inserted at the beginning of this book. While confirming the basic endosymbiont hypothesis, molecular investigations also revealed many surprises and raised new questions as to how evolution has shaped the modern-day organelles. Present-day mitochondrial genomes are extremely diverse (Gray, chapter 2), and the mitochondrial genomes of the land plants (reviewed by Fauron et al., chapter 6) are uniquely expanded over the ancestral type. The mitochondrial genomes, and even the relatively conserved plastid genomes, vary in coding content among land plant species (Maier and Schmitz-Linneweber, chapter 5; Fauron et al., chapter 6). The functional transfer of genes from organelle to nuclear genomes has occurred frequently and recently (on MOLECULAR BIOLOGY AND BIOTECHNOLOGY OF PLANT ORGANELLES 3 an evolutionary scale) in the plant lineages. Group I and group II introns are abundant in plant organelle genomes, but are found only rarely in the modern bacterial relatives of the organelle progenitors. The gain of introns by plant organelles was accompanied by the recruitment of an interesting spectrum of proteins that facilitate intron splicing (reviewed by Barkan, chapter 11). One of the greatest surprises of plant organelle biology was the C-to-U and U-to-C RNA editing process that occurs in both plastids and mitochondria. In many cases RNA editing is required to restore the coding of highly conserved amino acid sequences, but specific RNA editing sites are not always conserved, even among closely related plant species. The molecular mechanisms that underlie the selection and editing of these sites are only beginning to be understood (reviewed by Mulligan, chapter 9). 3. ORGANELLES AND THE NUCLEUS The endosymbiont origin of the organelles was accompanied by the transfer of significant genetic information to the nuclear genome. In the case of the mitochondria, this transfer may have been simultaneous with the origin of the organelle (Gray, chapter 2). The majority of organelle proteins are translated on cytosolic ribosomes, and sophisticated targeting and import machinery (reviewed by Glaser and Soll, chapter 14) is required to ensure these proteins find their ultimate destination in the cell. The analysis of complete nuclear genome sequences with organelle targeting prediction programs (reviewed by Colas des Francs-Small et al., chapter 7; Glasser and Soll, chapter 14) estimates that 10% and 15% of plant nuclear genes predict proteins targeted to mitochondria and chloroplasts, respectively. Organelle function is therefore dependent upon interactions between nuclear and organelle genetic systems. The identification of nuclear genes having organelle targeting signals does not always, however, provide information regarding the role of the gene or its product in the organelle. Systematic functional genomics approaches (reviewed by Colas des Francs-Small et al., chapter 7) will doubtless provide further insights. Forward genetic analysis has also identified nuclear genes key to organelle processes such as RNA processing, intron splicing, protein synthesis and protein complex assembly. (See chapters 10, 11, 12, 13 and 22 for examples.) Much remains to be learned regarding the regulation of the many nuclear genes relating to organelle function. The “retrograde” regulation of nuclear genes by signals originating in the organelle was first discovered in yeast (Parikh et al., 1987), and many examples of retrograde signalling have now been uncovered in plants. The reader is referred to recent reviews (McIntosh et al., 1998; Rodermel, 2001; Surpin et al., 2002; Pfannschmidt et al., 2003; Gray et al., 2003; Juszczuk and Rychter, 2003; Pfannschmidt, 2003; and Rodermel and Park, 2003) for details on these systems. The recent finding that some cases of CMS result from retrograde regulation of floral homeotic genes by the mitochondria (reviewed by Zubko, 2004; Chase and Gabay-Laughnan, chapter 22) indicates that the nuclear gene targets of organelle regulatory signals are likely to extend beyond those genes directly associated with organelle functions. 4 HENRY DANIELL AND CHRISTINE D. CHASE Organelle proteomics (reviewed by Colas des Francs-Small et al., chapter 7) provide an excellent complement to genetics and genome analysis for the study of organelle biogenesis and function. Organelle targeting signals are not yet predicted with high confidence. Some may be overlooked or misidentified, and there are several interesting observations of proteins targeted to both plastids and mitochondria. (See chapters 7 and 14 for examples.) Organelles are well suited to proteomic studies, because these membrane-bound components are relatively easy to separate from other cellular constituents. Recent studies of plant organelle proteomes have provided new insights into many aspects of organelle gene expression and metabolism. This, in turn, reveals new targets fo

Mitochondrial gene expression in developing male gametophytes of male-fertile and S male-sterile maize

Abstract Mitochondria play a critical role in the normal development of the plant male gametophyte and in the disruption of normal gametophyte development associated with cytoplasmically inherited male sterility (CMS). To investigate the role of mitochondria in these processes, the accumulation of mitochondrial gene transcripts and the accumulation of nuclear gene transcripts encoding mitochondrial proteins were investigated through male gametophyte development in normal maize and through the course of pollen abortion in CMS-S maize. Male gametophytes differing in developmental stage were isolated from male-fertile or male-sterile plants by sucrose density gradient centrifugation. Mature pollen was collected from dehiscent anthers of male-fertile plants. Aborted pollen, which collapsed during starch accumulation, was isolated from emergent tassels of CMS-S male-sterile plants. Microspores, developing pollen and mature pollen exhibited striking differences in mitochondrial transcript accumulation. Mature pollen lacked detectable mitochondrial transcripts. Aborted pollen of CMS-S plants contained abundant, intact transcripts of all mitochondrial genes studied, but prematurely degraded transcripts of several nuclear genes. Transcripts of the CMS-S associated mitochondrial open reading frames (orf355 and orf77) were detected from the early stages of microspore development through the aborted pollen stage. The implications of these findings are discussed in terms of the mitochondrial requirements for pollen function and the mechanism of pollen abortion in CMS-S maize.

PLEIOTROPIC EFFECTS OF A NUCLEAR RESTORER-OF-FERTILITY LOCUS ON MITOCHONDRIAL TRANSCRIPTS IN MALE-FERTILE AND S MALE-STERILE MAIZE

Abstract Cytoplasmic male sterility (CMS) is encoded by the plant mitochondrial genome and can be reversed by nuclear restorer-of-fertility(Rf) alleles. In the CMS-S system of maize, reproductive failure and fertility restoration are gametophytic, occurring during the starch-filling stages of pollen development. Transcripts of the CMS-S-associated mitochondrial open reading frames (orf355 and orf77) are present from the early stages of microspore development through the aborted pollen stage. To investigate the molecular basis of fertility restoration, we compared mitochondrial-transcript accumulation in aborting CMS-S pollen and in CMS-S pollen restored to fertility by the Rf3 nuclear allele. In the presence of the Rf3 allele, novel, shorter transcripts of the orf355-orf77, cob and atp6 mitochondrial genes were created, and the relative abundance of larger transcripts was decreased for each of these loci. The altered transcript patterns cosegregated with male fertility conditioned by the Rf3 allele. The novel cob and atp6 transcripts were also observed in leaf-tissues of both normal and S-cytoplasm plants carrying the Rf3 allele. These observations support the hypothesis that the Rf3 allele encodes, or regulates, a modifier of mitochondrial transcript (Mmt) activity that affects both CMS and essential mitochondrial gene transcripts.

Inheritance of organelle genomes in citrus somatic cybrids

Restriction fragment length polymorphisms (RFLPs) were used for the characterization of citrus organelle inheritance in somatic cybrids produced during six different citrus protoplast fusions. All the cybrids in this work inherited their mitochondrial genome from the embryogenic fusion partner (callus or cell suspension). In some of the combinations, non-parental bands were observed among the mitochondrial configurations. In contrast, the cybrids inherited plastid DNA from either the embryogenic or the nonembryogenic (leaf) fusion partner. The relative abundance of organelle DNAs in the embryogenic and leaf cells was in accordance with these inheritance patterns. Stochastic processes may therefore influence the outcome of somatic cell fusions with respect to organelle genomes.

Organization of ATPA coding and 3′ flanking sequences associated with cytoplasmic male sterility in Phaseolus vulgaris L.

SummaryA region of the mitochondrial genome associated with cytoplasmic male sterility (CMS) in Phaseolus vulgaris was flanked by two different repeated sequences designated x and y. The DNA sequence of the CMS-unique region and a portion of each flanking repeat was determined. Repeat x contained a complete coding copy of the F1 ATPase subunit A (atp A) gene, as well as an open reading frame (orf) predicting a protein of 209 amino acids. The TGA termination codon of the atpA gene and the ATG initiation codon of orf209 were overlapping. These reading frames were oriented with their 3′ ends proximal to the CMS-unique region. The CMS-unique region of 3736 nucleotides contained numerous orfs. The longest of these predicted proteins being of 239, 98 and 97 amino acids. The 3′ coding and 3′ flanking regions of orf98 were derived from an internal region of the higher plant chloroplast tRNA alanine intron. The region of repeat y immediately adjacent to the CMS-unique region contained the 111 carboxy-terminal coding residues of the apocytochrome b (cob) gene. This segment was oriented with its 5′ end proximal to the CMS-unique region, but cob gene sequences were not fused to an initiation codon within the unique region.

Mitochondrial RNA editing truncates a chimeric open reading frame associated with S male-sterility in maize

Abstract. Adjacent mitochondrial open reading frames orf355 and orf77 are associated with S cytoplasmic male sterility (CMS-S) in maize, but the mechanisms leading to collapse of developing CMS-S pollen are unknown. Sequence similarity between orf77 and the mitochondrial ATP synthase subunit 9 (atp9) locus led us to examine RNA editing in orf77 and atp9 transcripts of pre-collapse CMS-S microspores. Editing of atp9 was not influenced by the presence of orf77 transcripts. Sequence analysis of cDNA clones demonstrated that atp9 transcripts are fully edited in CMS-S microspores. Orf77 nucleotides corresponding to edited nucleotides in atp9 were either not edited or edited inefficiently within the context of orf77, perhaps due to limited conservation of flanking sequences between orf77 and atp9. However, eight of ten orf77 cDNA clones carried an unexpected terminating edit that truncated orf77 to predict a peptide of 17 amino acids (ORF17) sharing significant identity with the C-terminal transmembrane domain of the ATP9 protein.

Properties of the linear N1 and N2 plasmid-like DNAs from mitochondria of cytoplasmic male-sterile Sorghum bicolor.

SummaryThe linear N1 and N2 plasmid-like DNAs were recovered from mitochondria of the IS1112C line of cytoplasmic male-sterile (CMS) Sorghum bicolor (S. bicolor). Molecular clones containing internal sequences of these plasmids were constructed. These clones were used to probe Southern blots of mitochondrial genomes from six CMS and five male-fertile (MF) lines of S. bicolor, as well as Southern blots of IS1112C chloroplast, IS1112C nuclear and kafir nuclear genomes. We found no evidence for integrated copies of N1 or N2 in any of the mitochondrial, chloroplast or nuclear genomes probed in this study. Our clones did detect an N1-homologous transcript of 3.1 kb and N2-homologous transcripts of 3.9 and 1.4 kb in IS1112C mitochondrial RNA prepared from lines with and without nuclear, fertility-restoring genes.N1 and N2 DNAs were degraded by exonuclease III but were resistant to lambda exonuclease, presumably due to the presence of 5′ terminal proteins. We detected multimeric forms of N1 and N2 in Southern blots of unrestricted, IS1112C mitochondrial DNA (mtDNA). These forms apparently also had associated protein molecules.

Expression of CMS-unique and flanking mitochondrial DNA sequences in Phaseolus vulgaris L.

The expression of mitochondrial DNA sequences unique to a cytoplasmically male-sterile (CMS) line of Phaseolus vulgaris was investigated. RNA-blot hybridizations with strand-specific probes demonstrated CMS-unique transcripts (7.0, 6.8, 4.7, 3.3 and 2.8 kb) to be in the sense orientation with respect to the longest open reading frames within the CMS-unique region. Hybridizations revealed co-transcription of CMS-unique and upstream, atpA-coding sequences to generate the 6.8-kb RNA. However, hybridizations with CMS-unique and flanking DNA probes accounted for only 4.9 kb of the longest and most abundant (7.0 kb) CMS-unique transcript, providing indirect evidence for the involvement of a splicing process in the generation of this transcript. Sedimentation experiments demonstrated the association of 7.0- and 6.8-kb CMS-unique transcripts with polyribosomes in seedlings and floral buds of a CMS line and a line restored to fertility by the nuclear gene Fr2. However, steady-state levels of the 7.0- and 6.8-kb transcripts were decreased in the restored line relative to the CMS line.

Sorghum mitochondrialatp6: divergent amino extensions to a conserved core polypeptide

Sorghum mitochondrialatp6 occurs as one copy in the line Tx398 and as two copies in IS1112C. In IS1112C a repeated sequence diverged within theatp6 open reading frames. The two open reading frames (1137 bp,atp6-1; 1002 bp,atp6-2) share an identical conserved region of 756 bp but are flanked 5′ by divergent extensions of 246 (atp6-1) or 381 bp (atp6-2). Tx398 carried onlyatp6-2. The breakpoint of the repeated sequence of the conserved core region corresponds to the amino acid sequence Ser-Pro-Leu-Asp, which is the amino terminus of the proteolytically processed yeast ATP6. The 5′ extensions ofatp6-1 andatp6-2 were similar to those of rice and maize, respectively. Each open reading is transcribed, however nuclear background influenced transcriptional patterns ofatp6-2 in IS1112C.

Maize Centromere Mapping: A Comparison of Physical and Genetic Strategies

Centromere positions on 7 maize chromosomes were compared on the basis of data from 4 to 6 mapping techniques per chromosome. Centromere positions were first located relative to molecular markers by means of radiation hybrid lines and centric fission lines recovered from oat-maize chromosome addition lines. These centromere positions were then compared with new data from centric fission lines recovered from maize plants, half-tetrad mapping, and fluorescence in situ hybridizations and to data from earlier studies. Surprisingly, the choice of mapping technique was not the critical determining factor. Instead, on 4 chromosomes, results from all techniques were consistent with a single centromere position. On chromosomes 1, 3, and 6, centromere positions were not consistent even in studies using the same technique. The conflicting centromere map positions on chromosomes 1, 3, and 6 could be explained by pericentric inversions or alternative centromere positions on these chromosomes.