Cytoplasrnic male sterility (CMS) is a maternally inherited defect in the production of pollen, the male gamete of the flower. This sterility can be suppressed by nuclear Restorer of Fertility (Rf) genes that normally downregulate the expression of the CMS-associated novel mitochondrial gene. In Brassica napus, nap CMS and pol CMS are associated with related chimeric mitochondrial genes orf222 and orf224, respectively. CMS in both nap and pol is associated with a polar loss of locule development, loss of synchronous locule development and clumping of sporogenous tissue away from the tapetal cell layer, as well as secondary effects on petal and bud formation. In nap CMS, early accumulation of orf222 transcripts in the locule regions of developing anthers is associated with sterility, while the absence of orf222 transcripts from the locules is associated with fertility restoration. Accumulation of novel antisense transcripts of atp6 in a cell specific manner which matches that of sense transcripts of orf222 and atp6 in nap CMS anthers may be indicative of a post-transcriptional regulatory mechanism associated with CMS in flower buds. / Restoration of fertility in Brassica napus nap and pol CMS is associated with nuclearly encoded genes Rfn and Rfp, respectively. These restorers are very closely linked to one another, and may be allelic. Further efforts to isolate Rfp have narrowed the genomic region to approximately 105 kb of a syntenic region in Arabidopsis thaliana. Cosmid clones isolated from a library of Brassica rapa genomic DNA introgressed with Rfp have been successfully sorted into contigs through the application of the amplified fragment length polymorphism technique. The region to which Rfp is mapped is syntenic to a region of Arabidopsis DNA that is a duplication of a second location at the 23 megabase region of chromosome 1 of that genome. This region contains pentatricopeptide (PPR) motif-encoding genes that are highly related to other restorers of fertility of other species. By inference, Rfp from Brassica napus may encode PPR motifs. The PPR genes related to these previously characterized restorers of fertility are often found alongside the restorer genes existing as mini-clusters of several PPR-encoding genes. This is likely caused by selective pressure acting on PPR-encoding genes that resulted in diversification and multiplication of these genes. In addition, the PPR genes of this duplicated region are not syntenically located, whereas the non-PPR-encoding genes maintain their syntenic locations. The same is true for orthologous comparisons between Arabidopsis and other plant species. PPR genes are therefore malleable and capable of alteration in response to changing environmental pressures, such as the evolution of sterility inducing genes.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Doctor of Philosophy / Department of Agronomy / Tesfaye Tesso / Sweet Sorghum [Sorghum bicolor (L.) Moench] is a type of cultivated sorghum grown primarily for its sugar-rich stalks. Because of its high fermentable sugar content, the crop is widely recognized as an alternative feedstock source for bio-fuel production. The extent to which stalk sugar accumulation occurs may be determined by several factors including the sink size. Grain is the most important sink in sorghum and other grain crops. Three experiments were conducted in this study to determine the extent to which the grain sink can reduce sugar accumulation in the stalks, to test and validate a genetic system that allows development of sterile sweet sorghum hybrids, and to assess the potential of sugar-rich hybrids to overcome stalk rot diseases. The first experiment, based on 22 sweet sorghum genotypes, was undertaken to study the effect of eliminating the grain sink (removing the head prior to anthesis) on stalk juice yield, sugar accumulation, and biomass. The data showed that the grain sink had a significant effect on all traits measured. Elimination of the grain sink significantly increased oBrix % (17.8%), dry biomass (27.8%), juice yield (23.9%), and total sugar yield (43.5%). The second experiment was aimed at validating the role of A3 genetic male sterility system for producing sterile sweet sorghum hybrids. Ten sweet sorghum pollinator lines of variable sugar content were selected among the entries included in the previous experiment. The lines were crossed to four A1 and A3 cytoplasmic male sterile (CMS) lines using a Design II mating scheme. The A3 females did not have effective restorers so that the hybrids were expected to be sterile. The parental lines and corresponding hybrids were evaluated for biomass production, oBrix, juice and sugar yield using a randomized complete block design. All A3 hybrids were sterile and did not produce seed when heads were covered prior to pollination. The effect of grain sink represented by the A1 vs. A3 CMS were highly significant for Brix%, biomass, juice, and sugar yield. Comparison of parents vs. crosses component was highly significant, indicating marked heterosis effect for the traits. Both general (GCA) and specific (SCA) combining ability effects were also significant for all traits, indicating the role of both additive and dominance genetic effects in the inheritance of the characters. Earlier studies have shown positive relationships between stalk sugar concentration and stalk rot disease resistance in sorghum. Thus, the objective of the third experiment was to study the effects of the CMS mediated differential accumulation of stalk sugar on severity of charcoal rot disease caused by Macrophomina phaseolina. The experiment provided an opportunity to test the effect of variable stalk sugar in the same genetic backgrounds. The data indicated that hybrids produced from A3 cytoplasm were more resistant to charcoal rot (7.1cm lesion length) compared to those produced from the A1 hybrids (9.5 cm lesion length). The enhanced resistance of hybrids with higher sugar yield could have significant agronomic advantage in sugar based bio-fuel feedstock production.

No description available.

The vast majority of valuable agronomic traits are encoded polygenetically. Chloroplast genetic engineering offers an alternate approach to multigene engineering by allowing the insertion of entire pathways in a single transformation event, while being an environmentally friendly approach. Stable integration into the chloroplast genome and transcription of the phaA gene coding for β-ketothiolase was confirmed by Southern and northern blots. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with high enzyme activity. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines followed an accelerated anther developmental pattern, affecting their development and maturation, resulting in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affecting pollen grain and resulted in the irregular shape and collapsed phenotype. Reversibility of the male sterility phenotype was achieved by exposing the plants to continuous illumination, producing viable pollen and copious amounts of seeds. This is the first report of engineered cytoplasmic male sterility and offers a new tool for transgene containment for both nuclear and organelle genomes. Detailed characterization of transcriptional, posttranscriptional and translational processes of heterologous operons expressed via the chloroplast genome is reported here. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons, revealed that in most cases, only polycistronic mRNA was produced or polycistrons were the most abundant form and that they were not processed into monocistrons. Despite such lack of processing, abundant foreign protein accumulation was detected in these transgenic lines. Interestingly, a stable secondary structure formed from a heterologous bacterial intergenic sequence was recognized and efficiently processed, indicating that the chloroplast posttranscriptional machinery can indeed recognize sequences that are not of chloroplast origin, retaining its prokaryotic ancestral features. Processed and unprocessed heterologous polycistrons were quite stable even in the absence of 3'UTRs and were efficiently translated. Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of any cellular control. Finally, we observed abundant read-through transcription in the presence of chloroplast 3'UTRs. Such read-through transcripts were efficiently processed at introns present within native operons. Addressing questions about polycistrons, as well as the sequences required for their processing and transcript stability are essential for future approaches in metabolic engineering. Finally, we have shown phytoremediation of mercury by engineering the mer operon via the chloroplast genome under the regulation of chloroplast native and heterologous 5'UTRs. These transgenenic plants hyperexpress were able to translate MerA and MerB enzymes to levels detectable by coomassie stained gel. The knowledge acquired from these studies offer guidelines for engineering multigene pathways via the chloroplast genome.

Abstract Demographic history and selection affect patterns of genetic diversity in nature. Timing of growth, reproduction and dormancy are important traits targeted by natural selection, because they are crucial for survival of plants growing in boreal and temperate climates, as reproduction must occur when conditions are favorable and in outcrossing plants it must be synchronized to assure pollination. In addition to adaptation to local environments, evolutionarily diverged populations may contain genomic incompatibilities that result in sterile hybrids in crosses between populations. In several plant families within population crosses can also lead to male sterile progeny, because of conflict between maternally and bi-parentally inherited genomes. In this thesis I used DNA-sequence data to estimate the demographic history of nine Arabidopsis lyrata populations and present genetic variation in some key flowering time genes, evolution of which natural selection has shaped. By crossing experiments I explored genetics of reproductive fitness in hybrids between divergent populations. I found that local climatic conditions have resulted in directional selection in addition to the demographic effects of bottlenecks during colonization events. Flowering time genes have reduced diversity compared to reference loci, which indicates selective sweeps. Selection on nucleotide variation in flowering time genes was found in Scandinavian and Icelandic populations that can be explained by selective sweeps at flowering genes when these populations colonized northern habitats after the last glacial maximum. Cryptic cytoplasmic male sterility was found in a Norwegian population, for which North Carolinian population did not have fertility restorers. It was confirmed that there is only one fertility restorer locus, the genomic location of which was mapped to a 600 kb interval at the top of chromosome two. / Tiivistelmä Populaatioiden levittäytymishistoria ja luonnonvalinta vaikuttavat geneettiseen monimuotoisuuteen ja sen vaihteluun genomin eri osissa. Kukkimisen ja kasvun päättämisen ajoitus ovat tärkeitä luonnonvalinnan kohteena olevia ominaisuuksia, sillä ne ovat välttämättömiä kasvien selviytymiselle lauhkeissa ja pohjoisissa ilmastoissa. Paikallisiin olosuhteisiin sopeutumisen lisäksi populaatioiden erilaistuminen voi johtaa genomisiin yhteensopimattomuuksiin, joiden vuoksi populaatioiden väliset risteymät ovat lisääntymiskyvyttömiä. Monilla kasvisuvuilla myös populaation sisäiset risteytykset voivat johtaa koirassteriileihin jälkeläisiin johtuen konfliktista vain äidin puolelta ja molemmilta vanhemmilta periytyvien genomien välillä. Tässä väitöskirjassa selvitän DNA-sekvenssimuunteluun perustuen yhdeksän idänpitkäpalkopopulaation demografista eli levittäymis- ja populaatiorakennehistoriaa sekä luonnonvalinnan osuutta kukkimisaikaan vaikuttavien geenien evoluutiossa. Risteytyskokeiden avulla tutkin erilaistuneiden populaatioiden risteymäjälkeläisiä selvittääkseni niiden lisääntymiskelpoisuutta ja siihen vaikuttavia geenejä. Geneettinen muuntelu kukkimisgeeneissä oli vähäisempää kuin vertailugeeneissä, joka on merkki kukkimisgeeneihin kohdistuneesta suuntaavasta valinnasta. Kukkimisgeeneihin kohdistuvaa valintaa löytyi eniten skandinaavisista ja islantilaisesta populaatiosta, mikä on selitettävissä niihin kohdistuneella suuntavalla valinnalla aikana, jolloin kasvit levittäytyivät jääkauden jälkeen pohjoisiin elinympäristöihin. Norjalaisesta populaatiosta löytyi piilevä sytoplasminen koirassteriliteetti, jolle Pohjois-Carolinan populaatiolla ei ollut hedelmällisyyden palauttavia geenejä. Tutkimus vahvisti, että hedelmällisyyden palauttaa yksi geeni, joka sijaitsee 600 000 emäsparin kokoisella alueella kromosomin kaksi alkupäässä.

Un objectif central de la biologie évolutive est la compréhension des mécanismes qui conduisent à la formation de nouvelles espèces. Les stérilités hybrides constituent un type de barrières reproductives pouvant mener à la spéciation. Ce travail dissèque les bases génétiques d’une stérilité mâle observée chez l'hybride entre deux accessions naturelles d'Arabidopsis thaliana, Shahdara et Mr-0, lorsque Shahdara est le parent femelle. Par des approches génétiques et cytologiques, nous montrons que deux phénomènes interviennent dans cette stérilité. D'une part le cytoplasme de Shahdara induit une stérilité mâle cytoplasmique (CMS), en interaction avec plusieurs locus nucléaires. D'autre part, une létalité pollinique est due à plusieurs locus distorteurs de ségrégation (pollen killers). La stérilité de l'hybride résulte d'une liaison génétique entre les déterminants nucléaires de la CMS et les pollen killers. L'un des pollen killers a été localisé dans un intervalle de 70 Kb qui contient également des éléments nécessaires à la restauration de la CMS. Ce locus est complexe et présente de nombreuses variations structurales, notamment au niveau de gènes PPR. Ces résultats suggèrent que deux types de conflits génomiques, les distorteurs de ségrégation et la CMS, pourraient coévoluer dans des populations naturelles et conduire à l’élaboration de barrières reproductives au sein d'une même espèce. / Species differentiation and the underlying genetics of reproductive isolation are central topics in evolutionary biology. Hybrid sterility is one kind of reproductive barrier that can lead to differentiation between species. Here, we analyze the complex genetic basis of the intraspecific hybrid male sterility that occurs in offspring of two distant natural strains of Arabidopsis thaliana, Shahdara and Mr-0, with Shahdara as the female parent. Using genetic approaches as well as cytological observation of pollen viability, we demonstrate that this particular hybrid sterility results from two causes of pollen mortality. First, the Shahdara cytoplasm induces gametophytic cytoplasmic male sterility controlled by several nuclear loci. Second, several segregation distorters leading to allele-specific pollen abortion (pollen killers) operate in the hybrids. The complete sterility of the hybrid with the Shahdara cytoplasm results from the genetic linkage of the two causes of pollen mortality, i.e. CMS nuclear determinants and pollen killers. One pollen killer was localized in a 70 Kb interval which also contains restorer alleles for the CMS. This locus is complex and harbors many structural variations, particularly at PPR genes. Our results suggest that two types of genomic conflicts, CMS and segregation distorters, may coevolve in natural populations and contribute to reproductive isolation, and possibly to speciation.

Les stérilités mâles cytoplasmiques (SMC) résultent d'une incompatibilité nucléo-cytoplasmique. Le cytoplasme (presque toujours la mitochondrie) peut porter un gène de stérilité mâle, et le noyau peut restaurer la fertilité pollinique ou non. Les mécanismes physiologiques conduisant à la mort pollinique restent largement incompris. Plusieurs hypothèses ont été proposées, parmi lesquelles une déficience en ATP. Une SMC gamétophytique a été découverte chez A. thaliana. Une phase ouverte de lecture codant possiblement un peptide de 117 acides aminés, appelée orf117Sha, a été identifiée comme facteur de stérilité candidat.Au cours de ma thèse, j'ai cherché à valider le rôle de l'orf117Sha, et à comprendre comment une anomalie mitochondriale pouvait induire cette SMC. Aucune différence n'a pu être détectée au niveau de l'ARNm de l'orf117Sha entre les lignées stérile et restaurée, mais sa protéine semble accumulée uniquement dans la lignée stérile. La phénocopie par transgénèse de la SMC a suggéré un effet délétère de l'ORF117SHA dans les gamétophytes mâle et femelle.La description cytologique de la SMC montre une mort pollinique progressive à partir du stade binucléé. Auparavant, les mitochondries du pollen gonflent puis éclatent, et le développement s'arrête. L’utilisation de senseurs génétiquement encodés mesurant la concentration en ATP (ATeam) et l'état redox du glutathion (roGFP2-Grx1) a permis la mesure de ces facteurs en microscopie confocale, dans des tissus végétatifs et dans le pollen. La production d'ATP ne semble pas affectée dans la lignée stérile, contredisant l'hypothèse de l'ATP. Le glutathion mitochondrial est suroxydé dans la lignée stérile, à la fois dans les tissus végétatifs étudiés et le pollen, qui serait liée à la SMC car annulée par la restauration génétique de fertilité.Avec cette étude, j'apporte des arguments en faveur de l'orf117Sha dans l'induction de la SMC Sha, et je décris les évènements préalables à l'avortement du grain de pollen. Mes résultats permettent de mieux comprendre les évènements physiologiques conduisant à la mort du pollen. / This work aims at better understand the events leading to pollen abortion in a recently discovered gametophytic cytoplasmic male sterility (CMS) in Arabidopsis thaliana. Although CMS have been widely used in hybrid seed production in many crops, the physiological mechanisms leading to pollen death by the mitochondrial sterilizing genes in the permissive (maintainer) nuclear backgrounds are poorly understood. Association genetics previously identified orf117Sha as a candidate mitochondrial CMS-associated gene.In a first part, I analyzed the expression of the orf117Sha gene in sterile plants and in fertile plants carrying nuclear genes restoring male fertility. I observed unusual features of its mRNA, but detected no difference at this level between sterile and restored plants. Oppositely, the ORF117SHA protein seems to be accumulated specifically in the sterile line, supporting its role in CMS. A phenocopy attempt by transgenesis suggested a possible link between a female and male gametophytic lethality and the ORF117SHA, even though few individuals could be analyzed.In a second part, I observed pollen development in sterile plants and fertile controls using different cytological approaches. My results show a progressive pollen death starting from the binucleate stage in the sterile. Prior to abortion, pollen mitochondria swell before rupture, and the development stops. I used confocal microscopy combined with genetically encoded sensors to explore specific physiological features in pollen and vegetative tissues of sterile plants. With ATeam, which allows the assessment of ATP content in the cytosol, I could challenge the generally accepted hypothesis of an ATP deficiency leading to pollen abortion in CMS. Indeed, the ATP production does not seem to be affected in the sterile line. With a mitochondria-addressed roGFP2-Grx1, I was able to assess the redox state of the glutathione pool in vegetative tissues and in the male gametophyte. I observed an overoxydation of the glutathione pool in mitochondria of the sterile line, in vegetative tissue investigated and in the pollen grain. This overoxydation seems to be linked to the CMS as it is annihilated by the presence of restorer genes.My results pave the way for further exploration of the links between the sterility protein, mitochondrial morphology changes, mitochondrial overoxydation, and pollen development arrest and death in the A. thaliana CMS.