The Drosophila genome is thought to have five recA like proteins: Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3. In Drosophila Rad51/SpnA, XRCC3/SpnB, and Rad51 C/SpnD participate in homologous recombination repair. The function of DmRad51 D and DmXRCC2 are unknown. The goal of this project was to elucidate the function ofXRCC2 in Drosophila. RNA interference allowed us to knockdown the function XRCC2 and its possible binding partner Rad51D. It was seen the knocking down the function of either XRCC2 or Rad51D does not affect the viability of the fly. However, drug treatment data does not allow us to make any conclusions about how the knockdown ofXRCC2 affects the viability of the fly. RNA in-situ hybridization shows highly intricate and complex branching patterns for XRCC2, which resembles the embryonic tracheal system. Lastly, XRCC2 was purified to generate an antibody made to recognize the XRCC2 protein will help localize the XRCC2 protein in future studies as well as determine protein-protein interactions with XRCC2.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13073号 / 論農博第2843号 / 新制||農||1046(附属図書館) / 学位論文||H29||N5029(農学部図書室) / 33224 / 京都大学大学院農学研究科農学専攻 / (主査)教授 北島 宣, 教授 土井 元章, 教授 田尾 龍太郎 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

Recent advances in plant genetic engineering offer substantial benefits to farmers throughout the world. Genetic research has identified many exogenous genes that could considerably decrease production costs through transgene-mediated resistance to insect, viral, fungal and bacterial pathogens. Potato can be produced from true potato seed (TPS) through a sexual polyploidization step, known as 4x-2x hybridization. Little is known regarding the stability of transgenes through sexual polyploidization in potato, although studies have associated ploidy elevation with transgene silencing in plants such as Arabidopsis thaliana. In the present study, potato was transformed with two different transgenes, cry3Aa and PVYo cp, and transgene expression was analyzed through 4x-2x hybridization. Transgene introgression did not affect fertility or agronomic performance (tuber set, average tuber weight, total tuber yield) of the resulting 4x-2x hybrids; however, reduced seed germination was observed for several transgenic lines in an in vitro study. Ploidy elevation did not affect a highly expressed single copy cry3Aa transgene, simplex or duplex, transmitted through pollen to 4x-2x hybrids. By contrast, multiple copies of cry3Aa triggered significant transgene silencing in diploids and silencing was further pronounced upon pollen transmission to 4x-2x hybrids. Crosses between two, single insert plants demonstrated additional evidence that multiple cry3Aa transgenes resulted in reduced expression, as well as provided evidence for maternal effects on expression of the cry3Aa transgene. Finally, Cry3Aa expression levels of progeny derived from low expressing, multiple copy 4x-2x hybrids indicated that reduction of transgene number in progeny, through meiotic segregation, could increase Cry3Aa expression. The results suggest that 4x-2x hybridization using single copy, male parents can result in high expressing, transgenic 4x-2x hybrids while segregating for a low frequency of non-transgenic hybrids that create a "refuge" to inhibit development of resistance to transgenes in pest populations. / Ph. D.

ARGONAUTE proteins (AGOs) are generally accepted as key components of the post transcriptional gene silencing mechanism, also involved in plant antiviral defense. Except for reports on the antiviral roles of AGO1, AGO2 and AGO7 in Arabidopsis, the exact roles played by the individual AGOs in other plant species are largely unknown. This research focused on the identification and characterization of AGOs involved in antiviral RNAi response to various viruses in N. benthamiana. Based on the temporal and spatial distribution of AGO transcripts in 3 and 8-week old plant root, stem and leaf tissues, expressions of NbAGO mRNAs were found to vary with age and tissue specificity. Plant endogenous AGO mRNAs were knocked down through virus induced gene silencing techniques using the Tobacco rattle virus vector system and posteriorly challenged with a GFP-chimeric virus construct deficient of a silencing suppressor. Unlike in control non-silenced plants, the Tomato bushy stunt virus construct deficient of its P19 silencing suppressor was consistently seen to exhibit a strong fluorescence on N. benthamiana plants silenced for NbAGOs 2 and X. Similar results were also obtained upon silencing of NbAGO2 using hairpin vector techniques. Comparable observations were also made when Tobacco mosaic virus GFP constructs were agroinfiltrated on NbAGO2 silenced plants further hinting the antiviral defense roles played by these AGOs. Agroinfiltration of Foxtailmosaic virus, Sunnhemp mosaic virus, and Turnip crinkle virus GFP chimeric constructs on NbAGO2 silenced N. benthamiana plants, however did not result in accumulation of GFP indicating the AGO antiviral defense specificity to TBSV and TMV. The results also hinted at a role for AGO7. Collectively my findings suggest that the expression of AGOs in N. benthamiana is tissue and age dependent, and that unlike in the model plant Arabidopsis where the main antiviral AGO is thought to be AtAGO1; in N. benthamiana, NbAGOs 2 and X seem to be involved in an antiviral defense role against TBSV and TMV with other AGOs perhaps contributing.

Les complexes polyazaaromatiques de ruthéniumII, et en particulier le [Ru(bpy)3]2+, ont fait l’objet de nombreuses études fondamentales en photochimie et photophysique. Du fait de ses propriétés photophysiques, et entre autres grâce à son temps de vie de luminescence relativement long, le [Ru(bpy)3]2+ est devenu un composé modèle en photophysique. Dès les années 1970, et principalement grâce aux travaux de T.J. Meyer, la photophysique du [Ru(bpy)3]2+ a été étudiée en détail afin de permettre l’élaboration d’un modèle photophysique qui peut être valablement étendu aux autres complexes polyazaaromatiques de RuII. La caractérisation du complexe [Ru(bpy)2(dppz)]2+ et de ses interactions avec l’ADN a, elle, promu l’étude des complexes de RuII en présence de biomolécules et a encouragé la recherche pour l’utilisation de complexes de ruthénium comme photosondes en biochimie.<p>Dans ce cadre, le laboratoire de Chimie Organique et Photochimie de l’ULB s’est attaché au développement de complexes polyazaaromatiques de ruthéniumII se caractérisant par leur capacité à photoréagir avec certaines biomolécules. Ces complexes se caractérisent par l’utilisation de ligands fortement π-déficients, comme le 1,2,4,5,8-tétraazaphénanthrène (TAP). Nettement plus photooxydant que les complexes analogues au [Ru(bpy)3]2+, ces complexes photooxydants sont capables, sous irradiation, de donner lieu à un transfert d’électron depuis la base guanine de l’ADN vers le complexe excité. Les deux entités radicalaires ainsi formées peuvent ensuite réagir entre elles pour former un photoadduit au sein duquel un lien covalent lie irréversiblement un ligand TAP du complexe à la guanine.<p><p>Les travaux réalisés dans le cadre de cette thèse de doctorat s’inscrivent dans la poursuite de la recherche effectuée au sein du laboratoire autour de cette photoréaction. Deux axes majeurs ont été développés. Un premier axe de recherche a été dédié à l’étude fondamentale des propriétés photophysiques et photochimiques du photoadduit obtenu suite à la photoréaction du [Ru(TAP)3]2+ avec une base guanine. Cette étude photophysique fondamentale de l’adduit [Ru(TAP)2(TAP-GMP)] (présentée dans le deuxième chapitre) vise à caractériser sa photophysique afin de comprendre comment, sous irradiation, des biadduits entre un complexe de ruthénium et deux guanines sont observés, alors que les premières études réalisées sur les photoadduits indiquent que ceux-ci ne sont pas luminescents. Le second axe de recherche consiste en la mise au point de systèmes élaborés à base des complexes de ruthénium visant à contrôler leur photoréactivité dans un milieu biologique. Pour ce faire, les complexes de ruthénium photoréactifs ont été ancrés sur des molécules biologiques. D’une part, les complexes ont été conjugués sur des OAS, oligonucléotides anti-sens, afin de conférer aux conjugués résultants la possibilité de cibler une partie précise de l’ADN ou d’ARN, et mener, in fine, au blocage de l’expression d’un gène particulier. Ces conjugués ont déjà été étudiés par le passé dans notre laboratoire. Les résultats présentés ici (chapitre 3) permettent à la fois de mieux comprendre la photochimie des Ru-OAS en présence de leur cible spécifique, ainsi que de démontrer in vivo la validité de la stratégie de gene silencing envisagée depuis quelques années. D’autre part, des complexes de ruthénium ont été conjugués à des peptides ou plateformes en vue de leur permettre de pénétrer à l’intérieur des cellules (chapitre 4). Les complexes ne pouvant normalement pas traverser les membranes cytoplasmiques, nous avons démontré que l’ancrage de ceux-ci au peptide transvecteur TAT permet de les vectoriser dans le cytoplasme. Cette incorporation se fait vraisemblablement par endocytose. Lors de ces études, l’importance de la localisation finale du complexe au sein de la cellule a été mise en évidence. Afin de conférer une sélectivité de vectorisation dans des cellules données (pénétration active et selon la présence de récepteurs spécifiques à la surface membranaire), les complexes ont été ancrés sur une plateforme RAFT(RGD)4. Dans ce cas, nous avons démontré qu’une internalisation spécifique dans des cellules sur-exprimant l’intégrine αvβ3 est possible pour les conjugués Ru-RAFT(RGD)4. Finalement, des études ont été réalisées sur les complexes ancrés sur plateforme calixarènique. Les résultats présentés permettent de caractériser ces conjugués Ru-Calix afin d’orienter leur développement avant les études de vectorisation cellulaire. Grâce aux résultats obtenus, un design permettant aux complexes de conserver leur photoréactivité a pu être établi et servira pour les développements futurs. En sus de ces deux axes de recherche principaux, le premier chapitre de résultats et discussions porte quant à lui sur l’étude fondamentale des complexes [Ru(TAP)3]2+ et [Ru(TAP)2(phen)]2+ ;plus précisément, une étude complète du complexe [Ru(TAP)2(phen)]2+ dans l’acétonitrile et le butyronitrile en présence d’un composé calixarènique (développé dans l’équipe du Pr. Ivan Jabin) est présentée. Il appert que l’utilisation du calixarène permet de mettre en évidence des processus photophysiques et photochimiques complexes, qui n’avaient pas été détectés auparavant. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be protected. To achieve this several in vivo tools are needed for the study of this crop and the pathogens that infect it. Recently the grapevine genome has been sequenced and the next important step will be gene annotation and function using these in vivo tools. In this study the use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression and VIGS vector for heterologous protein expression and functional genomics in Nicotiana benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a molecular sequence comparison study. Results confirmed the separation of GVA variants into three groups, with group III (mild variants) being the most distantly related. It showed the high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III respectively. A collaboration study investigating the molecular divergence of GVA variants linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive control by the grapevine industry, was found to contain a 119 nt insert within the native ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA variants suggested that the components in the GVA genome that cause pathogenicity in V. vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana. The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength cDNA clones under control of CaMV 35S promoters. After several strategies were attempted, including a population cloning strategy for GTR1-2, none of the clones generated were able to replicate in N. benthamiana plants. A single amino acid substitution at position 13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce replication of the virus to below a detectable level. Two infectious clones of Israeli variants of GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were infectious, able to replicate, move systemically and induce typical GVA symptoms after agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5- ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector, 35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA- GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N. benthamiana both vectors showed similar GUS expression levels and photobleaching symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118. No GUS expression was observed for the gene exchange vector 35S-GVA-GR5- ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5- ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves after 4 months. This study showed that GVA can be used as gene insertion and gene exchange vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of GVA is not needed for long distance movement in grapevine. To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA clone was removed and subsequently substituted by the corresponding ORFs of four South African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA constructs were able to move systemically through the plant. At this stage no correlation could be found between severity of symptoms, the presence of the P163-M5 insert and the specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral genome or the host plant probably play a crucial role. This study contributed to the pool of available in vivo tools for study and improvement of the valuable grapevine crop. It also opened several exciting research avenues to pursue in the near future. / AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera L.) is ‘n baie belangrike landboukundige gewas wat beskerm moet word. Om die rede word verskeie in vivo gereedskap vir die bestudering van die wingerdplant, en die patogene wat dit infekteer benodig. Die wingerd genoom se volgorde is bepaal en dus is die volgende logiese stap om die gene te annoteer en funksie daaraan toe te skryf. In hierdie studie is die gebruik van Grapevine virus A (GVA), genus Vitivirus, familie Flexiviridae, as tydelike uitdrukking- en virus-geinduseerde geenuitdowingsvektor vir heteroloë proteïen uitdrukking en funksionele genoomstudies in Nicotiana benthamiana en V. Vinifera getoets. Vollengte genoomvolgordes van drie Suid-Afrikaanse variante van die virus (GTR1-1, GTG11-1 en GTR1-2) is gegenereer en in ‘n molekulêre volgorde vergelyking studie gebruik. Resultate het die verdeling van GVA variante in drie groepe, waar groep III die verste verwant is, bevestig. Dit het ook gewys dat die virus ‘n baie hoë molekulêre heterogeniteit het en dat oopleesraam 2 (ORF 2) die mees divers is. ‘n Samewerking studie waar die molekulêre diversiteit van GVA variante, gekoppel aan Shiraz siekte (SD), ondersoek is, is twee interessante variante van groep II beskryf, naamlik GTR1-2 en P163-M5 (Goszczynski et al., 2008). Groep II variante is vooraf gevind om nou verwant te wees aan die ontwikkeling van SD in wingerd. Die GTR1-2 variant is uit ’n vatbare wingerd plant, wat nooit SD-simptome vertoon het nie, geïsoleer (Goszczynski et al., 2007). In die ORF 2 van die P163-M5 variant, wat simptome van die ergste graad in N. benthamiana geïnduseer het, en ook deur die industrie as betroubare SD-positiewe kontrole gebruik word, is ’n 119 nt invoeging gevind. Die vergelykende analise wat uitgevoer is, het daarop gedui dat die determinante van patogenisiteit in die GVA genoom moontlik meer kompleks kan wees in V. vinifera as in N. benthamiana. Die drie Suid-Afrikaanse variante (GTR1-1, GTG11-1 en GTR1-2) is in afsonderlike vollengte cDNA klone, onder beheer van CaMV 35S promotors, aanmekaargesit. Nadat verskeie kloneringstrategieë, insluitend ’n populasie kloneringstrategie vir die GTR1-2 kloon, gebruik is, het geen een van die cDNA klone die vermoë besit om in N. benthamiana te repliseer nie. ’n Enkele aminosuur substitusie in posisie 13 (Tyr/Y Cys/C) in ORF 5 van die GTR1-2 kloon, het die replisering van die virus tot laer as ’n opspoorbare vlak verlaag. Twee infektiewe klone van Israeliese GVA variante (T7-GVAGR5 en T7-GVA118, verkry van M. Mawassi) is onder beheer van ‘n CaMV 35S promotor geplaas (35S-GVA-GR5 and 35S-GVA118). Beide klone het na agro-infiltrasie in N. benthamiana plante gerepliseer, sistemies beweeg en tipiese GVA simptome geinduseer. Hierdie twee klone het as raamwerk gedien vir verdere eksperimente in karakterisering van tydelike uitdrukkings- en VIGS vektore. Die gebruik van GVA as geen-insvoegingsvektor (35S-GVA118) en geen-vervangingsvektor (35S-GVA-GR5- ORF2+sgMP) is in N. benthamiana en V. vinifera vergelyk. Die geen-invoegingsvektor 35S-GVA118, was op die vollengte GVA genoom gebasseer. Die geen-vervangingsvektor 35S-GVA-GR5- ORF2+sgMP, was in hierdie studie gekonstrueer. Dit is gemaak eerstens deur eliminasie van ORF 2 in die 35S-GVA-GR5 kloon, en tweedens deur die invoeging van ’n subgenomiese promotor van die beweginsproteïen (sgMP) en unieke beperkings-ensiemsetels om klonering van transgene te fasiliteer. Beide vektore het in N. benthamiana vergelykbare GUS uitdrukkingsvlakke en fotobleikende simptome getoon na virus-geinduseerde NbPDS uitdowing. In V. Vinifera is beperkte GUS uitdrukkingsvlakke en VIGS fotobleikende simptome opgemerk met die geen-invoegingsvektor, 35S-GVA118. Geen GUS uitdrukking is in hierdie gasheerplant met die geen-vervangingsvektor opgemerk nie. Slegs een wingerdplant het fotobleikende simptome, na 4 maande in 3 sistemies geïnfekteerde blare gewys, na agroinfiltrasie van die 35S-GVA-GR5- ORF2-VvPDS+sgMP konstruk. Hierdie studie het bevestig dat GVA as geen-invoeging en geen-vervangingsvektor, vir heteroloë proteïenuitdrukking en VIGS, in N. benthamiana gebruik kan word, maar dit blyk of die gebruik daarvan in wingerd meer tot die floeëm weefsel beperk is. Hierdie studie wys vir die eerste keer dat ORF 2 nie nodig is vir langafstand beweging van die virus in wingerd nie. Om die moontlike rol van die P163-M5 119 nt invoeging en die GVA ORF 2 (met onbekende funksie), in die uitdrukking van simptome in plante te ondersoek, is ORF 2 van die 35SGVA- GR5 cDNA kloon verwyder en daaropvolgens vervang met die ooreenstemmende ORFs van vier Suid-Afrikaanse GVA variante. Na agro-infiltrasie in N. benthamiana blare, het al die chimeras die vermoë gehad om te repliseer, sistemies te beweeg en simptome te induseer. Geen korrelasie kon gevind word tussen die graad van simptome, die teenwoordigheid van die P163-M5 insersie en die spesifieke GVA ORF 2 teenwoordig in die chimeras nie, wat dus daarop dui dat ander faktore in die virusgenoom of die gasheerplant `n moontlike belangrike rol kan speel. Hierdie studie het bygedrae tot die beskikbare poel van in vivo gereedskap vir die bestudering en verbetering van die kosbare wingerdgewas. Dit het ook talle interessante navorsingsgeleenthede oopgemaak om in die nabye toekoms te betree.

Thesis (PhD (Plant Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Virus Induced Gene Silencing (VIGS) was optimized to allow for the study of starch metabolism. The plastidial inorganic pyrophosphatase gene, for which a mutant has never been identified, was studied using VIGS and it was found to have a broad role in this subcellular compartment. The accumulation of inorganic pyrophosphate limited the production of starch, carotenoids, chlorophyll, and increased the plants susceptibility to drought stress. These effects highlight the importance of this enzyme in maintaining a low intraplastidial concentration of PPi providing an environment which facilitates these anabolic processes. Several genes involved in starch synthesis and degradation were also targeted with the aim of establishing a system of multiple gene silencing for the study of metabolic pathways. One, two and three genes were successfully silenced using this system which was validated based on previously published data. Interestingly, simultaneous silencing of the two isoforms of disproportionating enzyme led to a novel phenotype as a large reduction in starch instead of the expected increase was observed. / No Afrikaans abstract available