Thesis (PhD (Plant biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In soils, the concentration of available phosphate (P) for plants is normally very low (ca. 1 µM in the soil solution), because most of the P combines with iron, aluminium and calcium to form relatively insoluble compounds. Inorganic P (Pi)-deficiency is thought to be one of the limiting factors of nitrogen fixation due to the high energy requirement for nitrogenase function of plants taking part in nitrogen fixation. Pideficiency has important implications for the metabolic Pi and adenylate pools of plants, which influence respiration and nitrogen fixation. An alternative route of pyruvate supply during Pi stress has been proposed involving the combined activities of phosphoenolpyruvate carboxylase (PEPc), malate dehydrogenase (MDH) and NAD-malic enzyme (ME) supplying pyruvate to the mitochondrion during Pi stress. Previously, three isoforms of PEPc were isolated from lupin nodules and roots, with two forms being nodule specific. The aim of this project was to determine the effect of Pi stress on these PEPc isoforms in Lupinus luteus at transcript and protein expression level with a view to produce genetically modified crops for nutrient-poor soils. Cytosolic P levels were measured over a time course to give an indication of temporal development of P stress in nodules. The changes in enzyme activities of PEPc, MDH and PK (pyruvate kinase) under P stress were measured and the downstream effect on amino and organic acid pools were analysed. Two novel PEPc isoforms, LUP1 (AM235211) and LUP2 (AM237200) were isolated from nodules, followed by transcriptional and protein expression analyses. Nodules under P stress had lower amounts of metabolically available Pi and as P stressed developed, the amount of Pi decreased. This decline in Pi levels was associated with lower growth, but higher biological nitrogen fixation (BNF). A greater proportion of root-nodule respiration was devoted to nutrient acquisition than to new growth. A typical P-stress response is higher anaplerotic carbon fixation via PEPc. However, in this study, no significant differences were found for PEPc, MDH or PK in P-stressed plants compared to P-sufficient plants which would lead to an increase in organic acids. An increase in key amino acids was reported along with unchanged levels of organic acids. These levels of organic and amino acid are in congruence with the increases in BNF under P-starvation. No significant differences were found in expression of PEPC1 or PEPC2 at 12 and 20 days for both P-sufficient and P-stressed plants which further supported the lack of engagement of the PEPc-MDH-ME bypass. PEPc activity appeared not to be regulated by gene expression or phosphorylation indicating that other posttranslational modifications such as a decrease in protein degradation may be of importance. / AFRIKAANSE OPSOMMING: Die konsentrasie van fosfaat (P) beskikbaar vir opname deur plante vanuit die grond is gewoonlik baie laag (in die omgewing van 1 µM) aangesien die P onoplosbare komplekse vorm met katione soos yster, aluminium en kalsium. ‘n Tekort aan anorganiese P (Pi) word gereken as een van die beperkende faktore van stikstofbinding as gevolg van die hoë energie behoefte wat nitrogenase plaas op plante wat van gefikseerde stikstof gebruik maak. Hierdie P-tekort het ook belangrike betrekking op die metaboliese fosfaat- en adenilaatpoele wat weer op hul beurt respirasie en stikstofbinding beÏnvloed. ‘n Alternatiewe roete van pirovaatvoorsiening aan mitochondria tydens fosfaatstres is voorgestel wat bestaan uit die aktiwiteite van fosfoenolpirovaat karboksilase (PEPc), malaat dehidrogenase en NAD-malaat ensiem. Vantevore is drie isovorme van PEPc uit Lupinus luteus wortelknoppies en wortels geïsoleer, met twee van die isovorme wat wortelknoppie-spesifiek was. The doel van hierdie projek was om die invloed van P-tekort op die transkripsie en proteien uitdrukkingsvlak van hierdie PEPc isovorme te bepaal met die doel van gemodifiseerde gewasse vir arm gronde ingedagte. Sitoplasmiese P konsentrasies is gemeet oor tyd om ‘n aanduiding te gee van die ontwikkeling van P-tekort oor tyd. Veranderinge in ensiemaktiwiteite van PEPc, MDH en pirovaatkinase (PK) is gemeet gedurende P-tekort as ook die moontlike effek van hierdie ensiemaktiwiteite op aminosuur en organiese suur poele. Twee nuwe PEPc isovorme, LUP1 (AM235211) en LUP2 (AM237200) is uit wortelknoppies geïsoleer en gekarakteriseer. Transkripsie en proteïenuitdrukking is geanaliseer. Wortelknoppies wat P-tekort behandeling ontvang het, het laer vlakke van metabolise beskikbare Pi gehad en soos die P-tekort ontwikkel het oor tyd, het die Pi vlakke gedaal. Hierdie afname in vlakke van Pi was geassosieer met laer groei, maar met ‘n toename in biologiese stikstofbinding. ‘n Groter proporsie van respirasie is toegestaan aan minerale opname as aan nuwe groei. ‘n Tipiese reaksie op P-tekort is hoër anaplerotiese koolstofbinding via PEPc. Alhoewel, in hierdie studie is geen gevind betekenisvolle verandering gevind in die aktiwiteite van PEPc, MDH en PK nie in plante wat P-tekort ervaar het nie. Verhoogde aktiwiteit van hierdie ensieme sou verhoogde organise suur konsentrasies tot gevolg hê. ‘n Toename in aminosuur konsentrasies is gevind tesame met onveranderde vlakke van organiese sure. Hierdie toename in aminosure word onderskryf deur die verhoogde biologiese stikstofbinding tydens P-tekort. Geen betekenisvolle verskille is gevind in die geenuitdrukking van pepc1 en pepc2 by beide 12 en 20 dae van P-tekort nie, wat verder die afwesigheid van die PEPc- MDH-ME alternatiewe roete beaam het. Dit blyk dat PEPc aktiwiteit nie deur geenuitdrukking of proteïenfosforilering beheer word nie, maar eerder dat ander posttranslasie modifikasies soos ‘n verlaagde afbraak van proteïen ‘n rol speel.

Stomata are small pores that are located on the surface of epidermal leaves, and they can regulate the uptake of CO2 and prevent water lose by opening and closing the pores. Stomata of plants can be regulated by external condition such as CO2, biotic and abiotic stresses and internal factors. CAM (crassulacean acid metabolism) plants adapt to hot and dry environments by closing stomata during the day and opening stomata during the cool night. However, it is still unclear how CAM plants open their stomata during the night and close them during the day. In this study, a number of factors were evaluated for their potential roles in promoting stomatal opening in the model CAM plant Kalanchoe laxiflora. Citrate is an important organic acid and it accumulates during the night in CAM plants. It is shown in this study that citrate promoted stomatal opening in detached leaf epidermis of Kalanchoe laxiflora. Further, the cytokinin zeatin is also shown to stimulate stomatal opening in detached leave of Kalanchoe laxiflora. Melatonin is an important regulator of circadian rhythms in mammals and has been implicated in regulation of plant abiotic stress responses. Melatonin was detected in the leaves of Kalanchoe laxiflora. It promoted stomatal opening in detached epidermis of Kalanchoe laxiflora. Together, these results suggest that stomata of Kalanchoe laxiflora respond to citrate and malate which are the main organic acids accumulate during nighttime and also to some signaling molecules (zeatin, melatonin, and serotonin) by opening stomata during dark period.

Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.

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