<p>Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets. </p><p>In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention. </p><p>I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release. </p><p>Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways. </p><p>Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.</p> / Dissertation

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation and salt, probably by stabilizing protein structures and lipid membranes. Trehalose-6-phosphate synthase 1 (TPS1) is a subunit of trehalose synthase complex in fungi / it plays a key role in the biosynthesis of trehalose. In this study, a TPS1 gene fragment in R. oryzae was cloned successfully by PCR with primers designed according to eight hypothetical proteins found from BLAST search which was performed by using S. cerevisiae TPS1 gene template. The full length of R. oryzae TPS1 gene (designated RoTPS1) was attained by RTPCR with primers specific to the 3&amp / #8242 / and 5&amp / #8242 / end of the RoTPS1 cDNA. The RoTPS1cDNA was composed of 2505 bps encoding a protein of 834 amino acids with a molecular mass of 93.8 kDa. The amino acid sequence has relatively high homology with the TPS1s of several other organisms. RoTPS1 was further characterized by transformation into S. cerevisiae tps1 mutant. In galactose media, the growth curves of wild type, tps1 mutant and transformant S. cerevisiae cells had a comparable pattern in general, tps1 mutant reached to a higher maximum cell concentration compared to the others and wild type had a slightly lower specific growth rate compared to the tps1 mutant and transformed cells. Trehalose levels of transformant and wild type cells were increased up to 37 mg/gdw in the stationary phase.

Trehalose (&amp / #945 / -D-glucopyranosyl-1,1-&amp / #945 / -D-glucopyranoside) is a non reducing disaccharide of glucose that occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant. Until recently, only few plant species, mainly desiccation tolerant &amp / #8216 / resurrection&amp / #8217 / plants, were considered to synthesize trehalose. Although most plant species do not appear to accumulate easily detectable amounts of trehalose, the discovery of genes for trehalose biosynthesis in Arabidopsis and in a range of crop plants suggests that the ability to synthesize trehalose is widely distributed in the plant kingdom. In this study, three wheat cultivars (Triticum aestivum L.) Tosun, Bolal (stress tolerant) and &Ccedil / akmak (stress sensitive) were analysed for the presence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from seeds and seedlings of three different wheat cultivars (Bolal, Tosun and &Ccedil / akmak). The trehalose amount was quantified by high performance liquid chromatography connected with refractory index detector. Effects of drought and salt stress on trehalose contents of wheat cultivars were studied at seedling level and trehalose analysis was achieved both on shoot and root tissues. It was found that trehalose had accumulated under salt and drought stress conditions in all wheat cultivars. The highest trehalose accumulation was detected in roots of Bolal cultivar under drought stress condition. Furthermore, trehalose metabolizing enzymes / trehalose-6-phosphate synthase (TPS) and trehalase enzyme activities were measured in roots and shoots of Bolal and &Ccedil / akmak cultivars under control, salt and drought stress conditions. The most interesting results that we found that TPS activity sharply increased under stress conditions. The activity of TPS in roots under drought stress condition was the highest and reached to 3-4 times of its activity under control condition. The increase in the activity of TPS showed parallelism with trehalose accumulation under stress condition. Trehalase activity in Bolal cultivar decreased under both salt and drought stress conditions, however there was no significant change in trehalase activity of &Ccedil / akmak variety.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Sugarcane is a tropical perennial grass species belonging to the Poaceae (true grasses) family. Mature sugarcane is comprised mostly of sugarcane stalks, which accumulate high amounts of sucrose, a fact that has led to its wide cultivation of sugarcane for sucrose production. Sugar yields from sugarcane have been improved in the past by either creating transgenic sugarcane or through using traditional breeding methods. Increasing sugar yields in sugarcane is still of interest and new cisgenic strategies are being considered to alleviate consumer concerns over transgenic plants. This thesis consists of two parts. The first was aimed at understanding the relation between trehalose-6-phosphate (T6P) synthesis and sucrose accumulation in sugarcane. In this study the E. coli genes involved in trehalose synthesis, otsA and otsB, were overexpressed in sugarcane in order to observe their effects on soluble sugar accumulation. Nine otsA and two otsB overexpressing lines were created, confirmed by gDNA insertion PCRs, sq-RT-PCR and immuno detection of encoded enzymes. Preliminary measurements of soluble sugars showed that four out of the nine otsA lines had significantly decreased and one line significantly increased sucrose concentrations. Correlating sq-RT-PCR results with soluble sugar measurements suggest that trehalose-6-phosphate synthase (TPS) expression affects sucrose levels in sugarcane, but further research of TPS activity is required before a conclusion can be reached. Further analysis of mature cane material in regard to relevant enzyme levels, carbohydrate levels and gene expression should contribute to more conclusive results. Three novel sugarcane TPS encoding sequences were isolated and proven to be functional through complementation of the growth defect in tps1Δ yeast grown on glucose as a carbon source. Sugarcane TPS isoforms named SoTPSa, SoTPSb and SoTPSc, were isolated by successful application of 5‟ RACE alongside standard PCR using primers based on other monocotyledonous TPS sequences. The encoded SoTPSa contains a 25 amino acid insertion within the partial TPP domain. The encoded SoTPSc contains a 126 amino acid long N terminal truncation, which removes one of the thirteen amino acids found within the active site of the TPS domain. Future characterization of the encoded enzymes will determine the effects of these modifications on TPS activity. The second part of this thesis describes initial efforts made in attempting to develop a cisgenic in vitro selectable marker system for sugarcane, S. officinarum callus, which uses a diphenylether type (DPE) herbicide as a selection agent and a sugarcane protoporphyrinogen oxidase (PPO) gene as a selection marker. Firstly the plastid targeted PPO from tobacco (NtPPO-1) was isolated and mutagenized, to mimic the double mutated Arabidopsis PPO, used by Li et al., (2003) in maize. However, sugarcane calli transformed with the double mutated NtPPO-1 and grown on media containing fomesafen herbicide, were incapable of regenerating. Future efforts will utilize a N-terminal sequence that is targeted to the plastid organelle, so as to ensure translocation of the enzyme to that subcellular location. Also, random mutations were induced in the NtPPO-1 gene to screen for mutations that confer DPE herbicide resistance, however this work is currently on hold until a heme deficient E. coli can be obtained. Secondly, attempts were made to isolate a putative sugarcane plastid targeted PPO gene, so as to eventually use this in developing a cisgenic strategy. 5‟ RACE was successful in revealing additional nucleotide sequence adding 1006 bp to the already known partial sugarcane PPO sequence. However the fragment isolated was still a partial sequence. / AFRIKAANSE OPSOMMING: Suikerriet is 'n tropiese meerjarige gras spesie wat deel is van die Poaceae (ware grasse) familie. Volwasse suikerriet bestaan hoofsaaklik uit suikerrietstamme, wat hoë hoeveelhede sukrose akkumuleer, 'n feit wat gelei het tot die wye verbouing van suikerriet vir sukrose produksie. In die verlede is suikeropbrengste vanuit suikerriet verbeter deur die skep van transgeniese suikerriet óf die gebruik van tradisionele teelmetodes. Toenemende suiker opbrengste in suikerriet is steeds van belang en nuwe cisgeniese strategieë word oorweeg om verbruikerskommer oor transgeniese plante te akkommodeer. Hierdie tesis bestaan uit twee dele. Die eerste deel is daarop gemik om die begrip van die verhouding tussen trehalose-6-fosfaat (T6P) sintese en sukrose ophoping in suikerriet te verstaan. In hierdie studie is die E. coli gene wat betrokke is in trehalose sintese, otsA en otsB, ooruitgedruk in suikerriet ten einde die uitwerking daarvan in die opgaar van oplosbare suiker te bestudeer. Nege otsA en twee otsB verhoogte uitdrukkings lyne is geskep, bevestig deur gDNA bygevoegde PKR, sq-RT-PKR en immuno opsporing van geïnkripteerde ensieme. Voorlopige metings van oplosbare suikers toon dat vier van die nege otsA lyne ʼn beduidende afname in sukrose vlakke en een lyn „n beduidende toegeneem in sukrose vlakke getoon het. Korrelerende sq-RT-PKR resultate met oplosbare suikermetings dui daarop dat trehalose-6- fosfaat sintese (TPS) geenuitdrukking sukrose vlakke sal affekteer, maar verdere navorsing van TPS aktiwiteit is nodig voordat 'n gevolgtrekking gemaak kan word. Verdere ontleding van volwasse riet materiaal met betrekking tot relevante ensiem vlakke, koolhidrate vlakke en geenuitdrukking, behoort by te dra tot meer volledige resultate. In hierdie studie is drie nuwe suikerriet TPS gene geïsoleer en dit is bewys as funksioneel deur die komplimentering van die groeidefek van tps1Δ gis, gegroei op glukose as 'n koolstof bron. Suikerriet TPS isoforme, genoem SoTPSa, SoTPSb en SoTPSc, is geïsoleer deur die suksesvolle toepassing van 5 'RACE, in kombinasie met standaard PKR, deur van spesiaal ontwerpte primers, gebaseer op ander eensaadlobbige TPS gene, gebruik te maak. Die gekodeerde SoTPSa bevat 'n 25 aminosuur invoeging binne-in die gedeeltelike TPP domein. Die gekodeerde SoTPSc bevat 'n 126 aminosuur lange N terminaal afkapping, wat een van die dertien aminosure binne die aktiewe terrein van die TPS domein verwyder. Toekomstige karakterisering van hierdie geïnkripteerde ensiemes sal die effek van hierdie veranderinge op TPS aktiwiteit bepaal. Die tweede deel van hierdie tesis beskryf die aanvanklike probeerslae wat gemaak is in 'n poging om „n cisgeniese in vitro selekteerbare merker vir suikerriet, S. officinarum kallus te ontwikkel. Hierin word gebruik gemaak van 'n difenylether tipe (DPE) onkruiddoder as 'n seleksie agent, en 'n suikerriet protoporphyrinogen oksidase (PPO) geen as 'n seleksie merker. In 'n poging om dit te bewerkstellig is daar eerstens plastied geteikende PPO van tabak (NtPPO-1) geïsoleer en geteikende mutagenese suksesvol daarop uitgevoer. Mutasies wat geinduseer is, is gegrond op die dubbele gemuteerde Arabidopsis PPO, wat gebruik was in mielies deur Li et al., (2003). Alhoewel die suikerriet kallus getransformeer is met die dubbele gemuteerde NtPPO-1 konstruk en geselekteer is op media wat fomesafen onkruiddoder bevat, was die kallus nie in staat om te regenereer nie. In toekomstige pogings sal probeer word om 'n N-terminale volgorde, geteiken op „n plastied organel, te benut sodat translokasie van die ensiem aan die plastied organel verseker kan word. So ook is toevallige mutasies veroorsaak in die NtPPO-1 gene om te soek vir nuwe mutasies wat DPE onkruiddoderweerstand verleen, maar hierdie werk is tans gestop totdat 'n heem gebrekkige E. coli mutant verkry kan word. Tweedens, is pogings aangewend om 'n vermeende suikerriet plastied geteikende PPO gene te isoleer, om uiteindelik te gebruik in die ontwikkeling van 'n cisgeniese strategie in suikeriet. 5 'RACE was suksesvol in die onthulling van bykomende nukleotiede volgorde deur 1006 bp by te voeg by die reeds bekende gedeeltelike suikerriet PPO fragment. Nie teenstaande is die fragment wat nuut geïsoleer is, steeds slegs 'n gedeeltelike volgorde volgens vergelykings met ander bekende plant PPO gene.

The project goals for this dissertation were to manipulate Populus trichocarpa source-sink relationships to optimize this woody crop species for specific agricultural traits such as increased growth rate, stress tolerance and/or improvements in overall biomass accumulation. We targeted specific tissues such as xylem, where alterations in the relationship of source and sink tissues can lead to the control of xylem cell deposition or of various wood properties. This led to the characterization of 165 protein-protein interactions and 20 protein-DNA interaction which constitute numerous woody tissue related subnetworks. One such network, centered on the DIVARACATA and RADIALIS INTERACTING FACTOR (PtrDRIF), identified PtrWOX13c as an interacting protein. Characterization of PtrWOX13c shows that it displays the ability to control promoters related to lignin biosynthesis genes and overexpression phenotypes show alterations in axillary branch activity. Genes which control the differentiation and specialization of cells such as members of the WOX family are also highly responsive to abiotic stress which can force major changes in plant metabolism and nutrient mobilization. ABA, a prominent plant phytohormone with known roles in the adaptation to stress has shown novel connections in the regulation of growth promoting complexes such as TOR through antagonistic regulatory actions of the SnRK2 protein kinase in Arabidopsis. Characterization of the core ABA signaling in P. trichocarpa has identified a regulatory clade A protein phosphatase which interacts with numerous PtrSnRK2 proteins and when overexpressed in hybrid poplar results in increased height and node production potentially by indirect control of growth promoting complexes like TOR through SnRK2 inhibition. This work has also demonstrated that in addition to the involvement of phytohormones in the regulation of plant development, sugar phosphates such as T6P can exert significant control of plant architecture. Together, these studies comprise the discovery and subsequent characterization of novel wood associated networks, hormone pathways and sugar signaling in the manipulation of P. trichocarpa source-sink relationships for the promotion of biomass accumulation. / PHD / Detailed analyses of gene activity in different tissues or under the influence of various environmental conditions have identified numerous genes that control desirable traits and plant characteristics. However, the activities and functions of the proteins produced from these genes is less understood. One of the ways proteins work is through the formation of complexes with other proteins. Using the commercially valuable tree Populus trichocarpa (poplar) as our research model, we have identified novel complexes of interacting proteins with the potential to sense and respond to the environment and to promote plant growth. We tested the function of some of the members of these newly discovered protein complexes using transgenic poplar. As a result, we revealed previously unknown functions for two poplar proteins: PtrWOX13c promoted increased branching and PtrHAB2 promoted an increase in tree height. Independent of these functional analyses of poplar proteins, we also tested the ability of a sugar phosphate, trehalose6-phosphate, known from previous work to regulate plant growth, for its ability to promote poplar growth. We found that reducing levels of trehalose-6-phosphate resulted in increased branch growth, similar to the impact of the PtrWOX13c protein. In summary, identification of new protein complexes is a valuable strategy for the discovery of proteins that can increase tree growth. Additionally, combining targeted changes in both proteins and regulatory sugars may be a promising path toward future crop improvement and tree domestication.