Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2005. / The current understanding of the regulation of sucrose accumulation is still incomplete even though many scientists have investigated this subject. Components of trehalose metabolism have been implicated in the regulation of carbon flux in bacteria, yeast and more recently in plants. With a view to placing trehalose metabolism in the context of cytosolic sugarcane sucrose metabolism and carbon partitioning we have investigated the metabolites, transcripts and enzymes involved in this branch of carbohydrate metabolism in sugarcane internodal tissues. Sugarcane internodal trehalose levels varied between 0.31 ± 0.09 and 3.91 ± 0.99 nmol.g-1 fresh weight (FW). From statistical analysis of the metabolite profile it would appear that trehalose does not directly affect sucrose accumulation, although this does not preclude involvement of trehalose- 6-phosphate in the regulation of carbon partitioning. The metabolite data generated in this study demanded further investigation into the enzymes (and their transcripts) responsible for trehalose metabolism. Trehalose is synthesised in a two step process by the enzymes trehalose-6-phosphate synthase (EC 2.4.1.15, TPS) and trehalose-6-phosphate phosphatase (EC 3.1.3.12, TPP), and degraded by trehalase (EC 3.2.1.28). Two novel sugarcane partial cDNAs that coded for trehalase (tre) and actin (required for normalisation in profiling experiments) were isolated and used along with partial transcripts for TPS and TPP to determine transcript levels in different tissue- and genotypes. A putative full-length SugTPS cDNA was isolated and characterised. Enzyme activities for TPS, TPP and trehalase were measured at levels of 2.7 nmol.min-1.mg-1protein, 8.5 nmol.min-1.mg-1protein and 6.2 nmol.min-1.mg-1protein respectively, from young internodal protein extracts of sugarcane, variety N19. TPP enzyme activity and transcript levels were higher in S. spontaneum than Saccharum interspecific hybrids. Kinetic analysis of TPP and trehalase activities were performed with the purpose of providing parameters for an in silico kinetic model of trehalose and sucrose metabolism. Three isoforms of TPP were identified and desingated TPPAI, TPPAII and TPPB. Both TPPA isoforms had pH optima of 6.0, and TPPB of pH 6.5. Apparent Km values were determined as 0.447 ± 0.007 mM for TPPAI, 13.82 ± 1.98 mM for TPPAII and 1.387 ± 0.18 mM for TPPB. Partial purification and characterisation of trehalase demonstrated dual pH optima of 3.5 and 6.0, with Km values between 0.345 and 0.375 mM. These data were used as the basis for a kinetic model of trehalose metabolism. A previously described kinetic model of cytosolic sucrose metabolism has been expanded to include the trehalose pathway (TPS, TPP and trehalase). The aim was to supplement the available information on cytosolic metabolism in sugarcane storage parenchyma, identify points of control between sucrose and trehalose metabolism, and provide a platform from which further experimental and in silico modelling can be launched. The model predicted trehalose in the same order of magnitude as those determined in the metabolite profiling experiments. The majority of control of flux over the trehalose pathway resided in the TPS step, with flux control coefficients > 70% of the total pathway. Incorporation of the trehalose branch into the original sucrose model showed that reactions from the original model significantly affected the steady-state attributes of the trehalose pathway. Due to the relatively low flux through the trehalose branch of the expanded model, complete recycling of trehalose, and the lack of allosteric regulation by trehalose-6-phosphate or trehalose on any of the reactions from the original sucrose model, incorporation of the trehalose branch had no significant effect on either steady-state cytosolic sucrose concentration or flux of sucrose into the vacuole. The expanded model affords a basis from which to further investigate trehalose metabolism in the context of plant sucrose accumulation.

Diagnoses of tuberculosis, 'TB,' currently rely upon non-specific techniques such as X-ray exams and acid-fast microscopy. Improved diagnostics would preferably consider specific bacterial processes to provide real-time readouts of disease burden and response to chemotherapy. This dissertation presents the cell-wall incorporation of trehalose analogues (fluorescent and radioactive) by the mycobacterial antigen 85 enzymes as a novel method to label the causative bacteria of TB, Mycobacterium tuberculosis (Mtb). The trehalose mycolyltransesterase enzymes (antigens 85A, B, and C (Ag85)) serve as essential mediators of cell envelope function and biogenesis in Mtb. We show that the Ag85 enzymes display activities so broad that they allow added non-natural carbohydrate probes to be incorporated into Mtb growing in vitro and within macrophages. Design and synthesis of a library of structurally-diverse analogs of the sugar trehalose (Tre) revealed that Ag85-enzymes catalyze esterification of a wide variety of non-natural Tre structures, even stereoisomers and those appended with charged or bulky groups (Chapter 2). A novel mass-spectrometry based Ag85 enzyme assay was developed and employed to screen the library of compounds against all three isoforms of Ag85 (Chapter 3). This screen revealed that the Ag85 enzymes exhibit preference for dissacharides over monosaccharides and a broad tolerance for most modified trehalose compounds. This activity assay also afforded full kinetic analysis and the discovery of a novel, covalent inhibitor of the Ag85 enzymes. The Ag85 activity assay informed the design of a fluorescent trehalose-based compound (FITC-Tre), which is the first, non-toxic, selective, small molecule probe for mycobacterial infection. FITC-Tre was acylated with mycolyl esters by growing mycobacteria, anchoring the probe in the cell envelope resulting in fluorescent bacteria (Chapter 4). Adding FITC-Tre to Mtb-infected macrophages allowed selective, fluorescent tagging of Mtb in vivo (Chapter 5). Colocalization studies with antibodies against a variety of phagosomal associated components have hinted at the possibility of FITC-Tre as readout of cellular trafficking of bacteria. ¹⁸F-trehalose, biotin-trehalose and rhodamine-trehalose are also substrates of Ag85. ¹⁸F-trehalose shows promise as Mtb selective PET probe in an infected rabbit model of tuberculosis. Future work with these probes may allow for fluorescent tracking of the Mtb during the macrophage infection process, as well as the ability to label Mtb in infected tissue. The functional differences between the three isoforms of Ag85, A, B and C, are not well understood and may have implications for the survival and persistence of mycobacteria within humans. The differences in substrate specificity and catalytic activity between the Ag85 isoforms (discussed in Chapter 3) has been further investigated (Chapter 6). Mutation of three secondary site amino acids from Ag85C into Ag85B afforded nearly a twenty-fold gain in enzyme activity. Mutation of the equivalent Ag85B residues into Ag85C triggered nearly a twenty-fold loss in activity. Dissection of the roles of these three amino acids helps to explain the previously reported large differences in catalytic activity between Ag85A, B and C. Overexpression of Ag85A, B and C under tetracycline regulation revealed that these enzymes differentially modulate incorporation of mycolates into the cell wall. The Ag85 enzymes are not functionally redundant, and instead serve unique purposes in cell wall biosynthesis. In summary, this research has demonstrated that the broad substrate tolerance of Ag85 enzymes, coupled with their extracellular location, opens the door to probes of mycobacterial infection using many imaging modalities.

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.

As leveduras são de relevante importância dentro da agroindústria sucroalcooleira devido sua participação no processo fermentativo de produção de álcool. Deste modo, faz-se necessário o conhecimento deste agente fermentativo com destaque para Saccharomyces cerevisiae, principal gênero. O objetivo deste trabalho foi estudar a linhagem de levedura S. cerevisiae Y904, exposta a condições não proliferantes, após fermentação em meio que sofreu adição de óleo vegetal e sua possível correlação com manutenção da viabilidade celular. Foram realizadas análises para contagem de unidades formadoras de colônias, viabilidade celular, concentração celular, nitrogênio total na levedura e no meio, carboidratos totais, trealose e glicogênio. As leveduras submetidas a condições não proliferantes apresentaram menores teores de carboidratos totais, com destaque para trealose e glicogênio, em relação às leveduras comerciais. Saccharomyces cerevisiae sofreu queda de viabilidade acentuada após 24 h em solução fisiológica, em condições não proliferantes, sob agitação de 90 rpm e temperatura de 30 ± 1°C, seguida de uma acentuada autólise a partir de 120 h (5°dia), provavelmente, devido ao teor de carboidratos de reserva da célula que se encontravam em valores extremamente baixos, da ordem de 0,15 mg de trealose em 100 mg da matéria seca e 4 mg de glicogênio em 100 mg da matéria seca. A partir desse ponto entraram em total desorganização celular. / Yeast is highly important in sugar and alcohol agroindustry due to its role in the fermentative process of alcohol production. Thus, it is necessary to know this microorganism, most specially the Saccharomyces cerevisiae, the main species. The objective of this work was to study the strain Y904 of the yeast Saccharomyces cerevisiae under non-proliferating conditions after fermentation in a medium in which it was added vegetable oil and verify its possible correlation with the maintenance of the cellular viability. Analyses were performed in order to determine colony forming units, cellular viability, cellular concentration, total nitrogen in yeast and in medium, total carbohydrates and trehalose and glycogen contents. The yeast submitted to non-proliferating conditions presented a lower content of total carbohydrates, specially trehalose and glycogen, when compared to commercial yeasts. The viability of the yeast Saccharomyces cerevisiae Y904 markedly decreased after 24 hours in physiological solution under non-proliferating conditions in a shaker for 90 rpm at 30 ± 1°C. It was observed an accentuated autolysis from the 120 th hour (5 th day) on. This was probably because of the very low content of the carbohydrates of reserve in the cells, 0.15 mg of trehalose and 4.0 mg of glycogen in 100 mg of dry weight. From this point the cells began a total cellular disorganization.

Entre centenas de espécies brasileiras de pirilampos, o Pyrearinus termitilluminans é o único cujo ciclo de vida se passa integralmente nos cupinzeiros luminosos localizados no Brasil central claramente observados durante a estação das chuvas. A emissão de luz nos elaterídeos tem a função de atração de presas para a alimentação. A bioluminescência desses animais desaparece nos meses de seca juntamente com a nuvem de presas aladas. Assim, o metabolismo de trealose aqui estudado poderia prover informações sobre da capacidade da larva de sobreviver em ambientes edáficos. As concentrações de trealose e glicose são determinadas nos extratos de larvas com um sistema DIONEX® de cromatografia iônica. A trealase foi medida com 17 mM de trealose. O glicogênio foi estimado com uma amiloglicosidase. Paralelamente o estresse oxidativo associado com a restrição de água foi monitorado através da determinação do TBARS, juntamente com a catalase, glutationa peroxidase e glutationa redutase. Nas larvas submetidas ao ensaio na câmara climática (25% de umidade) a trealase (159,69±10,95 mU/animal) estava 80 vezes mais alta do que a do grupo controle (2,02±1,41mU/animal). As larvas sob estresse apresentaram distintos níveis de trealose e glicose (29,85±3,20 µmol/animal e 18,27±0,82 µmol/animal, respectivamente) quando comparadas com o grupo controle (64,61±1,54 µmol/animal e 2,16±0,11 µmol/animal, respectivamente). A elevação dos níveis das enzimas antioxidantes (4, 4,3 e 2,4 vezes respectivamente) com níveis invariáveis de TBARS apontam para a ação antioxidante contra a produção elevada de espécies reativas de oxigênio. Os insetos submetidos à restrição hídrica perderam aproximadamente 35% do peso. O aumento da atividade da trealase, com concomitante decréscimo dos níveis de trealose, sugerem que esse açúcar poderia ser usado como fonte hídrica. Entretanto, os níveis de glicose, 10 vezes mais elevados no grupo sob estresse poderia ser usado na via de biossíntese de trealose uma vez que os níveis de glicogênio estão diminuídos. As mudanças no metabolismo de trealose e o desbalanço no equilíbrio redox, poderiam fornecer importantes informações fisiológicas desses insetos sob estresse hídrico. / The life cycle of Pyrearinus termitilluminans (Coleoptera: Elateridae) takes place totally into the so-called luminous termite mounds located in Central Brazil, which are clearly observed during the rainy season. Light emission by the elaterid larvae acts like a trap attracting flying insects. The bioluminescence disappears in the dry months together with the food supply. The trehalose metabolism study described here could provide information about larva capacity to survive throughout hard climatic changes. Trehalose and glucose concentrations are determined in the larva extracts with a DIONEX® ion chromatography system. The trehalase activity was measured with 17 mM trehalose. The glycogen level was estimated with amyloglucosidase. In parallel oxidative stress associated to water deprivation was evaluated through determination of TBARS and the catalase, glutathione peroxidase and glutathione reductase activities. In larvae submitted to dryness in a growth chamber (25% humidity) we have found 80-fold higher trehalase activity (159.69±10.95 mU/animal) than in the control group raised under room conditions (2.02±1.41 mU/animal). Stressed larvae showed distinct trehalose and glucose contents (29.85±3.20 µmol/animal and 18.27±0.82 µmol/animal, respectively) when compared with the control group(64.61±1.54 µmol/animal and 2.16±0.11 µmol/animal, respectively), whereas the glycogen level was lower (11.53±2.01 mg/animal and 28.26±2.31 mg/animal, respectively). Elevation of the antioxidant enzyme levels (4-fold, 4.3-fold and 2.4-fold respectively) with maintenance of TBARS pointed to depletion to exacerbated production of reactive oxygen species. Insects submitted to water restriction lost about 35% wet weight. The observed increase of trehalase activity and concomitant decrease of trehalose level suggest that trehalose could be used as a metabolic water source. Moreover, the 10-fold higher glucose level in the stressed group could be used by the trehalose biosynthetic pathway as the glycogen level decreases in parallel. The trehalose metabolic and redox balance changes described here may shed light on the yet unknown physiological mechanisms of larval elaterid adaptation to water stress.

Recovering deoxyribose nucleic acid (DNA) from items of evidence can provide critical information in criminal cases. Since the development of the polymerase chain reaction (PCR) and use of short tandem repeats (STR) to create unique profiles from an individual’s genome1, sampling items of evidence for the presence of DNA has become routine. Biological evidentiary specimens are commonly collected at crime scenes as well as sampled from collected items of interest by using a cotton swab which can then be easily stored and tested as needed. However, even with modern advances in technology and methods, large amounts of DNA can be either lost throughout processing or remain on the substrate used for collection of the sample, such as a cotton swab2. While many of the downstream processes of evidence evaluation have been vastly improved through the use of automated procedures, engineered buffers, and commercially available extraction kits, the front-end procedures are typically more technician dependent; it is an area in which opportunities to fine-tune techniques remain. The most recent change to generalized stain recovery occurred after Sweet et al. achieved an increased efficiency of recovery by using what they referred to as the “double swab technique”. The classic method of collection before this time used a single, wet cotton swab. Based on a need to increase the effective collection of DNA from saliva samples, the double swab method was developed. The classic method was modified by using a second, dry swab to collect remaining moisture deposited by the first, wet swab3. To continue the effort to maximize cellular and DNA recovery from cotton swabs the use of trehalose in the cotton swab wetting solution was explored. D-(+)-Trehalose dihydrate is a naturally occurring disaccharide composed of two alpha glucose molecules. An alpha, alpha-1, 1 bond connects the two molecules which lends high resistance to acid hydrolysis, giving the molecule unique properties. Specifically, these properties allow the compound to maintain stability even during exposure to high temperatures and in acidic conditions4,5. In nature, trehalose can be found in plants and small organisms where it is thought to act as a protectant against fluctuations in moisture and temperature. Synthesis and release of trehalose by lower life forms during stressed states shows protective properties to cellular integrity by inhibiting protein denaturation6. The objectives of this study are to investigate the use of trehalose as an additive in DNA collection processes. The experiments examine the ability of trehalose to increase efficiency of cellular release from cotton swabs during the elution step and compares trehalose to other common buffer additives, bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS), when utilized as a pre-treatment or moistening agent on the cotton swab. Two procedures were developed to test the ability of trehalose to increase efficiency of cellular and DNA release from cotton swabs. The first procedure tested trehalose at 0.2 molar (M) and 1 M concentrations as the incubating solution over1 hour and 18 hour time periods after which the cotton swab was eluted using a spin-x insert and centrifugation. Both eluate and cotton swab were then processed using ZyGEM direct lysis and quantified. Quantification results of the eluate and swabs incubated in trehalose solution were not significantly different from controls. However, it is apparent that a large portion of deposited DNA remained on the swabs even after elution and ZyGEM direct lysis. The second procedure tested trehalose against BSA and SDS as treatments to cotton swabs before DNA collection. A pre-treated group (solution was applied to the swab and dried overnight; DNA was deposited to the dried swab) and a moist group (solution was applied and DNA deposited immediately) were tested after deposition of a set volume of saliva cell suspension. Quantification and amplification results of SDS treated samples indicated significant differences of DNA recovery and average peak height of profiles compared to water and buffer controls. Trehalose samples did have some significant improvement in DNA yield; however, the addition of trehalose as a moistening agent for cotton swabs does not prove to be of forensic value.

Embora o congelamento apresente vantagens em relação a outros métodos de conservação de alimentos, o mesmo causa alterações sensoriais, principalmente em produtos de origem vegetal. O morango, uma fruta muito apreciada e com formas variadas de consumo, tem comportamento sazonal e apresenta como fator limitante para o congelamento sua estrutura frágil e sensível ao processo, o que causa alterações sensoriais. Tais alterações podem ser minimizadas com a incorporação de crioprotetores, como a trealose, um dissacarídeo que vem se destacando pelo seu efeito crioprotetor em produtos congelados e desidratados. O principal objetivo deste trabalho foi verificar o efeito crioprotetor da trealose incorporada através da impregnação a vácuo em soluções de diferentes concentrações (100, 300 e 500 g/L) em morangos submetidos ao congelamento e descongelamento. As condições de impregnação foram de 5 min, aplicando pressão de -650 mmHg e 10 min de tempo de relaxamento. As alterações provocadas pelo processo de impregnação bem como a verificação do efeito crioprotetor da trealose foram identificadas através de análise de cromatografia líquida de alta eficiência (High Performance Liquid Chromatography- HPLC), análise colorimétrica, análise de textura, determinação do teor de sólidos solúveis, determinação do teor de umidade e perda de massa (perda por gotejamento). Os resultados mostraram que a concentração da solução de trealose exerce influência significativa no teor de umidade, teor de sólidos solúveis e teor de trealose. As amostras tratadas com soluções mais concentradas não sofreram desidratação após descongelamento. O teor de trealose, após descongelamento, permaneceu constante em todas as amostras tratadas. Todas as amostras tiveram a mesma perda de massa após descongelamento (perda por gotejamento), porém a composição da massa diferiu entre elas. Amostras tratadas com a solução mais concentrada perderam sólidos enquanto que as amostras tratadas com a menos concentrada, perderam água. Na análise de textura, a introdução da trealose não influenciou a força máxima de pico nas amostras impregnadas. Após descongelamento, todas as amostras, exceto a tradada com solução de 500 g/L, sofreram amolecimento. A parte externa dos morangos não sofreu alterações de cor devido à introdução da trealose nem devido ao congelamento e descongelamento. Na parte interna dos frutos, ocorreram variações no parâmetro L* devido à impregnação e no parâmetro b*, devido ao congelamento e descongelamento. / Although freezing offers advantages to others food conservations process, it causes sensorial changes, mostly in vegetables products. The strawberry, a quite appreciated fruit, shows different ways of use, has seasonal behavior and is limited to freezing due the sensorial changes caused by its fragile structure to freezing process. These sensorial changes can be minimized by the incorporation of cryoprotectors, as trehalose, that is known by its cryoprotector effect during freezing and dehydration. The aim of this work was to verify the trehalose cryoprotector effect in frozen and thawed strawberries introduced by vacuum impregnation with different solutions (100, 300 and 500 g/L). The impregnation conditions were 5 min of applying pressure of -650 mmHg and after atmospheric pressure was restored, the sample was maintained within the solution for 10 min (these conditions were obtained from previous experiments). The alterations caused by the vacuum impregnation and the verification of the cryoprotector effect of trehalose were identified by High Performance Liquid Chromatography (HPLC), color analysis, texture analysis, soluble solids content, moisture content and drip loss. The results have shown that concentration of the trehalose solution had a significant influence on the moisture content, soluble solids and trehalose content of impregnated strawberries. The samples treated with more concentration solutions did not dehydrated after thawing. The trehalose content was the same in all treated samples after thawing. All the samples showed the same drip loss due to thawing although the composition of the mass was different among the samples. Samples treated with the most concentration solution lost trehalose while the sample treated with the least concentration solutions lost water. The introduction of trehalose did not affect the maximum peak force of the impregnated samples. The freezing and the thawing process caused the softening of the samples. This effect was not observed on the sample treated with solution of 500 g/L. The introduction of trehalose did not cause significant differences in all color parameters measured on the outside of the strawberries after impregnation and thawing. In the inside of the samples, there were variation in the L* parameter caused by the vacuum impregnations and in the b* parameter caused by the freezing and thawing process.

The aim of this study was cloning of wheat (Triticum aestivum L. cv. Bayraktar) Trehalose-6-phosphate synthase gene and examining of gene expression pattern of wheat seedlings in response to salt and drought stress conditions using Wheat GeneChip (Affymetrix). In this study, 10-days old wheat seedlings were subjected to the salt (350 mM NaCl) and drought stress (20% PEG) for 24 hours, then root and leaf tissues were used for wheat TPS gene cloning and microarray studies. RACE (Rapid Amplification of cDNA Ends) was used to determine cDNA sequence of wheat TPS gene, TaTPS. The ORF of TaTPS encodes a putative protein of 859 amino acids with a predicted molecular weight (MW) of 96.7 kDa and an isoelectric point (pI) of 5.97. Based on tblastx, TaTPS showed great similarity with other plants TPS genes. In root tissue, expression of TaTPS increased under drought stress while no change was observed under salt stress. In leaf tissue, both salt and drought treatments repressed the expression of TaTPS. Microarray study was used to monitor transcript abundance in salt and drought stressed wheat. Data analyses were determined by using GCOS 1.4 and GeneSpring GX10. The genes encoding ferritin, Lipid transfer protein, LEA/Dehydrin, early nodulin, cold regulated protein and germin like proteins were upregulated at least 10-fold under salt and drought stress conditions. In addition, salt and drought stresses induced the expression of genes identified as DREB, ERF, NAC, MYB, and HSF, suggesting existence of various transcriptional regulatory pathways under salt and drought stresses.

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. Very little is currently known about the molecular mechanisms required for pathogenicity of S. nodorum, despite its major impact on Australian agriculture. S. nodorum is a polycyclic pathogen. Rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate within 2-3 weeks. Several cycles of infection are needed to build up inoculum for the damaging infection of flag leaves and heads, sporulation is therefore a critical component of the infection cycle of S. nodorum; our aim is to determine the genetic and biochemical requirements for sporulation for development of control of the pathogen. Disease progression of S. nodorum on wheat cv. Amery was monitored by light microscopy to determine the time point when pycnidia development began. Early pycnidia development was evident 12 days post-infection. This information was used to guide a genomics and a metabolomics based approach to determine the requirements for sporulation in S. nodorum. The genomics approach utilised two cDNA libraries created from sporulating and non-sporulating cultures. EST frequency was used to determine highly expressed genes under the two developmental states. Gene expression from the most highly represented genes during sporulation were confirmed using quantitative PCR. A gene encoding an arabitol 4-dehydrogenase (Abd1), was mutagenised, in its absence sporulation was reduced by approximately 20%. The metabolomics approach isolated metabolites from both in planta infection and in vitro growth. Rapid changes in the abundance of metabolites were detected during the onset of sporulation. Key fungal metabolites identified include mannitol and trehalose. The concentration of both mannitol and trehalose increased dramatically in concert with pycnidia formation. Both mannitol and trehalose have also been linked to pathogenicity in filamentous fungi. Creation of deletion mutants of the gene encoding trehalose 6-phosphate synthase showed the synthesis of trehalose is required for full sporulation of S. nodorum in planta and in vitro.