Tesis sobre el tema "Plant proteins"

Humans obtain approximately 70% of their dietary protein from plant sources on a global basis. In developing countries, vegetable protein intake is higher than in developed countries (Lusas and Rhee, 1986). Cereals, pulses and oilseeds are not only very important plant foods in the human diet, but also the main components of feeds for livestock, which can be considered as source of dairy products and meat for humans (Lasztity and Hidvegi, 1983). Cereals contribute the major dietary source of carbohydrates, and a substantive source of protein, vitamins, and minerals. Oilseeds are one of the main sources of lipid, and pulses supply protein and / or lipid. In many countries, including developed and developing countries, wheat products are consumed as a major component of the diet. Wheat flour is one of the most important foods in many countries in the world. Wheat grain contains 6-20% protein, 63-77% starch, approximately 2% fat, 2.0-2.7% crude fiber and 1.4-2.0% ash, depending in part on variety and class, and on environmental conditions during growth (Pomeranz, 1988). In many Asian countries, rice is the main cereal in the diet.

Thesis (Ph. D.)--School of Molecular and Microbial Biosciences, Faculty of Science, University of Sydney, 2004.
Chapter headings on separately inserted unnumbered cream coloured leaves. Bibliography: leaves 122-135.

The phenolic compounds as food components represent the largest group of secondary metabolites in plant foods. The phenolic compounds, e.g. chlorogenic acid (CQA), are susceptible to oxidation by enzymes specially, polyphenol oxidase (PPO) and at alkaline conditions. Both enzymatic and non-enzymatic oxidations occur in the presence of oxygen and produce quinone, which normally further react with other quinone to produce colored compounds (dimers), as well as is capable of undergoing a nucleophilic addition to proteins. The interactions of proteins with the phenolic compounds have received considerable attention in the recent years where, plant phenolic compounds have drawn increasing attention due to their antioxidant properties and their noticeable effects in the prevention of various oxidative stress associated diseases. Green coffee beans are one of the richest sources of chlorogenic acids. Therefore, a green coffee extract would provide an eligible food relevant source for phenolic compounds for modification of proteins. The interaction between 5-CQA and amino acid lysine showed decrease in both free CQA and amino acid groups and only a slight effect on the antioxidative capacity depending on the reaction time was found. Furthermore, this interaction showed a large number of intermediary substances of low intensities. The reaction of lysine with 5-CQA in a model system initially leads to formation of 3-CQA and 4-CQA (both are isomers of 5-CQA), oxidation giving rise to the formation of a dimer which subsequently forms an adduct with lysine to finally result in a benzacridine derivative as reported and confirmed with the aid of HPLC coupled with ESI-MSn. The benzacridine derivative containing a trihydroxy structural element, was found to be yellow, being very reactive with oxygen yielding semiquinone and quinone type of products with characteristic green colors. Finally, the optimal conditions for this interaction as assessed by both the loss of CQA and free amino groups of lysine can be given at pH 7 and 25°C, the interaction increasing with incubation time and depending also on the amount of tyrosinase present. Green coffee bean has a higher diversity and content of phenolics, where besides the CQA isomers and their esters, other conjugates like feruloylquinic acids were also identified, thus documenting differences in phenolic profiles for the two coffee types (Coffea arabica and Coffea robusta). Coffee proteins are modified by interactions with phenolic compounds during the extraction, where those from C. arabica are more susceptible to these interactions compared to C. robusta, and the polyphenol oxidase activity seems to be a crucial factor for the formation of these addition products. Moreover, In-gel digestion combined with MALDI-TOF-MS revealed that the most reactive and susceptible protein fractions to covalent reactions are the α-chains of the 11S storage protein. Thus, based on these results and those supplied by other research groups, a tentative list of possible adduct structures was derived. The diversity of the different CQA derivatives present in green coffee beans complicates the series of reactions occurring, providing a broad palette of reaction products. These interactions influence the properties of protein, where they exposed changes in the solubility and hydrophobicity of proteins compared to faba bean proteins (as control). Modification of milk whey protein products (primarily b-lactoglobulin) with coffee specific phenolics and commercial CQA under enzymatic and alkaline conditions seems to be affecting their chemical, structural and functional properties, where both modifications led to reduced free amino-,thiol groups and tryptophan content. We propose that the disulfide-thiol exchange in the C-terminus of b-lactoglobulin may be initiated by the redox conditions provided in the presence of CQA. The protein structure b-lactoglobulin thereupon becomes more disordered as simulated by molecular dynamic calculation. This unfolding process may additionally be supported by the reaction of the CQA at the proposed sites of modification of -amino groups of lysine (K77, K91, K138, K47) and the thiol group of cysteine (C121). These covalent modifications also decreased the solubility and hydrophobicity of b-lactoglobulin, moreover they provide modified protein samples with a high antioxidative power, thermally more stable as reflected by a higher Td, require less amount of energy to unfold and when emulsified with lutein esters, exhibit their higher stability against UV light. The MALDI-TOF and SDS-PAGE results revealed that proteins treated at alkaline conditions were more strongly modified than those treated under enzymatic conditions. Finally, the results showed a slight change in emulsifying properties of modified proteins.
Für die Verbesserung von Nahrungsmitteleigenschaften können Modifikationen an verschiedenen Inhaltsstoffen vorgenommen werden. Beispielsweise werden bereits Proteine miteinander verknüpft und bilden sogenannte „Crosslinks“ oder vernetzte Biomoleküle. Diese werden für die Herstellung fester, viskoelastischer Produkte, die zum Verdicken als auch zum Stabilisieren von Emulsionen oder Schäumen eingesetzt werden, genutzt. Da die Verbraucher sich Zunehmens mit gesundheitsfördernden Lebensmitteln befassen, ist das Einbringen von gesundheitsfördernden Inhaltsstoffen wie z.B. phenolische Verbindungen, immer mehr in den Fokus der Forschung gerückt. Demnach ist das wissenschaftliche Bestreben phenolische Verbindungen in die Vernetzung von Proteinen mit einzubeziehen und deren positive Wirkungen (antioxidativ) auszunutzen, vorteilhaft. Als Phenole werden Verbindungen bezeichnet, die eine oder mehrere Hydroxygruppen am Benzolring aufweisen. Phenole liegen in der Enolform vor, da diese, bedingt durch den Erhalt des aromatischen Benzolringes, energetisch begünstigt ist. Kaffeesäure ist eine Hydroxyzimtsäure und in Kaffeebohnen zu finden. Der am häufigsten anzutreffende Ester besteht aus Kaffee- und Chinasäure. Der einfachste Vertreter ist die Chlorogensäure (5-Caffeoylchinasäure, 5-CQA), die in vielen Pflanzenteilen enthalten ist. Chlorogensäure und ihre Derivate besitzen ebenfalls antioxidative Eigenschaften. Zusätzlich wirken sie auf Enzyme, die an entzündlichen- oder allergischen Reaktion teilnehmen, inhibierend. Während Verarbeitungs- und Lagerungsprozessen können phenolische Komponenten pflanzlicher Lebensmittel mit den Aminosäuren der Proteine in Lebensmitteln reagieren. Solche Reaktionen können die physikalisch-chemischen Eigenschaften von Proteinen verändern und deren ernährungsphysiologische Wertigkeit vermindern. Proteine weisen verschiedene reaktive Seitengruppen (Sulfhydryl-, Hydroxyl-, Aminogruppen) auf, mit denen sie über kovalente und nicht-kovalente Wechselwirkungen mit Phenolen Verbindungen eingehen können. Zu den nicht-kovalenten Verbindungen gehören u. a. Wasserstoffbrückenbindungen, hydrophobe Wechselwirkungen und Ionenbindungen. Phenole (z.B. Chlorogensäuren) können bei Anwesenheit von Sauerstoff enzymatisch bzw. nichtenzymatisch oxidiert werden. Die Reaktionsprodukte (Chinone) bilden anschließend mit reaktiven Thiol- bzw. Aminogruppen von Proteinen Addukte. Die Erfassung dieser verschiedenen Facetten von Interaktionen stellt somit die primäre Forschungsaufgabe im Rahmen dieser Arbeit. Die primäre Aufgabe der vorliegenden Arbeit besteht demzufolge in der Etablierung der Analysen- und der Charakterisierungsmöglichkeiten solcher Wechselwirkungen (Bindung) pflanzlicher Verbindungen bzw. deren Reaktionsprodukten mit Proteinen u.a. über massenspektrometrische Methoden. Da die Wechselwirkung mit Proteinen auch zu Veränderungen der Proteinstruktur führt, können deren funktionelle Eigenschaften auch verändert sein. Dies soll anhand der Messung von isolierten Proteinen die an der Wechselwirkung beteiligt sind, nachgewiesen werden. Anschließend sollen über Docking-Untersuchungen die entsprechenden Bindungsstellen näher charakterisiert werden. Durch die vorliegenden Ergebnisse wurden mögliche Reaktionen von phenolischen Verbindungen mit Proteinen, näher charakterisiert. Es wurde festgestellt, dass die Apfelsorte Braeburn über die höchste PPO- Enzymaktivität beim gleichzeitigen niedrigen CQA Gehalt im Vergleich zu den anderen untersuchten Sorten verfügt. Die PPO/Tyrosinase modulierte Reaktionen zwischen CQA und Lysine wurden in Abhängigkeit der vorherrschenden Bedingungen optimiert und die Reaktionsprodukte analysiert. In dem zweiten Teil wurden solche Reaktionsmöglichkeiten in den Grünen Kaffeebohnen lokalisierte und modelliert. Dazu wurden die sortenabhängige CQA-Zusammensetzung ermittelt und die möglichen Reaktionen mit den Hauptspeicherproteinen des Kaffees dargestellt. Im letzten Teil wurden dann diese Reaktionen mit Molkenproteinen simuliert und Einflüsse auf die Struktur und die funktionellen Eigenschaften erfasst. Die Ergebnisse belegen eine umfangreiche und sehr heterogene Adduktbildung mit den Aminoseitenketten des Lysins und Cysteins. Ein Katalog der unterschiedlichen Reaktionsprodukte wurde erstellt und am Protein modelliert. Die entsprechende Veränderung an die Proteinstruktur wurde experimentell belegt und der Einfluss wurde in den technofunktionelle Eigenschaften (wie die Löslichkeit, Emulgierbarkeit usw.) wiederspiegelt. Ein Anstieg des antioxidativen Potentials der Proteine wurde erreicht und diese so modifizierten Proteine wurden weiter zur Stabilisierung und Produktentwicklung getestet. Die ersten Ergebnisse eröffnen Nutzungsmöglichkeiten der modifizierten Proteine zur Verkapselung von bioaktiven Sekundären Pflanzenstoffen.

Cytoplasmic male sterility (CMS) in plants is associated with mitochondrial dysfunction. We have proposed in this study that the mitochondrial-encoded chimeric peptide thought to be responsible for cytoplasmic male sterility in Polima system could function as a dominant male sterility inducer when expressed in the nucleus and targeted to the mitochondria. Transgenic plants expressing such mitochondrial targeting constructs exhibited reduction of pollen production that was characterized in fertile Westar (nap ) and restored fertile Westar (pol) plants by homeotic transformation of floral organs and in male-sterile Westar (pol) plants by a reduction in pollen production with shortening of the stamens. Genetic and molecular analysis has shown that the phenotypic changes were correlated with the effective genetic transmission of the inserted transgene through female gametes. Most significantly, we have found that differences in floral morphology induced by transgene expression between pol CMS and fertile Westar plants might be related to differences in transcriptional activity of the APETALA3 MADS box gene. We suggest that the alterations in floral morphology that accompany CMS in several plant species might be due to effects of mitochondria on transcriptional activity of floral organ identity genes.

Plant viruses post a significant risk to both global food security, and industrial agriculture, however very little is known regarding their molecular mechanisms. Despite intensive study since the discovery of a multitude of plant virtual movement proteins, it remains unknown how they transverse the plasmodesmata, and thus move between cells. The CMV virus is widespread, infecting over a thousand plant species, and yet the means by which the movement protein CMV 3a associates to cellular membranes, targets itself and viral genomes to plasmodesmata have not been described. This study initially attempted to purify the CMV 3a protein from bacterial expression for structural and biophysical studies to examine viral protein and host membrane interactions. The study also began mapping the CMV 3a protein surface to investigate protein localisation and membrane attachment in planta, identifying structural features, including two potentially amphipathic helices which bear further investigation for potential roles in membrane association. Finally, this thesis examined the potential for the lipid modification S-acylation (Palmitoylation) as a membrane anchor, across a range of viral movement proteins. Describing this modification of viral movement proteins for the first time, S-acylation was demonstrated to not only be widespread, but potentially play different roles across a range of plant virus movement systems. This information is vital for the advancement of the field's understanding of the cell to cell movement of plant viruses, and the potential development of control strategies; and hence the safeguarding of global food security.

Phytoplasmas are insect-transmitted plant pathogenic bacteria that dramatically alter plant development. Phytoplasma virulence protein (effector) SAP54 mediates degradation of host MADS-box transcription factors (MTFs) via 26S proteasome shuttle protein RAD23 to abolish normal flower development and produce leaf-like flowers (phyllody). Phyllodies are common symptoms in phytoplasma-infected plants worldwide. Why do phytoplasmas degrade MTFs and induce phyllody? Are changes in host plant morphology adaptive and benefit phytoplasma spread? Because phytoplasmas rely on their insect (leafhopper) vectors for transmission from plant to plant, I hypothesized that the vegetative tissues of the leaf-like flowers render plants more attractive to the insect vectors that will aid phytoplasma dispersal in nature. I discovered that the induction of phyllody is genetically linked with enhanced insect egg-laying preference on the infected plants that exhibit the leaflike flower phenotype. However, SAP54 enhances insect colonisation of plants independently from floral transition and the changes in plant morphology. Interestingly, male leafhoppers are required for the preference of females to lay eggs on SAP54 plants. Moreover, SAP54 suppresses insect induced plant responses in sex-specific manner by selectively downregulating male-induced defence and secondary metabolism pathways. Furthermore, I identified four MTFs that are expressed in plant leaves and play important roles in egg-laying preferences by leafhoppers and demonstrate sex-specific regulation by SAP54. Taken together, phytoplasma effector SAP54 enhances insect vector colonisation of plants by suppression of insect-induced plant responses independent of developmental changes. This is likely to occur by targeting MTFs – a conserved regulators of both plant development as well as plant defence against herbivorous insects. In addition to developmental changes, degradation of MTFs by SAP54 may result in modulation of male-induced plant responses to attract female insects for egg-laying and aid phytoplasma spread in nature.

Among bioindustries, agribusiness is one of the most important application of biotechnology. The aim of this work is the design and production of recombinant proteins to improve food quality. Food quality comprises various aspects such as the conservation process, generally understood as shelf-life, the physical (e.g. color, texture, taste) and nutritional properties of food or the ability to produce food suitable for people with food intolerances or specific diseases. Since sucrose over-intake has been implicated in different pathologies such as obesity, caries, and diabetes mellitus, our first research topic was a non-calorigenic protein sweetener. Among the sweet-tasting proteins isolated until now, brazzein seems to be the most promising one. The limited availability of the natural brazzein source has rendered large-scale production of brazzein from its natural source not viable. Recombinant DNA technology provides a feasible and cheaper alternative for mass production. Therefore, we develop an efficient method for brazzein inducible or constitutive expression in the GRAS (Generally Recognized As Safe) yeast Pichia Pastoris; about 200 mg of brazzein was obtained from a 1.5 liter fermentation volume. A safe and low cost purification process was optimized taking advantage of cation exchange chromatography, resulting in a final brazzein purity of 99.98 %. The purified protein was then characterized by several techniques: mass spectrometry, calorimetry and NMR analysis were performed to evaluate respectively the mass/post-translational modifications present in the final product, and the thermal stability of brazzein; NMR experiments were also performed to confirmed the structural similarity of the recombinant brazzein with the wild-type brazzein extracted from the natural plant; a taste panel assay for sweet taste control allowed us to determine the sweetness potency of the produced recombinant protein; finally, we evaluated the allergenic potential of brazzein by sequence analysis and pepsin digestion. With regard to an improvement of the shelf-life and of the physical properties of food we focused our attention on a protein suitable for frozen food applications. In particular we are interested in the inhibition of ice recrystallization, a phenomenon by which larger ice crystals grow at the expense of smaller ones at temperature close to 0 °C. The ability of ISPs (Ice Structuring Proteins; that protect organisms that live in sub-zero environments from the deleterious effects of ice crystals’ formation in cells) to retard ice recrystallization makes them suitable for the food industry as natural ice modulators for cold storage of frozen products such as ice cream. In particular, plant ISPs are good inhibitors of the ice-recrystallization process compare to other ISPs (e.g. fish and insect ISPs). Therefore, we identified a promising wheat (Triticum aestivum) ISP and we did the first steps towards the development of an expression method for its industrial production. To perform preliminary studies we expressed this ISP in different Escherichia coli strains, obtaining a very low quantity of the recombinant protein, which precipitated in the pellet during the purification process. Hence, in order to obtain high levels of soluble recombinant ISP, we moved to the P. pastoris expression system. We constitutively expressed different form of this protein: with or without an histidine tag at the C-terminus, and with or without the Saccharomyces cerevisiae α-factor secretion signal. Thanks to experiments on a small scale we determined that only the wild type protein with the α-factor was expressed, and that its observed molecular weight is comparable with the expected one. Future development will be the scale-up of the process, the optimization of a purification protocol, and the characterization of the produced protein. Finally, in order to study the possible applications of an ISP as ingredient in the food industry or as cryoprotectant in medicine (another scope for ISPs application), we set up an experimental approach to evaluate the ice recrystallization inhibition activity and the cryopreservation ability of different ISP sources (wheat extract, and type I and II fish ISPs).
Tra le bioindustrie quella agroalimentare rappresenta uno dei campi più importanti di applicazione delle biotecnologie. Obiettivo di questo lavoro è la progettazione e produzione di proteine ricombinanti al fine di migliorare la qualità del cibo. La qualità può interessare diversi aspetti come ad esempio il processo di conservazione intesa come shelf-life (‘vita di scaffale’, ossia il periodo durante il quale un prodotto può essere tenuto presso un punto vendita al dettaglio senza che vengano alterate le sue qualità), le proprietà fisiche (es. colore, consistenza, sapore) e nutrizionali dell’alimento, o la possibilità di produrre cibi adatti a persone con intolleranze alimentari o con particolari malattie. Dato che un eccessivo consumo di saccarosio è implicato in varie patologie come ad esempio l’obesità, le carie e il diabete, il nostro primo tema di ricerca si è focalizzato su una proteina dolcificante non-calorica. Tra le proteine dolcificanti isolate finora, la brazzeina sembra essere la più promettente. La disponibilità limitata della fonte naturale di brazzeina ha reso economicamente non sostenibile la sua produzione su scala industriale a partire dalla fonte naturale. La tecnologia del DNA ricombinante costituisce un’alternativa praticabile e più economica per la produzione su ampia scala. Abbiamo pertanto sviluppato un metodo efficiente per l’espressione inducibile o costitutiva della brazzeina nel lievito GRAS (Generally Recognized As Safe) Pichia Pastoris; circa 200 mg di brazzeina sono stati ottenuti da un volume di fermentazione pari a 1.5 litri. Abbiamo inoltre ottimizzato un processo di purificazione sicuro ed economico mediante cromatografia a scambio cationico, ottenendo la brazzeina con una purezza finale pari al 99.98 %. La proteina purificata è stata poi caratterizzata mediante divere tecniche: spettrometria di massa, analisi calorimetriche e NMR sono state effettuate per valutare rispettivamente la massa e le modifiche post-traduzionali presenti nel prodotto finale, e la stabilità termica della proteina; analisi NMR sono state effettuate anche per confermare la similarità di struttura della proteina prodotta per via ricombinante, con quella della proteina wild-type direttamente estratta dalla pianta; un saggio per determinare l’intensità del gusto dolce ci ha permesso di definire la ‘sweetness potency’ della brazzeina ricombinante; abbiamo infine analizzato il potenziale allergenico della brazzeina mediante analisi della sua sequenza amminoacidica e digestione con l’enzima gastrico pepsina. Per quanto riguarda il miglioramento della shelf-life e delle proprietà fisiche dei cibi, abbiamo concentrato la nostra attenzione su una proteina interessante per l’applicazione in prodotti surgelati. In particolare siamo interessati all’inibizione della ricristallizzazione, fenomeno che causa la formazione di grandi cristalli di ghiaccio a spese di cristalli piccoli a temperature vicine ai 0 °C. L’abilità delle ISPs (Ice Structuring Proteins; proteine che proteggono gli organismi che vivono a temperature inferiori allo zero dagli effetti deleteri della formazione di cristalli di ghiaccio nelle cellule) di inibire il fenomeno della ricristallizzazione le rende interessanti per l’industria alimentare come naturali modulatori del ghiaccio per la conservazione di prodotti surgelati come ad esempio il gelato. In particolare, è stato visto che le ISPs di pianta sono degli ottimi inibitori della ricristallizzazione del ghiaccio. Abbiamo pertanto identificato una promettente ISP di grano (Triticum aestivum) e abbiamo fatto i primi passi verso lo sviluppo di un metodo di espressione per la sua produzione a livello industriale. Al fine di effettuare degli studi preliminari abbiamo espresso la ISP in diversi ceppi di Escherichia coli, ottenendo però una piccolissima quantità di proteina ricombinante che, oltretutto, precipita nel pellet durante il processo di purificazione. Quindi, al fine di ottenere alti livelli di ISP solubile, siamo passati al sistema di espressione costitutiva in P. pastoris. La proteina è stata espressa in diverse forme: con e senza coda di istidine al C-terminale, e con e senza il fattore di secrezione α-factor di Saccharomyces cerevisiae. Grazie a esperimenti su piccola scala abbiamo determinato che solamente la proteina wild type con l’α-factor viene espressa, e che il suo peso molecolare osservato è comparabile con quello atteso. Sviluppi futuri riguarderanno lo scale-up del processo di espressione, l’ottimizzazione di un protocollo di purificazione e la caratterizzazione della proteina prodotta. Per concludere, al fine di studiare la possibile applicazione di una ISP come ingrediente nell’industria alimentare o come crioprotettore in medicina (altra possibile applicazione delle ISPs) abbiamo messo a punto un protocollo sperimentale per valutare l’attività di inibizione della ricristallizzazione e l’abilità di criopreservazione di differenti fonti di ISPs (estratto di grano, ISPs di tipo I e II di pesce).

Thesis (Ph. D.)--University of Kentucky, 2004.
Title from document title page (viewed on May 6, 2010). Document formatted into pages; contains: vii, 84 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 71-82).

A three part study is presented which examines the functional properties of plant proteins as they relate to textural and stability characteristics of protein-replaced meat emulsions. In the first chapter, the effects of cooking time (25 or 50 min) and temperature (70 or 95°C) on texture, microstructure and cook stability of a model meat emulsion system containing soy or canola protein isolate were investigated. The plant proteins were added either dry or rehydrated at replacement levels of 33.3% and 66.7% of the meat protein. Instrumental texture profile analysis and stability data revealed several complex interactions between experimental variables; however, level of protein replacement was predominant, with decreased firmness and increased yield resulting from increased replacement of meat protein. Thermorheological profiles of emulsions and protein dispersions demonstrated that the development of elasticity of all-meat emulsions during heating was essentially complete at 75-80°C, while the elasticity of canola or soy protein dispersions continued to rise with heating to 95°C. A meat emulsion containing canola protein displayed characteristics of the all-meat emulsion and canola protein dispersion thermoprofiles, but the increased structure formation from the canola protein at higher heating temperatures did not fully compensate for an initial decrease in elasticity that resulted from the loss of meat protein. Although there were slight differences in the fat particle distributions of the emulsions containing plant protein, the distributions had similar shapes, where particles larger than 50 micrometers approximated a normal distribution, and were thought to be relatively intact fat cells while the number of particles with diameters of 10-50 micrometers increased in an essentially logarithmic manner as size decreased. The microstructure of the proteinaceous matrix was affected primarily by protein source, replacement level and cooking conditions. In chapter 2, thermally induced gelation (72°C, 30 min heating) and emulsification properties of unmodified and succinylated canola protein isolate (54% and 84% modification of free amino groups) were examined over a wide range of pH values (pH 3.5-11.0) and sodium chloride concentrations (0.0-0.7M). Succinylation improved the gelation ability of canola isolate. For the unmodified isolate, gels formed at only 4 of 18 combinations of pH and NaCl concentration, while 12 gels formed from each level of succinylation under the same conditions. Above pH 6.5, succinylated protein formed gels only in the presence of NaCl. In general, the firmest gels were obtained with the moderate level of succinylation. Translucent and opaque gels responded differently to Theological tests and were related in different ways to the physicochemical and Theological properties of protein dispersions. The viscoelastic properties of the translucent gels were affected mainly by protein solubility and hydrophobicity, while those of the opaque gels were related to solubility, hydrophobicity, zeta potential and apparent viscosity of protein dispersions. The types of bonds involved in gel formation and stability were tentatively identified as hydrophobic interactions and hydrogen bonds. With the succinylated isolates, gels were formed in the presence of calcium ions at a concentration an order of magnitude less than was required for similar gel strengths with NaCl, which has implications for exploiting the gelation ability of succinylated proteins in products where high concentrations of NaCl are undesirable. Both emulsification activity and emulsion stability were increased by succinylation, but exhaustive succinylation was not required to produce a significant improvement in these properties. Emulsification activity was related to protein solubility, hydrophobicity, zeta potential and flow behavior of protein dispersions, while emulsion stability appeared to be mainly a measure of resistance to creaming and was related to protein solubility, zeta potential, apparent viscosity of protein dispersions, and the difference in density between the aqueous and oil phases. The third chapter examined the relationship between textural measurements of canola isolate gels obtained by means of a puncture test with an Instron tester, and fundamental rheological parameters obtained from nondestructive dynamic shear measurements with a Weissenberg Rheogoniometer. Although the force required to rupture the gels, as measured by the puncture test, was poorly correlated with the viscoelastic parameters, the slope of the force-deformation curves to the point of rupture was well correlated with the storage and loss moduli of the gels. In addition, the area under the force-deformation curves to rupture followed a curvilinear relationship with the loss tangent of the gels. The response of translucent and opaque gels to the two types of rheological tests was not identical, which indicated that gel microstructure is an influential factor when evaluating gel rheological properties by destructive or nondestructive methods.
Land and Food Systems, Faculty of
Graduate

The plant non-specific lipid transfer proteins (nsLTPs) are known for the ability to transfer different lipids in vitro, but their in vivo functions have not yet been elucidated. They seem to play a role in the defense against biotic and abiotic stresses; the gene expression of nsLTPs is often upregulated when exposed to stresses. Further, two different nsLTPs have been shown to affect the lipid composition of the plant cuticle, a structure acting as a protective barrier. However, more evidence is needed to prove this hypothesis and to pinpoint their exact role in this process. In this thesis I have shown that the nsLTPs are found in all land plants, but not in any of the studied algae. This supports a role in defense response, since protection against dehydration, radiation, pathogens and other stresses played a crucial role when plants adapted to a life on land. Characterization of the nsLTPs in early diverging land plant revealed that even though the amino acid similarity towards nsLTPs in flowering plants is not very high, the main properties of the proteins are still the same (Paper I). This includes the protein structure, which consists of α-helices surrounding a lipid binding cavity, a conserved pattern of cysteine residues involved in disulphide bonds and a signal sequence directing the protein to the  extracellular space. Further, the expression of nsLTPs in the moss Physcomitrella patens was shown to respond to stresses, and construction of an YFP-LTP fusion protein confirmed the localization to the periphery of the cell in planta (Paper II). Heterologous expressed Physcomitrella nsLTPs were also shown to have the ability to bind lipids and to be very heat stable, features previously only studied in nsLTPs from flowering plants. By examining the presence of a cuticle in Physcomitrella, a correlation between the nsLTPs´ lipid binding ability and the lipid composition of the cuticle could be found, which further strengthens the involvement of nsLTPs in transfer of lipids for cuticle construction. In the flowering plant Arabidopsis thaliana, I showed that several of the nsLTPs followed the same expression pattern when examining data from different tissues, stress treatments, hormones, chemical treatments and developmental stages, but also that four of the genes were undergoing alternative splicing resulting in different isoforms of the proteins (Paper III). Based on their expression patterns, the genes could be divided into three different coexpression networks. By examining other genes similarly expressed, each network could be designated to a putative function: Transfer of lipids for synthesis of the cuticle, suberin layer and sporopollenin, respectively. In Paper IV, these hypotheses were tested in vivo by examining knockout mutants of several nsLTPs in Arabidopsis. The involvement in sporopollenin deposition could be confirmed; two of the knockout lines showed collapsed pollen grains. Further, two other lines showed an increased seed coat permeability due to an altered lipid composition of the suberin layer. Together, the results support a role for nsLTPs in construction of the protecting barriers in all land plants.

Secreted surface proteins are an innate immune defense component employed by animals to inhibit invading microbes. Surface proteins have not been documented in plants, even though the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum, and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete pathogen Peronospora tabacina. We investigated the site of phylloplanin biosynthesis using biochemical techniques. These techniques included radiolabeling of detached trichome glands, radiolabeling of epidermal peels, analysis of leaf water washes of various Nicotiana plants, and examination of guttation fluid, leaf vein contents, and extracellular fluid. From these experiments, we tentatively conclude that phylloplanins are produced by hydathodes, or an unknown surface secreting system, but not by glandular secreting trichomes. Future experiments with the phylloplanin promoter, whose elucidation is described herein, and its fusion to a reporter gene (GUS or GFP), will undoubtedly provide further insight into the location of phylloplanin biosynthesis and deposition. We suggest that the hydrophobic nature of phylloplanins aids in their dispersal over the leaf surface. Phylloplanins constitute a first-point-of-contact, rapid response, innate immune deterrent to pathogen establishment on N. tabacum leaf surfaces, and are the first studied representatives of a novel protein class in the plant kingdom. Further study of leaf surface proteins is justified to understand further their roles in plant defense, and to investigate their potential in agricultural biotechnology. Additionally, we describe miscellaneous observations we have made during the course of this research. Low molecular mass proteins (as yet uncharacterized) are washed from leaf surfaces of sunflower, soybean, and other plants. Pathogenesis-related (PR-)-5a, a known antifungal protein, was found to be present on the leaf surfaces of healthy plants, although its function there remains unknown. A phylloplanin homologue from Arabidopsis appears to be antibacterial. Further study of this protein is warranted. We note that proteins can also be recovered from N. tabacum root surfaces, or the rhizoplane, but we have not further characterized these proteins. In summary, novel surface-accumulated proteins, termed phylloplanins, and the gene encoding these have been discovered in N. tabacum. An antifungal function for phylloplanins is reported, and evidence was found for a unique mechanism of surface deposition.

The nuclear envelope (NE) is a double lipid bilayer enclosing the eukaryotic genome. The metazoan nucleoskeleton includes the peripheral lamina and the internal nucleoskeleton. The lamina is composed of a network of intermediate filament (IF) proteins called lamins, as well as lamin- and/or chromatin-binding inner nuclear membrane (INM) proteins. The components of the metazoan lamina lack sequence homologues in plants. There is however evidence of a network of nuclear filamentous proteins underlying the NE. This study aims to characterise a novel family of NE-associated proteins (NEAP) in the model plant, Arabidopsis thaliana. The family consists of four proteins, AtNEAP1-4 conserved in plants restricted to the angiosperm clade. Their expression is ubiquitous with up-regulation in embryo, inflorescence and guard cells. NEAP protein structure consists of extensive coiled-coil (CC) domains, followed by a nuclear localisation signal (NLS) and a C-terminal transmembrane (TM) domain. Confocal microscopy shows that fluorescent protein tagged NEAP proteins localise to the nuclear periphery as part of highly immobilised stable complexes. Domain deletion mutants confirm the presence of functional NLS and TM domains, while their CC nature causes insolubility under high ionic salt and Triton X-100 conditions similar to other IF-like proteins. AtNEAP2 and AtNEAP3 interact with themselves as well as with AtNEAP1 and each other. NEAP proteins also interact with the classical and mid-SUN domain families. NEAP proteins also cause mis-localisation of the plant nuclear matrix constituent protein 1 from the nuclear periphery to the nucleoplasm. An A. thaliana cDNA library screen identified a basic leucine zipper transcription factor (TF), AtbZIP18 as a novel interactor of AtNEAP1. This is a first description of a chromatin-binding protein partner of the plant INM. Single and double NEAP knockout and knockdown mutants analysed displayed various defects in nuclear size, shape and positioning in different tissues. Therefore NEAP proteins appear to be involved in regulating nuclear morphology in plants. Thus as novel nuclear IF-like proteins that interact with a chromatin binding TF and have functions in regulating nuclear morphology, NEAP proteins are putative components of the plant lamina anchored at the INM.

Wheat gluten, wheat gliadin, and corn zein agricultural proteins were evaluated as multifunctional additives that: (1) provided reinforcement, (2) improved thermal stability, and (3) lowered the cost of polymer composites. Wheat proteins were utilized in two polymer matrices: poly(vinyl alcohol) (PVA) and synthetic cis-1,4-polyisoprene rubber (IR). The proteins were hydrolyzed and dispersed in the polymer matrix, where they cooperatively self-assembled into nanostructures called amyloids. Amyloids have the potential for high rigidity and stability due to high β-sheet content. In Chapter II, trypsin hydrolyzed wheat gluten (THWG) proteins were incubated in aqueous PVA solutions, then the composite solutions were air dried and compression molded into films. Anisotropic protein aggregates formed through a typical mechanism of β-sheet self-assembly, where a greater molding time and pressure and/or a lower PVA molecular weight allowed for more protein aggregation. The larger protein structures provided less reinforcement. In Chapters III and IV, THWG and trypsin hydrolyzed gliadin (THGd), a component protein in wheat gluten, were compounded in synthetic polyisoprene rubber to form nanocomposites. The reinforcement correlated to the protein β-sheet content and varied with protein concentration, protein batch preparation, processing temperature, and compounding time. The isotropic β-sheet containing structures were very thermally stable, even under harsh rubber compounding conditions. By optimizing the processing parameters uniform protein dispersion and optimal IR reinforcement were achieved, although the protein and IR phases had poor compatibility. In Chapter V, the THGd-IR composites were cured using a typical cure package and molding process. Protein aggregation into nanostructured β-sheets was observed during the curing process. Rubber reinforcement increased as a function of protein concentration and curing time. In Chapter VI, a hydrophobic protein (zein) was substituted for the hydrophilic protein (gliadin) used previously to improve protein-IR compatibility. The zein protein was better at reinforcing IR, while gliadin improved mechanical stability. Both zein and gliadin improved the thermal stability of IR. The results from Chapters II-VI showed an interesting concept: in situ filler formation in polymer matrices where the choice of protein, polymer, and processing conditions influenced the final morphology and composite properties.
Doctor of Philosophy
We use plastics every day for a wide range of applications, from food packaging to automobile tires. Many of these plastics are composite materials, called “polymer composites,” meaning they are made of two or more chemically distinct materials where one material is a polymer. For reference, a polymer is a long chain molecule made of many (“poly-”) units (“- mer”). Polymer composites often contain additives which modify the properties of the polymer. For example, many soft polymers, such as tire rubber, need to be made stiffer and so a “reinforcing additive” is used to improve the stiffness of the rubber. Many composite materials are made stiffer so less material can be used. This process is called “lightweighting.” The automotive industry and food packaging industry use this process to reduce weight and fuel costs. In this research, plant proteins are tested as reinforcing additives in polymer composites. Plant proteins, such as wheat gluten, are abundant, non-toxic, sustainable, and can self-assemble into extremely small, stiff structures. For these reasons, plant proteins offer an environmentally friendly alternative to typical reinforcing additives. This dissertation shows that plant proteins can reinforce two polymers with very different properties. The first polymer is poly(vinyl alcohol) (PVA), which is biodegradable, hydrophilic (i.e., “water loving”), and is commonly used in flexible food packaging. The second polymer is synthetic cis-1,4-polyisoprene rubber (IR), which is non-biodegradable, hydrophobic (i.e., “water fearing”), and is commonly used in automotive tires. In Chapters II-V, the wheat gluten protein is hydrolyzed, i.e., chemically “chopped” into short chain peptides, to encourage the self-assembly of the plant protein into small, stiff structures. The self-assembled protein structures survive typical industrial processing techniques, such harsh rubber compounding conditions which involve high heat, pressure, and shear forces (i.e., the material is pushed in opposing directions). In Chapter VI, full corn and wheat proteins are incorporated into IR using standard industrial mixing and curing processes. The corn and wheat proteins reinforce the synthetic rubber and inhibit the degradation of the chemical structure of cured rubber under high heat. At certain protein concentrations, the proteins improve the elasticity and lessen the permanent deformation in the polymer composite. Together, Chapters II-VI show that proteins from diverse plant sources can be used to improve the performance of polymers with dissimilar properties.

Thesis (Ph. D.)--West Virginia University, 2009.
Title from document title page. Document formatted into pages; contains ix, 125 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 98-125).

Studies in this thesis concern expression of one class of small HSPs (sHSPs) in field grown desert plant species and the isolation of a new HSP gene encoding an sHSP targeted to plant mitochondria. Expression of class I, cytosolic sHSPs was assessed in three desert species: Screwbean Mesquite, Baja Fairyduster, and Sweet Acacia. Total leaf protein, and if available, flower and pod protein, was extracted from samples and analyzed by SDS-PAGE and Western blotting. Sweet Acacia showed strong sHSP expression in leaves with an apparent diurnal pattern of increased expression in the hotter PM. Screwbean Mesquite pods showed significant sHSP expression, which was not correlated to temperature. The isolation and sequence analysis of a gene encoding a mitochondrion-localized sHSP from Arabidopsis was completed. Comparisons to other plant sHSPs verified it was most similar to other mitochondrial-localized sHSPs from plants.

A liquid artificial diet system, which was suitable for bioassay of added compounds, was developed for the glasshouse potato aphid, Aulacorthum solani. The diet supported normal growth and reproduction of this insect. Once established, the artificial diet bioassay system was used to test potential insecticidal activities of a variety of proteins found naturally occurring in plants. Effects on survival, development and fecundity were measured. The lectin found in snowdrop, Galanthus nivalis agglutinin (GNA) was found to significantly reduce the fecundity of A. solani, in terms of parthenogenetic nymph production, when administered in artificial diets at the 0.1% w/v level. No significant reductions in survival were found, although GNA administered in vitro did appear to slow the development of A. solani. Transgenic potato plants expressing GNA were used in a growth room trial to show that the reduction in fecundity with the in vitro trials could be reproduced in planta. Aphids feeding on the GNA-expressing potatoes had a significantly lower cumulative nymph production than those feeding on non- transformed plants. The transgenic plants had no effect on the survival of A. solani. The GNA-expressing plants were tested in a larger scale glasshouse trial and resulted in a significantly slower buildup of aphids when compared to control potatoes, thus confirming the results of the artificial diet bioassays and in planta growth room trials. Immunohistochemical studies were performed to detect the presence of GNA in the gut lumen of A. solani fed on artificial diet containing 0.1% w/v GNA; the lectin was observed to be selectively concentrated in the region of the epithelial membrane in the stomach, suggesting that binding to surface carbohydrates or glycoproteins was taking place. Binding to the gut surface has been suggested to mediate lectin toxicity in higher animals, and other insects. A synergistic effect was observed with transgenic potatoes expressing a double construct encoding GNA and bean chitinase (BCH); A. solani cumulative nymph production on these plants was significantly reduced compared to aphids feeding on control and GNA-only expressing plants. However, interestingly, BCH-only expressing plants did not significantly affect the fecundity of A. solani, although a slight reduction in nymph production was observed. On the basis of reports in the literature that suggested that chitin-binding lectins were toxic to insects, an attempt to isolate the gene encoding the chitin-binding stinging nettle lectin was made. RNA was extracted from nettle rhizomes and used to prepare a cDNA library. Successful library construction was verified. PGR methods and a primary screen of the library were used in an attempt to locate the gene.

The NET protein superfamily is a recently discovered family of novel, plant-specific actin binding proteins. The identification of this family represents a significant discovery as the plant cytoskeleton is not identical to the animal cytoskeleton and plant cells show plant specific processes and subcellular structures which rely on actin. There is a need for plant specific proteins which are capable of modelling actin within plant cells. The NET family comprises thirteen proteins in Arabidopsis thaliana, which share the NET actin binding domain (found at the N-terminal end of each protein). Based on the C-terminal domains of the proteins, the family can be separated into four groups, each with a particular localisation. The localisations of these proteins are frequently within plant specific cell types, such as pollen tubes, guard cells or roots, or to plant specific cell structures such as plasmodesmata. It is thought that these proteins may be involved in modelling the actin cytoskeleton at junctions between actin and membranes (either cell membranes or membrane bound structures such as the endoplasmic reticulum). The NET1 proteins are a group of four proteins, each consisting of an N-terminal actin binding domain and C-terminal coiled-coil domains. NET1a was the first protein to be discovered in a high-throughput screen of plant proteins for novel localisations carried out by Karl Oparka, where it was shown to bind to filaments. Work by Calcutt (Calcutt 2009) showed the filaments to be the actin cytoskeleton. The aim of this thesis has been to complete the characterisation of all Group 1 NET proteins, building on the analysis of NET1a by Calcutt (Calcutt 2009) and to investigate the possible functions of the NET1 subfamily proteins. All four proteins have been shown to be capable of actin binding and to be expressed in, and locate to structures within, the roots of A. thaliana. NET1a has been linked to plasmodesmata, and the combined absence of NET1a and NET1b in the plant results in a cumulative, long root phenotype. Theories to explain this phenotype are suggested here, although the validation and testing of these will form the basis of future work.

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Chloroplasts are cell organelles responsible for photosynthesis. Although chloroplast have their own genome it is not sufficient to encode all the proteins which are located there. Most of the proteins are imported from the cytosol through the so called toc/tic pathway. It has been recently showed that Arabidopsis CAH1 is transported to the chloroplast through the secretory route in a fully new pathway. It has also been demonstrated that the N-terminal signal peptide of CAH1 targets it to the ER where the protein gets glycosylated. Structure of the Arabidopsis CAH1 suggests that its C-terminus might be responsible for targeting the protein to the chloroplast. By expressing N- and C-terminal labeled CAH1 we show that the expression level of the N-terminal labeled form is high and the majority of the labeled protein is localized in the chloroplast. By contrast, the C-terminal labeled CAH1 expressed weakly if at all, and due to the low expression level immunolocalization of the protein is difficult. We also demonstrate that the strong expression level of the N-terminal labeled CAH1 makes it feasible to affinity purify the glycosylated protein for structural studies.

PHELAN, THOMAS JOSEPH, Genetic and Molecular Analysis of Plant Nuclear Matrix Proteins. (Under the direction of Steven L. Spiker.)The eukaryotic nucleus is composed of DNA, RNA and protein, encapsulated by a nuclear envelope. DNA is compacted up to ten thousand times in order to be packaged into the nucleus. The nucleus must maintain order in the presence of a very high density and variety of protein and RNA. The nuclear matrix is a proteinaceous network thought to provide structure and organization to the nucleus. We believe that relatively stable interactions of nuclear molecules with the nuclear matrix are key to organization of the nucleus. Numerous "Matrix Attachment Region" DNA elements (MARs), have been isolated from plants, animals, and fungi. Evidence suggests that these MARs attach to the nuclear matrix, delimiting loops of chromosomal DNA. In studies of transgenic plants and animals, MARs have been shown to give important advantages to organisms transformed with genes flanked by these elements. Unlike most DNA elements, no specific sequence elements have been identified in MAR DNAs. Partly due to the insolubility of the matrix, and to the heterogeneity of MAR DNA, very few of the protein components of the nuclear matrix have been identified. This work presents analysis the proteins of the plant nuclear matrix. We have characterized a set of related proteins from the model plant Arabidopsis that associate with MAR DNA in vitro. These proteins appear to be similar to the NOP56/NOP58 family of proteins previously identified in several eukaryotic organisms. The NOP56/NOP58 proteins are thought to be involved in modifications of ribosomal RNA. Binding studies presented in this work suggest that these plant proteins may participate in RNA/DNA/protein complexes in the nucleus.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology." Discipline: Biology; Department/School: Biology.

Homopteran insect pests can cause severe economical damage to crop plants by both direct physical means and as vectors of plant viral diseases. They are notoriously difficult insects to control by conventional methods, primarily due to their ability to evolve resistance-breaking biotypes within a relatively short time period. The production of genetically modified crop plants, expressing insecticidal genes, offers a novel method of control for a wide range of insect species. Once suitable gene products, such as plant- derived proteins, have been identified as having insecticidal effect against specific insects in vitro, their effect can be determined in vivo by expressing the relevant gene in transgenic plants. Insect feeding trials were carried out to determine the effects of incorporating a range of plant-derived proteins into artificial diets fed to planthopper, leafliopper and aphid pests and to aphids in planta. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soybean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stal) when incorporated into artificial diet at 0 1% {w/v}, 0-1% (w/v) and 0 08% {w/v} levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephottetix cinciteps Uhler) and the peach potato aphid {Myzus persicae Sulzer). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects. The mechanism of action of all three effective proteins was examined using BPH as a model insect. As judged by honeydew production, the proteins all had a deterrent effect on insect feeding. However, subsequent toxic effects are also indicated. When fed sub-optimal concentrations of effective proteins in combination no synergistic or additive effects were observed, indicating that pyramiding the genes of these effective proteins would be of no advantage in protecting the crop against BPH.

Investigations of plant mitochondrial protein import have been very limited and few reports exist in the literature. The work presented in this thesis describes the development of an in vitro Zea mays mitochondrial protein import system, which has enabled the import of plant nuclear-encoded mitochondrial proteins to be examined. This Z.mays import system was characterised and partially optimised with the Nicotiana plumbaginifolia manganese superoxide dismutase (MnSOD). Results suggest that the energy requirements for protein import into plant mitochondria are similar to those of Saccharomyces cerevisiae and Neurospora crassa, requiring both an energised inner mitochondrial membrane and ATP. The inclusion of 1,10-phenanthroline inhibited the processing, but not the import of MnSOD and indicated that the processing activity within Z.mays mitochondria was dependent upon the presence of metal ions. The plant mitochondrial processing protease may therefore be similar to the characterised S.cerevisiae and N.crassa matrix processing protease. The import of the Z.mays adenine nucleotide translocator (ANT) protein was then investigated. Unlike the ANT of S.cerevisiae and N. crassa, this plant ANT was synthesised as a precursor protein, which was processed upon import into mitochondria isolated from both Z.mays and Solanum tuberosum. The subsequent isolation of an S.tuberosum ANT cDNA clone PANT-1, enabled the import of a second plant ANT to be investigated. Results corroborated the findings previously obtained with the Z.mays ANT and it was therefore concluded that the import of plant ANT proteins are distinctly different from those of S.cerevisiae and N.crassa.

Jasmonates (JAs) play a major role in plant defense against herbivores while some caterpillar species use effectors in their labial saliva to suppress the induction of JA-mediated defense responses. On the other hand, activation of plant defense is associated with slowed plant growth which is controlled by gibberellins (GA) and growth repressor DELLA proteins. Recent studies have shown that DELLA proteins play an important role in plant stress response and are involved in the crosstalk between JA and GA pathways. However, the role of DELLA proteins in plant-insect interactions remains unclear. In this study, wild-type Arabidopsis, wild-type sprayed with GA and a quadruple-della Arabidopsis mutant (quad-della) were subject to herbivory by beet armyworm Spodoptera exigua caterpillars with either intact or impaired labial salivary secretions. Wild-type Arabidopsis, Arabidopsis + GA and the quad-della mutant showed a JA burst in response to herbivory. This was reflected in increased transcript levels of the JA-dependent gene markers, such as AtPDF1.2, AtLOX2 and AtVSP2. A caterpillar saliva-specific pattern of JA hormone levels (JA, JA-isoleucine, OPDA) were observed in the wild-type background but not in the quad-della mutant, suggesting that DELLAs are involved in plant response to caterpillar saliva, probably by mediating the crosstalk between JA- and salicylic acid (SA)-dependent pathways. Additionally, high constitutive expression of the SA pathway marker gene AtPR1 was observed in the quad-della mutant but not in wild-type Arabidopsis, which indicates that DELLAs play a role in maintaining the homeostasis of SA signalling by repressing its constitutive induction.
Les Jasmonates (JAs) font une partie importante dans la défense des plantes contre les herbivores. Certaines espèces de chenilles utilisent des effecteurs dans leur salive labiale pour supprimer l'induction de réponses de défense induites par les JAs. En revanche, l'activation de défense de la plante est associée au ralentissement de la croissance des plantes. La croissance des plantes est contrôlée par les gibbérellines (GA) et les répresseurs de la croissance, les protéines DELLA. Des études récentes ont montré que les protéines DELLA font partie de la réponse des plantes au stress et participent à la diaphonie entre les voies métaboliques du JA et GA. Cependant, le rôle des protéines DELLA reste incertain. Dans cette étude, Arabidopsis type sauvage, Arabidopsis type sauvage traités avec une pulvérisation du GA, et le mutant quadruple-della (quad-della) souffert des attaques par le herbivore Spodoptera exigua. Les chenilles sont normales ou avec les facultés affaiblies dans les glandes salivaires. Les trois groupes des plantes ont montré une explosion du JA en réponse aux herbivores. Cette réponse a été reflétée dans les niveaux de transcription des gènes dépendant de JA, comme AtPDF1.2, AtLOX2 et AtVSP2. Un motif spécifique à la salive des niveaux des hormones JA (JA, JA-isoleucine, OPDA) a été observé dans le type sauvage mais pas dans les mutants. Ce résultat suggère que DELLAs sont impliquées dans la réponse de la plante à la salive probablement par la médiation de la diaphonie entre les voies métaboliques du JA et du acide salicylique (SA). Ailleurs, une forte expression du gène AtPR1, qui est partie de la voie d'SA, a été observé dans le mutant quad-della. Ce résultat suggère que DELLAs sont impliquées dans l'homéostasie de la signalisation de SA en réprimant son expression constitutive.

Orientadores: Gonçalo Amarante Guimarães Pereira, Julio Cezar de Mattos Cascardo
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ?? 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.

Eukaryotic organisms require the ability to respond to their environments. They do so by utilizing signal transduction pathways that allow for signals to effect final biological responses. Many times, these final responses require new gene expression events that have been stimulated or repressed within the nucleus. Thus, much of the understanding of signal transduction pathways converges on the understanding of how signaling affects gene expression alterations (Kumar et al., 2004). The regulation of gene expression involves the modification of chromatin between condensed (closed, silent) and expanded (open, active) states. Histone modifications, such as acetylation, can determine the open versus closed status of chromatin. The PHD (Plant HomeoDomain) finger is a structural domain primarily found in nuclear proteins across eukaryotes. This domain specifically recognizes the epigenetic marks H3K4me2 and H3K4me3, which are di- and tri-methylated lysine 4 residues of Histone H3 (Loewith et al., 2000; Kuzmichev et al., 2002; Vieyra et al. 2002; Shiseki et al., 2003; Pedeux et al., 2005, Doyon et al., 2006). It is estimated that there are ~150 proteins that contain the PHD finger in humans (Solimon and Riabowol, 2007). The PHD finger is conserved in yeast and plants, however an analysis of this domain has only been performed done in Arabidopsis thaliana (Lee et al., 2009). The work presented in this report aims to extend the analysis of this domain in plants by identifying the PHD fingers of the crop species Oryza sativa (rice). In addition, a phylogenetic analysis of all PHD fingers in Arabidopsis and rice was undertaken. From these analyses, it was determined that there are 78 PHD fingers in Arabidopsis and 70 in rice. In addition, these domains can be categorized into classes and groups by defining features within the conserved motif. In a separate study, I investigated the function of two of the PHD finger proteins from Arabidopsis, ING1 (INhibitor of Growth1) and ING2. In humans, these proteins can be found in complexes associated with both open and closed chromatin. They facilitate chromatin remodeling by recruiting histone acetyltransferases and histone deacetylases to chromatin (Doyon et al., 2006, Pena et al., 2006). In addition, these proteins recognize H3K4me2/3 marks and are believed to be â interpretersâ of the histone code (Pena et al., 2006, Shi et al., 2006). To understand the function of ING proteins in plants, I took a reverse genetics approach and characterized ing1 and ing2 mutants. My analysis revealed that these mutants are altered in time of flowering, as well as their response to nutrient and stress conditions. Lastly, I was able to show that ING2 protein interacts in vitro with SnRK1.1, a nutrient/stress sensor (Baena-Gonzalez et al., 2007). These results indicate a novel function for PHD proteins in plant growth, development and stress response.
Master of Science