2 Abstract ARP2/3 protein complex is formed from seven proteins (ARP2, ARP3 and ARC1- ARPC5) with a relatively conserved structure. ARP2/3 complex branches and nucleates new actin filaments. This thesis focuses on the study of the role and importance of the individual subunits of the complex ARP2/3 in plants. One of the principal aims of this work is to determine whether complex ARP2/3 may at least partially maintain its role when one or more of the subunits are not available. Furthermore if the individual subunits play another, plant-specific role and if the subunits are functionally equivalent in the complex. The main way how to achieve this objective is the analysis of multiple mutants of Arabidopsis thaliana in subunits of ARP2/3 complex. After comparing several phenotypes of mutant lines it is obvious that all the subunits are functionally equivalent. A loss of ARPC5 subunit usually manifests the strongest phenotypic expression. On the contrary, loss ARPC3 and ARPC2b subunits have weak phenotypic manifestations. Because some phenotypes, such as phenotype distorted trichomes was detected only in some mutant lines, whereas the phenotype of faster roots gravitropic response or vacuolar system fragmentation that was detected in all analyzed mutants suggests, that different subunits play varying roles...

Evolutionary-developmental study of membrane proteins Mgr. Stanislav Vosolsobě Abstract Using a plethora of experimental approaches for phylogenetical and functional study on several membrane signalling proteins, I brought new evidences supporting a hypothesis that the molecular evolution of protein families is a highly dynamic, not conservative, process. In DREPP family of calcium-binding peripherally-associated plasma-membrane proteins I found a broad flexibility in protein-membrane binding manners coupled with a many independent duplication of this Euphyllophyta-clade specific plant gene. In three families of auxin transporting proteins, PIN-FORMED, LAX and PILS, I showed that emergences of these proteins are uncorrelated and placed on different levels of the plant kingdom phylogenetic tree. However these proteins ensure very fundamental plant morphogenetic processes, like cell differentiation, organ formation or tropisms, with strong effects of their deleterious mutations, I found many gene radiations and losses on a all taxonomic levels in these families, evidencing that key and shared physiological processes may be realised by genes touched by a recently undergoing evolution. Evolutionary-developmental synthesis of a functional and phylogenetic data must be done with caution due to high risk of...

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<p dir="ltr">Exocytosis is crucial for delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces, playing a vital role in normal plant development as well as responses to biotic and abiotic stresses. One key molecular player, the exocyst, is an octameric protein complex that tethers secretory vesicles to the plasma membrane (PM). Chapter 1 is a literature survey that introduces the function of the exocyst, as well as the characterization of Endosidin2 (ES2), a synthetic molecule that targets the EXO70 subunit of exocyst. This chapter also defines existing knowledge gaps in the profiling of cargo proteins trafficked by the exocyst and the identification of novel modulators of exocytosis. Chapter 2 employs a comparative proteomics approach to examine the changes of PM proteome of root cells following ES2-treatment. Proteins with decreased abundance at the PM were considered candidate cargo proteins of ES2-targeted trafficking and several were validated with quantitative live-cell imaging. Chapter 3 describes the use of ES2 as a tunable and reversible chemical genetics tool as demonstrated by the development and deployment of a large-scale mutant screen in Arabidopsis that identified 70 <u>ES2</u>-hyper<u>s</u>ensitive mutants (<i>es2s</i>). Among these, candidate mutations for 14 non-allelic lines were mapped and reported. T-DNA insertion lines were subsequently screened as alternative alleles to identify causal mutations. In Chapter 4, the causal mutation of <i>es2s-15-12</i> was confirmed as <i>ArgJ</i> with a second T-DNA insertion mutant allele as well as genetic and chemical complementation. <i>ArgJ</i> encodes an enzyme in the arginine biosynthesis pathway. It was demonstrated that arginine biosynthesis deficiency synergizes with ES2 to inhibit root growth in Arabidopsis. Root growth in <i>argj</i> mutants was not hypersensitive to other inhibitors with different modes of action, such as LatB, ES9-17, and BFA. Additionally, roots of <i>argj-1</i> displayed a reduced abundance of PIN2 at the apical PM in epidermal cells; however, PIN2 polar distribution was not further reduced by ES2 treatment. Our findings point to a functional connection between arginine metabolism and exocytosis. Chapter 5 discusses potential future directions and experiments, including technological advances and the testing of new hypotheses. Overall, this study presents a detailed application of chemical genetics to dissect the exocytosis process in Arabidopsis and uncovers novel modulators of exocytosis in plants.</p>

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Department of Biotechnology and Food Technology, Faculty of Applied Sciences, Durban University of Technology, South Africa,2009. / Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide. / CSIR

We have studied the effects of exogenous salt on whole plant and suspension culture cells of the halophytic tomato Lycopersicon pennellii. Under low salt conditions (2.9 dS/M) plants showed enhanced (halophytic) growth (107% of control). At moderate (7.5 dS/M) and high (18.5 dS/M) salt levels, salt stress reduced growth to about 78% and 40% of control respectively. Salt-induced changes in root mRNAs were analyzed via two-dimensional PAGE of cell free translation (CFT) products. We have identified 14 proteins whose levels were enhanced by exogenous salt. One of these proteins was unique to low salt induced halophytic growth. This system allowed for discrimination between proteins up-regulated at all salt levels and those up-regulated only during salt stress induced growth reduction. Ten proteins were identified whose levels were reduced by exogenous salt. Once again, one could identify a subset of proteins whose levels were reduced only under salt stressed conditions. Proteins identified in this study are candidates for roles in growth maintaining stress adaptive metabolism in L.pennellii. These data underscore the complexity of the genetic control of salt metabolism in higher plants. The effects of exogenous salt on protein synthesis and accumulation were studied in suspension cultures of L.pennellii. Two salt levels were applied to the cells. Under low salt conditions (LS, 10 mM), L.pennellii cells showed enhanced (halophytic) growth. Under high salt conditions (HS, 50 mM), the cells showed reduced (salt-stressed) growth. Changes in proteins with time were analyzed by a combination of cell free translation, in vivo labeling and total accumulated protein. In vivo labeling studies showed that the pattern of steady state protein synthesis was disrupted shortly after addition of salt. High salt induced greater disruption in the pattern. Over time, the steady state levels of most proteins shifted back towards those of the unstressed-control. However, the level of several proteins remained altered. Analysis of proteins whose levels increased with exogenous salt showed differences in the response patterns that may allow for discrimination between proteins involved in growth maintaining and stress shock responses.

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In order to understand and manipulate carbon flux to sucrose one needs to consider not only its biosynthetic pathways, but also the competing sinks for carbon in various parts of the plant and at different stages of development. The cell wall and sucrose is known to be the major sinks for carbon in young and mature tissues of sugarcane. UDP-Glucose is a central metabolite in the synthesis of both sucrose and most of the cell wall polysaccharides (including cellulose, hemicellulose and pectic polymers) and manipulation of the flux into either of the cell wall components could therefore cause an increase of flux toward one or more of the competing sinks. In the present study UDP-Glucose dehydrogenase (UGD) activity was chosen for down regulation as it catalyzes the rate limiting step in the biosynthesis of the precursors of both hemicellulose and pectin, a major competing sink for assimilated carbon. Transgenic sugarcane lines with repressed UGD activity showed significantly increased sucrose accumulation in all internodes which was highly correlated with reduced UGD activity. Sucrose phosphate synthase had increased activation which suggests an alteration in carbon flux toward sucrose. The reduction of carbon flux through UGD was compensated for by an increase in the activity of the myo-inositol oxygenation pathway (MIOP), an alternative pathway for the synthesis of cell wall matrix precursors. The increased activity of the MIOP resulted in increased total uronic acids and pentoses in the cell wall. Total cell wall glucose was also increased which is a further indication of altered carbon metabolism.

Thesis (PhD(Agric) (Horticulture))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Mealiness, a soft, dry textural disorder of ‘Forelle’ pear (Pyrus communis L.), is a problem for the South African fruit export industry. Soft, dry textural disorders seem to be related to changes in cell wall breakdown. The aim of this work was, therefore, to investigate the occurrence of mealiness‐associated changes in the cell wall and elucidate the mechanism by which mealiness occurs in ‘Forelle’ pear, as well as to characterise cell wall changes occurring during normal ripening. Mealy ‘Forelle’ tissues had significantly lower total galacturonic acids associated with the middle lamella (water‐ and CDTA‐soluble fractions). The water‐soluble pectin of mealy tissues was depolymerised at an earlier stage of ripening. The widespread disintegration of cell‐to‐cell adhesion in mealy cell walls only, suggests that the middle lamella and the plasmodesmata are more broken down. In mealy ‘Forelle’ tissues there was no indication of less broken down high molecular weight polyuronides in the CDTA fraction, normally associated with these dry, soft textures. The pectins from mealy tissues were more broken down and both mealy and non‐mealy tissue polyuronides depolymerised. Furthermore, there was a lack of light toluidine staining in the larger air spaces, which would indicate such water‐insoluble pectins. These data suggest that the formation of high molecular weight pectate gels is unlikely in mealy ‘Forelle’ pear. The slight increase in the galactose content in mealy tissues in CDTA‐ and Na2CO3‐soluble fractions and slight decrease in the 1 M KOH glycan fraction during later stages of ripening (6+11, 9+7, 9+11; weeks at ‐0.5°C plus days at 15°C) may indicate that galactose loosely interlinked into the glycan fraction broke down sooner for mealy tissues. This didn’t increase molecular size profiles in the CDTA fraction. Arabinose content was slightly higher in the 4 M KOH fraction and slightly lower in mealy tissues of water‐ and CDTA fractions. This did not influence the molecular weight of the glycans compared to those in the nonmealy tissues. ‘Forelle’ data therefore seem to be more congruent with a decrease in intercellular adhesion as the mechanism by which mealiness occurs, rather than the formation of high molecular weight pectins taking up the cellular fluid. ‘Forelle’ pear water‐soluble pectin content increases with increased ripening. High amounts of watersoluble pectin and low amounts of Na2CO3‐soluble pectin suggests that solubilisation of rhamnogalacturonan‐I pectins must have taken place during early ripening (at a fruit firmness of > 4.7 kg (7.9mm tip). Galactose and glucose in the pectin fraction dramatically decreased after fruit ripened to a firmness of 4.5 kg, whereafter they remained unchanged. This was also the period in which fruit softened the most and the biggest increase in pectin water‐solubility occurred. It is not known whether these events are coincidental, or linked causally. Rhamnose and arabinose extractability increased in the water fraction and xylose, fucose and mannose increased in glycan fractions with ripening. The biggest changes in polyuronide solubilisation and depolymerisation occurred in water‐ and CDTA fractions between storage and ripening durations of 3+7 (4.7 kg) and 6+4 (2.7 kg). / AFRIKAANSE OPSOMMING: Melerigheid, ʼn sagte droë tekstuur afwyking van ‘Forelle’ pere (Pyrus communis L.), is ʼn probleem vir die Suid Afrikaanse vrugte uitvoerbedryf. Sagte, droë tekstuur afwykings blyk betrekking te hê op selwandafbraak veranderinge. Die doel van die studie was dus om die melerigheid‐geassosieerde veranderinge in die selwand te ondersoek, sowel as om vas te stel wat die meganisme betrokke is by melerigheid ontwikkeling in ‘Forelle’ pere. Die selwand veranderinge gedurende normale rypwording is ook gekarakteriseer. Melerige ‘Forelle’ weefsel het betekenisvol laer totale galakturoonsuur wat geassosieer is met die middellamella (water‐ en CDTA‐oplosbare fraksies). Die water‐oplosbare pektien van melerige weefsel was op ʼn vroeër stadium van rypwording gedepolimeriseer. Die wydverspreide disintegrasie van sel‐tot‐sel adhesie, slegs in melerige selwande, dui aan dat die middellamella en die plasmodesmata meer afgebreek is. Daar is geen indikasie van hoë molekulêre massa poliuroniedes in die CDTA fraksie van melerige ‘Forelle’ weefsel, wat gewoonlik geassosieer word met droë, sagte teksture nie. Die pektiene van melerige weefsel was meer afgebreek en melerige en nie‐melerige weefsel se poliurone was gedepolimeriseer. Daar was ook geen ligte toluïdien verkleuring in die groter intersellulêre lugruimtes nie, wat ʼn aanduiding sou wees van wateronoplosbare pektiene. Hierdie data dui dus aan dat die vorming van hoë molekulêre pektien jel in melerige ‘Forelle’ pere onwaarskynlik is. Die klein toename in galaktose inhoud in die CDTA‐ en Na2CO3‐ oplosbare fraksies en ʼn klein afname in 1 M KOH glikaan fraksie tydens latere rypheidstadiums (6+11, 9+7, 9+11; weke by ‐0.5°C plus dae by 15°C), kan beteken dat los verweefde galaktose in die glikaan fraksie vroeër afgebreek het in melerige weefsels. Die molekulêre grootte profiel is nie verander in die CDTA fraksie nie. Arabinose inhoud was bietjie hoër in die 4 M KOH fraksie en bietjie laer in melerige weefsel van die water‐ en CDTA fraksies. Die molekulêre massa van die glikane was klaarblyklik onbeïnvloed hierdeur. ‘Forelle’ data blyk dus meer saam te stem met die meganisme waar ʼn vermindering in intersellulêre adhesie ʼn rol speel in melerigheid, eerder as die meganisme waar hoë molekulêre pektien selvloeistowwe bind. ‘Forelle’ peer water‐oplosbare pektieninhoud neem toe met toenemende rypheid. Hoë vlakke wateroplosbare pektien en lae vlakke Na2CO3‐oplosbare pektien stel voor dat die oplossing van rhamnogalakturonan‐I pektiene gedurende vroeë rypwording moes plaasgevind het (by ʼn fermheid van > 4.7 kg (7.9mm punt). Galaktose en glukose in die pektienfraksie het drasties verminder nadat vrugte tot ʼn fermheid van 4.5 kg ryp geword het, waarna hul onveranderd gebly het. Dit was ook die periode waarin vrugte die meeste sag geword het en die grootste toename in poliuronied wateroplosbaarheid gevind is. Dit is nie bekend of die gebeure toevallig of oorsaaklik verbind is nie. Rhamnose en arabinose ekstraheerbaarheid het vermeerder in die water fraksies, en xylose, fukose en mannose het vermeerder in die glikaan fraksies gedurende rypwording. Die grootste verandering in oplosbaarheid en depolimerisasie het plaasgevind in die water‐ en CDTA fraksies tussen opberging en rypwordingsperiodes van 3+7 (4.7 kg) en 6+4 (2.7 kg).

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