Thèse de doctorat : Biologie cellulaire et moléculaire des plantes : Strasbourg 1 : 2008. / Titre provenant de l'écran-titre. Bibliogr. 16 p.. Notes bibliogr.. Index.

Thesis (Ph. D.)--University of Queensland, 2001. / Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 161-172). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

NTPDases (Apyrases) (EC 3.6.1.5) require divalent cations (Mg2+, Ca2+) for hydrolysis of di- and triphosphate nucleotides, but do not hydrolyze monophosphate nucleotides. They are insensitive to inhibitors of F-type, P-type, and V-type ATPases and are categorized as E-type ATPases. They are grouped in the GDA_CD39 superfamily. Seven NTPDases (AtAPY1-7) have been cloned from Arabidopsis. In this work, AtAPY1 or AtAPY2 tagged with C-terminal green fluorescence protein (GFP) and driven by their respective native promoter displayed Golgi apparatus localization. These GFP constructs can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, which indicates their accuracy and functionality in localization studies. Furthermore, both AtAPY1 and AtAPY2 can complement the Saccharomyces cerevisiae Golgi-localized GDA1 mutant by rescuing its aberrant protein glycosylation phenotype. The GFP tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity in the mutants confirming that they both also have functional competency. Loss-of-function apy1, apy2 and APY1RNAi apy2 mutants showed higher levels of galactose in the cell wall monosaccharide analysis. However, the efficiency of the galactose transport was not altered APY1RNAi apy2 mutants. AtAPY3 through 7 all displayed intracellular localization by transiently expressed C-terminal tagged YFP in the onion epidermal cells. AtAPY3 showed a subcellular localization distinct from the others. Biochemical analyses showed that AtAPY3 prefers to hydrolyze NTP more than NDP. AtAPY4 resides in the cis-Golgi. It has fairly weak NTPDase activity but can still rescue some part of the phenotypic defects in Golgi luminal NTPDases mutants. AtAPY5 is a strong NDPase and has a broad spectrum of substrate preferences. It can fully restore phenotypic defects in Golgi luminal NTPDases in yeast. AtAPY6 and AtAPY7 are ER and Golgi associated. However, the expression of these two enzymes cannot be detected in the Saccharomyces cerevisiae host, which prevents further analysis. Taken together these results reveal that the current seven APYRASE members are intracellulary associated with Golgi/ER or unknown vesicles. They all display typical NTPDase enzyme activities that can hydrolyze di- or triphosphate nucleotides in the cells. / text

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

A sorghum gene SbLRR2, which is predicted to encode a simple extracellular leucine-rich repeat (LRR) protein, was previously isolated among a collection of fungal pathogen-induced sorghum cDNA clones generated by suppression subtractive hybridization. Phylogenetic analysis revealed that they are distinct from the simple extracellular LRR proteins reported previously. Subcellular localization analysis demonstrated that the SbLRR2-EYFP fusion protein was targeted to the extracellular space in tobacco leaf cells. Peptide N-Glycosidase F treatment revealed that the SbLRR2 is N-glycosylated with non-fucosylated oligosaccharides when transiently expressed in Nicotiana benthamiana leaves. Functional analysis was performed in SbLRR2 over-expression (OE) Arabidopsis plants which showed enhanced resistance against the necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola. In addition, the OE lines were found to have elevated expression of several jasmonate acid (JA)-associated genes and higher endogenous JA contents. Hence, the SbLRR2-mediated defense responses in transgenic Arabidopsis are likely to be dependent on JA-signaling through increased JA production. On the other hand, the OE lines remained susceptible to Pseudomonas syringae pv. tomato as the wild type plants. Consistently, there was no up-regulation of salicylic acid (SA) defense marker gene expression or SA levels in the OE lines. Using yeast two-hybrid analysis, SbLRR2 was further shown to interact with Arabidopsis hypersensitive-induced response protein 1. Such interaction may suppress hypersensitive response which is known to enhance necrotrophic pathogen invasion. These data suggested a positive regulatory role of SbLRR2 in plant defense. Further phenotypic analysis of transgenic SbLRR2 revealed its novel role in enhancing lead [Pb(II)] tolerance in Arabidopsis. OE-lines were showed to alleviate Pb(II)-induced root inhibition, reduce the accumulation of Pb(II), and enhance transcription of AtPDR12 which was previously shown to function as a potential Pb(II) efflux pump contributing to Pb(II) detoxification. However, all the Pb(II) tolerance responses were abolished when SbLRR2 was transformed into the atpdr12 mutant. Meanwhile, the extracellular localization of SbLRR2 was shown to be essential for the enhanced Pb(II) tolerance in transgenic Arabidopsis. Together, these results indicated that SbLRR2-mediated Pb(II) tolerance was dependent on AtPDR12 via Pb(II) extrusion. Further investigations revealed the Pb(II)-induced transcriptional activation of SbLRR2 and several highly conserved AtPDR12 homologs in sorghum seedlings, suggesting the possibilities of a common molecular mechanism for Pb(II) tolerance in diverse plant species. Finally, an iTRAQ-based LC-MS/MS quantitative proteomics approach was used to investigate of lead responses in Arabidopsis. A total of 114 proteins showed significant changes in protein abundance with 58 up-regulated and 56 down-regulated proteins. Analysis of changes in the protein profile revealed that the photosynthesis, photorespiration and protein biosynthesis in Arabidopsis were inhibited under lead toxicity. On the other hand, abundances of proteins involved in the antioxidant system, glucosinolate-myrosinase system and JA biosynthesis pathway were elevated upon Pb(II) treatment. Further investigation revealed that Pb(II) stress induced a rapid increase of JA contents in Arabidopsis whereas a JA biosynthesis deficient mutant (AOS) showed hypersensitivity to Pb(II) toxicity, strongly implicating a significant role of JA in Pb(II) response. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Not available / text

A network of three classes of proteins consisting of bHLH and MYB transcription factors and a WD40 repeat protein - TRANSPARENT TESTA GLABRA1 (TTG1) act in concert to activate trichome initiation and patterning in Arabidopsis. These proteins also regulate the flavonoid-based pigment biosynthetic pathway in almost all higher plants including Arabidopsis. Using TTG1-YFP translational fusions, I show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using conditional transgenic alleles I demonstrate that TTG1 directly regulates the same genes as GL3. In vivo binding of GL3, GL1 and TTG1 to the promoters of GL2, TTG2, CPC and ETC1 establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, I confirm that TTG1 interacts with the GL3 (bHLH) and GL1 (Myb) proteins in vivo, forming a complex. The loss of members of the TTG1 complex through mutation, affects the subcellular distribution of other complex members. Using particle bombardment, I show that TTG1, GL3, GL1 and GL2 do not move between adjacent epidermal cells while CPC does move to neighboring cells. These data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf. In addition, I also show that GL3 is recruited to its own promoter in a GL1-independent manner, which results in decreased GL3 expression, suggesting the presence of a GL3 negative auto-regulatory loop. Expression studies using GL3-GR (GL3-glucocorticoid receptor) and TTG1-GR fusions reveal direct regulation of the late anthocyanin biosynthetic genes, but not of early biosynthetic genes. Taken together, our results provide insights on the molecular mechanisms by which the combinatorial TTG1-bHLH-MYB regulatory complexes activate and repress both developmental and biosynthetic pathways in Arabidopsis.

ATP can serve as a signal molecule in the extracellular space to regulate biological processes and physiological effects in plant and animal cells. In mammalian cells, the level of extracellular ATP (eATP) is regulated by ectoapyrases, which can hydrolyze extracellular ATP to ADP and ADP to AMP. In this dissertation, I describe the important role of two Arabidopsis apyrases in the regulation of plant growth. Seven apyrases have been identified in Arabidopsis thaliana. The genes for two of these seven apyrases, APY1 and APY2, which have high sequence similarity, were cloned and characterized previously. The function of APY1 and APY2 was analyzed by T-DNA insertional mutant lines. The double knockout (DKO) apyrase pollen displayed a complete block of pollen germination, which implicated this step as the cause of the lethality of apyrase double knockout mutants. The vast majority of the mutant pollen grains were identical to wild-type in their nuclear state, and were viable as assayed by metabolic activity and plasma membrane integrity. Pollen tube elongation was inhibited by suppression of apyrase activity using anti-apyrase antibodies or by chemical inhibitors of apyrases. Etiolated hypocotyls overexpressing APY1 (with expression driven by a constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter) exhibited faster growth rates compared to wild-type plants. Because of the lethality of apy1apy2 double mutants, RNA interference (RNAi) was performed as an alternative approach to posttranscriptionally silence the expression of apyrases. The suppression of apyrases in the RNAi lines resulted in a dwarf phenotype in overall vegetative growth and dramatically reduced growth in primary root and etiolated hypocotyls. In addition, the RNAi mutant plants lacked a well-defined meristematic zone and had a greatly reduced elongation zone in the primary root. Previously, promoter-GUS fusions showed that high expression of apyrase was associated with areas of rapid growth and regions with high auxin levels. Abnormal auxin accumulation was found in the proximal regions of the primary roots of RNAi mutant plants, which demonstrated that the absence of apyrase results in disrupted auxin distribution. Other phenotypes in RNAi mutant plants, such as less lateral root formation and more adventitious roots, could also be associated with abnormal auxin distribution. The investigation of the subcellular localization of apyrases showed that some fraction of apyrase was localized on cell periphery. These results suggest that the expression of APY1 and APY2 is essential for plant growth. They favor the hypothesis that Arabidopsis apyrases, like their homologs in animals, control the levels of ATP in the extracelluar space, and this control allows them to act as key regulators in growth.