Growth and development of plant body is dependent on correct and effective integration of information about current deployment of its body parts, as well as on perception and transduction of inputs from environment. Multiple developmental processes within plant body are determined by specific and tightly controlled distribution of molecule with unique signaling mission within plant development - auxin. Spatial distribution of auxin is co-determined by plethora of tightly controlled processes, and the polar auxin transport plays unique role among them. PIN proteins are the plant-specific family of secondary transporters driving movement of auxin across membranes. With their frequent asymmetrical localization within cells, specific expression patterns in developing tissues and their reactiveness to external cues they secure unique, dynamic and asymmetric distribution of auxin within the plant body. This dissertation thesis is focused on characterization of the role different PIN proteins play in determining cellular auxin homeostasis and consequent formation of auxin gradients. Controlled overexpression of PIN proteins in tobacco cells showed, that PIN4 and to some extent also PIN6, function as the direct auxin efflux carriers. In the cellular auxin transport they play the role analogous to other...

The evolution of auxin homeostasis mechanisms Ph.D. thesis Roman Skokan, 2021 Abstract The streptophyte lineage consists of land plants (embryophytes) and several groups of primarily freshwater green algae called charophytes. While the phytohormone auxin is a conserved regulator of land plant development, little has been known of the possible origins of auxin response mechanisms in charophytes. We found that one of these mechanisms, the cellular auxin efflux via the PIN family of transport proteins, is most likely a deeply conserved feature in streptophytes. Additionally, we investigated the state of conservation in the green lineage (Viridiplantae) of the gene families known to be involved in auxin transport in land plants. We revealed that some families are deeply conserved outside land plants, but not others. We also helped uncover a unique radiation within the PIN family in Charophyceae. Striving to uncover the native significance of auxin transport in charophytes, we discovered a growth response to exogenously-applied auxin in Closterium, though the effort to produce stable mutant lines in the native PIN homolog is still underway. Altogether, we brought important insights into the evolution of auxin transport and response in the streptophyte lineage, though many questions still remain.

This dissertation comprises a series of studies designed to explore the associations between plants and the endophytic fungi they harbor in their above-ground tissues. By viewing endophyte diversity in ecologically and economically important hosts through the lenses of phylogenetic biology, microbiology, and biotechnology, this body of work links plant ecology with newly discovered symbiotic units comprised of endophytic fungi and the bacteria that inhabit them.This work begins with a large-scale survey of endophytic fungi from native and non-native Cupressaceae in Arizona and North Carolina. After isolating over 400 strains of endophytes, I inferred the evolutionary relationships among these fungi using both Bayesian and parsimony analyses. In addition to showing that native and introduced plants contained different endophytes, I found that the endophytes themselves harbor additional microbial symbionts, recovering members of the beta- and gamma-proteobacterial orders Burkholderiales, Xanthomonadales, and Enterobacteriales and numerous novel, previously uncultured bacteria. This work finds that phylogenetically diverse bacterial endosymbionts occur within living hyphae of multiple major lineages of ascomycetous endophytes.A focus on 29 fungal/bacterial associations revealed that bacterial and fungal phylogenies are incongruent with each other and did not reflect the phylogenetic relationships of host plants. Instead, both endophyte and bacterial assemblages were strongly structured by geography, consistent with local horizontal transmission. Endophytes could be cured of their bacterial endosymbionts using antibiotics, providing a tractable experimental system for comparisons of growth and metabolite production under varying conditions. Studies of seven focal fungal/bacterial pairs showed that bacteria could significantly alter growth of fungi at different nutrient and temperature levels in vitro, and that different members of the same bacterial lineages interact with different fungi in different ways.Focusing on one isolate, I then describe for the first time the production of indole-3-acetic acid (IAA) by a non-pathogenic, foliar endophytic fungus (Pestalotiopsis neglecta), suggesting a potential benefit to the host plant harboring this fungus. I show that this fungus is inhabited by an endohyphal bacterium (Luteibacter sp.) and demonstrate that mycelium containing this bacterium produces significantly more IAA in vitro than the fungus alone. I predict that the general biochemical pathway used by the fungal-endohyphal complex is L-tryptophan-dependent and measure effects of IAA production in vivo, focusing on root and shoot growth in tomato seedlings.

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Plant growth, development, and environmental responsiveness are dependent on hormone-induced gene expression. This dissertation reports multiple interactions between the plant hormones auxin and ethylene and investigates their contribution to the gravitropic response, elongation growth, adventitious root formation, callus and tracheary element initiation and growth, and flower development. Four mutants of tomato (Lycopersicon esculentum, Mill.) altered in either hormone production or hormone response were used to test the involvement of ethylene and auxin. These mutants included diageotropica (dgt) which is auxin-resistant, Never-ripe (Nr), which is ethylene-resistant, epinastic (epi), which overproduces ethylene and lazy-2 (lz-2), which exhibits a phytochrome-dependent reversed-gravitropic response. Additionally, a double mutant between Nr and dgt was constructed and tested. Gravitropism was studied as an exemplary process involving both auxin and ethylene. Mutant analysis demonstrated that ethylene does not play a primary role in the gravitropic response via the currently known ethylene response pathways. However, ethylene can modify the gravitropic response, e.g. the delayed gravitropic response of the dgt mutant can be restored with exceedingly low concentrations of ethylene and ethylene synthesis- and ethylene-action inhibitors can partially inhibit the graviresponse. The role of gravity in tracheary element (TE) production was tested in microgravity (during a space shuttle flight) and in hypergravity (centrifugation). A correlation was found between gravitational force and the production of TEs, with decreased numbers of TEs produced in microgravity and increased numbers produced in response to hypergravity. Increased production of TEs by dgt in both increased and reduced gravity indicates that gravity regulates vascular development via a DGT-dependent pathway involving auxin. Combination of both the Nr and dgt mutations in a double mutant leads to plants which exhibit the reduction of auxin-sensitivity typical of dgt as well as a delay in fruit ripening typical of Nr. The reduced gravitropic response of the dgt mutant was restored to wild-type levels in the double mutant confirming a complex role for ethylene in the gravitropic response. Abnormal floral organ development was observed in a subset of double mutant flowers.These data demonstrate multiple connections between auxin and ethylene during development and provide further insight into their cellular interactions. / Graduation date: 2001

Les annexines sont définies comme étant des protéines qui se lient de manière calcium-dépendante aux phospholipides membranaires chargés négativement. Elles ont été associées à différents processus biologiques tels les réponses des plantes aux stress biotiques et abiotiques. Nous avons identifié une annexine végétale, appelée Ntann12, dont l’expression est induite après infection des plantes par la bactérie Rhodococcus fascians. Ntann12 possède les domaines caractéristiques des annexines et se lie aux phospholipides chargés négativement, de manière calcium-dépendante. L’expression de Ntann12 est très abondante dans les cellules différentiées des racines, où la protéine a été détectée par immunolocalisation dans le cytosol et dans le noyau. Des analyses par western blot ont montré que l’accroissement relatif de la quantité de protéines liées aux membranes est positivement corrélé à l’augmentation de la concentration en Ca2+. Au niveau physiologique, l'expression de Ntann12 est induite par l’apport exogène d’auxine. Elle est contrôlée dans les racines par un signal induit par la lumière, et provenant des parties aériennes. Le transport polaire de l'auxine a été identifié comme étant le processus cellulaires nécessaires à l'expression de Ntann12 dans les racines. En outre, cette expression est réprimée par les stress salin, osmotique et hydrique. Ces résultats suggèrent que l’annexine Ntann12 est impliquée dans le métabolisme de l’auxine. / Annexins are defined as calcium-binding proteins, and they have been associated in plants with different biological processes such as responses to biotic and abiotic stress. Ntann12 expression is induced upon infection of tobacco plant by R. fascians. Ntann12 possesses the conserved annexin repeat with the sequence for type II Ca2+-binding site and recombinant as well as native Ntann12 binds to negatively charged phospholipids in a Ca2+-dependent manner. It is mainly expressed in root differentiated cells where the protein was immunolocalized in the cytosol and in the nucleus. Ntann12 was examined by western blot in both microsomal and cytosolic fractions from tobacco roots cells, and was detected in both the cytosol and microsome. The relative increase of Ntann12 proteins associated with the microsome is coupled with an increase in Ca2+ concentration. At the physiological level, Ntann12 expression is induced by exogenous application of auxin, and was found to be regulated in the root system by a light-induced signal coming from plant aerial part and polar auxin transport was identified to be the cellular process required for Ntann12 expression in root cells. Furthermore, Ntann12 expression is down-regulated by salt, osmotic and water stress. These results collectively suggest that the annexin Ntann12 is implicated in auxin metabolism.

Telomerase is the key enzyme synthesizing telomeric DNA in most eukaryotic organisms. In mammals, telomerase expression is abundant in the germline cells but is undetectable in most other differentiated organs. Intensive studies of telomerase have focused on human cancerous cells, where over 90% of all cancerous tissues examined have telomerase activity. In wild-type Arabidopsis, telomerase expression is abundant in reproductive organs and dedifferentiated tissues such as flowers, siliques and calli but barely detectable in vegetative tissues (both rosette and cauline leaves). In this study, a biochemical screen strategy was developed for isolation of telomerase activating mutants in Arabidopsis thaliana. Through screening of Arabidopsis activation-tagged lines by a PCR-based TRAP assay, two tac (for telomerase activator) mutants were isolated. RT-PCR analysis of AtTERT expression revealed that different mechanisms are involved in alternating telomerase activity in tac1 and tac2. We cloned and characterized the TAC1 gene. TAC1 encodes a single zinc finger protein and acts synergistically with auxin to induce telomerase expression without altering cell cycles. Telomere length was unperturbed in the mutant, but other phenotypes, such as altered root development and the ability of cells to grow in culture without exogenous auxin, indicated that TAC1 not only is part of the previously reported link between auxin and telomerase expression, but also potentiates other classic responses to this phytohormone. DNA microarrays were used to analyze the expression profile of the tac1 mutant and revealed that several drought-induced genes were up-regulated 3 to 10 fold in the tac1-1D mutant. RT-PCR analysis further confirmed this up-regulation for five of these genes. Investigation of root growth also indicated that tac1-1D roots were ~20% longer relative to wild-type. Further experiments demonstrated that over-expression of TAC1 does confer drought tolerance, but not salt tolerance. In addition, our preliminary result showed that treatment with a low concentration of IAA could induce drought tolerance in wild-type Arabidopsis. Although plants with constitutive expression of telomerase have no practical utility, the ability of TAC1 to confer drought tolerance could have significant agricultural applications.

The auxins, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and naphthaleneacetic acid (NAA), promotes the formation of adventitious root in hypocotyls of soybean (Glycine max). IBA significantly promotes the formation of adventitious roots more than IAA and NAA . The activity of anionic pI 3.7 peroxidase (POX) and cationic pI 8.5 POX were inhibited by exogenous auxins during the induction of adventitious root on day 2. Besides, the activity of pI 5.3 POX was enhanced by IBA during the initiation stage on day 4. The increase of the activity of pI 5.3 POX was accompanied by the increase of H2O2 levels. In the previous researches, it shows that the promoter of pI 8.5 POX gene contains both ARF/AuxRE and CATATGGMSAUR motifs that are responded to auxins. In this studies, the pI 5.3 POX gene, which responses to auxins a day or two later, contains only ARF/AuxRE motif. The regulation of pI 5.3 POX gene is probably initiation phase-dependent. The results suggest that anionic pI 5.3 POX produces significant amount of H2O2 through the binding of auxin to POX and mediate the auxin signaling pathway leading to plant growth .

A review of the literature indicated that the purine cytokinin 6-(benzylamino)purine (SA) may be converted to a wide range of metabolites. Although the functional significance of these metabolites remains obscure, cytokinin physiologists have essentially classed them as either active or inactive. Inactivation of cytokinins is considered to proceed via catabolic oxidation (side-chain cleavage), or N-conjugation with glucose or alanine moieties. The literature survey was hampered by the confusing array of synonyms which have been coined for cytokinin metabolites. Accordingly, a working system of (semi-systematic) abbreviations was devised which accommodated all groups and classes of purine cytokinins. Prior to commencing metabolic interactive studies, it was necessary to resolve the contentious issue associated with the successful extraction of cytokinin nucleotides. Five-week-old soybean callus was fed [8[-14]C]BA and subsequently extracted using four widely used cytokin in extraction techniques. Techniques compared were a modified Bieleski method, 80% ethanol with tissue homogenisation, 80% ethanol without homogenisation, and boiling ethanol. All four procedures produced similar results, showing that all metabolites of SA, including the nucleotide, were adequately extracted. It was concluded that the extraction of nucleotides with Bieleski solvents did not warrant the inconvenience. Auxins have been shown to interact with cytokinins in the regulation of many physiological processes, although little is known of the mechanisms of interaction which proceed at the metabolic level. Previous investigators have shown that auxin promoted cytokinin degradation through catabolic oxidation, Shoot-apex and seed derived cell suspensions of Dianthus zevheri subsp. natalensis were incubated with [8[-14] C]BA for between 30 minutes and 48 hours in the presence of both low (2 mg l-1) and high (4 mg 1¯¹) levels of exogenously supplied 2,4-dichlorophenoxyacetic acid (2,4-D), In both systems, the auxin 2,4-D was shown to promote SA inactivation through 7-glucosylation (N-conjugation). This observation represents the first report of auxin-promoted cytokinin N-conjugate formation. The auxin effect on metabolism was transient in the case of shoot-apex, but not in seed-derived systems over a 48 hour period. Formation of the 7-glucoside of SA was dose-dependent in apex-derived cultures. Further studies were undertaken with indole-3-acetic acid (lAA) and α-naphthaleneacetic acid (NAA). It was found that auxin-promoted 7-glucosylation of SA was only minimally effected by these two auxins. In comparable studies with soybean suspension cultures (Glycine max cv. Acme), 2,4-D-promoted catabolic oxidation was observed between 18 and 48 hours, following application of phytohormones. The main catabolite was tentatively identified as adenosine-5'-monophosphate (AMP), based on chromatographic characteristics. Carrot (Daucus carota) cell suspensions similarly supplied with 2,4-D and SA maintained a large active cytokinin pool. Neither substantial oxidative nor Nconjugative processes were observed. Instead, there was a transient effect by 2,4- D on the relative formation of the riboside and the 7- and 9-glucosides of SA. The effect of auxin on the metabolism of SA thus varied with the species and system investigated. Generally, auxin promoted (rather than inhibited), the formation of inactivated metabolites and catabolites of SA, possibly by the induction of relevant enzyme systems. Transient auxin effects on the metabolism of SA are discussed in relation to the role of the auxin/cytokinin balance in the induction of developmental processes. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.

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