Phytohormone auxin plays an important role in regulating plant development. Directional (polar) cell-to-cell auxin transport creates auxin gradients within plant tissues, which trigger a specific developmental response. The vast majority of available data concerns angiosperms. Lower land plants have been much less explored in this regard, but the important auxin-related mechanisms (including polar auxin transport) are already present in mosses. To uncover the origins of auxin action, one must focus on green algae, especially of clade Streptophyta, which are the direct ancestors of all land plants. In this study, the possible effects of auxins, both native and synthetic, were investigated on two algae: basal, unicellular Chlorella lobophora and advanced, filamentous Spirogyra sp. The latter received comparably more attention, since it belongs to a clade now acknowledged as a sister group to land plants. Chlorella lobophora culture growth was irresponsive to synthetic auxin NAA. The average Spirogyra sp. cell length was, however, changed by auxins at high concentrations. By conducting accumulation assays of radioactively labelled auxins and HPLC analysis, auxin metabolism and transport was investigated in Spirogyra sp. This alga was able to metabolize the plant-native IAA, but not synthetic auxins...

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Auxin is phytohormone that regulates several developmental processes and environmental responses. One of the most well-described outcome of the auxin signalling pathway is regulation of gene transcription. Aux/IAA proteins play an important role in this process, acting as transcriptional repressors. Recent studies revealed that several root growth responses are too rapid to be explained by changes in the level of transcription. The correlation between the amount of Aux/IAAs and the root growth rate suggests that these proteins might be involved in root growth regulation, especially during rapid growth responses that are not associated with transcriptional reprogramming. This work is focused on one of the 29 Arabidopsis Aux/IAA proteins - the IAA17/AXR3 protein. First, we produced stable transgenic lines of Arabidopsis thaliana expressing different combinations of fluorescently labelled AXR3-1 proteins and/or fused to subcellular localization tags under the control of different tissue-specific promoters, in order to characterize the subcellular localization of the studied protein. Subsequent visualization by confocal microscopy methods confirmed information about the role of IAA17/AXR3 protein in root growth responses, its involvement in auxin signalling, and gravitropism. Next, we showed that the...

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Il mais è uno dei cereali maggiormente diffusi perché utilizzato in ambito alimentare umano e animale, per la produzione di materiale biodegradabile e di bioetanolo. I processi fisiologici che coordinano la crescita della cariosside vengono regolati principalmente dall’auxina che agisce a livello trascrizionale e post-traduzionale. Attraverso analisi comparative tra il mutante de18 (defective endosperm 18), deficitario nella produzione di acido indolo-3-acetico (IAA), e del suo corrispettivo wild-type, è stato possibile individuare i geni coinvolti nella determinazione delle dimensioni della cariosside. In particolare, sono state effettuate analisi morfologiche e di quantificazione dell’amido su cariossidi de18 e wild type negli stadi precoci di sviluppo. E’ stato inoltre allestito un esperimento di RNA sequencing sull’endosperma dei due genotipi a 8 e 12 DAP (Days After Pollination). L’analisi dei geni differenzialmente espressi attraverso la classificazione GO (Gene Ontology) ha permesso di studiare l’effetto della carenza di auxina sull’espressione genica. Nel mutante si riscontra l’attivazione tardiva della sintesi dell’amido e l’incremento delle proteine di riserva. Inoltre, la carenza di auxina determina una riduzione dell’attività mitotica ed endoreduplicativa, confermata dalla repressione dell’attività di geni legati al ciclo cellulare. / Maize is one of the world’s leading cereal grains due to its diverse functionality as a food source for both humans and animals, as well as a source of raw materials and biofuel. The physiological processes responsible for the growth of the kernel are regulated mainly by auxin acting at the transcriptional and post-translational level. Through comparative analysis between mutant de18 (defective endosperm 18), defective in indol-3-acetic acid (IAA) production, and its wild-type B37, it has been possible to identify the genes involved in the determination of the final seed size. Morphological analysis and quantification of starch were done on seed mutant and wild-type in the early stages of their development. Finally, an RNA sequencing analysis was carried out on mutant and wild-type endosperm at 8 and 12 DAP (Days After Pollination) and differentially expressed genes were classified by Gene Ontology. Down-regulation of genes related to sugar metabolism suggested a delayed activation of starch biosynthesis. This finding was confirmed by the determination of starch content that was lower in the mutant endosperms respect to the normal in the early stages of gran filling (12 and 16 DAP). The reduced auxin level affected the mitotic and endoreduplication activities as suggested by the repression of genes involved in the cell cycle.

Evolution of developmental genes is an important mechanism for plant morphological evolution. The LRL genes are an ancient group of bHLH transcription factors that positively regulate root hair development in angiosperms. Here I show that, in the moss Physcomitrella patens, two LRL genes are present and they positively regulate rhizoid and caulonema (a rhizoid-like cell type) development. GUS-transcriptional reporter plants show that both PpLRL1 and PpLRL2 are expressed in tissues giving rise to caulonemata. Loss-of-function mutants in either PpLRL1 or PpLRL2 led to defective rhizoid and caulonema development, and the Pplrl1 Pplrl2 double loss-of-function mutants completely lack rhizoids and caulonemata. Consistent with this, gain-of-function mutants show enhanced rhizoid and caulonema development. In addition, I show that the stimulatory effects of auxin and low phosphate on the development of rhizoids and/or caulonemata required PpLRL gene function. Together, these results show that LRL genes are conserved, positive regulators in tip-growing rooting cell development in land plants. To elucidate whether LRL genes belong to part of a conserved gene network, I use qRT-PCR to determine the transcriptional interaction between LRL genes and the Class I RSL genes, which is another group of conserved regulators of rhizoids and root hairs. Comparing the LRL-RSL network between P. patens and A. thaliana reveals that LRL and Class I RSL genes are transcriptionally independent of each other in P. patens but one LRL gene is transcriptionally downstream of Class I RSL genes in A. thaliana. This suggests that the gene network controlling tip-growing rooting cell development has changed since mosses and angiosperms last shared a common ancestor.

Ribosomes, large enzymatic complexes containing an RNA catalytic core, drive protein synthesis in all living organisms. 80S cytoplasmic eukaryotic ribosomes are comprised of four rRNAs and approximately 80 ribosomal proteins (r-proteins). R-proteins are encoded by gene families with large families (average of twelve members) predominating in mammals and smaller families (two to seven members) in plants. Increased ribosome heterogeneity is possible in plant ribosomes due to multiple transcriptionally active members in each family, whereas, in mammalian r-protein gene families, only one member is typically active. Multiple functional paralogs provide for greater plasticity in response to environmental/developmental cues, as well as, increasing the possibility of individual paralogs procuring or retaining extraribosomal functions. This research investigated the effects of r-protein mutations on plant growth and development. Through RNA interference (RNAi) mediated knockdown (KD) of type I (cytoplasmic: RPS15aA/D and F) and type II (non-cytosolic: RPS15aB and E) RPS15a family members I was able to confirm the delineation between the two types. Subcellular localization of the type I isoforms was nuclear/nucleolar while localization of type II isoforms was non-mitochondrial and probably cytosolic. Illumina sequencing of two r-protein mutant transcriptomes, pfl1 (rps18a) and pfl2 (rps13a), identified a novel set of up and down regulated genes, previously unknown or linked to r-protein mutants. The 20 genes identified were classified into four groups (1) plant defense, (2) transposable elements, (3) nitrogen metabolism and (4) genes with unknown function. Illumina miRNOME analysis revealed no changes in the miRNA profile of pfl1 and pfl2 plants. These data do not support the previously proposed theory that a disruption in ribosome biogenesis (by decreased r-protein synthesis) disrupts miRNA-mediated degradation of a range of auxin response genes. Finally, a novel double r-protein mutant, rps18a:HF/RPL18B, presented a late flowering/thickened bolt phenotype not seen in a rps13a:HF/RPL18B mutant, suggesting that RPS18A has an extraribosomal role in plant growth and development in Arabidopsis.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Jason J. Griffin / Heat and drought tolerance make shantung maple (Acer truncatum) and caddo sugar maple (A. saccharum) good candidates for midwestern landscapes. Improving cutting propagation or mound layering techniques could increase the availability of these species. The influence of time of year, cutting position, and auxin concentration, formulation, and solvent on rooting of stem cuttings of shantung maple was investigated. Semi-hardwood cuttings rooted best (55%). Generally, rooting percentage decreased as indole-3-butyric acid (IBA) concentration increased. Cutting position, auxin formulation, and solvent did not affect rooting. Mean root number and mean root length were unaffected by treatments. Results suggest semi-hardwood cuttings and low IBA concentrations [< 2500 ppm (0.25%)] promote rooting. Auxin concentration influenced rooting of caddo and shantung maple mound layered shoots. Rooting peaked at 15,000 ppm (1.5%) IBA for both caddo (71%) and shantung maples (34%). Mean root number for caddo, but not shantung, increased as IBA concentration increased. Differences in mean root length were not significant. Growers may now propagate caddo maple by mound layering. For shantung maple propagation, stem cuttings are recommended. Propagation substrates can strongly influence rooting success of stem cuttings. Eastern redcedar (Juniperus virginiana) chips (ERC) have been suggested as a propagation substrate component. This report investigated ERC as a perlite substitute in a 3 perlite: 1 sphagnum peat moss (v/v) rooting substrate. Stem cuttings of spreading euonymus (Euonymus kiautschovicus), forsythia (Forsythia x intermedia), English ivy (Hedera helix), lantana (Lantana camara), and coleus (Solenostemon scutellarioides) were rooted in substrates containing increasing concentrations of ERC hammer milled to pass a 4.8 mm (0.19 in) screen. All species rooted well (≥95%) in all substrates except forsythia which rooted poorly in all substrates (8% to 36%). ERC did not affect mean root number or mean root length in any species except spreading euonymus where mean root number peaked at 0% and 100% ERC content and mean root length decreased with increasing ERC content. Bulk density, container capacity, and total porosity increased as ERC replaced perlite. Physical properties of all substrates were suitable for cutting propagation. ERC can effectively replace perlite in rooting substrates for many ornamental species.