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<p><strong>ABSTRACT</strong></p> <p>As plants are sessile organisms, detecting the presence of neighboring plants and exhibiting competitive behavior to acquire limiting resources is crucial. One of the ways plants respond to belowground competition is by allocation to fine roots in order to acquire the limited resources. However, this phenotypic plasticity can be costly as it assigns resources away from reproduction. Being able to recognize the relatedness of one′s neighbours and preferentially compete with strangers is a beneficial trait that can minimize the costs of competition with relatives and increases inclusive fitness. Many studies have looked at the association between resource availability and competition in plants while others have observed kin recognition in several plants species. However, no one has yet studied the effect of resource availability on kin recognition in plants. Here, I looked at root architecture to test if there is an association between kin recognition and nutrient availability in <em>Cakile edentula</em>.</p> <p>I found that the root system architecture is highly plastic and complex, showing variable responses to neighbour identity signals and resource availability. The results from the four experiments demonstrate that the responses of <em>C. edentula </em>to neighbour relatedness are dependent on nutrient availability. Additionally, this study also indicates that kin recognition in <em>C. edentula</em> does not require root contact; instead it occurs through a signal found in soluble compounds excreted from plants, possibly root exudates, as observed in <em>Arabidopsis thaliana</em> <em>(</em>Biedrzycki et al. 2010).</p> <p>In conclusion, this study provides novel findings regarding the dynamics of root behavior in response to nutrient availability and the relatedness of neighbours.</p> / Master of Science (MSc)

Circadian clocks are biochemical mechanisms that allow eukaryotic and some prokaryotic organisms to coordinate their physiology with daily environmental changes. It enables organisms to increase their fitness by taking advantage of beneficial environmental conditions while also avoiding or restricting certain sensitive processes during harsh conditions. Similarly, post-translational histone modifications allow eukaryotic organisms to regulate gene expression in response to environmental or developmental factors. Some post-translational modifications of histones are associated with active transcription while others are associated with repressed transcription depending upon the location, type and degree of modification. Trimethylation of lysine 4 on the N-terminal tail of histone H3 (H3K4me3) near a gene's promoter has been linked to active transcription of that gene in several organisms. The purpose of the current study was to investigate whether the amount of H3K4me3 at promoters of three specific genes shows a circadian rhythm in Chlamydomonas reinhardtii, a unicellular green alga. Two of the genes had previously been shown to display a circadian rhythm of expression with opposite phase (LHCBM6 and JMJD6-like2), while the third gene is constitutively expressed (RACK1). Quantitative PCR was used to determine the amount of immunoprecipitated H3K4me3 over a circadian cycle. It was hypothesized that H3K4me3 amount at the JMJD6-like2 and LHCBM6 promoter would show a circadian rhythm with a phase correlating directly with the phase of each gene’s rhythm of expression. Conversely, the H3K4me3 amount at the RACK1 promoter was predicted to not show a circadian rhythm, as the gene is constitutively expressed. Instead, results showed that H3K4me3 amount exhibits a circadian rhythm with identical phase for all three genes. ANOVA confirmed that the rhythms were not significantly different between the three genes. General histone H3 amount at promoters did not show a circadian rhythm across any of the three genes. Since recent genome-wide studies in mouse liver revealed a circadian rhythm of H3K4me3 amount with identical phase at the promoter of many genes with diverse expression, the findings presented here suggest that C. reinhardtii might show a similar global regulation of rhythmic H3K4me3 as in mice and that, therefore, this feature has been preserved during eukaryotic evolution.

The plant hormone cytokinin plays essential roles in many aspects of growth and development. The cytokinin signal is transmitted by a multistep phosphorelay to the members of two functionally antagonistic classes of Arabidopsis response regulators (ARRs): the type-B ARRs (response activators) and type-A ARRs (negative-feedback regulators). Previous studies have shown that mutations in AXR1, encoding a subunit of the E1 enzyme in the related to ubiquitin (RUB) modification pathway, leads to decreased cytokinin sensitivity. This research shows that the cytokinin resistance of axr1 seedlings is suppressed by loss-of-function of type-A ARRs and that the cytokinin resistance caused by ectopic expression of ARR5, a type-A ARR family member, is enhanced in axr1 background. Based on the established role of the RUB pathway in ubiquitin-dependent proteolysis, these data suggested that AXR1 promotes the cytokinin response by facilitating type-A ARR degradation. Indeed, both genetic (axr1 mutants) and chemical (MLN4924) suppression of RUB E1 increased ARR5 stability, suggesting that the ubiquitin ligase that promotes ARR5 proteolysis requires RUB modification for optimal activity. In addition, ARR1, a type-B ARR family member, also accumulated in the axr1 mutant background, suggesting that AXR1 regulates primary cytokinin signaling at multiple levels.

<p>Phenylalanine (Phe) is a proteinogenic aromatic amino acid that also serves as a precursor for numerous primary and secondary metabolites in plants. Phe is synthesized from chorismate, the final product of the shikimate pathway. In plants, Phe is predominantly synthesized in the plastids via the arogenate pathway, while most Phe-derived compounds are produced in the cytoplasm, requiring exportation of Phe from plastids to the cytosol. Here, we provided genetic evidences that a<i> Petunia hybrida</i> plastidial cationic amino acid transporter (PhpCAT) participates in the exportation of Phe from plastids, as well as regulation of carbon flux through Phe biosynthesis.</p> <p> By using reverse genetics, we demonstrated that a petunia phenylpyruvate aminotransferase (PhPPY-AT) is able to convert phenylpyruvate to Phe in the cytosol <i>in vivo</i>, and that a cytosolic chorismate mutase (CM2), which converts chorismate to prephenate, directs carbon flux from the plastidial Phe biosynthesis pathway towards the cytosolic pathway. Downregulation of <i>PhPPY-AT</i> and <i>PhCM2</i> resulted in significant decreases in Phe levels and emission of Phe-derived volatiles in petunia flowers, respectively. Metabolic flux analysis showed that the carbon flux through the cytosolic Phe biosynthesis pathway is significantly lower in <i>PhCM2</i> RNAi petunia flowers relative to wild type control. We also demonstrated that the conversion of prephenate to phenylpyruvate in the cytosol is catalyzed by a cytosolic prephenate dehydratase (PDT) produced from an alternative transcription start site of a known plastidial arogenate dehydratase (ADT). These results suggest that a microbial-like phenylpyruvate pathway for Phe biosynthesis operates in the cytosol of plant cells and the cytosolic pathway splits from the plastidial pathway at chorismate.</p> <p> To evaluate the metabolic potential of the cytosolic phenylpyruvate pathway, <i>PhCM2 </i>overexpressing transgenic petunia plants were generated. Unexpectedly, Phe levels and emission of Phe-derived volatiles were both reduced, even though the flux through the cytosolic pathway was increased relative to wild type control. Electron microscopy, metabolic profiling and metabolic flux analysis revealed that the number of leucoplasts, starch levels and flux through the plastidial pathway were all reduced in <i>PhCM2</i> overexpression lines, while the concentrations of auxin and its biosynthetic intermediate, indole-3-pyruvic acid (IPA), were elevated. Overexpression of Arabidopsis aminotransferase VAS1, which converts IPA to Trp, in <i>PhCM2</i> overexpression petunia background recovered Phe levels and Phe-derived volatiles emission. These results indicate that there exists a metabolic crosstalk between cytosolic Phe production and Trp-dependent auxin biosynthesis .</p> <p> Our research completed the post-chorismate cytosolic Phe biosynthesis pathway in plants and revealed possible metabolic crosstalk between cytosolic Phe production and auxin biosynthesis in plant cells, providing targets for future genetic modification of metabolites in plants.</p>

Plant cell wall is critical for the regulation of cell shape, cell growth, and responses to abiotic stress and pathogen infection. The plant cell wall is composed of several monosaccharides including glucose, galactose, mannose, xylose, fucose, rhamnose, and arabinose. Arabinose-containing polymers account for ~20 % of the total cell wall saccharides in rice and Arabidopsis. Arabinose is a plant-specific monosaccharide that is required for the decoration of several cell wall polysaccharides, including rhamnogalacturonan I (RGI)-arabinan, arabinoxylan, and rhamnogalacturonan II (RGII). Arabinose is also involved in the modification of some cell wall glycoproteins, including arabinogalactan-proteins (AGPs), extensins, and leucine-rich repeat extensin (LRX) proteins. In addition, arabinose is conjugated to signaling peptides like CLAVATA3 and some cytoplasmic arabinosylated flavonols, such as quercetin 3-O-l-arabinoside and myricetin. The only known enzyme in the final step of the arabinose de novo biosynthesis pathway is the Golgi-localized UDP-D-xylose 4-epimerase (MUR4), which converts UDP-xylose to UDP-arabinose. There is a 50% reduction of cell wall arabinose in mur4 mutant, indicating that other enzymes may also be involved in the de novo biosynthesis pathway. Under salt stress, mur4 mutant plants exhibit reduced root elongation and abnormal cell-cell adhesion. The roles of three MUR4 paralogs, MURL, DUR, and MEE25, in arabinose biosynthesis and salt stress tolerance are described. Data are also shown regarding the importance of AGPs in salt tolerance. Analysis of higher order mutants of mur4 with its three paralogs reveals that the three proteins also contribute to the biosynthesis of UDP-Ara and are critical for root elongation. The salt-hypersensitivity of the mur4 mutant is rescued by exogenous arabinose or gum Arabic (a commercial AGP product). Taken together, my work reveals the importance of arabinose metabolism in salt stress tolerance and provides new insights into the enzymes involved in UDP-Ara biosynthesis in plants. Plants have evolved cell-wall integrity sensing and signaling pathways to maintain cell-wall homeostasis in response to stress conditions, but the cellular components involved in the perception and transduction of cell-wall signals are largely unknown. I found that the cell wall-localized leucine-rich repeat extensins (LRX) 3/4/5 interact with RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23 to transduce cell wall signals. Mature RALF22/23 peptides convey signals to the plasma membrane-localized FERONIA (FER) to induce intracellular stress responses. The lrx345 and fer mutants and RALF22/23 overexpressing transgenic plants display similar phenotypes, including retarded growth and increased sensitivity to salt stress. These results suggest that LRX3/4/5, RALF22/23, and FER function as a module to regulate plant growth and salt stress tolerance. Further analyses show that the LRXs-RALF-FER module negatively regulates the accumulation of the phytohormones jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA), and the simultaneous over-accumulation of these stress hormones can be detrimental to plants. Based on genetic and biochemical data, we propose that salt-induced perturbations of the cell wall may be sensed by the LRXs, triggering the release of RALF peptides in the extracellular space; these peptides are then perceived by FER, inducing its internalization and down-regulating its function as part of a homeostatic mechanism to halt growth and to acclimate to salt stress through the activation of ABA, JA and SA signaling. Taken together, my work offers valuable insights into how salt stress is sensed in the apoplast by the LRXs-RALFs-FER signaling module, which subsequently modulates hormone signaling to establish a homeostatic mechanism coordinating growth and stress responses. In brief, my study contributes to the understanding of the role of MUR4 family of enzymes in plant arabinose biosynthesis and the role of arabinose-containing macromolecules in salt stress sensing and adaptation.<br>

<p>Plant biomass is an abundant source of renewable energy, but the efficiency of its conversion into liquid fuels is low. One reason for this inefficiency is the recalcitrance of biomass to extraction and saccharification of cell wall polysaccharides. This recalcitrance is due to the complex and rigid structure of the plant cell wall. A better understanding of the genes effecting cell wall composition in bioenergy crops could improve feedstock quality and increase conversion efficiency. To identify genetic loci associated with biomass quality traits, we utilized genome-wide association studies (GWAS) in an 840-line <i>Sorghum</i> diversity panel. We identified several QTL from these GWAS including some for lignin composition and saccharification. Linkage disequilibrium (LD) analysis suggested that multiple polymorphisms are driving the association of SNPs within these QTL. Sequencing and further analysis led to the identification of a SNP within the coding region of a gene encoding phenylalanine ammonia-lyase (PAL) that creates a premature stop codon and co-segregates with an increase in the ratio of syringyl (S) to guaiacyl (G) lignin. A comparison of net PAL activity between lines with and without the mutation revealed that this mutation results in decreased PAL activity. </p>

Flavonoids are plant secondary metabolites that have significant biochemical and physiological roles. Biosynthesis of these compounds involves several modifications, most predominantly glucosylation, which is catalyzed by glucosyltransferases (GTs). A signature amino acid sequence, the PSPG box, is used to identify putative clones and has been shown to be involved in UDP-glucose binding. Site-directed mutagenesis is used to answer questions regarding the structure and function of this family of enzymes, particularly what allows some GTs to be more selective towards some substrates than others. The grapefruit (Citrus paradisi) flavonol-3-O-glucosyltransferase (CpF3GT) is specific for flavonol substrates and will not glucosylate anthocyanidins. Comparison of the CpF3GT sequence with that of Vitis vinifera GT, which glucosylates both flavonols and anthocyanidins, provided the basis for the amino acid substitution of proline 145, alanine 374, and alanine 375 in CpF3GT to threonine, aspartate, and glycine, respectively, to test the affect on GT’s affinity for flavonoid substrates.