The multidrug and toxic compounds extrusion (MATE) family includes hundreds of functionally uncharacterised proteins from bacteria and all eukaryotic kingdoms except the animal kingdom, that function as drug/toxin::Na⁺ or H⁺ antiporters. In <i>Arabidopsis thaliana</i> the MATE family comprises 56 members, one of which is NIC2 (Novel Ion Carrier 2). Using heterologous expression systems including <i>Escherichia coli</i> and <i>Saccharomyces cerevisiae</i>, and the homologous expression system of <i>Arabidopsis thaliana</i>, the functional characterisation of NIC2 was performed. It has been demonstrated that NIC2 confers resistance of <i>E. coli</i> towards the chemically diverse compounds such as tetraethylammonium chloride (TEACl), tetramethylammonium chloride (TMACl) and a toxic analogue of indole-3-acetic acid, 5-fluoro-indole-acetic acid (F-IAA). Therefore, NIC2 may be able to transport a broad range of drug and toxic compounds. In wild-type yeast the expression of NIC2 increased the tolerance towards lithium and sodium, but not towards potassium and calcium. In <i>A. thaliana</i>, the overexpression of NIC2 led to strong phenotypic changes. Under normal growth condtions overexpression caused an extremely bushy phenotype with no apical dominance but an enhanced number of lateral flowering shoots. The amount of rossette leaves and flowers with accompanying siliques were also much higher than in wild-type plants and the senescence occurred earlier in the transgenic plants. In contrast, RNA interference (RNAi) used to silence NIC2 expression, induced early flower stalk development and flowering compared with wild-type plants. In additon, the main flower stalks were not able to grow vertically, but instead had a strong tendency to bend towards the ground. While NIC2 RNAi seedlings produced many lateral roots outgrowing from the primary root and the root-shoot junction, NIC2 overexpression seedlings displayed longer primary roots that were characterised by a 2 to 4 h delay in the gravitropic response. In addition, these lines exhibited an enhanced resistance to exogenously applied auxins, i.e. indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) when compared with the wild-type roots. Based on these results, it is suggested that the NIC2 overexpression and NIC2 RNAi phenotypes were due to decreased or increased levels of auxin, respectively. The Pro_NIC2:GUS fusion gene revealed that NIC2 is expressed in the stele of the elongation zone, in the lateral root cap, in new lateral root primordia, and in pericycle cells of the root system. In the vascular tissue of rosette leaves and inflorescence stems, the expression was observed in the xylem parenchyma cells, while in siliques it was also in vascular tissue, but as well in the dehiscence and abscission zones. The organ- and tissue-specific expression sites of NIC2 correlate with the sites of auxin action in mature Arabidopsis plants. Further experiments using Pro_NIC2:GUS indicated that NIC2 is an auxin-inducible gene. Additionally, during the gravitropic response when an endogenous auxin gradient across the root tip forms, the GUS activity pattern of the Pro_NIC2:GUS fusion gene markedly changed at the upper side of the root tip, while at the lower side stayed unchanged. Finally, at the subcellular level NIC2-GFP fusion protein localised in the peroxisomes of <i>Nicotana tabacum</i> BY2 protoplasts. Considering the experimental results, it is proposed that the hypothetical function of NIC2 is the efflux transport which takes part in the auxin homeostasis in plant tissues probably by removing auxin conjugates from the cytoplasm into peroxisomes. / &quot;Multidrug and Toxic Compounds Extrusion&quot; (MATE) &ndash; Proteine sind Membranproteine, die eine Vielzahl komplexer und giftiger Substanzen transportieren können. Sie sind weit verbreitet und kommen in Bakterien und Höheren Organismen mit Ausnahme des Tierreichs vor. Insgesamt gibt es hunderte von bisher kaum untersuchten Genen dieser Familie, die eine hohe Sequenzhomologie aufweisen. In der Pflanze Arabidopsis thaliana wurden 56 Gene der MATE - Familie zugeordnet. Eines von ihnen, der &quot;Novel Ion Carrier 2&quot; (NIC2) wurde näher charakterisiert. Dafür wurden heterologe Expressionssysteme wie Bakterien (Escherichia coli) und Hefe (Saccharomyces cerevisiae) genutzt und transgene Pflanzen (Arabidopsis thaliana) hergestellt. Es wurde gezeigt, dass NIC2 Bakterien eine Resistenz gegenüber mehreren giftigen Stoffen verlieh. In Hefe erhöhte NIC2 die Salztoleranz gegenüber Lithium und Natrium, aber nicht gegenüber Kalium und Kalzium. Das deutet darauf hin, dass NIC2 diese Stoffe transportieren kann und so zur Entgiftung beziehungsweise erhöhter Stresstoleranz beiträgt. In Pflanzen führte die Überexpression von NIC2 zu dramatischen Änderungen im Wachstum. Die Pflanzen waren buschig ohne zentralen Blütenstand, hatten jedoch eine höhere Anzahl von Blättern und Blüten und längere Wurzeln mit einer im Vergleich zu den Wildtyppflanzen verzögerten gravitropen Antwort. In Gegensatz dazu entwickelten Pflanzen, in denen die Expression von NIC2 gehemmt wurde, früh einen zentralen Blütenstand, der allerdings nicht gerade wuchs, sondern die Tendenz hatte, sich zum Boden zu biegen. Das Wurzelsystem bestand aus einer Hauptwurzel und vielen sekundären Wurzeln und war im Vergleich zu den Wildtyppflanzen besser entwickelt. Vermutlich kann die Wuchsform auf einen veränderten Gehalt des Pflanzenhormons Auxin zurückgeführt werden. Die Expression von NIC2 wird durch Auxin induziert. Experimente, in denen die Aktivität eines Gens mit Hilfe eines Reportergens nachgewiesen wird, zeigten, dass NIC2 in Wurzeln, Blättern, Blütenstielen, Blüten und Schoten aktiv ist. Innerhalb der Zelle ist NIC2 in Peroxisomen lokalisiert. Peroxisomen sind kleine Organellen, die eine Rolle im Hormonstoffwechsel spielen können, wie z.B. im Fall von Auxinen. Die Daten sprechen dafür, dass NIC2 eine Funktion beim Auxintransport und somit bei der Auxin-Homöostase hat.

This thesis describes the characterization of the Arabidopsis thaliana mutant terminal flower2 (tfl2), the cloning of the corresponding gene, and the analysis of TFL2 function in plant development. The tfl2 mutant is pleiotropic, exhibiting early floral induction in both long and short day conditions, a terminating inflorescence and dwarfing. TFL2 was isolated using a positional cloning strategy, and was found to encode a homolog to HETEROCHROMATIN PROTEIN1 (HP1), previously identified in yeast and animals where it is involved in gene regulation at the level of chromatin, as well as in the structural formation of constitutive heterochromatin. Investigating the light response during seedling photomorphogenesis I found that the tfl2 hypocotyl is hypersensitive to red and far-red light and that tfl2 is impaired in phytochrome mediated light responses such as the shade avoidance response. In the tightly regulated transition to flowering, we have shown that tfl2 might contribute to the interpretation of both external signals such as light and temperature as well as endogenous cues, via FCA, in the autonomous pathway. The Arabidopsis inflorescence meristem is indeterminate, and TFL2 possibly acts to maintain this indeterminate fate by repression of the floral meristem genes APETALA1 and AGAMOUS. In yeast two hybrid experiments TFL2 was shown to interact with IAA5, a protein with suggested functions in auxin regulation. Further, in tfl2 mutants the levels of the auxin indole-3-acetic acid decrease with age in aerial tissues, suggesting a function of TFL2 in regulation of auxin homeostasis and response. In summary, TFL2 contributes to regulation of several aspects of plant development, in accordance with the mutant phenotype and the identity of the TFL2 protein.

Au cours du développement des plantes, l'auxine contrôle de nombreux processus dont notamment la dominance apicale, le phototropisme, la phyllotaxie, la formation des racines latérales et le développement des fruits. Le métabolisme, la perception ainsi que la signalisation de l'auxine ont majoritairement été étudiés chez Arabidopsis. Afin d'élucider la fonction de cette hormone au cours du développement des fruits, nous avons utilisé la tomate comme plante modèle. En effet la tomate est à la fois une espèce référence pour la famille des Solanacées mais également pour les plantes à fruits charnus. Les gènes Aux/IAA jouent un rôle déterminant dans la voie de signalisation auxinique en formant notamment un complexe avec l'un des récepteurs de cette hormone et en réprimant l'activité des facteurs de transcriptions de type ARF. Au cours de ce travail, nous avons caractérisé la famille multigènique des Aux/IAA chez la tomate. Elle est composée de 25 membres que nous avons nommés en référence à ceux d'Arabidopsis. Le niveau d'expression des gènes Aux/IAA est variable en fonction du gène, de l'organe ou du tissu considéré. L'expression de plusieurs de ces gènes est régulée à la fois par l'auxine et l'éthylène, ce qui suggère que les Aux/IAA sont reliés aux voies de signalisation de ces deux hormones. L'élucidation de la fonction des Aux/IAA de tomate est réalisée à travers la caractérisation de plantes transgéniques avec une attention particulière portée aux lignées montrant des phénotypes affectant le développement et la qualité du fruit ou présentant un intérêt pour le dialogue entre l'auxine et l'éthylène. Parmi ces lignées, les plantes sous-exprimant le gène Sl-IAA27 présentent une altération du développement des fleurs et des fruits. De plus, la diminution de l'expression de Sl-IAA27 entraine la sous-expression de gènes impliqués dans la voie de biosynthèse de la chlorophylle se traduisant par une diminution de la teneur en chlorophylle dans les feuilles. Ces résultats montrent la diversité fonctionnelle des Sl-IAA et souligne le rôle de régulateur joué par l'auxine au cours du développement du fruit.

The development of plants challenged by environmental stress alters plant architecture through several pathways, including those involving plant hormone responses and reactive oxygen species (ROS) production. Auxin, a phytohormone associated with every aspect of development, and abscisic acid (ABA), a phytohormone involved in abiotic stress responses, both interact with ROS. These ROS are used as secondary messengers to activate transcription of abiotic stress genes, and also in developmental responses such as cell elongation. To understand the mechanisms involved in the abiotic stress response and how the response intersects with auxin, ABA, and ROS, I examined COPPER INDUCED IN LEAVES 1 (<i>CIL1</i>) from <i>Brassica carinata</i> and its Arabidopsis orthologue, AUXIN INDUCED IN ROOTS 12 (AIR12). Expression of both genes increases in response to auxin and recent work has placed both <i>CIL1</i> and AIR12 within a family of plant-specific cytochrome b561 proteins thought to be involved with transmission of ROS signals. This suggests a link between auxin and ROS production resulting from abiotic stress. Antisense <i>CIL1 B. carinata</i> plants produced fewer lateral roots and were resistant to salinity stress during vegetative growth. Mutant air12 plants showed a 50% reduction in lateral root number, lateral root length, and H2O2 root distribution. Growth in the presence of H2O2 was able to restore lateral root length to control levels. In silica analysis of the <i>CIL1</i> and AIR12 amino acid sequences detected an attachment site for glucosylphosphatidylinositol, predicting that the protein is targeted to the extracellular leaflet of the plasma membrane where it could be cleaved and released into the apoplast. Subcellular localization using p35S::GFP-CIL1 and p35S::GFP-AIR12 translational fusions confirmed that CIL1 and AIR12 localize to the plasma membrane and are released into the apoplast. Organ localization of AIR12 using the pAIR12::GFP-AIR12 construct in stably transformed Arabidopsis showed fusion protein accumulation in the apex of the primary root and in the vascular tissue. Fusion protein also localized to cells flanking emerging lateral roots. Investigation of pAIR12::GUS Arabidopsis showed GUS accumulation in the apex of elongating lateral roots. I demonstrate that AIR12 is an extracellular protein and that air12 seedlings are susceptible to salt stress, but not osmostic stress and display increased and decreased sensitivity to ABA during germination and primary root elongation, respectively, suggesting that AIR12 acts downstream of abiotic stress recognition.

The development of plants challenged by environmental stress alters plant architecture through several pathways, including those involving plant hormone responses and reactive oxygen species (ROS) production. Auxin, a phytohormone associated with every aspect of development, and abscisic acid (ABA), a phytohormone involved in abiotic stress responses, both interact with ROS. These ROS are used as secondary messengers to activate transcription of abiotic stress genes, and also in developmental responses such as cell elongation. To understand the mechanisms involved in the abiotic stress response and how the response intersects with auxin, ABA, and ROS, I examined COPPER INDUCED IN LEAVES 1 (<i>CIL1</i>) from <i>Brassica carinata</i> and its Arabidopsis orthologue, AUXIN INDUCED IN ROOTS 12 (AIR12). Expression of both genes increases in response to auxin and recent work has placed both <i>CIL1</i> and AIR12 within a family of plant-specific cytochrome b561 proteins thought to be involved with transmission of ROS signals. This suggests a link between auxin and ROS production resulting from abiotic stress. Antisense <i>CIL1 B. carinata</i> plants produced fewer lateral roots and were resistant to salinity stress during vegetative growth. Mutant air12 plants showed a 50% reduction in lateral root number, lateral root length, and H2O2 root distribution. Growth in the presence of H2O2 was able to restore lateral root length to control levels. In silica analysis of the <i>CIL1</i> and AIR12 amino acid sequences detected an attachment site for glucosylphosphatidylinositol, predicting that the protein is targeted to the extracellular leaflet of the plasma membrane where it could be cleaved and released into the apoplast. Subcellular localization using p35S::GFP-CIL1 and p35S::GFP-AIR12 translational fusions confirmed that CIL1 and AIR12 localize to the plasma membrane and are released into the apoplast. Organ localization of AIR12 using the pAIR12::GFP-AIR12 construct in stably transformed Arabidopsis showed fusion protein accumulation in the apex of the primary root and in the vascular tissue. Fusion protein also localized to cells flanking emerging lateral roots. Investigation of pAIR12::GUS Arabidopsis showed GUS accumulation in the apex of elongating lateral roots. I demonstrate that AIR12 is an extracellular protein and that air12 seedlings are susceptible to salt stress, but not osmostic stress and display increased and decreased sensitivity to ABA during germination and primary root elongation, respectively, suggesting that AIR12 acts downstream of abiotic stress recognition.

Light is the fundamental source of energy and information throughout the plant life cycle. Light signals regulate plant architecture and branching, key processes that determine biomass production and grain yield. Low red (R) to far-red (FR) light ratios (R:FR) perceived by phytochromes serve as a warning signal about impending competition for light resources and lead to shade avoidance responses (SARs), including reduced branching. The R:FR regulates branching in both a bud autonomous and non-bud autonomous manner, however a detailed mechanistic understanding of the process remains unclear. We hypothesized that high R:FR promotes bud outgrowth by differentially regulating branching-related genes (transcriptome) within the axillary bud and that increased apical dominance under low R:FR or with phyB deficiency is mediated by auxin or other novel signal/s. We analyzed the branching phenotype of Arabidopsis Columbia-60000 ecotype in response to different R:FR treatments and conducted a microarray study to identify early (within 3 hours) changes in the transcriptome of buds from different rosette positions in response to altered R:FR. Physiological experiments were also conducted to determine if auxin concentration, transport rate, sensitivity, and establishment of an auxin transport stream were important in determining the branching phenotype of shade avoiding plants. The results revealed that the duration of low R:FR determines plant architecture and the branching phenotype and that bud outgrowth is regulated by the R:FR in a spatial and temporal manner. Low R:FR promoted the elongation of branches at top rosette nodes while it suppressed the outgrowth of axillary buds at lower nodes. High R:FR could reverse the effects of previous low R:FR by promoting the outgrowth of buds from lower axils within 24 hours of treatment. Transcriptomic analysis revealed that the R:FR differentially regulated the expression of genes related to hormone biosynthesis/transport/signaling, cell-cycle regulation and cell wall modification. Cis-elements responsive to light and hormone signaling pathways were overrepresented in several gene clusters. Apical dominance related studies discovered that loss of phyB function results in a slower auxin transport rate, fewer xylem parenchyma cells, and reduced sensitivity to auxin. These results, in addition to estimates of correlative inhibition, suggested that auxin is at least partially responsible for increased apical dominance under low R:FR or with phyB deficiency, but may be acting in conjunction with other undefined regulators.

No description available.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

No description available.