Light is one of the major environmental factors that controls plant development, through a process known as photomorphogenesis. Plants perceive light via photoreceptors, and the information used to direct a myriad of developmental responses. Analysis of mutants defective in photomorphogenic responses elucidates the complex interactions between light and plants. Previous genetic screens have yielded a class of mutants which exhibit exaggerated responses to ambient light, designated shl (seedling hyper-responsive to light). The following work encompasses the identification of five new shl mutants, a detailed examination of one of these mutants (shl7), and of the SHL7 gene. The mutants were isolated in a low-white light screen of seedlings derived from T-DNA mutagenesis. Each of the mutants exhibits a heritable hyper-responsive phenotype in low-white light, but displays minimal effects in darkness. For each, a putative site of T-DNA insertion has been located. In addition to a low-white light phenotype, the shl7 mutant exhibits a mild hyper-responsive phenotype to 670 nm red and 735 nm far-red light, but significant hyper-responses to 420 nm blue light. SHL7 encodes a small, unique, and previously undescribed protein annotated as At4g04925. GFP protein fusion analysis indicates that the protein is localized to mitochondria.

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Circadian rhythmsare biochemical, physiological, and behavioral processes display oscillations of oughly 24-hour, which existing in both prokaryotes and eukaryotes. Circadian rhythms improve fitness of organisms in both constant and changing environments. The cryptochrome (CRY)and PAS-containing proteins are light sensors and key elements of the circadian system in eukaryotic organisms. Photolyases and cryptochromes are evolutionarily related flavoproteins which perform distinct physiological functions. Photolyases are evolutionarily ancient enzymes that activated by light and repairing UV-induced DNA damage. Although cryptochromes share structural similarity with the DNA photolyases, they lack the DNA repair activity. CRYs are key elements of mammal circadian system, and play roles in light sensing in insects and plants to entrain circadian rhythms. The PAS domains are widely distributed in proteins across all kingdoms of life and act as signal modules. They are common in photoreceptors and transcriptional regulators of eukaryotic circadian clock components including bHLH-PAS proteins (BMAL, CYC,CLK and NPAS2) and PER in animals, PHY and ZTL in plants, WC-1, 2and VVD in fungi. They are mainly involved in protein-protein interaction and light sensing functions. The CRY/PHR superfamily consists of 7 major subfamilies: CPD class I and CPD class II PHRs, (6-4) PHR, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the superfamily evolved primarily under strong purifying selection (average ω = 0.0178), it experienced strong episodic positive selection at some periods of evolution. The level of variation is subfamily-and domain-specific. The homologs with apparent circadian functions (i.e., plant and animal CRY) are significantly more conserved than the other photolyases. Photolyases were lost in eukaryotic groups like placental mammals, suggesting that natural selection apparently became weaker in the late stage of evolutionary history. The phylogenetic trees of fish Cry features two major clusters, which correspond to Cry1and Cry2. Teleost species possess extra copies of Cry1 due to fish-specific genome duplication (FSGD), and formed 3 clades of Cry1. Clade1B of Cry1(π= 0.129 ±0.062) is more conserved than the other paralogs (πrange from 0.173to 0.195). Test of positive selection revealed that fish cryptochromes evolved under strong purifying selection (average ω= 0.0066).Different fishes preserved different Cry duplicates that associated with reciprocal gene loss, thus generated the diverse circadian molecular mechanisms. The level of DNA variation in the PAS-containing proteins appears to be subfamily-specific. The animal PAS-containing homologs are more polymorphic than the plant and fungal homologs. Although the whole superfamily evolved primarily under strong purifying selection (average ω range from 0.0030to 0.1164), it experienced strong positive selection at some periods of the evolution. Although the PAS domains from different proteins vary in sequence and length, they maintain a fairly conserved 3D structure. The 3D fold of PAS domains is determined by only 8 conserved residues which shared by all subfamilies. The evolutionary time estimates showed that plant and animal Cry, WC-1& 2, bHLH-PAS proteins and Per originated in the Neoproterozoic Era (~1000 –542 Mya), plant Phy and ZTL evolved in the Paleozoic (541 –252 Mya), which might be a result of adaptation to the global climate and light regime changes. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 131-146).

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No description available.

The circadian clock is an endogenous timer that oscillates with a period of approximately 24 hours and is reset upon environmental time cues such as the daily light/ dark or temperature cycles. Chlamydomonas reinhardtii is an ideal model organism for research on the circadian clock, because it shows several well-characterized behaviors that exhibit a circadian rhythm. Its circadian rhythm of phototaxis (swimming toward light) has been automated. Former action spectrum studies using the circadian phototaxis rhythm as an indicator surprisingly found that pulses of blue light were not effective in resetting the circadian clock of dark-adapted cells. This may have been because of the particular strain used in the study (the cell wall-deficient strain CW15). It may also have been due to the additional phase shift caused by the act of placing the cultures into the monitoring machine at particular times during their circadian cycle. This additional phase shift was most likely the result of the white background light present when monitoring the rhythm of phototaxis. The phototaxis monitoring process was improved by using narrow-wavelength LEDs specific for phototaxis as test lights and by omitting the background light between test light cycles. This study demonstrates that the modifications prevent any phase shifts due to the cultures being placed into the monitoring machine. Using a further improved experimental set-up and the wild-type strain CC124, this study unambiguously shows that blue light of 440nm is effective in resetting the circadian clock in Chlamydomonas reinhardtii. Because of this difference in blue light response to the earlier study, the action spectrum of the entire visible light range was also evaluated. Effective wavelengths for resetting the circadian clock in wild-type C. reinhardtii were found to occur at 400nm, 440nm, 540nm, and 640-660nm, corresponding to near UV-A, blue, green, and red light, respectively. With the exception of 440nm, these findings are congruent with previous action spectrum studies for the cell wall-deficient strain CW15.

The mammalian suprachiasmatic nuclei (SCN) are located in the ventral part of the hypothalamus and orchestrate circadian rhythms in physiology and behaviour. The ~20.000 neurones of the murine SCN express key molecular clock components including the Cryptochrome (Cry1/2) and Period (Per1/2/3) genes and their protein products CRY1/2 and PER1/2/3. Using different mouse models, this work demonstrates that with disrupted expression of CRY in the after-hours (Afh/Afh) mouse, cells of the ventral part of the SCN (vSCN) have a propensity to desynchronise. They receive increased GABAergic inputs and are less excitable during the projected night but not during the day compared to congenic wildtype (+/+). The linkage between CRY protein expression and the reduced excitability at night is supported by recordings from SCN cells of Cry2 deficient mice (Cry2-/-), which exhibit similar electrophysiological behaviour. Luminometrical recordings of single cell Per2 expression confirms the involvement of GABAergic signalling in both, maintaining a coherent rhythm in synchronised SCN cells from +/+ controls and the propensity of Afh/Afh SCN cells to desynchronise. A mechanism by which neuronal excitability is regulated in mammals, is the modulation of activity of small-conductance Ca2+-activated K+ (SK) channels. Western blot analysis demonstrates the expression of SK2 and SK3 channel protein in SCN neurones. Functionally, we show with whole cell electrophysiology, calcium imaging and luminometry how SK channels regulate the levels of intracellular calcium ([Ca2+]i) from day to night. In the more hyperpolarised SCN network of the Afh/Afh genotype at night, SK channel activity is altered and contributes to the lower single cell excitability. Vasoactive intestinal polypeptide (VIP) and its cognate receptor, VPAC2, are synthesised by SCN neurones and this intercellular signal facilitates coordination of suprachiasmatic neuronal activity. How the loss of VPAC2 receptor signalling affects the electrophysiology of SCN neurones and their response to excitatory inputs is unclear. Here we made patch clamp recordings of SCN neurones in brain slices prepared from animals that do not express VPAC2 receptors (Vipr2-/- mice) as well as non-transgenic animals (Vipr2+/+ mice). While Vipr2+/+ SCN neurones exhibit coordinated day-night variation in their electrical state, Vipr2-/- neurones do not and instead manifest a range of states during both day and night. We find that Vipr2+/+ neurones vary the membrane threshold potential at which they start to fire actions potentials from day to night, while Vipr2-/- neurones lack this variation. This is due to Vipr2-/- neurones lacking a voltage-gated sodium current. Subsequently we determine that this aberrant temporal control of neuronal state and excitability alters appropriate neuronal responses to a neurochemical mimic of the light-input pathway to the SCN. Conclusively, these results highlight the critical role intercellular signalling plays in the activity of individual neuronal state and their response to neural input as well as ensemble activity and function of the suprachiasmatic neural network.

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<strong>Chapter 1</strong> introduces the concept of spin, how spins interact, and how the spin state in a radical pair can affect the outcome of a chemical reaction between the unpaired electrons. The computational methodology for simulating such radical pairs is also discussed. <strong>Chapter 2</strong> discusses anisotropy in the singlet recombination yield of a radical pair in a carotenoid-porphyrin-fullerene triad, containing many hyperfine couplings. The singlet yield was calculated as a function of the direction of an applied magnetic field, using symmetry in the molecule to reduce the size of the problem. The symmetry reduction was partially successful, however it was not possible to include all the hyperfine couplings in the molecule. <strong>Chapter 3</strong> introduces a radical pair located on a flavin ligand and a tryptophan residue in the protein cryptochrome, and discusses the spin-relaxation mechanism of singlet-triplet dephasing. Magnetic field effect curves, describing the formation of a secondary radical pair as a function of applied magnetic field, were found to be broader in longer-lived radical pairs, due to dephasing caused by spin-selective recombination to the singlet ground state. Additional singlet-triplet dephasing may occur due to hopping of one of the unpaired electrons, between a zone of strong exchange interaction and a zone of negligible exchange interaction, although this is an incomplete description of the spin-relaxation. <strong>Chapter 4</strong> discusses the effect of rotational tumbling on spin-relaxation in the flavin-tryptophan radical pair in cryptochrome. Simulations indicated that the resulting modulation of anisotropic hyperfine couplings contributed modestly to spinrelaxation during transient absorption measurements, but was insufficient to explain the lack of an experimental low-field effect, or to explain the width of the experimental magnetic field effect curves as a function of magnetic field strength. <strong>Chapter 5</strong> discusses magnetic field effects on the mutual annihilation of a pair of triplet excitons in tetracene and anthracene crystals. The experimental singlet recombination yield was found, for the first time, to be modulated as a function of the direction of a applied magnetic field as weak as 2 mT. Simulations indicated that this anisotropy arose due to the zero field splitting of the electronic state in each triplet exciton. The direction of the external magnetic field altered the singlet component of the eigenstates of the Hamiltonian, and therefore altered the timeaverage of the singlet probability of a triplet exciton pair. This is different to the already established mechanism under a strong magnetic field, where the anisotropy arises from level crossings of eigenstates.