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.

This study is concerned with the responses of internode extension rate in white-light-grown Sinapis alba seedlings to light: the kinetics, nature and organ localization of the photoreceptors and possible transduction chains. Phytochrome status was modified either by means of red, or far-red, light pulses given at the end of the photoperiod, or by supplementing white fluorescent light with different fluence rates of far-red light during the photoperiod. The status of specific blue light-absorbing photoreceptor(s) was modified by supplementing a background of blue-deficient light with different fluence rates of blue light. Low Pfr/P established in the leaves induced a promotion of internode extension rate that persisted for ca 24 h after the plant returned to high Pfr/P. The effect of Pfr/P during dark periods is due mainly to this persistent response. Several fluence-rate-dependent responses were revealed: (a), blue light reaching the leaves modulates the responsiveness of the internode to a reduction of Pfr/P restricted to the internode itself; (b), blue light reaching the leaves inhibits internode extension in subsequent darkness; (c), for a given period in darkness, internode growth rate is higher if Pfr/P is reduced before, rather than at the beginning of darkness, or at the beginning of darkness, rather than later; (d), the transition from light to darkness causes a rapid reduction (lag 10 min) of internode extension rate. A correlation (kinetics and localization) between the extracellular activity of one moderately acidic peroxidase isoform extracted from the internodes, and the effect of Pfr/P on internode extension rate is presented. The activity of another extracellular peroxidase isoform was unaffected by Pfr/P, but showed a rapid increase after wounding. This treatment caused no significant effects on the isoform controlled by Pfr/P. Present findings are discussed in relation to the ecological significance and the physiological basis of the control of internode extension growth by light.

No description available.

Graduation date: 1998

The ability of plants to not only detect but also adjust to their environment is crucial for their survival. The genes involved in photomorphogenesis – developmental changes in response to light – and their regulation have long been of interest to researchers. While the phytochrome and cryptochrome photoreceptors have been isolated and partially characterized, the downstream components of the light signaling pathway which transmit the perceived light signals and regulate gene expression are still being discovered. A negative regulator of photomorphogenesis, DET1 (de-etiolated 1), was discovered in a mutant screen for plants that develop a light grown phenotype in the dark. DET1 is nuclear localized, but its exact function remains unknown. Two contrasting mechanisms for the role of DET1 in the regulation of gene expression have been proposed based on studies of the tomato and human orthologs of DET1. In order to reveal the mechanism and molecular context of DET1 action, suppressor mutant screens were employed to discover additional genes acting in conjunction with DET1 (designated as TED genes). In this research, new genomic methods were developed and employed to identify the genes underlying the ted1-1SD and ted2-1D suppressor mutations. A long hypocotyl QTL and suppression of the det1-1 dark grown phenotype by the Bensheim (Be-0) ecotype of Arabidopsis mapped to the HAT4 gene, a homeoboxdomain leucine-zipper transcription factor involved in shade-avoidance responses. Sequence analysis uncovered two functionally distinct alleles of HAT4 in the Be-0 alleles of HAT4 compared to the genomic standard Columbia (Col-0) ecotype. Expression analysis showed that in addition to negative autoregulation by itself, HAT4 is also negatively regulated by DET1. The ted2-1D mutation was mapped to a 57 Kbp interval on chromosome I containing three likely candidate genes. Suppression of the det1-1 phenotype by ted2-1D is overdominant which is highly unusual and typically associated with hybrid vigor or heterosis traits. The discovery of the genes underlying the ted1-1SD and ted2-1D suppressor mutations have furthered the understanding of the role for DET1 in regulation of photomorphogenesis as well as mechanisms involved in overall gene regulation during light signaling.

As sessile organisms, coordination of development and reproduction in a dynamic, and often stressful, environment presents a particular challenge for plants. Rapid processing of internal and external cues by complex signal transduction pathways leads to stimulus-appropriate physiological responses on an organismal scale. In plants, myriad signaling pathways are mediated by calcium (Ca2+) signals, and it is thought that different stimuli elicit unique patterns of Ca2+ influx into cells (termed Ca2+ ‘signatures’) that encode information important for proper physiological responses. Encoding of information in the form of Ca2+ signatures requires that decoding elements be present in cells to direct downstream cellular processes. This role is filled by Ca2+-binding proteins that serve as Ca2+ sensors. Interestingly, plant genomes encode multiple expanded families of Ca2+ sensors not found in animal genomes. Among these, the calmodulin (CaM)-like proteins (CMLs) are represented by a 50 member family in Arabidopsis. On the basis of structural homology, CMLs are predicted to function like conserved CaM, however, little work has been done to address this question. Biochemical characterization of CML39 indicates that it possesses structural properties consistent with function as a Ca2+ sensor. Analysis of transgenic CML39 loss-of-function (cml39) mutants revealed that CML39 is important for proper seedling establishment in the absence of exogenous metabolisable carbon as cml39 seedlings entered a state of developmental arrest shortly after germination. cml39 mutants also exhibited a conditional ‘de-etiolated’ phenotype when grown in complete darkness and exaggerated hypocotyl elongation under a short-day light regime. Genetic data suggest that CML39 functions in signaling pathways downstream of light perception, and this idea is supported by the observation that CML39 iii is expressed in light-sensing tissues, and that subunit 5 of the COP9 signalosome, a protein critical for photomorphogenesis, was identified as a putative target of CML39. Collectively, results show that CML39 is Ca2+ sensor that serves a critical regulatory role during seedling establishment when sucrose is limited, and importantly, further underscore the pervasiveness of Ca2+ signaling in plant growth and development. / Thesis (Ph.D, Biology) -- Queen's University, 2013-05-29 18:16:25.769

Les plantes disposent de mécanismes rapides d'adaptation de leur physiologie et de leur développement à des conditions environnementales changeantes. Leur mise en œuvre dépend largement d’une capacité de reprogrammation de l'expression des gènes qui implique généralement des changements continus de l'épigénome. Chez de nombreux organismes, différentes voies d’enlèvement de la monoubiquitination de l’histone H2B (H2Bub) participent d'une part à faciliter la transcription des gènes par l'ARN polymérase II et d'autre part à éviter l'établissement d'un état permissif à la transcription par enlèvement de domaines enrichis en H2Bub sur des régions répétées telles que les séquences télomériques. Cette thèse a porté sur l’étude des voies régulant la marque chromatinienne H2Bub chez les plantes, dont la connaissance des mécanismes de contrôle dynamique est très fragmentaire. Une nouvelle ubiquitine protéase de l'espèce Arabidopsis thaliana a été identifiée comme étant un homologue fonctionnel de Ubp8, une protéine associée à l'élongation de la transcription au sein d'un module de déubiquitination d'H2Bub du complexe SAGA chez S. cerevisiae. L'identification et la caractérisation fonctionnelle de trois composants de ce module chez A. thaliana a révélé qu'il agit sur des milliers de gènes, suggérant son implication dans des mécanismes basaux de la transcription. Dans une seconde partie, il a été observé que l'abondance de deux sous-unités de ce module est régulée au cours de la photomorphogenèse par DET1, un acteur central de la signalisation de la lumière. Cette transition développementale permet l'adaptation du métabolisme et de la morphologie de la plante en réponse à la première exposition à la lumière, notamment via l'établissement de l'activité photosynthétique. La régulation post-traductionnelle du module de déubiquitination de l'histone H2B pourrait permettre d'ajuster son activité aux changements d'activité transcriptionnelle de la cellule au cours de cette transition. Une approche génétique a également permis d'identifier une redondance fonctionnelle partielle entre l'activité du module de déubiquitination et UBP26, une seconde déubiquitinase d'H2Bub connue pour son implication dans la répression des gènes PHERES1 et FLC par une activité Polycomb ainsi que de certains éléments transposables. Ces analyses ont permis de révéler une influence positive de UBP26 sur l'établissement d'un état répressif à la transcription sur des centaines de gènes et également dans certains contextes hétérochromatiniens. Collectivement, ce travail a permis de disséquer les spécificités et les redondances fonctionnelles de deux voies de déubiquitination de l'histone H2B portées par des complexes protéiques distincts. / Plants utilize rapid mechanisms to adapt their physiology and development to changing environments. Their triggering depends greatly on gene expression reprogramming leading to important changes in the epigenome. In numerous organisms, different pathways remove monoubiquitination of histone H2B (H2Bub) to facilitate gene transcription by RNA polymerase II, with H2Bub removal on repeated genomic regions, such as telomeres, prohibiting establishment of a transcription permissive state. This thesis aims to better characterize the pathways that regulate the chromatin mark H2Bub in plants. A new ubiquitin protease from Arabidopsis thaliana was identified as a potential homolog of Ubp8, a protein associated with transcription elongation within a deubiquitination module of the SAGA complex in S. cerevisiae. Identification and functional characterization of the three components of the deubiquitination module in A. thaliana reveals its action on thousands of genes, suggesting a role in basal transcription mechanism. Secondly, it was shown that the quantity of two subunits from this module is regulated during photomorphogenesis by DET1, a central protein involved in light signaling. This developmental transition allows adaptation of metabolism and morphology of the plant in response to the first light exposure, notably during photosynthesis establishment. The post-translational regulation of the histone H2Bub deubiquitination module may allow its adjustment to changes in cell transcription needs during this transition. A genetic approach identified a partial functional redundancy between the deubiquitination module activity and UBP26, a second H2Bub deubiquitinase known to repress the genes PHERES1 and FLC by a Polycomb activity and certain transposable elements. These analyses revealed a positive influence from UBP26 on establishing a repressive transcriptional state on hundreds of genes and on some heterochromatinian contexts. Collectively, this work dissected specificities and functional redundancies of two H2Bub deubiquitination pathways driven by distinct protein complexes.

No description available.

Plants modulate their growth and development according to the prevailing light conditions. To detect light signals plants have an array of photoreceptors including the phytochromes which monitor the red and far-red light regions of the light spectrum. Phytochromes regulate gene expression in response to light in part by physically interacting with nuclear-localized bHLH transcription factors called PHYTOCHROME INTERACTING FACTORS (PIFs). PIFs are known to function as negative regulators of photomorphogenesis. Here we show that PIF1, the PIF family member with the highest affinity for phys, is degraded after pulses or continuous red, far-red or blue light in a phytochrome dependent manner. In etiolated seedlings, phyA plays a dominant role in regulating the degradation of PIF1 after a pulse of red, far-red or blue light; while phyB, phyD and other phys also influence PIF1 degradation after prolonged illumination. PIF1 interacted with phyA and phyB in a blue light-dependent manner, and the interactions with phys are necessary for the light-induced degradation of PIF1. In response to red, far-red or blue light treatments PIF1 is rapidly phosphorylated, poly-ubiquitinated and degraded via the ubiquitin/26S proteasomal pathway. In addition, we show that PIF1 negatively regulates photomorphogenesis at the seedling stage. The overexpression of a light-stable truncated form of PIF1 causes constitutively photomorphogenic phenotypes in the dark. pif1 seedlings displayed more open cotyledons and slightly reduced hypocotyl length compared to wild type under diurnal (12h light/12h dark) blue light conditions. Double mutant analyses demonstrated that pif1phyA, pif1phyB, pif1cry1 and pif1cry2 have enhanced cotyledon opening compared to the single photoreceptor mutants under diurnal blue light conditions. Taken together, these data suggest that PIF1 functions as a negative regulator of photomorphogenesis and that light-activated phys induce the degradation of PIF1 through the ubi/26S proteasomal pathway to promote photomorphogenesis. / text

Graduation date: 1994