COMPARATIVE ANATOMY OF TWO CULTIVATED SPECIES OF COLEUS BLUMEI BENTH. (LAMIACEAE) LEAVES WITH ORNAMENTAL VALUE

The paper presents a comparative study concerning the leaf anatomy of two ornamentally by leaves varieties of Coleus blumei Benth.: Coleus blumei ‘Black Dragon’ and C. blumei ‘Violet Tricolor’. The comparative study discloses both similarities and differences in the leaf anatomical structure of the two Coleus species. Anatomically, the leaves of the studied species are quite similar in the basic structure but differences appear concerning the petiole shape, the non glandular and glandular trichomes types, diversity, structure and density on the petiole and lamina surface, the number of vascular bundles in the petiole. Both species have a bifacial leaf and a homogenous, hypostomatic mesophyll

The evolution of RNA interference system, blue light sensing mechanism and circadian clock in Rhizophagus irregularis give insight on Arbuscular mycorrhizal symbiosis

La symbiose mycorhizienne arbusculaire (MA) est une association formée par les racines des plantes et les champignons mycorhiziens arbusculaires (CMA). Ces champignons sont les plus anciens symbiotes des plantes et ils sont apparus il y a au moins 460 millions d'années avec l'émergence et l'évolution des plantes terrestres. Les CMA sont également les partenaires symbiotiques des plantes les plus répandus dans les écosystèmes et ils peuvent s’associer avec plus de 80% des espèces de plantes vasculaires. Les CMA appartiennent à une lignée fongique primitive dont la position phylogénétique est encore en débat. Les CMA sont des microorganismes biotrophes obligatoires qui dépendent entièrement du carbone provenant de la photosynthèse des plantes hôtes, autrement dit, les CMA ne peuvent assimiler le carbone qu’on association avec les racines des plantes. En échange, les CMA aident les plantes à absorber divers nutriments essentiels du sol, tels que le phosphore. En effet, les CMA absorbent les nutriments et ils les transportent à travers leurs hyphes jusqu’aux cellules des racines dans lesquelles ils forment une structure appelée arbuscule. L'allocation des nutriments et les voies métaboliques interconnectées entre le champignon et l'hôte ont subi une pression sélective en tant que partenaires symbiotiques. En plus, les hyphes de des CMA agissent comme une niche écologique pour divers microbes du sol tels que les bactéries et les champignons, formant ainsi le pivot de la rhizosphère des racines. La symbiose MA est un élément essentiel pour comprendre la physiologie des plantes ainsi que l'écosystème. Malgré les rôles cruciaux des CMA dans les écosystèmes, leur génétique et leur évolution demeure méconnues. Le système d'interférence de l'ARN (ARNi), le mécanisme de détection de la lumière bleue et l'horloge circadienne sont des mécanismes importants qui sont impliqués dans la régulation de l’expression des gènes chez les champignons. Bien que son rôle reconnu dans la régulation des gènes et la traduction des protéines, en particulier dans la symbiose telle que celle des nématodes, le système ARNi n’a jamais été étudié chez les CMA. Pareil pour le cas du mécanisme de détection de la lumière bleue. Seules quelques études ont montré que la lumière bleue peut affecter la germination des spores et la croissance des hyphes des CMA, cependant son mécanisme n'a pas été décrit. Dans le cas de l’horloge circadienne, même si le rythme circadien est omniprésent chez les champignons et que le rythme diurne de la croissance des hyphes a été reporté dans les CMA dans une étude sur le terrain, le mécanisme demeure méconnu. Le génome et le transcriptome du CMA modèle Rhizophagus irregularis isolat DAOM 197198, étaient publiquement disponibles et ils ont été exploité dans mon projet. L'objectif de ma thèse de doctorat visait donc à étudier l'évolution du système ARNi, du mécanisme de détection de la lumière bleue et de l'horloge circadienne dans le génome de R. irregularis à l'aide d'approches biologiques et bioinformatiques. Les objectifs spécifiques étaient de: 1) déterminer si le système ARNi est conservé dans le génome de R. irregularis et d’analyser les traits évolutifs de ses protéines; 2) décrire le mécanisme de détection de la lumière bleue dans le génome R. irregularis ; 3) étudier le mécanisme circadien fongique dans le génome de R. irregularis. J'ai analysé les données génomiques et transcriptomiques pour rechercher les mécanismes conservés du système ARNi de R. irregularis et de certaines espèces de CMA qui lui sont apparentées. Deux phases du cycle de vie de R. irregularis (la phase de la germination des spores et la phase symbiotique avec des racines) ont été utilisées pour déterminer les profils d'expression des gènes en utilisant la PCR quantitative par transcriptase inverse (qPCR). J'ai identifié des traits évolutifs particuliers dans le système ARNi de R. irregularis, tels que le transfert de gènes horizontal (HGT) d’un gène important codant la protéine ribonucléase III, d’origine des cyanobactéries qui n’a jamais été observé chez aucun eucaryote. J'ai également trouvé et identifié un ancien mécanisme de détection de la lumière bleue corrélé à l'horloge circadienne. J’ai trouvé que le gène frequency est conservé dans le génome R. irregularis et que son expression est influencée par l’exposition à la lumière bleue. Ce qui est intéressant est que la protéine la plus importante de l’horloge circadienne (FRQ) n’a jamais été retrouvée dans d’autres lignées primitives fongiques, y compris chez Mucoromycotina, un sous-embranchement fongique considéré comme le plus proche des CMA. Les résultats de mon projet de doctorat a significativement contribuer à la progression de nos connaissances sur les mécanismes importants qui régulent l’expression des gènes chez les CMA qui sont des partenaires symbiotiques des racines des plantes et les plus anciens et les plus répandus dans les écosystèmes. Mes résultats apportent également de nouvelles informations sur le transfert des gènes entre les cyanobactéries et les CMA, et ils ont élargi les connaissances de l'évolution du gène frq chez les champignons. De plus, la présence de gène frq dans le génome de R. irregularis ouvre la voie à l’étude de la chronologique de la symbiose MA, qui peut être le modèle intéressant d’holobiontes des plantes.Arbuscular mycorrhizal (AM) symbiosis is formed by plant roots and arbuscular mycorrhizal fungi (AMF) which are the oldest symbiotic partners of plants and have evolved at least 460 million years ago with the emergence and evolution of land plants. AMF are also the most ubiquitous symbiotic partner of plants as they can colonize more than 80% of vascular plant species. AMF are an early diverged fungal lineage whose phylogenetic position is still under debate. AMF are obligatory plant root symbionts which depend on a source of carbon from the photosynthesis of host plants. In exchange, AMF help plants to absorb various essential soil nutrients, such as phosphorus and transfer these nutrients through their hyphae which have grown into and colonized plant root cells in which they form a structure called an arbuscule. The nutrient allocation and interlocked metabolic pathways between the fungus and the host underwent selective pressure as symbiotic partners. Moreover, AMF hyphae act as an ecological niche for various soil bacteria and other fungi, thus forming the backbone of the rhizospheric part of the plant. AM symbiosis is an essential element to understand plant physiology and ecosystem. Despite the crucial roles of AMF in ecosystems, their genetics and evolution are far from being understood. The RNA interference (RNAi) system, the blue light sensing mechanism and the circadian clock are important mechanisms which regulate expression of various genes in fungi. Although it has an acknowledged role in gene regulation, especially with symbiosis as well as reflected selective pressures on core proteins in the cases of other symbiotic organisms such as nematode worms, the RNAi system has never been considered in AMF. The same is true for the case of blue light sensing mechanisms. Only a few studies showed that blue light can affect spore germination and hyphae growth of AMF, but the mechanism was not addressed. In the case of the circadian clock, even though circadian rhythms are ubiquitous in fungi and a diurnal rhythm of hyphae growth was reported in AMF during a field level study, the mechanism was unknown. Currently, the genome and transcriptome of the model AM fungus Rhizophagus irregularis isolate DAOM 197198, are publicly available and they were used in my studies. The objective of my Ph.D. project was therefore to study the evolution of the RNAi system, blue light sensing mechanisms and the circadian clock in R. irregularis genome using both bioinformatic and molecular biological approaches. The specific objectives of my Ph.D. project were: 1) to investigate whether the RNAi system is conserved in the genome of R. irregularis and explore the evolutionary traits in its core proteins; 2) to describe the blue light sensing mechanism in the genome of R. irregularis; and 3) to search for a fungal circadian mechanism in the genome of R. irregularis. I surveyed genomic and transcriptomic data to search for conserved elements of the RNAi system of R. irregularis and its relatives. Two life stages of the R. irregularis lifecycle (germination of spores without roots and established mycorrhizal symbiosis) were investigated for gene expressional profiles using reverse-transcriptase quantitative PCR (qPCR). I identified particular evolutionary traits in R. irregularis RNAi system, such as horizontal gene transfer (HGT) of its core gene coding ribonuclease III protein from autotrophic cyanobacteria, which has never been reported in any eukaryotes so far. I also found and identified an ancient mechanism of blue light sensing which is related to circadian clock. It was intriguing to find a conserved core gene (frequency) that responds to light exposure in the genome of this underground plant-root symbiont. At the same time, the circadian clock core component (FRQ) was not found in other basal fungal lineages including Mucoromycotina, a fungal subphylum which is considered as the closest relative of AMF. The outcome of my Ph.D. project advanced our knowledge on important mechanisms which regulate the expression of various genes in the oldest and most ubiquitous symbiotic partner of plants. My results also provide new insight on the intimacy between cyanobacteria and AMF which resulted in a unique HGT in the RNAi system. It also expands the knowledge of evolution of the circadian frq gene in fungi. Furthermore, the presence of circadian clock and output genes in R. irregularis opens the door to the chronological study of AM symbiosis, which can be used as a model for the plant holobiont

Antiphase light and temperature cycles disrupt rhythmic plant growth : the Arabidopsis jetlag

Light and temperature are important determinants of plant growth and development. Plant elongation is stimulated by positively increasing differences between day and night temperature (+DIF, phased cycles). In contrast, a negative temperature difference (-DIF, antiphased cycles) reduces elongation growth. In chapter 1the different responses of plants to light and temperature are described. We focus on how light and temperature are perceived and integrated with physiological and molecular pathways to control plant development and architecture. As both light and temperature converge at the circadian oscillator attention is given to temperature entrainment and temperature compensation of the Arabidopsis circadian clock. Finally we discuss the importance of temperature effects on plant growth for horticulture. -DIF is frequently applied in commercial greenhouses to inhibit unwanted elongation of crops. Despite the economic importance, the response of plants to -DIF was poorly understood. Using Arabidopsis thaliana,our research aimed to understand the mechanisms underlying the -DIF response. The main questions that needed to be answered at the start of this project were: (1) At what time during the diurnal day is growth affected by -DIF? (2) What are key genes in the diurnal signalling pathways that result in reduced growth under -DIF? (3) Is the temporal effect of -DIF on growth linked to the circadian clock, and if so how? To answer question 1 (when is growth affected by -DIF?) it was important to develop a monitoring system through which growth could be analysed over the full day, including the dark period. This would allow us to determine how growth proceeds over the day and whether there is a specific period of the day at which growth is most affected by the -DIF regime. Elongation in plants is not constant throughout the day, but exhibits a diurnal rhythm. However, the effect of treatments on growth is usually scored as a cumulative effect after many days. Thus the precise relationship between environmental changes and the daily cycles in the growth of the plant remain mostly unnoticed. More detailed analysis can reveal whether the window of growth or the growth rate itself is affected by the environmental conditions. For this purpose, OSCILLATOR, a growth monitoring system, which allows the analysis and parameterisation of diurnal growth of rosette plants was constructed. The demonstration and validation of OSCILLATOR as growth monitoring system is described in chapter 2. The system consists of IR sensitive cameras and allows time-lapse imaging and subsequent analysis of leaf growth and leaf movement of Arabidopsis, tomato and petunia. We use this system to examine how fluctuating diurnal temperature cycles affect leaf movement in different Arabidopsis ecotypes, demonstrating that this approach allows comparison of various genotypes through parameterisation of rhythmic growth. The analysis by OSCILLATOR showed that diurnal growth is accompanied by a cyclic movement of the growing leaves, and parameters (phase and amplitude) of this diurnal leaf movement can be used as a proxy for growth rate. This facilitated the characterisation of the effect of -DIF on growth. To answer question 2 (what are key genes affected by -DIF) we tested many different mutants impaired in either light signalling, hormone perception, or hormone biosynthesis and studied their response to -DIF in comparison with wild-type plants. Chapter 3 describes how, using this approach, we unravel the light and hormonal signalling processes that mediate the effect of -DIF on leaf movement. Pharmacological treatments combined with the genetic screens identify ethylene signalling as limiting for leaf growth and movement under -DIF. We demonstrate that specifically the activity of the ethylene biosynthesis gene ACC synthase 2activity in the petiole relates to the -DIF leaf phenotype. In addition, the effect of -DIF on ethylene sensitivity and biosynthesis is shown to depend on active PHYB. To further characterise how light and hormone signalling affect growth under -DIF, we set out to identify factors limiting cell elongation. In chapter 4, local cell elongation in the hypocotyl is linked to local auxin signalling capacity. We demonstrate that ethylene, similar to its role in rosette leaves, becomes limiting in this tissue under -DIF as a result of reduced auxin production. While previously overall auxin was shown to be reduced in Arabidopsis inflorescence tissue developed under -DIF, we now demonstrate that it is mainly the effect of tissue specific auxin signalling that limits growth under -DIF. Moreover, we show that auxin can complement growth inhibition under -DIF in wild-type plants but not in ethylene signalling or biosynthesis mutants, placing the effect of auxin on growth upstream of ethylene. Downstream, ethylene signalling activates the growth promoting transcription factor PIF3, which is known to activate genes controlling cell elongation. In contrast, PIF5 acts upstream, possibly regulating the input of the signalling cascade. Remarkably, PIF4, which is a main regulator of heat induced hypocotyl elongation, is not required for the response to -DIF. To answer question 3 (does -DIF affect the clock?) we used luciferase reporter plants and developed a unique luminometer set-up with which we could monitor gene promoter activity in mature rosette plants under different diurnal light regimes. This system was used in penultimate chapter 5 where we demonstrate that an altered function of the circadian clock under -DIF is responsible for altered output processes identified in the other chapters. Analysis of expression patterns of core clock genes under diurnal conditions reveals that -DIF reduces the amplitude of most clock genes and differentially shifts the phase of core clock components. The magnitude and direction of these shifts differ for each clock gene, suggesting that -DIF alters the coordination within the circadian clock itself. We subsequently showed that the phase shifts occurring under -DIF relate to a temperature compensation mechanism controlled by GI. GIwas previously identified to be required for temperature compensation in the amplitude of clock controlled genes at low and high temperature. Moreover, GIwas identified to be responsible for the effect of -DIF on the phase of clock genes. Indeed, gi loss-of-function mutants are insensitive to the effects of -DIF on growth. We demonstrate that under –DIF starch biosynthesis during the day, and starch degradation rates at night are altered. Carbohydrate availability during the night is essential for growth and therefore part of the sugars generated during the photoperiod are stored as starch. Throughout the night this starch is degraded in a controlled rate, which is adjusted to the predicted length of the dark period. The starch degradation rate under different photoperiod lengths is therefore tightly controlled by the circadian clock in anticipation of the expected dawn, to prevent running out of carbohydrates at the end of the night. Indeed, under -DIF starch metabolism is disturbed, resulting in an apparent starch shortage at the end of the night. This was monitored by activation of a reporter gene for carbohydrate starvation under -DIF. Furthermore, the phase of leaf movement of starch mutants under control (+DIF) conditions resembles the phase of wild-type plants developing under -DIF, indicating that the carbohydrate status of a plants determines rhythmic leaf movement. In chapter 6the results obtained in this thesis are discussed and a conceptual model that aims to integrate all findings with recently published literature is proposed. In this model, -DIF affects growth by directly affecting the phase and amplitude of clock genes, which in turn control downstream processes such as starch metabolism and hormone signalling pathways. The auxin and ethylene signalling pathways affected by -DIF show significant crosstalk and interconnect with the circadian clock at several positions, by direct interaction with the PIFs, which are regulated by PHYB, of which transcription is under circadian control. Therefore, special focus is given to the unique position of the photoreceptor PHYB in this model. PHYB is essential for PIF protein stability and in addition is an important component for light entrainment of the clock. Finally we discuss the potential applications of the results described for horticulture and speculate on possible ways to improve the efficiency of DIF like treatments.</p