Functional analysis of various conserved domains of NPH3 involved in phototropism in Arabidopsis thaliana

Abstract only availablePhototropism - the directional growth (curvature) for a plant towards light, is a very important adaptive response in plants in order for them to maximize photosynthesis. Blue light triggers phototropic response in Arabidopsis thaliana via the dominant photoreceptor phototropin 1 (phot1). Genetic studies have identified several genes that encode phot1 interacting proteins. Of these, currently, only NON PHOTOTROPIC HYPOCOTYL 3 (NPH3) is known to be absolutely required for phototropism. nph3 mutants are completely aphototropic, resembling phot1 null mutants. NPH3 is a phosphoprotein containing a BTB and a coiled coil domain, and the dephosphorylation of this protein into its active state in light is known to be entirely phot1 dependant. Yet little is known of the role of NPH3 as a mediator in the phototropic signal-response pathway. To better understand the role of conserved domains of NPH3 in phototropism, a range of serial deletions and mutants of NPH3 were generated, driven by its own native NPH3 promoter and the constitutive CaMV 35S promoter in nph3-6 and wild-type Col-0 backgrounds respectively. All truncated and mutant NPH3 proteins were also translationally fused with a green fluorescent protein (GFP). Multiple T3 homozygous transgenic lines were evaluated by comparing average angles of hypocotyl curvature with those of aphotoropic nph3-6 and wild-type Col-0. Over-expression of NPH3 or different portions of NPH3 in Col-0 resulted in reduced phototropism. Selective expression of the NPH3 domains under the native promoter could not complement the null nph3-6 phenotype. The alterations in the subcellular localization of these transgenic lines were also investigated using confocal fluorescence microscopy.NSF Plant Genomics Internship @ M

Physiological responses of a hyper-phototropic mutant to various light stimuli [abstract]

Abstract only availableFaculty Mentor: Mannie Liscum, Biological SciencesPhysiological responses of a hyper-phototropic mutant to various light stimuli Phototropic responses are the directional curvature of organs in response to differences in light intensity and/or quality. The experimental plant is the hyper-phototropic hypocotyl () mutant that is associated specifically with phototropism and is hyper-responsive to blue light stimulation. In wild-type seedlings the response is dependent upon the fluence of blue light (number of incident of photons) used to stimulate seedlings. In this analysis seedlings were exposed to several fluence rates of blue light. Results from these experiments suggest that the phenotypes are specific to lower fluence rates. Additionally, red light which has been shown to enhance the phototropic response to blue light was used as a pretreatment to assess whether is altered in its response to red light as well as blue. Results from this latter analysis show that mutant seedling still exhibit a higher degree of curvature than control samples, suggesting the red light enhances the phototropic response of as well. Together these experiments suggest that affects a repressor of phototropin 1-dependent phototropism in low blue light conditions.University of Missouri--Columbia. Office of Undergraduate ResearchPlant Genomics Internship @ M

Natural variation in phenotypes associated with phototropins among geographically isolated populations of Arabidopsis thaliana [abstract]

Abstract only availableIn Arabidopsis thaliana several phenotypes are controlled by blue light including chloroplast relocation movements and bending of the seedling stem in phototropism. These responses are controlled by the phototropins, phot1 and phot2. Chloroplasts move towards the brightest area in a plant cell under low light intensity to maximize light interception (accumulation response), but toward darker areas under high intensity light to minimize photo-damage (avoidance response). Phot1 and phot2 control the accumulation movement, but phot2 alone controls the avoidance response. Phototropism in response to weak blue light is controlled solely by phot1. Here, we have tested seventeen different accessions of A. thaliana for phenotypes uniquely associated with phot1 and phot2 and examined variation in phot1 and phot2 sequence data for a small subset. Accessions were chosen to better understand the ecological context of variation in phototropin function in nature.National Science Foundation grant to M. Liscu

Genome-Wide Identification, Functional Analysis and Expression Profiling of the Aux/IAA Gene Family in Tomato

Auxin is a central hormone that exerts pleiotropic effects on plant growth including the development of roots, shoots, flowers and fruit. The perception and signaling of the plant hormone auxin rely on the cooperative action of several components,among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. In this study, we identified and comprehensively analyzed the entire Aux/IAA gene family in tomato (Solanum lycopersicum), a reference species for Solanaceae plants, and the model plant for fleshy fruit development. Functional characterization using a dedicated single cell system revealed that tomato Aux/IAA proteins function as active repressors of auxin-dependent gene transcription, with, however, different Aux/IAA members displaying varying levels of repression. Phylogenetic analysis indicated that the Aux/IAA gene family is slightly contracted in tomato compared with Arabidopsis, with a lower representation of non-canonical proteins. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. The data presented here shed more light on Sl-IAA genes and provides new leads towards the elucidation of their function during plant development and in mediating hormone cross-talk

Phototropins maintain robust circadian oscillation of PSII operating efficiency under blue light

The circadian system allows plants to coordinate metabolic and physiological functions with predictable environmental variables such as dusk and dawn. This endogenous oscillator is comprised of biochemical and transcriptional rhythms that are synchronized with a plant's surroundings via environmental signals, including light and temperature. We have used chlorophyll fluorescence techniques to describe circadian rhythms of PSII operating efficiency (Fq′/Fm′) in the chloroplasts of Arabidopsis thaliana. These Fq′/Fm′ oscillations appear to be influenced by transcriptional feedback loops previously described in the nucleus, and are induced by rhythmic changes in photochemical quenching over circadian time. Our work reveals that a family of blue photoreceptors, phototropins, maintain robust rhythms of Fq′/Fm′ under constant blue light. As phototropins do not influence circadian gene expression in the nucleus our imaging methodology highlights differences between the modulation of circadian outputs in distinct subcellular compartments

Determination of phototropism by UV-B radiation

UV-B phototropism in etiolated Arabidopsis seedlings has only been shown recently and needs further exploration. Here we elaborate on how to generate a customized setup with a unilateral UV-B light source, the required plant materials, different growth substrates, and a framework for data analysis

Automation of ORIGEN2 calculations for the transuranic waste baseline inventory database using a pre-processor and a post-processor

The purpose of the work described in this report was to automate ORIGEN2 calculations for the Waste Isolation Pilot Plant (WIPP) Transuranic Waste Baseline Inventory Database (WTWBID); this was done by developing a pre-processor to generate ORIGEN2 input files from WWBID inventory files and a post-processor to remove excess information from the ORIGEN2 output files. The calculations performed with ORIGEN2 estimate the radioactive decay and buildup of various radionuclides in the waste streams identified in the WTWBID. The resulting radionuclide inventories are needed for performance assessment calculations for the WIPP site. The work resulted in the development of PreORG, which requires interaction with the user to generate ORIGEN2 input files on a site-by-site basis, and PostORG, which processes ORIGEN2 output into more manageable files. Both programs are written in the FORTRAN 77 computer language. After running PreORG, the user will run ORIGEN2 to generate the desired data; upon completion of ORIGEN2 calculations, the user can run PostORG to process the output to make it more manageable. All the programs run on a 386 PC or higher with a math co-processor or a computer platform running under VMS operating system. The pre- and post-processors for ORIGEN2 were generated for use with Rev. 1 data of the WTWBID and can also be used with Rev. 2 and 3 data of the TWBID (Transuranic Waste Baseline Inventory Database)

Mathematically modelling the dynamics of cholesterol metabolism and ageing

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the UK. This conditionbecomes increasingly prevalent during ageing; 34.1% and 29.8% of males and females respectively, over 75years of age have an underlying cardiovascular problem. The dysregulation of cholesterol metabolism isinextricably correlated with cardiovascular health and for this reason low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol (HDL-C) are routinely used as biomarkers of CVD risk. Theaim of this work was to use mathematical modelling to explore how cholesterol metabolism is affectedby the ageing process. To do this we updated a previously published whole-body mathematical model ofcholesterol metabolism to include an additional 96 mechanisms that are fundamental to this biologicalsystem. Additional mechanisms were added to cholesterol absorption, cholesterol synthesis, reversecholesterol transport (RCT), bile acid synthesis, and their enterohepatic circulation. The sensitivity of themodel was explored by the use of both local and global parameter scans. In addition, acute cholesterolfeeding was used to explore the effectiveness of the regulatory mechanisms which are responsible formaintaining whole-body cholesterol balance. It was found that our model behaves as a hypo-responderto cholesterol feeding, while both the hepatic and intestinal pools of cholesterol increased significantly.The model was also used to explore the effects of ageing in tandem with three different cholesterolester transfer protein (CETP) genotypes. Ageing in the presence of an atheroprotective CETP genotype,conferring low CETP activity, resulted in a 0.6% increase in LDL-C. In comparison, ageing with a genotypereflective of high CETP activity, resulted in a 1.6% increase in LDL-C. Thus, the model has illustrated theimportance of CETP genotypes such as I405V, and their potential role in healthy ageing

The Arabidopsis JAGGED LATERAL ORGANS (JLO) gene sensitizes plants to auxin

Plant growth and development of new organs depend on the continuous activity of the meristems. In the shoot, patterns of organ initiation are determined by PINFORMED (PIN)-dependent auxin distribution, while the undifferentiated state of meristem cells requires activity of KNOTTED LIKE HOMEOBOX (KNOX) transcription factors. Cell proliferation and differentiation of the root meristem are regulated by the largely antagonistic functions of auxin and cytokinins. It has previously been shown that the transcription factor JAGGED LATERAL ORGANS (JLO), a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) family, coordinates KNOX and PIN expression in the shoot and promotes root meristem growth. Here we show that JLO is required for the establishment of the root stem cell niche, where it interacts with the auxin/PLETHORA pathway. Auxin signaling involves the AUX/IAA co-repressor proteins, ARF transcription factors and F-box receptors of the TIR1/AFB1–5 family. Because jlo mutants fail to degrade the AUX/IAA protein BODENLOS, root meristem development is inhibited. We also demonstrate that the expression levels of two auxin receptors, TIR1 and AFB1, are controlled by JLO dosage, and that the shoot and root defects of jlo mutants are alleviated in jlo plants expressing TIR1 and AFB1 from a transgene. The finding that the auxin sensitivity of a plant can be differentially regulated through control of auxin receptor expression can explain how different developmental processes can be integrated by the activity of a key transcription factor

Characterization of the cork oak transcriptome dynamics during acorn development

Background: Cork oak (Quercus suber L.) has a natural distribution across western Mediterranean regions and is a keystone forest tree species in these ecosystems. The fruiting phase is especially critical for its regeneration but the molecular mechanisms underlying the biochemical and physiological changes during cork oak acorn development are poorly understood. In this study, the transcriptome of the cork oak acorn, including the seed, was characterized in five stages of development, from early development to acorn maturation, to identify the dominant processes in each stage and reveal transcripts with important functions in gene expression regulation and response to water. Results: A total of 80,357 expressed sequence tags (ESTs) were de novo assembled from RNA-Seq libraries representative of the several acorn developmental stages. Approximately 7.6 % of the total number of transcripts present in Q. suber transcriptome was identified as acorn specific. The analysis of expression profiles during development returned 2,285 differentially expressed (DE) transcripts, which were clustered into six groups. The stage of development corresponding to the mature acorn exhibited an expression profile markedly different from other stages. Approximately 22 % of the DE transcripts putatively code for transcription factors (TF) or transcriptional regulators, and were found almost equally distributed among the several expression profile clusters, highlighting their major roles in controlling the whole developmental process. On the other hand, carbohydrate metabolism, the biological pathway most represented during acorn development, was especially prevalent in mid to late stages as evidenced by enrichment analysis. We further show that genes related to response to water, water deprivation and transport were mostly represented during the early (S2) and the last stage (S8) of acorn development, when tolerance to water desiccation is possibly critical for acorn viability. Conclusions: To our knowledge this work represents the first report of acorn development transcriptomics in oaks. The obtained results provide novel insights into the developmental biology of cork oak acorns, highlighting transcripts putatively involved in the regulation of the gene expression program and in specific processes likely essential for adaptation. It is expected that this knowledge can be transferred to other oak species of great ecological value.Fundação para a Ciência e a Tecnologi