The Function and Regulation of Photobodies in Phytochrome Signaling

Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant Arabidopsis thaliana, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies.

Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.

Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (PBG), and one in which it cannot (NGB). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the NGB line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in NGB regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.

In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); hmr mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak hmr allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, SON OF HEMERA (SOH). The soh mutations rescue all of the phenotypes associated with the weak hmr allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null soh alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.

In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, SON OF HEMERA, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.

Belief and recollection of autobiographical memories.

In three experiments, undergraduates rated autobiographical memories on scales derived from existing theories of memory. In multiple regression analyses, ratings of the degree to which subjects recollected (i.e., relived) their memories were predicted by visual imagery, auditory imagery, and emotions, whereas ratings of belief in the accuracy of their memories were predicted by knowledge of the setting. Recollection was predicted equally well in between- and within-subjects analyses, but belief consistently had smaller correlations and multiple regression predictions between subjects; individual differences in the cognitive scales that we measured could not account well for individual differences in belief. In contrast, measures of mood (Beck Depression Index) and dissociation (Dissociative Experience Scale) added predictive value for belief, but not for recollection. We also found that highly relived memories almost always had strong visual images and that remember/know judgments made on autobiographical memories were more closely related to belief than to recollection.

Road Traffic Injury Prevention Initiatives: A Systematic Review and Metasummary of Effectiveness in Low and Middle Income Countries.

BACKGROUND: Road traffic injuries (RTIs) are a growing but neglected global health crisis, requiring effective prevention to promote sustainable safety. Low- and middle-income countries (LMICs) share a disproportionately high burden with 90% of the world's road traffic deaths, and where RTIs are escalating due to rapid urbanization and motorization. Although several studies have assessed the effectiveness of a specific intervention, no systematic reviews have been conducted summarizing the effectiveness of RTI prevention initiatives specifically performed in LMIC settings; this study will help fill this gap. METHODS: In accordance with PRISMA guidelines we searched the electronic databases MEDLINE, EMBASE, Scopus, Web of Science, TRID, Lilacs, Scielo and Global Health. Articles were eligible if they considered RTI prevention in LMICs by evaluating a prevention-related intervention with outcome measures of crash, RTI, or death. In addition, a reference and citation analysis was conducted as well as a data quality assessment. A qualitative metasummary approach was used for data analysis and effect sizes were calculated to quantify the magnitude of emerging themes. RESULTS: Of the 8560 articles from the literature search, 18 articles from 11 LMICs fit the eligibility and inclusion criteria. Of these studies, four were from Sub-Saharan Africa, ten from Latin America and the Caribbean, one from the Middle East, and three from Asia. Half of the studies focused specifically on legislation, while the others focused on speed control measures, educational interventions, enforcement, road improvement, community programs, or a multifaceted intervention. CONCLUSION: Legislation was the most common intervention evaluated with the best outcomes when combined with strong enforcement initiatives or as part of a multifaceted approach. Because speed control is crucial to crash and injury prevention, road improvement interventions in LMIC settings should carefully consider how the impact of improvements will affect speed and traffic flow. Further road traffic injury prevention interventions should be performed in LMICs with patient-centered outcomes in order to guide injury prevention in these complex settings.