Daytime temperature is sensed by phytochrome B in Arabidopsis through a transcriptional activator HEMERA.

Ambient temperature sensing by phytochrome B (PHYB) in Arabidopsis is thought to operate mainly at night. Here we show that PHYB plays an equally critical role in temperature sensing during the daytime. In daytime thermosensing, PHYB signals primarily through the temperature-responsive transcriptional regulator PIF4, which requires the transcriptional activator HEMERA (HMR). HMR does not regulate PIF4 transcription, instead, it interacts directly with PIF4, to activate the thermoresponsive growth-relevant genes and promote warm-temperature-dependent PIF4 accumulation. A missense allele hmr-22, which carries a loss-of-function D516N mutation in HMR's transcriptional activation domain, fails to induce the thermoresponsive genes and PIF4 accumulation. Both defects of hmr-22 could be rescued by expressing a HMR22 mutant protein fused with the transcriptional activation domain of VP16, suggesting a causal relationship between HMR-mediated activation of PIF4 target-genes and PIF4 accumulation. Together, this study reveals a daytime PHYB-mediated thermosensing mechanism, in which HMR acts as a necessary activator for PIF4-dependent induction of temperature-responsive genes and PIF4 accumulation

Investigating the Functional Relationship of Photobodies and Phytochrome Interacting Factors in Phytochrome B Signaling

Photobodies are plant subnuclear membraneless compartments, composed of the highly conserved red and far-red light receptor and temperature sensor, Phytochrome B (PHYB). Since the discovery of photobodies in 1999, many studies have tried to elucidate the exact function of photobodies in phytochrome B signaling, but function of photobodies has remained frustratingly elusive. Active PHYB interacts with a key family of bHLH transcription factors known as Phytochrome Interacting Factors (PIFs) which drive light and temperature signaling within Arabidopsis. The relationship of PIFs and photobodies suggest several possible mechanisms of regulation. My dissertation research investigates how the spatial compartmentalization of PHYB to photobodies regulates PHYB signaling outputs by using PIF5 as a model and how components like PIF5 are recruited and influence photobody formation. In the first chapter, I review what is known about Phytochrome B in its structure, signaling, composition, photobody formation and possible hypotheses of function. To better understand photobodies as biomolecular condensates and PIFs as components of the photobody, discusses concepts of formation such as liquid-liquid phase separation, multivalency, composition and functions of biomolecular condensates that have been applied to emerging plant biomolecular condensate studies. In the second chapter, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR5 (PIF5) to photobodies. Surprisingly, PHYB exerts opposing roles in both degrading and stabilizing PIF5. Altering photobody size by overproducing PHYB created different responses. A moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal a signaling mechanism where photobody formation spatially segregates two antagonistic PHYB signaling actions: one for PIF5 stabilization in photobodies and PIF5 degradation in the surrounding nucleoplasm. These two compartments enables an environmentally-sensitive, counterbalancing mechanism to titrate PIF5 output signaling. In the third chapter, I investigate the scaffold and client model of photobodies where PHYB is a scaffold required for formation and PIF5 is a client which is recruited to photobodies. I show that PIF5 acts as a client of photobodies. I found that PIF5 is required for maintain the size of photobodies and affects the dynamics of PHYB diffusion between the photobody and surrounding nucleoplasm. I also found that the amount of PIF5 relative to PHYB in photobodies may alter the stability of active PHYB photobody formation. Finally, in the fourth chapter I investigate how PIF5 is recruited to photobodies by examining the structural features of PIF5. PIFs have a conserved Active Phytochrome B binding motif (APB) that interacts with the N-terminal of PHYB. PIF5 also has a predicted prion-like domain which is involved in liquid-liquid phase separation. I found that the C-terminal end of PIF5 containing the predicted prion-like domain is not required for photobody recruitment. I also found that while the APB of PIF5 alone is not sufficient for recruitment to the photobody, it confers specificity to PIF5 photobody localization. Interestingly the bHLH domain may play an important role in photobody interaction as well. Together, my dissertation reveals a complex role of photobodies as a site to control signaling outputs of their components like PIF5. I also reveal that clients of photobodies like PIF5, have specific recruitment to photobodies and can alter photobody dynamics the concentrations of the scaffold PHYB in relation to the client PIF5 may be critical for proper maintenance. This work advances our knowledge of the significance biomolecular condensate function in plants and the complexities of regulating signaling that can be applied to future work in the field within both animal and plants

Daytime temperature is sensed by phytochrome B in Arabidopsis through a transcriptional activator HEMERA.

Ambient temperature sensing by phytochrome B (PHYB) in Arabidopsis is thought to operate mainly at night. Here we show that PHYB plays an equally critical role in temperature sensing during the daytime. In daytime thermosensing, PHYB signals primarily through the temperature-responsive transcriptional regulator PIF4, which requires the transcriptional activator HEMERA (HMR). HMR does not regulate PIF4 transcription, instead, it interacts directly with PIF4, to activate the thermoresponsive growth-relevant genes and promote warm-temperature-dependent PIF4 accumulation. A missense allele hmr-22, which carries a loss-of-function D516N mutation in HMR's transcriptional activation domain, fails to induce the thermoresponsive genes and PIF4 accumulation. Both defects of hmr-22 could be rescued by expressing a HMR22 mutant protein fused with the transcriptional activation domain of VP16, suggesting a causal relationship between HMR-mediated activation of PIF4 target-genes and PIF4 accumulation. Together, this study reveals a daytime PHYB-mediated thermosensing mechanism, in which HMR acts as a necessary activator for PIF4-dependent induction of temperature-responsive genes and PIF4 accumulation

Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B.

Photoactivated phytochrome B (PHYB) binds to antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to regulate hundreds of light responsive genes in Arabidopsis by promoting PIF degradation. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (AD) consisting of a hydrophobic activator motif flanked by acidic residues. A PIF3mAD mutant, in which the activator motif is replaced with alanines, fails to activate PIF3 target genes in Arabidopsis, validating the functions of the PIF3 AD in vivo. Intriguingly, the N-terminal photosensory module of PHYB binds immediately adjacent to the PIF3 AD to repress PIF3's transactivation activity, demonstrating a novel PHYB signaling mechanism through direct interference of the transactivation activity of PIF3. Our findings indicate that PHYB, likely also PHYA, controls the stability and activity of PIFs via structurally separable dual signaling mechanisms

Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B.

Photoactivated phytochrome B (PHYB) binds to antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to regulate hundreds of light responsive genes in Arabidopsis by promoting PIF degradation. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (AD) consisting of a hydrophobic activator motif flanked by acidic residues. A PIF3mAD mutant, in which the activator motif is replaced with alanines, fails to activate PIF3 target genes in Arabidopsis, validating the functions of the PIF3 AD in vivo. Intriguingly, the N-terminal photosensory module of PHYB binds immediately adjacent to the PIF3 AD to repress PIF3's transactivation activity, demonstrating a novel PHYB signaling mechanism through direct interference of the transactivation activity of PIF3. Our findings indicate that PHYB, likely also PHYA, controls the stability and activity of PIFs via structurally separable dual signaling mechanisms

Analysis of Outcomes in Ischemic vs Nonischemic Cardiomyopathy in Patients With Atrial Fibrillation A Report From the GARFIELD-AF Registry

IMPORTANCE Congestive heart failure (CHF) is commonly associated with nonvalvular atrial fibrillation (AF), and their combination may affect treatment strategies and outcomes