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Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear membraneless organelles named photobodies (PBs). However, the function of PBs in PHYB signaling remains frustratingly elusive. Here, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) to PBs. Surprisingly, PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while 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 that PB formation spatially segregates two antagonistic PHYB signaling actions – PIF5 stabilization in PBs and PIF5 degradation in the surrounding nucleoplasm – which could enable an environmentally sensitive, counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses.

 

Photobodies (PBs) are membraneless subnuclear organelles that self-assemble via concentration-dependent liquid-liquid phase separation (LLPS) of the plant photoreceptor and thermosensor phytochrome B (PHYB). The current PHYB LLPS model posits that PHYB phase separates randomly in the nucleoplasm regardless of the cellular or nuclear context. Here, we established a robust Oligopaints method inArabidopsisto determine the positioning of individual PBs. We show surprisingly that even in PHYB overexpression lines – where PHYB condensation would be more likely to occur randomly – PBs positioned at twelve distinct subnuclear locations distinguishable by chromocenter and nucleolus landmarks, suggesting that PHYB condensation occurs nonrandomly at preferred seeding sites. Intriguingly, warm temperatures reduce PB number by inducing the disappearance of specific thermo-sensitive PBs, demonstrating that individual PBs possess different thermosensitivities. These results reveal a nonrandom PB nucleation model, which provides the framework for the biogenesis of spatially distinct individual PBs with diverse environmental sensitivities within a single plant nucleus.

 

Light initiates chloroplast biogenesis inArabidopsisby eliminating PHYTOCHROME-INTERACTING transcription FACTORs (PIFs), which in turn de-represses nuclear photosynthesis genes, and synchronously, generates a nucleus-to-plastid (anterograde) signal that activates the plastid-encoded bacterial-type RNA polymerase (PEP) to transcribe plastid photosynthesis genes. However, the identity of the anterograde signal remains frustratingly elusive. The main challenge has been the difficulty to distinguish regulators from the plethora of necessary components for plastid transcription and other essential chloroplast functions, such as photosynthesis. Here, we show that the genome-wide induction of nuclear photosynthesis genes is insufficient to activate the PEP. PEP inhibition is imposed redundantly by multiple PIFs and requires PIF3’s activator activity. Among the nuclear-encoded components of the PEP holoenzyme, we identify four light-inducible, PIF-repressed sigma factors as anterograde signals. Together, our results elucidate that light-dependent inhibition of PIFs activates plastid photosynthesis genes via sigma factors as anterograde signals in parallel with the induction of nuclear photosynthesis genes.

 

Abstract 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.