Photobody localization of Arabidopsis (Arabidopsis thaliana) phytochrome B (phyB) fused to green fluorescent protein (PBG) correlates closely with the photoinhibition of hypocotyl elongation. However, the amino-terminal half of phyB fused to green fluorescent protein (NGB) is hypersensitive to light despite its inability to localize to photobodies. Therefore, the significance of photobodies in regulating hypocotyl growth remains debatable. Accumulating evidence indicates that under diurnal conditions, photoactivated phyB persists into darkness to inhibit hypocotyl elongation. Here, we examine whether photobodies are involved in inhibiting hypocotyl growth in darkness by comparing the PBG and NGB lines after the red light-to-dark transition. Surprisingly, after the transition from 10 mmol m 22 s 21 red light to darkness, PBG inhibits hypocotyl elongation three times longer than NGB. The disassembly of photobodies in PBG hypocotyl nuclei correlates tightly with the accumulation of the growth-promoting transcription factor PHYTOCHROME-INTERACTING FACTOR3 (PIF3). Destabilizing photobodies by either decreasing the light intensity or adding monochromatic far-red light treatment before the light-to-dark transition leads to faster PIF3 accumulation and a dramatic reduction in the capacity for hypocotyl growth inhibition in PBG. In contrast, NGB is defective in PIF3 degradation, and its hypocotyl growth in the dark is nearly unresponsive to changes in light conditions. Together, our results support the model that photobodies are required for the prolonged, light-dependent inhibition of hypocotyl elongation in the dark by repressing PIF3 accumulation and by stabilizing the far-red light-absorbing form of phyB. Our study suggests that photobody localization patterns of phyB could serve as instructive cues that control light-dependent photomorphogenetic responses in the dark.

Plant phytochromes are thought to transduce light signals by mediating the degradation of phytochrome-interacting transcription factors (PIFs) through the N-terminal photosensory module, while the C-terminal module, including a histidine kinase-related domain (HKRD), does not participate in signaling. Here we show that the C-terminal module of Arabidopsis phytochrome B (PHYB) is sufficient to mediate the degradation of PIF3 specifically and to activate photosynthetic genes in the dark. The HKRD is a dimerization domain for PHYB homo and heterodimerization. A D1040V mutation, which disrupts the dimerization of HKRD and the interaction between C-terminal module and PIF3, abrogates PHYB nuclear accumulation, photobody biogenesis, and PIF3 degradation. By contrast, disrupting the interaction between PIF3 and PHYB’s N-terminal module has little effect on PIF3 degradation. Together, this study demonstrates that the dimeric form of the C-terminal module plays important signaling roles by targeting PHYB to subnuclear photobodies and interacting with PIF3 to trigger its degradation.

The yeast Chk2/Chk1 homolog Rad53 is a central component of the DNA damage checkpoint system. While it controls genotoxic stress responses such as cell cycle arrest, replication fork stabilization and increase in dNTP pools, little is known about the consequences of reduced Rad53 levels on the various cellular endpoints or about its roles in dealing with chronic vs. acute genotoxic challenges. Using a tetraploid gene dosage model in which only one copy of the yeast RAD53 is functional (simplex), we found that the simplex strain was not sensitive to acute UV radiation or chronic MMS exposure. However, the simplex strain was sensitized to chronic exposure of the ribonucleotide reductase inhibitor hydroxyurea (HU). Surprisingly, reduced RAD53 gene dosage did not affect sensitivity to HU acute exposure, indicating that immediate checkpoint responses and recovery from HU-induced stress were not compromised. Interestingly, cells of most of the colonies that arise after chronic HU exposure acquired heritable resistance to HU. We also found that short HU exposure before and after treatment of G2 cells with ionizing radiation (IR) reduced the capability of RAD53 simplex cells to repair DSBs, in agreement with sensitivity of RAD53 simplex strain to high doses of IR. We propose that a modest reduction in Rad53 activity can impact the activation of the ribonucleotide reductase catalytic subunit Rnr1 following stress, reducing the ability to generate nucleotide pools sufficient for DNA repair and replication. At the same time, reduced Rad53 activity may lead to genome instability and to the acquisition of drug resistance before and/or during the chronic exposure to HU. These results have implications for developing drug enhancers as well as for understanding mechanisms of drug resistance in cells compromised for DNA damage checkpoint.