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.

Plant genomes are extremely sensitive to, and can be developmentally reprogrammed by environmental light cues. Here using rolling-circle amplification of gene-specific circularizable oligonucleotides coupled with fluorescence in situ hybridization, we demonstrate that light triggers a rapid repositioning of the Arabidopsis light-inducible chlorophyll a/b-binding proteins (CAB) locus from the nuclear interior to the nuclear periphery during its transcriptional activation. CAB repositioning is mediated by the red/far-red photoreceptors phytochromes (PHYs) and is inhibited by repressors of PHY signalling, including COP1, DET1 and PIFs. CAB repositioning appears to be a separate regulatory step occurring before its full transcriptional activation. Moreover, the light-inducible loci RBCS, PC and GUN5 undergo similar repositioning behaviour during their transcriptional activation. Our study supports a light-dependent gene regulatory mechanism in which PHYs activate light-inducible loci by relocating them to the nuclear periphery; it also provides evidence for the biological importance of gene positioning in the plant kingdom.

Warm temperature is postulated to induce plant thermomorphogenesis through a signaling mechanism similar to shade, as both destabilize the active form of the photoreceptor and thermosensor phytochrome B (phyB). At the cellular level, shade antagonizes phyB signaling by triggering phyB disassembly from photobodies. Here we report temperature-dependent photobody localization of fluorescent protein-tagged phyB (phyB-FP) in the epidermal cells of Arabidopsis hypocotyl and cotyledon. Our results demonstrate that warm temperature elicits different photobody dynamics than those by shade. Increases in temperature from 12 °C to 27 °C incrementally reduce photobody number by stimulating phyB-FP disassembly from selective thermo-unstable photobodies. The thermostability of photobodies relies on phyB’s photosensory module. Surprisingly, elevated temperatures inflict opposite effects on phyB’s functions in the hypocotyl and cotyledon despite inducing similar photobody dynamics, indicative of tissue/organ-specific temperature signaling circuitry either downstream of photobody dynamics or independent of phyB. Our results thus provide direct cell biology evidence supporting an early temperature signaling mechanism via dynamic assembly/disassembly of individual photobodies possessing distinct thermostabilities.

Summary: Calreticulin (CRT) is an endoplasmic reticulum‐resident calcium‐binding molecular chaperone that is highly conserved in multi‐cellular eukaryotes. Higher plants contain two distinct groups of CRTs: CRT1/CRT2 and CRT3 isoforms. Previous studies have shown that bacterial elongation factor Tu receptor (EFR), a pattern‐recognition receptor that is responsible for pathogen‐associated molecular pattern‐triggered immunity, is a substrate for Arabidopsis CRT3, suggesting a role for CRT3 in regulating plant defense against pathogens. Here we report that Arabidopsis CRT2 is another regulator of plant innate immunity. Despite significantly increased salicylic acid levels and constitutive expression of the systemic acquired resistance‐associated marker genes PR1, PR2 and PR5, transgenic plants over‐expressing CRT2 displayed reduced resistance to virulent Pseudomonas syringae pv. tomato DC3000 (PstDC3000). A ⁴⁵Ca²⁺ overlay assay and a domain‐swapping experiment further demonstrated that the negatively charged C‐terminal tail of CRT2 is responsible for its high calcium‐binding capacity and function in regulating the endogenous salicylic acid level. In addition, over‐expression of the His173 mutant of CRT2 greatly enhanced plant defense against PstDC3000, supporting the existence of a self‐inhibition mechanism that can counteract the effects of salicylic acid‐dependent immune responses. These results suggest that CRT2 functions through its N‐terminal domain(s) as a self‐modulator that can possibly prevent the salicylic acid‐mediated runaway defense responses triggered by its C‐terminal calcium‐buffering activity in response to pathogen invasion.

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.