Dimerization and blue light regulation of PIF1 interacting bHLH proteins in Arabidopsis

Phytochrome Interacting Factor 1 (PIF1), a basic helix-loop-helix (bHLH) protein, functions as a negative regulator of various facets of photomorphogenesis. To indentify PIF1-interacting proteins, we performed yeast two-hybrid screening using PIF1 as a bait and identified a group of proteins including PIF1 itself, PIF3 and long hypocotyl in far-red 1 (HFR1), an atypical HLH protein. Directed yeast two-hybrid interaction assays showed that PIF1 can form heterodimers with all other PIFs as well as with HFR1. PIF1 and PIF3 interacted with each other in both in vitro and in vivo co-immunoprecipitation assays. PIF1-PIF3 heterodimer also bound to a G-box DNA sequence element in vitro. To understand the biological significance of these interactions, a pif1pif3 double mutant was obtained and characterized. Analyses of the single and double mutants showed that PIF3 plays a prominent role in repressing photomorphogenesis under continuous blue light conditions. pif1 and pif3 showed additive phenotypes more prominently under discontinuous blue light conditions. Similar to PIF1, PIF3 was also rapidly phosphorylated, poly-ubiquitylated and degraded in response to blue light. PIF3 also interacted with phytochromes in response to blue light. A PIF3 mutant defective in interaction with both phyA and phyB displayed reduced degradation under blue light, suggesting that phy-interaction was necessary for the blue light-induced degradation of PIF3. Taken together, these data suggest a combinatorial control of photomorphogenesis by bHLH proteins in response to light in Arabidopsis.     

The Arabidopsis phytochrome-interacting factor PIF7, together with PIF3 and PIF4, regulates responses to prolonged red light by modulating phyB levels

We show that a previously uncharacterized Arabidopsis thaliana basic helix-loop-helix (bHLH) phytochrome interacting factor (PIF), designated PIF7, interacts specifically with the far-red light-absorbing Pfr form of phyB through a conserved domain called the active phyB binding motif. Similar to PIF3, upon light exposure, PIF7 rapidly migrates to intranuclear speckles, where it colocalizes with phyB. However, in striking contrast to PIF3, this process is not accompanied by detectable light-induced phosphorylation or degradation of PIF7, suggesting that the consequences of interaction with photoactivated phyB may differ among PIFs. Nevertheless, PIF7 acts similarly to PIF3 in prolonged red light as a weak negative regulator of phyB-mediated seedling deetiolation. Examination of pif3, pif4, and pif7 double mutant combinations shows that their moderate hypersensitivity to extended red light is additive. We provide evidence that the mechanism by which these PIFs operate on the phyB signaling pathway under prolonged red light is through maintaining low phyB protein levels, in an additive or synergistic manner, via a process likely involving the proteasome pathway. These data suggest that the role of these phyB-interacting bHLH factors in modulating seedling deetiolation in prolonged red light may not be as phy-activated signaling intermediates, as proposed previously, but as direct modulators of the abundance of the photoreceptor.     

Heat Shock–Induced Fluctuations in Clock and Light Signaling Enhance Phytochrome B–Mediated Arabidopsis Deetiolation

Moderately warm constant ambient temperatures tend to oppose light signals in the control of plant architecture. By contrast, here we show that brief heat shocks enhance the inhibition of hypocotyl growth induced by light perceived by phytochrome B in deetiolating Arabidopsis thaliana seedlings. In darkness, daily heat shocks transiently increased the expression of PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9 and markedly enhanced the amplitude of the rhythms of LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression. In turn, these rhythms gated the hypocotyl response to red light, in part by changing the expression of PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5. After light exposure, heat shocks also reduced the nuclear abundance of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and increased the abundance of its target ELONGATED HYPOCOTYL5 (HY5). The synergism between light and heat shocks was deficient in the prr7 prr9, lhy cca1, pif4 pif5, cop1, and hy5 mutants. The evening element (binding site of LHY and CCA1) and G-box promoter motifs (binding site of PIFs and HY5) were overrepresented among genes with expression controlled by both heat shock and red light. The heat shocks experienced by buried seedlings approaching the surface of the soil prepare the seedlings for the impending exposure to light by rhythmically lowering LHY, CCA1, PIF4, and PIF5 expression and by enhancing HY5 stability.     

Diurnal dependence of growth responses to shade in Arabidopsis: role of hormone, clock, and light signaling

We investigated the diurnal dependence of the hypocotyl-growth responses to shade under sunlight-night cycles in Arabidopsis thaliana. Afternoon shade events promoted hypocotyl growth, while morning shade was ineffective. The lhy-D, elf3, lux, pif4 pif5, toc1, and quadruple della mutants retained the response to afternoon shade and the lack of response to morning shade while the lhy cca1 mutant responded to both morning and afternoon shade. The phyB mutant, plants overexpressing the multidrug resistance-like membrane protein ABCB19, and the iaa17/axr3 loss-of-function mutant failed to respond to shade. Transient exposure of sunlight-grown seedlings to synthetic auxin in the afternoon caused a stronger promotion of hypocotyl growth than morning treatments. The promotion of hypocotyl growth by afternoon shade or afternoon auxin required light perceived by phytochrome A or cryptochromes during the previous hours of the photoperiod. Although the ELF4-ELF3-LUX complex, PIF4, PIF5, and DELLA are key players in the generation of diurnal hypocotyl-growth patterns, they exert a minor role in the control of the diurnal pattern of growth responses to shade. We conclude that the strong diurnal dependency of hypocotyl-growth responses to shade relates to the balance between the antagonistic actions of LHY-CCA1 and a light-derived signal.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.