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.     

Nitric oxide regulates DELLA content and PIF expression to promote photomorphogenesis in Arabidopsis

The transition from etiolated to green seedlings involves a shift from hypocotyl growth-promoting conditions to growth restraint. These changes occur through a complex light-driven process involving multiple and tightly coordinated hormonal signaling pathways. Nitric oxide (NO) has been lately characterized as a regulator of plant development interacting with hormone signaling. Here, we show that Arabidopsis (Arabidopsis thaliana) NO-deficient mutant hypocotyls are longer than those from wild-type seedlings under red light but not under blue or far-red light. Accordingly, exogenous treatment with the NO donor sodium nitroprusside and mutant plants with increased endogenous NO levels resulted in reduced hypocotyl length. In addition to increased hypocotyl elongation, NO deficiency led to increased anthocyanin levels and reduced PHYB content under red light, all processes governed by phytochrome-interacting factors (PIFs). NO-deficient plants accordingly showed an enhanced expression of PIF3, PIF1, and PIF4. Moreover, exogenous NO increased the levels of the gibberellin (GA)-regulated DELLA proteins and shortened hypocotyls, likely through the negative regulation of the GA Insensitive Dwarf1 (GID1)-Sleepy1 (SLY1) module. Consequently, NO-deficient seedlings displayed up-regulation of SLY1, defective DELLA accumulation, and altered GA sensitivity, thus resulting in defective deetiolation under red light. Accumulation of NO in wild-type seedlings undergoing red light-triggered deetiolation and elevated levels of NO in the GA-deficient ga1-3 mutant in darkness suggest a mutual NO-GA antagonism in controlling photomorphogenesis. PHYB-dependent NO production promotes photomorphogenesis by a GID1-GA-SLY1-mediated mechanism based on the coordinated repression of growth-promoting PIF genes and the increase in the content of DELLA proteins.     

HFR1, a phytochrome A-signalling component,acts in a separate pathway from HY5, downstream of COP1 in Arabidopsis thaliana

HFR1, a basic helix-loop-helix protein, is known to be required for a subset of phytochrome A (phyA)-dependent photoresponses. To investigate the role of HFR1 in light signalling, we have examined the genetic interaction between HFR1 and HY5, a positive regulator of light signalling, and COP1, a repressor of photomorphogenesis. Double mutant analysis suggests that HFR1 mediates phyA-dependent inhibition of hypocotyl elongation independently of HY5. HFR1 was shown to be necessary for a subset of cop1-triggered photomorphogenic phenotypes in the dark, including inhibition of hypocotyl elongation, gravitropic hypocotyl growth, and expression of the light-inducible genes CAB and RBCS. Phenotypic analysis of the triple mutant cop1hy5hfr1 indicated that both HFR1 and HY5 are required for cop1-mediated photomorphogenic seedling development in darkness, consistent with their additive roles in phyA-dependent signalling. Taken together, these results suggest that HFR1 might act downstream of COP1, in a separate pathway from HY5, to mediate photomorphogenesis in Arabidopsis.     

Integration of light signaling with photoperiodic flowering and circadian rhythm

PLANT DE-ETIOLATION IS TRIGGERED BY LIGHT SIGNALS Light is arguably the most important resource for plants, and plants have evolved an array of photosensory pig- ments enabling them to develop optimally in a broad range of ambient light conditions. The ph…     

HYPERSENSITIVE TO RED AND BLUE 1 and its C-terminal regulatory function control FLOWERING LOCUS T expression

The red and far-red light-absorbing phytochromes and UV-A/blue light-absorbing cryptochromes regulate seedling de-etiolation and flowering responses. The signaling steps that mediate the photoreceptor regulation on key flowering genes remain largely unknown. We report that a previously identified photomorphogenic mutant, hypersensitive to red and blue 1 (hrb1), flowered late and showed attenuated expression of FLOWERING LOCUS T (FT) over both long days and short days. Transgenic plants that overexpress the full-length HRB1, or its C-terminal half, flowered early and accumulated more FT messages under short-day conditions. The transgenic plants also displayed hyposensitive de-etiolation phenotypes, and the expression of these phenotypes requires the action of PIF4. The double mutant of hrb1/cry2 showed a flowering phenotype and an FT expression pattern similar to hrb1 under long-day conditions, suggesting that HRB1 may function downstream of cry2 under long-day conditions. In contrast, hrb1/phyB-9 showed a flowering phenotype and an FT expression pattern similar to phyB-9 over both long days and short days, indicating a modulatory role of HRB1 in the flowering pathway mediated by phyB. Overexpression of HRB1 did not affect the expression of the central clock oscillators, TOC1 and CCA1. HRB1 therefore represents a signaling step that regulates FT expression downstream of red and blue light perception.     

DFL2, a new member of the Arabidopsis GH3 gene family, is involved in red light-specific hypocotyl elongation

A new GH3-related gene, designated DFL2, causes a short hypocotyl phenotype when overexpressed under red and blue light and a long hypocotyl when antisensed under red light conditions. Higher expression of this gene was observed in continuous white, blue and far-red light but the expression level was low in red light and darkness. DFL2 gene expression was induced transiently with red light pulse treatment. DFL2 transgenic plants exhibited a normal root phenotype including primary root elongation and lateral root formation, although primary root elongation was inhibited in antisense transgenic plants only under red light. The adult phenotypes of sense and antisense transgenic plants were not different from that of wild type. DFL2 promoter activity was observed in the hypocotyl. Our results suggest that DFL2 is located downstream of red light signal transduction and determines the degree of hypocotyl elongation.     

shl, a New set of Arabidopsis mutants with exaggerated developmental responses to available red, far-red, and blue light

The interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light-a threshold condition in which photomorphogenetic signaling pathways are only partially active-for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light. shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant and phyB mutant backgrounds but may be dependent on CRY1 for phenotypic expression in blue light. The shl2 phenotype was partially dependent on PHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent on HY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by CRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC, PHYD, and PHYE).     

BBX32, an Arabidopsis B-Box protein, functions in light signaling by suppressing HY5-regulated gene expression and interacting with STH2/BBX21

A B-box zinc finger protein, B-BOX32 (BBX32), was identified as playing a role in determining hypocotyl length during a large-scale functional genomics study in Arabidopsis (Arabidopsis thaliana). Further analysis revealed that seedlings overexpressing BBX32 display elongated hypocotyls in red, far-red, and blue light, along with reduced cotyledon expansion in red light. Through comparative analysis of mutant and overexpression line phenotypes, including global expression profiling and growth curve studies, we demonstrate that BBX32 acts antagonistically to ELONGATED HYPOCOTYL5 (HY5). We further show that BBX32 interacts with SALT TOLERANCE HOMOLOG2/BBX21, another B-box protein previously shown to interact with HY5. Based on these data, we propose that BBX32 functions downstream of multiple photoreceptors as a modulator of light responses. As such, BBX32 potentially has a native role in mediating gene repression to maintain dark adaptation.     

Control of hypocotyl elongation in Arabidopsis thaliana by photoreceptor interaction

In order to test the interaction of different phytochromes and blue-light receptors, etiolated seedlings of wild-type Arabidopsis thaliana (L.) Heynh., a phytochrome (phy) B-overexpressor line (ABO), and the photoreceptor mutants phyA-201, phyB-5, hy4-2.23n, fha-1, phyA-201/phyB-5, and phyA-201/hy4-2.23n were exposed to red and far-red light pulses after various preirradiations. The responsiveness to the inductive red pulses is primarily mediated by phyB which is rather stable in its far-red-absorbing form as demonstrated by a very slow loss of reversibility. Without preirradiation the red pulses had an impact on hypocotyl elongation only in PHYA mutants but not in the wild type. This indicates a suppression of phyB function by the presence of phyA. Preirradiation with either far-red or blue light resulted in an inhibition of hypocotyl elongation by red pulses in the wild type. Responsiveness amplification by far-red light is mediated by phyA and disappears slowly in the dark. The extent of responsiveness amplification by blue light was identical in the wild type and in the absence of phyA, or the cryptochromes cryl (hy4-2.23n) or cry2 (fha-1). Therefore, we conclude that stimulation of phyB by blue light preirradiation is either mediated by an additional still-unidentified blue-light-absorbing pigment or that phyA, cry1 and cry2 substitute for each other completely. Both blue and red preirradiation established responsiveness to red pulses in phyA-201/phyB-5 double mutants. These results demonstrate that inhibition of hypocotyl elongation by red pulses is not only mediated by phyB but also by a phytochrome(s) other than phyA and phyB. Received: 21 July 1998 / Accepted: 7 December 1998     

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.