The Arabidopsis endosperm is a temperature-sensing tissue that implements seed thermoinhibition through phyB.

Seed thermoinhibition, the repression of germination under high temperatures, prevents seedling establishment under potentially fatal conditions. Thermoinhibition is relevant for phenology and agriculture, particularly in a warming globe. The temperature sensing mechanisms and signaling pathways sustaining thermoinhibition are unknown. Here we show that thermoinhibition in Arabidopsis thaliana is not autonomously controlled by the embryo but is rather implemented by the endosperm. High temperature is sensed through endospermic phyB by accelerating its reversion from the active signaling Pfr form into the inactive Pr form, as previously described in seedlings. This leads to thermoinhibition mediated by PIFs, mainly PIF1, PIF3 and PIF5. Endospermic PIF3 represses the expression of the endospermic ABA catabolic gene CYP707A1 and promotes endospermic ABA accumulation and release towards the embryo to block its growth. Furthermore, endospermic ABA represses embryonic PIF3 accumulation that would otherwise promote embryonic growth. Hence, under high temperatures PIF3 exerts opposite growth responses in the endosperm and embryo.

The Sequential Action of MIDA9/PP2C.D1, PP2C.D2, and PP2C.D5 Is Necessary to Form and Maintain the Hook After Germination in the Dark.

During seedling etiolation after germination in the dark, seedlings have closed cotyledons and form an apical hook to protect the meristem as they break through the soil to reach the surface. Once in contact with light, the hook opens and cotyledons are oriented upward and separate. Hook development in the dark after seedling emergence from the seed follows three distinctly timed and sequential phases: formation, maintenance, and eventual opening. We previously identified MISREGULATED IN DARK9 (MIDA9) as a phytochrome interacting factor (PIF)-repressed gene in the dark necessary for hook development during etiolated growth. MIDA9 encodes the type 2C phosphatase PP2C.D1, and pp2c-d1/mida9 mutants exhibit open hooks in the dark. Recent evidence has described that PP2C.D1 and other PP2C.D members negatively regulate SMALL AUXIN UP RNA (SAUR)-mediated cell elongation. However, the fundamental question of the timing of PP2C.D1 action (and possibly other members of the PP2C.D family) during hook development remains to be addressed. Here, we show that PP2C.D1 is required immediately after germination to form the hook. pp2c.d1/mida9 shows reduced cell expansion in the outer layer of the hook and, therefore, does not establish the differential cell growth necessary for hook formation, indicating that PP2C.D1 is necessary to promote cell elongation during this early stage. Additionally, genetic analyses of single and high order mutants in PP2C.D1, PP2C.D2, and PP2C.D5 demonstrate that the three PP2C.Ds act collectively and sequentially during etiolation: whereas PP2C.D1 dominates hook formation, PP2C.D2 is necessary during the maintenance phase, and PP2C.D5 acts to prevent opening during the third phase together with PP2C.D1 and PP2C.D2. Finally, we uncover a possible connection of PP2C.D1 levels with ethylene physiology, which could help optimize hook formation during post-germinative growth in the dark.

Molecular convergence of clock and photosensory pathways through PIF3-TOC1 interaction and co-occupancy of target promoters.

A mechanism for integrating light perception and the endogenous circadian clock is central to a plant's capacity to coordinate its growth and development with the prevailing daily light/dark cycles. Under short-day (SD) photocycles, hypocotyl elongation is maximal at dawn, being promoted by the collective activity of a quartet of transcription factors, called PIF1, PIF3, PIF4, and PIF5 (phytochrome-interacting factors). PIF protein abundance in SDs oscillates as a balance between synthesis and photoactivated-phytochrome-imposed degradation, with maximum levels accumulating at the end of the long night. Previous evidence shows that elongation under diurnal conditions (as well as in shade) is also subjected to circadian gating. However, the mechanism underlying these phenomena is incompletely understood. Here we show that the PIFs and the core clock component Timing of CAB expression 1 (TOC1) display coincident cobinding to the promoters of predawn-phased, growth-related genes under SD conditions. TOC1 interacts with the PIFs and represses their transcriptional activation activity, antagonizing PIF-induced growth. Given the dynamics of TOC1 abundance (displaying high postdusk levels that progressively decline during the long night), our data suggest that TOC1 functions to provide a direct output from the core clock that transiently constrains the growth-promoting activity of the accumulating PIFs early postdusk, thereby gating growth to predawn, when conditions for cell elongation are optimal. These findings unveil a previously unrecognized mechanism whereby a core circadian clock output signal converges immediately with the phytochrome photosensory pathway to coregulate directly the activity of the PIF transcription factors positioned at the apex of a transcriptional network that regulates a diversity of downstream morphogenic responses.

Branching of the PIF3 regulatory network in Arabidopsis: roles of PIF3-regulated MIDAs in seedling development in the dark and in response to light.

Plants need to accurately adjust their development after germination in the underground darkness to ensure survival of the seedling, both in the dark and in the light upon reaching the soil surface. Recent studies have established that the photoreceptors phytochromes and the bHLH phytochrome interacting factors PIFs regulate seedling development to adjust it to the prevailing light environment during post-germinative growth. However, complete understanding of the downstream regulatory network implementing these developmental responses is still lacking. In a recent work, published in The Plant Cell, we report a subset of PIF3-regulated genes in dark-grown seedlings that we have named MIDAs (MISREGULATED IN DARK). Analysis of their functional relevance using mutants showed that four of them present phenotypic alterations in the dark, and that each affected a particular facet of seedling development, suggesting organ-specific branching in the signal that PIF3 relays downstream. Furthermore, our results also showed an altered response to light in seedlings with an impaired PIF3/MIDA regulatory network, indicating that these factors might also be essential to initiate and optimize the developmental adjustment of the seedling to the light environment.

Phytochrome-imposed oscillations in PIF3 protein abundance regulate hypocotyl growth under diurnal light/dark conditions in Arabidopsis.

Arabidopsis seedlings display rhythmic growth when grown under diurnal conditions, with maximal elongation rates occurring at the end of the night under short-day photoperiods. Current evidence indicates that this behavior involves the action of the growth-promoting bHLH factors PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) at the end of the night, through a coincidence mechanism that combines their transcriptional regulation by the circadian clock with control of protein accumulation by light. To assess the possible role of PIF3 in this process, we have analyzed hypocotyl responses and marker gene expression in pif single- and higher-order mutants. The data show that PIF3 plays a prominent role as a promoter of seedling growth under diurnal light/dark conditions, in conjunction with PIF4 and PIF5. In addition, we provide evidence that PIF3 functions in this process through its intrinsic transcriptional regulatory activity, at least in part by directly targeting growth-related genes, and independently of its ability to regulate phytochrome B (phyB) levels. Furthermore, in sharp contrast to PIF4 and PIF5, our data show that the PIF3 gene is not subject to transcriptional regulation by the clock, but that PIF3 protein abundance oscillates under diurnal conditions as a result of a progressive decline in PIF3 protein degradation mediated by photoactivated phyB, and consequent accumulation of the bHLH factor during the dark period. Collectively, the data suggest that phyB-mediated, post-translational regulation allows PIF3 accumulation to peak just before dawn, at which time it accelerates hypocotyl growth, together with PIF4 and PIF5, by directly regulating the induction of growth-related genes.

Functional profiling identifies genes involved in organ-specific branches of the PIF3 regulatory network in Arabidopsis.

The phytochrome (phy)-interacting basic helix-loop-helix transcription factors (PIFs) constitutively sustain the etiolated state of dark-germinated seedlings by actively repressing deetiolation in darkness. This action is rapidly reversed upon light exposure by phy-induced proteolytic degradation of the PIFs. Here, we combined a microarray-based approach with a functional profiling strategy and identified four PIF3-regulated genes misexpressed in the dark (MIDAs) that are novel regulators of seedling deetiolation. We provide evidence that each one of these four MIDA genes regulates a specific facet of etiolation (hook maintenance, cotyledon appression, or hypocotyl elongation), indicating that there is branching in the signaling that PIF3 relays. Furthermore, combining inferred MIDA gene function from mutant analyses with their expression profiles in response to light-induced degradation of PIF3 provides evidence consistent with a model where the action of the PIF3/MIDA regulatory network enables an initial fast response to the light and subsequently prevents an overresponse to the initial light trigger, thus optimizing the seedling deetiolation process. Collectively, the data suggest that at least part of the phy/PIF system acts through these four MIDAs to initiate and optimize seedling deetiolation, and that this mechanism might allow the implementation of spatial (i.e., organ-specific) and temporal responses during the photomorphogenic program.

The highly immunogenic enolase and Hsp70p are adventitious Candida albicans cell wall proteins.

Screening cDNA libraries with polyclonal antibody preparations against Candida albicans yeast or mycelial cell walls resulted in isolation of several positive clones. Some of them encoded enolase; others encoded a protein of the 70 kDa heat-shock protein family (Hsp70p), etc. The presence of these cytosolic proteins in the cell wall of actively growing C. albicans was discovered by analytical (SDS-PAGE and Western blot) and cytological (indirect immunofluorescence) experiments. Supplementation of cell cultures with papulacandin B, an antibiotic that inhibits formation of the beta-glucan skeleton, resulted in the release of enolase to the supernatant fluids; this release was prevented when 0.6 M KCl was present as an osmotic stabilizer. The cell wall of C. albicans incorporated exogenously added proteins (enolase and Escherichia coli and C., albicans cytosolic proteins). The presence in the C. albicans cell wall of enolase, Hsp70p, and probably other intracellular proteins that are highly immunogenic might help the fungal cells to evade the host defences, and consequently could represent a survival mechanism for C. albicans 'in vivo'.