Light, in a quality-and quantity-dependent fashion, induces nuclear import of the plant photoreceptors phytochrome, promotes interaction of phytochrome A (phyA) and phyB with transcription factors including phytochrome interacting factor 3 (PIF3), and is thought to trigger a transcriptional cascade to regulate the expression of ;2500 genes in Arabidopsis thaliana. Here, we show that controlled degradation of the transcription factor PIF3 is a major regulatory step in light signaling. We demonstrate that accumulation of PIF3 in the nucleus in dark requires constitutive photomorphogenesis 1 (COP1), a negative regulator of photomorphogenesis, and show that red (R) and far-red light (FR) induce rapid degradation of the PIF3 protein. This process is controlled by the concerted action of the R/FR absorbing phyA, phyB, and phyD photoreceptors, and it is not affected by COP1. Rapid light-induced degradation of PIF3 indicates that interaction of PIF3 with these phytochrome species is transient. In addition, we provide evidence that the poc1 mutant, a postulated PIF3 overexpressor that displays hypersensitivity to R but not to FR, lacks detectable amounts of the PIF3 protein. Thus, we propose that PIF3 acts transiently, and its major function is to mediate phytochrome-induced signaling during the developmental switch from skotomorphogenesis to photomorphogenesis and/or dark to light transitions.

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.

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Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (l max = 730 nm) and inactive Pr (l max = 660 nm) forms in a red/far-red-dependent fashion and regulates, as molecular switch, many aspects of lightdependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein-protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB-yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB Ser86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red lightinduced nuclear import and interaction of phyB Ser86Asp -YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB-green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.

The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.

Studies of de novo cytokinin biosynthesis in isopentenyltransferase (ipt)-transformed Arabidopsis thaliana, involving in vivo deuterium labeling and mass spectrometry, showed that the biosynthetic rate of zeatinriboside-5 -monophosphate was around 66-fold higher than that of isopentenyladenosine-5 -monophosphate (iPMP), the proposed primary product of the Agrobacterium ipt. Double tracer analysis, using [ 2 H6] isopentenyladenosine and deuterium oxide, provided evidence for an alternative, iPMPindependent, biosynthetic pathway for zeatin-type cytokinins, present in both ipt-expressing and wild-type Arabidopsis thaliana. Reduction of the biosynthetic flux in the alternative pathway by use of mevastatin, an inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase, indicated a terpenoid origin for the side-chain precursor of the iPMP independent pathway. C ytokinins are an important class of plant growth regulators, defined by their ability to promote cell division in tissue culture in the presence of auxins (1, 2). Virtually all naturally occurring cytokinins identified to date are adenine species substituted at N 6 with an isoprenoid or aromatic side chain. In this text, cytokinins will refer solely to the isoprenoid cytokinin bases and their sugar conjugates. Cytokinins affect many plant developmental processes including cell division, cell differentiation, chlorophyll senescence, and apical dominance (3).Efforts to elucidate the biosynthetic origin of cytokinins in plants have been inconclusive. Early suggestions that tRNA degradation could be the major source of free, active cytokinins (4) were disproved when calculations of tRNA turnover rates showed that a tRNA-independent de novo biosynthetic pathway also must be present in plants (5). A major breakthrough was the discovery of a cytokinin biosynthetic enzyme in the slime mold, Dictyostelium discoideum (6). Cell-free extracts from this organism can convert AMP and dimethylallyl-pyrophosphate (DMAPP) to the free cytokinins isopentenyladenosine-5Ј-monophosphate (iPMP) and the corresponding nucleoside (isopentenyladenosine, iPA). This finding, and studies on the metabolism of isopentenyl-type cytokinins (7,8), led to the proposal that iPMP is also the primary cytokinin intermediate in plants, and zeatin cytokinins are formed by hydroxylation of iPMP and its derivatives (9, 10) ( Fig. 1).Later, the product of the T-DNA gene 4 (ipt) of the crown gall-forming bacterium, Agrobacterium tumefaciens, also was described as a DMAPP:AMP isopentenyltransferase (ipt) (11,12). AMP was found to be the preferred adenylic substrate for the Agrobacterium ipt enzyme but searches for alternative side-chain donors have been limited. However, when de novo biosynthesis in crown gall tissue of Vinca rosea was traced with 14 C-adenine, in vivo production of iPMP was undetectable, whereas zeatinriboside-5Ј-monophospate (ZMP) production was strong (13). After studies of 14 C-isopententyladenine metabolism in this system, it was proposed that the very low levels of iPMP were caused by rapi...

The cryptochrome blue light photoreceptors mediate various photomorphogenic responses in plants, including hypocotyl elongation, cotyledon expansion, and control of flowering time. The molecular mechanism of cryptochrome function in Arabidopsis is becoming increasingly clear, with recent studies showing that both CRY1 and CRY2 are localized in the nucleus and that CRY2 is regulated by blue light-dependent phosphorylation. Despite these advances, no positive cryptochrome signaling component has been identified to date. Here, we demonstrate that a novel Ser/Thr protein phosphatase (AtPP7) with high sequence similarity to the Drosophila retinal degeneration C protein phosphatase acts as an intermediate in blue light signaling. Transgenic Arabidopsis seedlings with reduced AtPP7 expression levels exhibit loss of hypocotyl growth inhibition and display limited cotyledon expansion in response to blue light irradiation. These effects are as striking as those seen in hy4 mutant seedlings, which are deficient in CRY1. We further demonstrate that AtPP7 transcript levels are not rate limiting and that AtPP7 probably acts downstream of cryptochrome in the nucleus, ensuring signal flux through the pathway. Based on our findings and recent data regarding cryptochrome action, we propose that AtPP7 acts as a positive regulator of cryptochrome signaling in Arabidopsis.