Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, ...despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500–630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.
Cryptochromes are blue light-activated photoreceptors found in multiple organisms with significant similarity to photolyases, a class of light-dependent DNA repair enzymes. Unlike photolyases, ...cryptochromes do not repair DNA and instead mediate blue light-dependent developmental, growth, and/or circadian responses by an as yet unknown mechanism of action. It has recently been shown that Arabidopsis cryptochrome-1 retains photolyase-like photoreduction of its flavin cofactor FAD by intraprotein electron transfer from tryptophan and tyrosine residues. Here we demonstrate that substitution of two conserved tryptophans that are constituents of the flavin-reducing electron transfer chain in Escherichia coli photolyase impairs light-induced electron transfer in the Arabidopsis cryptochrome-1 photoreceptor in vitro. Furthermore, we show that these substitutions result in marked reduction of light-activated autophosphorylation of cryptochrome-1 in vitro and of its photoreceptor function in vivo, consistent with biological relevance of the electron transfer reaction. These data support the possibility that light-induced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.
Cryptochromes are blue-light receptors controlling multiple aspects of plant growth and development. They are flavoproteins with significant homology to photolyases, but instead of repairing DNA they ...function by transducing blue light energy into a signal that can be recognized by the cellular signaling machinery. Here we report the effect of cry1 and cry2 blue light receptors on primary root growth in Arabidopsis thaliana seedlings, through analysis of both cryptochrome-mutant and cryptochrome-overexpressing lines. Cry1 mutant seedlings show reduced root elongation in blue light while overexpressing seedlings show significantly increased elongation as compared to wild type controls. By contrast, the cry2 mutation has the opposite effect on root elongation growth as does cry1, demonstrating that cry1 and cry2 act antagonistically in this response pathway. The site of cryptochrome signal perception is within the shoot, and the inhibitor of auxin transport, 1-N-naphthylphthalamic acid, abolishes the differential effect of cryptochromes on root growth, suggesting the blue-light signal is transmitted from the shoot to the root by a mechanism that involves auxin. Primary root elongation in blue light may thereby involve interaction between cryptochrome and auxin signaling pathways.
In Crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) is subject to day-night regulatory phosphorylation of a conserved serine residue in the plant enzyme's N-terminal ...domain. The dark increase in PEPC-kinase (PEPC-k) activity is under control of a circadian oscillator, via the enhanced expression of the corresponding gene (1). The signaling cascade leading to PEPC-k up-regulation was investigated in leaves and mesophyll cell protoplasts of the facultative, salt-inducible CAM species, Mesembryanthemum crystallinum. Mesophyll cell protoplasts had the same PEPC-k activity as leaves from which they were prepared (i.e., high at night, low during the day). However, unlike C4 protoplasts (2), CAM protoplasts did not show marked PEPC-k up-regulation when isolated during the day and treated with a weak base such as NH4Cl. Investigations using various pharmacological reagents established the operation, in the darkened CAM leaf, of a PEPC-k cascade including the following components: a phosphoinositide-dependent phospholipase C (PI-PLC), inositol 1,4,5 P (IP3)-gated tonoplast calcium channels, and a putative Ca2+/calmodulin protein kinase. These results suggest that a similar signaling machinery is involved in both C4 (2, 3) and CAM plants to regulate PEPC-k activity, the phosphorylation state of PEPC, and, thus, carbon flux through this enzyme during CAM photosynthesis.
Phosphoenolpyruvate carboxylase (EC 4.1.1.31: PEPC) was characterized in de-embryonated
Sorghum seeds, focusing on the interaction between metabolites and posttranslational control of the enzyme by ...phosphorylation. Two PEPC polypeptides (108 and 110 kDa) were resolved by SDS/PAGE and shown to increase, in parallel with PEPC activity during seed germination. PEPC displayed very low
K
m values for PEP (90 μM) and inhibition constant (IC
50) for
l-malate (75 μM) in desalted protein extracts from de-embryonated dry seeds. The inhibition of PEPC by 0.16 mM
l-malate, pH 7.3, decreased from 70 to 30%, along with a consistent increase in IC
50 (75–220 μM) after 5 days of germination. PEPC phosphorylation was established both in vivo, after imbibing the seeds with
32Pphosphate, and in vitro in reconstituted assays. A PEPC kinase (PEPCk) was partially purified from seed protein extracts by blue dextran agarose chromatography and shown to be independent of calcium and to phosphorylate both seed and recombinant C
4 PEPC from
Sorghum on the enzyme’s N-terminal domain. Seed germination, PEPC accumulation and phosphorylation were severely inhibited in the presence of NaCl in the imbibing medium, although PEPCk content was not altered. However, in vitro, NaCl had no effect on both PEPCk activity and PEPC phosphorylation. On the other hand,
l-malate was a potent inhibitor of seed PEPCk activity in in vitro assays. Since NaCl also decreased the rate of
l-malate consumption in the imbibing grain, the salt inhibition of PEPC phosphorylation was suggested to be due to the concentration-dependent blocking of PEPCk activity in vivo by this compound. Consistent with these data, germination and PEPC phosphorylation were inhibited, while PEPCk levels were not altered, when seeds were germinated in the presence of
l-malate.
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