Polycomb group (PcG) proteins are evolutionarily conserved in animals and plants, and play critical roles in the regulation of developmental gene expression. Here we show that the Arabidopsis Polycomb repressive complex 2 (PRC2) subunits CURLY LEAF (CLF), EMBRYONIC FLOWER 2 (EMF2) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) repress the expression of FLOWERING LOCUS C (FLC), a central repressor of the floral transition in Arabidopsis and FLC relatives. In addition, CLF directly interacts with and mediates the deposition of repressive histone H3 lysine 27 trimethylation (H3K27me3) into FLC and FLC relatives, which suppresses active histone H3 lysine 4 trimethylation (H3K4me3) in these loci. Furthermore, we show that during vegetative development CLF and FIE strongly repress the expression of FLOWERING LOCUS T (FT), a key flowering-time integrator, and that CLF also directly interacts with and mediates the deposition of H3K27me3 into FT chromatin. Our results suggest that PRC2-like complexes containing CLF, EMF2 and FIE, directly interact with and deposit into FT, FLC and FLC relatives repressive trimethyl H3K27 leading to the suppression of active H3K4me3 in these loci, and thus repress the expression of these flowering genes. Given the central roles of FLC and FT in flowering-time regulation in Arabidopsis, these findings suggest that the CLF-containing PRC2-like complexes play a significant role in control of flowering in Arabidopsis.

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Bioorthogonal chemistry provides an exciting new strategy to visualize protein expression, track protein localization, measure protein activity, and identify protein interaction partners in living systems.[1] Two steps are typically involved in this approach: 1) the incorporation of a bioorthogonal group into a protein through either a biochemical pathway or semisynthesis; 2) a site-specific reaction between the protein that carries the bioorthogonal group and a cognate small-molecule probe. Although a plethora of methods have been developed to address the first step, such as non-sense suppression mutagenesis, [2] expressed protein ligation, [3] metabolic engineering, [4] and tagging-via-substrate, [5] only a small number of bioorthogonal reactions are known for the second step. These site-specific reactions include the acid-catalyzed nucleophilic addition of hydrazine to a ketone or aldehyde, [6] Staudinger ligation, [7] Cu I -catalyzed azide-alkyne 1,3-dipolar cycloaddition (click chemistry), [8] strain-promoted azide-alkyne 1,3-dipolar cycloaddition, [9] and the oxidative coupling of aniline.[10] To fully realize the potential of bioorthogonal chemistry in probing protein function, there is an urgent need for the discovery of additional bioorthogonal reactions with robust reaction attributes. Herein, we report a bioorthogonal, photoinducible 1,3-dipolar cycloaddition reaction that allows rapid and highly selective modification of proteins carrying a diaryl tetrazole group in biological media.Forty years ago, Huisgen and co-workers reported a photoactivated 1,3-dipolar cycloaddition reaction between 2,5-diphenyltetrazole and methyl crotonate.[11] A concerted reaction mechanism was proposed, whereby the diaryl tetrazole undergoes a facile cycloreversion reaction upon photoirradiation to release N 2 and generate in situ a nitrile imine dipole, which cyclizes spontaneously with an alkene dipolarophile to afford a pyrazoline cycloadduct (Scheme 1). The photolysis of diaryl tetrazoles was found to be extremely efficient upon UV irradiation at 290 nm, with quantum yields in the range 0.5-0.9.[12] Despite its robust mechanism, this photoactivated reaction has seen very few applications in the past four decades.[13]In our initial studies, we identified an extremely mild photoactivation procedure in the use of a hand-held UV lamp from UVP (UVM-57, 302 nm, 115 V, 0.16 amps). Under these mild conditions, the solvent compatibility, functional-group tolerance, regioselectivity, and yield of the photoactivated 1,3-dipolar cycloaddition reaction were excellent. [14] We then examined the reaction kinetics by incubating a tetrazole peptide with acrylamide in phosphate-buffered saline (PBS) at pH 7.5 under UV light (302 nm; see Figure S1 in the Supporting Information). We found that the photolysis of the tetrazole peptide to generate the nitrile imine intermediate was extremely rapid, with a first-order rate constant k 1 = 0.14 s À1 ; the subsequent cycloaddition with the dipolarophile acrylamide proceeded very efficiently, w...

We report a tetrazole-based, photoclick chemistry that can be employed to selectively functionalize an alkene genetically encoded in a protein inside E. coli cells. The reaction involved the treatment of E. coli cells with cell-permeable tetrazoles followed by a brief photo irradiation at 302 nm (4 min) and an overnight incubation at 4 degrees C. This in vivo alkene functionalization procedure was simple, straightforward, and nontoxic to E. coli cells. Additionally, fluorescent adducts were formed, facilitating the monitoring of the reaction in vivo. This reaction should offer a new tool for the study of alkene-containing proteins in living systems.