Mediator, a central coregulator of transcription, has been identified as a large protein complex in eukaryotes ranging from yeast to man. It is therefore remarkable that Mediator has not yet been identified within the plant kingdom. Here we identify Mediator in a plant, Arabidopsis thaliana. The plant Mediator subunits typically show very low homology to other species, but our biochemical purification identifies 21 conserved and six A. thaliana-specific Mediator subunits. Most notably, we identify the A. thaliana proteins STRUWWELPETER (SWP) and PHYTOCHROME AND FLOWERING TIME 1 (PFT1) as the Med14 and Med25 subunits, respectively. These findings show that specific plant Mediator subunits are linked to the regulation of specialized processes such as the control of cell proliferation and the regulation of flowering time in response to light quality. The identification of the plant Mediator will provide new tools and insights into the regulation of transcription in plants.

Multiple steps of plant growth and development rely on rapid cell elongation during which secretory and endocytic trafficking via the trans -Golgi network (TGN) plays a central role. Here, we identify the ECHIDNA (ECH) protein from Arabidopsis thaliana as a TGN-localized component crucial for TGN function. ECH partially complements loss of budding yeast TVP23 function and a Populus ECH complements the Arabidopsis ech mutant, suggesting functional conservation of the genes. Compared with wild-type, the Arabidopsis ech mutant exhibits severely perturbed cell elongation as well as defects in TGN structure and function, manifested by the reduced association between Golgi bodies and TGN as well as mislocalization of several TGN-localized proteins including vacuolar H + -ATPase subunit a1 (VHA-a1). Strikingly, ech is defective in secretory trafficking, whereas endocytosis appears unaffected in the mutant. Some aspects of the ech mutant phenotype can be phenocopied by treatment with a specific inhibitor of vacuolar H + -ATPases, concanamycin A, indicating that mislocalization of VHA-a1 may account for part of the defects in ech . Hence, ECH is an evolutionarily conserved component of the TGN with a central role in TGN structure and function.

A variety of experimental tests have been applied to the methylene-blue-sensitized photooxidation of amino acids to distinguish between singlet oxygen and non-singlet oxidation mechanisms. Conventional flash photolysis and laser photolysis were used to measure the rate constants for the quenching of excited triplet sensitizer and singlet oxygen by the amino acids histidine. tryptophan and methionine and the nucleotide guanosine-5'-monophosphate. In the case of histidine, the rate constants alone rule out an oxidation mechanism involving direct reaction with excited dye. With the other amino acids, and with guanosine monophosphate, the oxidation rates might be accounted for by either mechanism. The inhibition of the photooxidation of both tryptophan and methionine as well as histidine by the singlet-oxygen quenchers N3-and tetramethylethylene suggests that these reactions occur via a singlet-oxygen mechanism. A newly developed test of singlet oxygen reactions involving a comparison of photooxidation rates in normal and perdeuterated solvents has been used to establish that the photooxidation of tryptophan proceeds primarily by a singlet-oxygen mechanism. These experiments appear to constitute the first proof that singlet oxygen is involved in the photooxidation of the three amino acids tryptophan, methionine and histidine. I N T R O D U C T I O NINSIGHT into the mechanism of photodynamic action has been seriously hampered by the lack of knowledge concerning the primary events in the dye-sensitized photooxidations of biologically important compounds in the presence of oxygen[ 1,2].Although it has been amply demonstrated that oxygen is involved[ 1,2], the nature of the primary oxidizing species still remains to be identified. In trying to deduce the mechanism of any dye-sensitized photooxidation, there are at least two major pathways to be considered, including: (a) direct reaction between sensitizer triplets and the oxidizable substrate, followed eventually by reaction with oxygen, and (b) reaction between oxidizable substrates and singlet oxygen which has been generated by energy transfer from triplet-state sensitizers.In pathway (a) there are many possible mechanisms whereby oxygen can participate in the processes following the initial reaction between dye triplet, 3 S , and substrate. For example, molecular oxygen may reoxidize semireduced dye intermediates. Alternatively, it may act as an electron acceptor and cause irreversible formation of oxidized products by electron transfer from half-oxidized substrate molecules (X') as follows:3s+x-+ s -+ xx*+o, + x+ + 0,-2 0 , -+ 2 H + + 02+H20,