Conserved phenylalanine residues are required for blue-shifting of cyanobacteriochrome photoproducts.

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Both phytochromes and CBCRs reversibly convert between dark-stable and photoproduct states upon photoisomerization of their linear tetrapyrrole (bilin) chromophores. While most phytochromes convert between a red-absorbing dark state and a far-red-absorbing photoproduct, CBCRs exhibit spectral responses spanning the entire near-ultraviolet and visible spectrum. For example, red/green CBCRs such as AnPixJ and NpR6012g4 exhibit a red-absorbing dark state similar to that of phytochrome, but photoconversion yields a green-absorbing photoproduct. "Teal-DXCF" CBCRs convert from blue- or green-absorbing dark states to yield photoproducts with very narrow absorption in the teal region of the spectrum (approximately 500 nm). The recent determination of a crystal structure of AnPixJ in its red-absorbing dark state led to the proposal that movement of a Trp residue (the "lid Trp") upon photoconversion would allow solvation of the photoproduct, thereby producing a large blue-shift. We find that substitution of the lid Trp has little effect on the NpR6012g4 photoproduct. Instead, two Phe residues conserved in red/green and teal-DXCF CBCRs are essential for determining photoproduct absorption in both CBCR groups with no significant influence on the dark-adapted state. We propose that these Phe residues constrain chromophore movement after primary photoisomerization. This work supports a trapped-twist mechanism for generating both red/green and teal-DXCF photoproducts.

Phycoviolobilin formation and spectral tuning in the DXCF cyanobacteriochrome subfamily.

Phytochromes are red/far-red photosensory proteins that regulate adaptive responses to light via photoswitching of cysteine-linked linear tetrapyrrole (bilin) chromophores. The related cyanobacteriochromes (CBCRs) extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. CBCRs and phytochromes share a conserved Cys residue required for bilin attachment. In one CBCR subfamily, often associated with a blue/green photocycle, a second Cys lies within a conserved Asp-Xaa-Cys-Phe (DXCF) motif and is essential for the blue/green photocycle. Such DXCF CBCRs use isomerization of the phycocyanobilin (PCB) chromophore into the related phycoviolobilin (PVB) to shorten the conjugated system for sensing green light. We here use recombinant expression of individual CBCR domains in Escherichia coli to survey the DXCF subfamily from the cyanobacterium Nostoc punctiforme. We describe ten new photoreceptors with well-resolved photocycles and three additional photoproteins with overlapping dark-adapted and photoproduct states. We show that the ability of this subfamily to form PVB or retain PCB provides a powerful mechanism for tuning the photoproduct absorbance, with blue-absorbing dark states leading to a broad range of photoproducts absorbing teal, green, yellow, or orange light. Moreover, we use a novel green/teal CBCR that lacks the blue-absorbing dark state to demonstrate that PVB formation requires the DXCF Cys residue. Our results demonstrate that this subfamily exhibits much more spectral diversity than had been previously appreciated.

Neonicotinoid insecticides: oxidative stress in planta and metallo-oxidase inhibition.

Neonicotinoids not only control insect pests but also sometimes independently alter plant growth and response to stress. We find that imidacloprid, thiacloprid, acetamiprid, thiamethoxam, and clothianidin but not nitenpyram and dinotefuran induce foliar lesions and peroxidative damage in soybean ( Glycine max ) seedlings assayed with the 3,3'-diaminobenzidine stain. The chloropyridinyl-carboxylic acid (COOH) but not the -carboxaldehyde (CHO) metabolites induce peroxidative damage but in a different pattern. Surprisingly, the chlorothiazolyl -CHO and -COOH metabolites induce chlorosis but no clear superimposable peroxidative damage or cell death. Four metallo-oxidases known to modulate reactive oxygen species were not sensitive in vitro to the parent neonicotinoid itself but were to several CHO and COOH metabolites and related compounds, with a sensitivity order of CHO > COOH and tyrosinase > xanthine oxidase and aldehyde oxidase > catalase. Although metallo-oxidase inhibition does not correlate overall with lesion formation, it may play an as yet unknown role in plant response to neonicotinoids.

Neonicotinoid insecticides induce salicylate-associated plant defense responses.

Neonicotinoid insecticides control crop pests based on their action as agonists at the insect nicotinic acetylcholine receptor, which accepts chloropyridinyl- and chlorothiazolyl-analogs almost equally well. In some cases, these compounds have also been reported to enhance plant vigor and (a)biotic stress tolerance, independent of their insecticidal function. However, this mode of action has not been defined. Using Arabidopsis thaliana, we show that the neonicotinoid compounds, imidacloprid (IMI) and clothianidin (CLO), via their 6-chloropyridinyl-3-carboxylic acid and 2-chlorothiazolyl-5-carboxylic acid metabolites, respectively, induce salicylic acid (SA)-associated plant responses. SA is a phytohormone best known for its role in plant defense against pathogens and as an inducer of systemic acquired resistance; however, it can also modulate abiotic stress responses. These neonicotinoids effect a similar global transcriptional response to that of SA, including genes involved in (a)biotic stress response. Furthermore, similar to SA, IMI and CLO induce systemic acquired resistance, resulting in reduced growth of a powdery mildew pathogen. The action of CLO induces the endogenous synthesis of SA via the SA biosynthetic enzyme ICS1, with ICS1 required for CLO-induced accumulation of SA, expression of the SA marker PR1, and fully enhanced resistance to powdery mildew. In contrast, the action of IMI does not induce endogenous synthesis of SA. Instead, IMI is further bioactivated to 6-chloro-2-hydroxypyridinyl-3-carboxylic acid, which is shown here to be a potent inducer of PR1 and inhibitor of SA-sensitive enzymes. Thus, via different mechanisms, these chloropyridinyl- and chlorothiazolyl-neonicotinoids induce SA responses associated with enhanced stress tolerance.

S-Arachidonoyl-2-thioglycerol synthesis and use for fluorimetric and colorimetric assays of monoacylglycerol lipase.

We report the first synthesis of 2-thioglycerol and S-arachidonoyl-2-thioglycerol (the thioester analog of the endocannabinoid 2-arachidonoylglycerol) in an eight or nine step procedure with a yield of approximately 25% and establish the use of this substrate for maleimide-based fluorescent and dithiobis(2-nitrobenzoic acid)-based colorimetric assays of human recombinant monoacylglycerol (MAG) lipase (hMAGL) and human brain membrane MAG hydrolase activity. Inhibitor structure-activity relationships observed here for hMAGL and 2-ATG correlate well (r(2)=0.93, n=9) with earlier findings for mouse brain MAG hydrolase with non-thiol substrates.