Immunocytochemical determination of the subcellular distribution of ascorbate in plants

Ascorbate is an important antioxidant in plants and fulfills many functions related to plant defense, redox signaling and modulation of gene expression. We have analyzed the subcellular distribution of reduced and oxidized ascorbate in leaf cells of Arabidopsis thaliana and Nicotiana tabacum by high-resolution immuno electron microscopy. The accuracy and specificity of the applied method is supported by several observations. First, preadsorption of the ascorbate antisera with ascorbic acid or dehydroascorbic acid resulted in the reduction of the labeling to background levels. Second, the overall labeling density was reduced between 50 and 61% in the ascorbate-deficient Arabidopsis mutants vtc1-2 and vtc2-1, which correlated well with biochemical measurements. The highest ascorbate-specific labeling was detected in nuclei and the cytosol whereas the lowest levels were found in vacuoles. Intermediate labeling was observed in chloroplasts, mitochondria and peroxisomes. This method was used to determine the subcellular ascorbate distribution in leaf cells of plants exposed to high light intensity, a stress factor that is well known to cause an increase in cellular ascorbate concentration. High light intensities resulted in a strong increase in overall labeling density. Interestingly, the strongest compartment-specific increase was found in vacuoles (fourfold) and in plastids (twofold). Ascorbate-specific labeling was restricted to the matrix of mitochondria and to the stroma of chloroplasts in control plants but was also detected in the lumen of thylakoids after high light exposure. In summary, this study reveals an improved insight into the subcellular distribution of ascorbate in plants and the method can now be applied to determine compartment-specific changes in ascorbate in response to various stress situations

The aesthetic stance - on the conditions and consequences of becoming a beholder

What does it mean to be an aesthetic beholder? Is it different than simply being a perceiver? Most theories of aesthetic perception focus on 1) features of the perceived object and its presentation or 2) on psychological evaluative or emotional responses and intentions of perceiver and artist. In this chapter I propose that we need to look at the process of engaged perception itself, and further that this temporal process of be- coming a beholder must be understood in its embodied, contextual and dynamic speci- ficity. Through both phenomenological and neuroscientific explorations I analyze what is characteristic about a more “aesthetic stance” and argue that there is a certain asym- metry between beholder and beheld, which has to do with a disengagement of goal- directed action, and which allows for other kinds of perceptual involvement than in a more “practical stance”. It is a multi-disciplinary project integrating a sensorimotor no- tion of aesthetic affordances, 18th century philosophy, and large-scale brain network findings. What ensues is a new dynamic framework for future empirical and theoretical research on aesthetic perception

The oxidation of dehydroascorbic acid and 2,3-diketogulonate by distinct reactive oxygen species

l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H2O2), 'superoxide' (a ∼50 : 50 mixture of [Formula: see text] and [Formula: see text]), hydroxyl radicals (•OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1O2). Products were monitored electrophoretically. DHA quenched H2O2 far more effectively than superoxide, but the main products in both cases were 4-O-oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H2O2, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical •OH yield; it yielded oxalate, threonate, and OxT. 1O2 had no effect on DHA. DKG was oxidatively decarboxylated by H2O2, Fenton mixture, and 1O2, forming a newly characterised product, 2-oxo-l-threo-pentonate (OTP; '2-keto-l-xylonate'). Superoxide yielded negligible OTP. Prolonged H2O2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H2O2, Fenton mixture, or 1O2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H2O2 and superoxide; DKG more readily by 1O2 The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product 'fingerprints' are analytically useful, indicating which ROS are acting in vivo

Intron-mediated gusA expression in tritordeum and wheat resulting from particle bombardment

The promoterless maize ubiquitin first exon and intron fragment can drive gusA expression in immature tritordeum inflorescences and immature wheat scutella. In fluorescence assays, this fragment induces gusA expression in tritordeum inflorescences to 50 times higher than background. The activity of the complete promoter, exon and intron cassette was up to 20 000-fold higher than background but the maize ubiquitin promoter in isolation had very low activity. A construct with the maize alcohol dehydrogenase first exon and intron had low activity, visible in histochemical assays. Both intron sequences have promoter-like features and in the ubiquitin intron there is a sequence homologous to the opaque-2-binding box. We suggest that the combination of these elements may explain the promoter activity detected in these introns