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

Biosphere-atmosphere exchange of ammonia

Substantial progress has been made in the last eight years in the understanding and quantification of ammonia exchange between the atmosphere and biosphere. Much of the work has been linked to the joint EC/EUROTRAC subproject BIATEX (BIosphere ATmosphere EXchange), which has served as the main European forum for work in this area. In the mid-1980s there was still much confusion and uncertainty over the rate and direction of ammonia fluxes with different ecosystems; although the results of isolated studies were available, there was no clear overview of the key factors affecting ammonia fluxes. Work since that time has highlighted the dominant effects of ecosystem type and management, as well as humidity and wetness, on ammonia exchange. Ammonia is a key component of plant metabolism, so that ammonia emission may occur from plants in relation to nitrogen nutrition and plant growth stage. In contrast, ammonia is highly soluble and may be efficiently captured by leaf cuticles and surface w etness allowing large deposition velocities. The consequence is that ammonia exchange is bi-directional over agricultural ecosystems, though for most semi-natural ecosystems dry deposition dominates, being a significant component of the total atmospheric nitrogen input. The work within BIATEX has focused in more detail on the processes controlling these differences and, using the results of both micrometeorological and controlled environment measurements, has developed new models that are able to provide the synthesis necessary to predict ammonia fluxes. Long term and regional estimates of ammonia net exchange are still uncertain, though the models developed now provide the necessary framework to guide future measurements