differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aimed to obtain more information about these functions by studying the substrate and product specificities of both isoenzymes. Analyses of the products formed from linoleic acid confirmed that LOX-I oxygenated at C9, and LOX-2 at C13. When testing more complex substrates, it was found that both LOX-1 and LOX-2 were capable of metabolizing esterified fatty acids. K,,, values from both isoenzymes for free fatty acids were much lower than for esterified fatty acids (7-35-fold for LOX-I versus 2-Wold for LOX-2). Interestingly, LOX-I showed significantly higher K,,, values for esterified fatty acids than did LOX-2. This was reflected by analyses of the products formed from di-and tri-linoleoylglycerol ; LOX-2 formed higher amounts of oxygenated polyunsaturated fatty acids within the esterified lipids than did LOX-I, with a corresponding larger extent of oxygenation.In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extracts from barley after different periods of germination for LOX-derived products. The results indicated that esterified fatty acids were preferentially metabolized by LOX-2 activity. Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (1 3s)-hydroxy derivatives were formed, indicating the in vivo action of LOX-2. These data show that LOX-2 is capable of oxygenating storage lipids and suggest that during the onset of germination LOX-2 may be involved in oxygenation of esterified polyunsaturated fatty acids in barley seeds. We suggest that the oxygenation of these lipids precedes the onset of their catabolism and that the degradation product, (9Z,IlE,13S)-13-hydroxy-octadecadienoic acid, serves as an endogenous substrate for Boxidation and therefore as a carbon source for the growing barley embryo.

Expression of lipoxygenase was studied in barley (Hordeum distichum L.) embryos during germination. Total lipoxygenase activity was high in quiescent grains, dropped during the 1st d of germination, and subsequently increased to a leve1 similar to that in quiescent grains. The contribution of two isoenzymes, lipoxygenases 1 (LOX-1) and 2 (LOX-2), was studied at the activity, protein, and mRNA levels. Activity ratios of the two isoforms were determined via the ratio of 9-and 13-hydroperoxides, which are formed from linoleic acid. lsoenzyme protein levels were determined using specific monoclonal antibodies. mRNA levels were studied using the specific cDNA probes LoxA and LoxC, which correspond to LOX-1 and LOX-2, respectively. The major difference in temporal expression of LOX-1 and LOX-2 was observed in quiescent grains. At this stage, LOX-1 contributed almost exclusively to total lipoxygenase activity. LOX-2 activity rapidly increased until d 2 of germination.From this time point onward, LOX-1 and LOX-2 showed similar patterns at both activity and protein levels. The tissue distribution of the two isoenzymes in the germinating embryo was closely similar, with the highest expression levels in leaves and roots. The levels of LOX-1 and LOX-2 may be regulated mainly pretranslationally, as suggested by the similarity of the protein and mRNA patterns corresponding to the two isoforms.

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To investigate the influence of oxygen concentrations in the root system on plant development, young cucumber plants were grown during three weeks on stone wool blocks. A continuous flow of nutrient solution (0.75 L h-1), containing 0.5, 3.5 or 10 mg L-1 dissolved oxygen, was led through the substrates. Already after 3 days, the impact of the various oxygen levels in the root systems became evident. Plant development was reduced when plants were subjected to the lowest oxygen concentration. In time, increasing differences in leaf area were observed when plants were grown under different oxygen levels, showing largest leaf area at 10 mg L-1 oxygen. Also, root mass was significantly reduced when plants were grown under low oxygen conditions. Bacteria and other microorganisms that consume oxygen can reduce oxygen levels. Oxygen consumption was detected in samples that were taken at different places in greenhouses, e.g. irrigation water storage tanks, irrigation supply system and substrates. During the winter period, the contribution of microorganisms in oxygen consumption was low. However, in April oxygen consumption in the samples was significantly higher. Sometimes, oxygen in nutrient solution, present in the substrates, was fully consumed within 30 minutes, indicating that roots would suffer anoxia. Frequent refreshment of nutrient solution in the irrigation supply system had a major impact on oxygen levels. Continuous measurements showed that during the day oxygen levels in the irrigation system were high just after refreshment of the nutrient solution, but between two refreshments oxygen levels in the system dropped towards very low oxygen values. If technically feasible, in future the control of oxygen levels in root systems may become a new tool for growers to manage cultivation in horticulture, in addition to light, temperature, carbon dioxide, nutrition and water.

Associations between lipoxygenases (Lox) and 14-3-3 proteins were demonstrated by two different methods. First, immunoprecipitation experiments, using isoenzyme-specific monoclonal Lox antibodies, showed that 14-3-3 proteins coprecipitate with 13-Lox, but not with the 9-Lox from barley. Second, interactions between 13-Lox and 14-3-3 were established by surface plasmon resonance studies, showing that 13-Lox binds with 14-3-3 proteins in a concentration-dependent manner. The interactions between 14-3-3 proteins and 13-Lox may reveal their role during plant development.z 2000 Federation of European Biochemical Societies.

To study the conservation of peroxisomal targeting signals, we have determined the intracellular localization of human peroxisomal catalase when expressed in yeast. Using immunofluorescence, differential centrifugation and immunoelectron microscopy, we show that the protein is targeted to the peroxisomes of the heterologous cell and assembled in its active tetrameric form. These data show the conservation of the catalase targeting signal and import specificity between human and yeast peroxisomes.

Recently, we have demonstrated by two different methods that lipoxgenases (LOXs) and 14-3-3 proteins form interactions in barley embryos [Holtman, Roberts, Oppedijk, Testerink, van Zeijl and Wang (2000) FEBS Lett. 474, 48-52]. It was shown by both co-immunoprecipitations and surface-plasmon resonance experiments that 13-LOX, but not 9-LOX, forms interactions with 14-3-3 proteins. In the present report we show that the presence of 13-LOX and 14-3-3 proteins was established in high-molecular-mass complexes. Amounts of 13-LOX and 14-3-3 proteins in high-molecular-mass fractions increased during germination, but were reduced after dephosphorylation of protein extracts or competition with the 14-3-3-binding peptide P-Raf-259, indicating that 13-LOX and 14-3-3 proteins interact in a phosphorylation-dependent manner.

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