No abstract

No abstract

No abstract

A procedure is described for the rapid (<5 min) isolation of purified, physiologically active chloroplasts from Pisum sativum L. Mitochondrial and microbody contamination is substantially reduced and broken chloroplasts are excluded by washing through a layer containing a treated silica sol. On average the preparations contain 93% intact chloroplasts and show high rates of (14)CO2 fixation and CO2-dependent O2 evolution (over 100 μmol/mg chlorophyll(chl)/h); they are also able to carry out light-driven incorporation of leucine into protein (4 nmol/mg chl/h). The amino-acid contents of chloroplasts prepared from leaves and from leaf protoplasts have been determined. Asparagine is the most abundant amino acid in the pea chloroplast (>240 nmol/mg chl), even thought it is proportionately lower in the chloroplast relative to the rest of the cell. The chloroplasts contain about 20% of many of the amino acids of the cell, but for individual amino acids the percentage in the chloroplast ranges from 8 to 40% of the cell total. Glutamic acid, glutamine and aspartic acid are enriched in the chloroplasts, while asparagine, homoserine and β-(isoxazolin-5-one-2-yl)-alanine are relatively lower. Leakage of amino acids from the chloroplast during preparation or repeated washing was ca. 20%. Some differences exist between the amino-acid composition of chloroplasts isolated from intact leaves and from protoplasts. In particular, γ-aminobutyric acid accumulates to high levels, while homoserine and glutamic acid decrease, during protoplast formation and breakage.

In plants, the primary input of nitrogen (obtained from the soil or from symbiotic dinitrogen fixation) occurs through the assimilation of ammonia into organic form. Synthesis of glutamine (via glutamine synthetase) is the major, and possibly exclusive, route for this process, and there is little evidence for the participation of glutamate dehydrogenase. A variety of reactions distribute glutamine nitrogen to other compounds, including transfer to amino nitrogen through glutamate synthase. In many plants asparagine is a major recipient of glutamine nitrogen and provides a mobile reservoir for transport to sites of growth; ureides perform a similar function in some legumes. Utilisation of transport forms of nitrogen, and a number of other metabolic processes, involves release of ammonia, which must be reassimilated. In illuminated leaves, there is an extensive flux of ammonia released by the photorespiratory cycle, requiring continuous efficient reassimilation. Aspects of ammonia recycling and related amide metabolism in higher plants are reviewed.