Abstract
Mesophyll protoplasts were isolated from the leaves of wheat and corn seedlings. After purification the protoplasts were judged to be free of contaminating proteases in the isolation enzymes based on specific activity of the proteases in comparison to leaf tissue and their response to inhibitors that “differentiated” between leaf and isolation enzyme proteases. Wheat protoplasts showed rates of photosynthesis of 95 to 100 micromoles O2 per milligram chlorophyll per hour, while corn exhibited rates of 35 to 85 micromoles O2 per milligram chlorophyll per hour, indicating the intactness of the chloroplasts within the protoplasts. These chloroplasts were isolated from the protoplasts using the procedure of Robinson and Walker (1979 Arch Biochem Biophys 196: 319-323). Yields of 91 and 82% intact chloroplasts were obtained from wheat and corn, respectively, based on the distribution of ribulose bisphosphate carboxylase in wheat and NADP-malate dehydrogenase in corn. Vacuoles were obtained from the protoplasts using a modification of the techniques of Wagner and Siegelman (1975 Science 190: 1298-1299) and Saunders (1979 Plant Physiol 64: 74-78). The vacuoles were at least 98% free of protoplast contamination as determined by assaying for “marker” enzymes of chloroplasts, mitochondria, and endoplasmic reticulum. Assuming one vacuole per protoplast, the vacuoles contained 4% of the soluble protein of the protoplasts in wheat and 8% in corn. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuoles. Although the isolated chloroplasts always retained about 13% of the proteolytic activity of the protoplasts, this could be accounted for by that which became associated with the chloroplasts during their isolation.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arnon D. I. COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. Plant Physiol. 1949 Jan;24(1):1–15. doi: 10.1104/pp.24.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boller T., Kende H. Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol. 1979 Jun;63(6):1123–1132. doi: 10.1104/pp.63.6.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Edwards G. E., Robinson S. P., Tyler N. J., Walker D. A. Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates. Plant Physiol. 1978 Aug;62(2):313–319. doi: 10.1104/pp.62.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson H. S., Hatch M. D. Properties and regulation of leaf nicotinamide-adenine dinucleotide phosphate-malate dehydrogenase and 'malic' enzyme in plants with the C4-dicarboxylic acid pathway of photosynthesis. Biochem J. 1970 Sep;119(2):273–280. doi: 10.1042/bj1190273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Robinson S. P., Walker D. A. Rapid separation of the chloroplast and cytoplasmic fractions from intact leaf protoplasts. Arch Biochem Biophys. 1979 Sep;196(2):319–323. doi: 10.1016/0003-9861(79)90584-8. [DOI] [PubMed] [Google Scholar]
- Saunders J. A. Investigations of vacuoles isolated from tobacco: I. Quantitation of nicotine. Plant Physiol. 1979 Jul;64(1):74–78. doi: 10.1104/pp.64.1.74. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wittenbach V. A. Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings. Plant Physiol. 1978 Oct;62(4):604–608. doi: 10.1104/pp.62.4.604. [DOI] [PMC free article] [PubMed] [Google Scholar]