Purification and characterization of hydroxypyruvate reductase from cucumber cotyledons

Hydroxypyruvate reductase (HPR), a marker enzyme of peroxisomes, has been purified to homogeneity from cotyledons of light-grown cucumber seedlings (Cucumis sativus var. Improved Long Green). In addition, the peroxisomal location of both HPR and serine-glyoxylate aminotransferase has been confirmed in cucumber cotyledons. The isolation procedure involved Polymin-P precipitation, a two-step precipitation with ammonium sulfate (35 and 50% saturation), affinity chromatography on Cibacron Blueagarose, and ion-exchange chromatography on DEAE-cellulose. HPR was purified 541-fold to a final specific activity of 525 ± 19 micromoles per minute per milligram of protein. Enzyme homogeneity was established by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was 91 to 95 kilodaltons, approximately double the apparent subunit molecular weight of 40,500 ± 1,400. With hydroxypyruvate as substrate, the pH optimum was 7.1 and K_m values were 62 ± 6 and 5.8 ± 0.7 micromolar for hydroxypyruvate and NADH, respectively. With glyoxylate as substrate, the pH optimum was 6.0, and the K_m values for glyoxylate and NADH were 5700 ± 600 and 2.9 ± 0.5 micromolar, respectively. Antibodies to HPR were raised in mice (by the ascites tumor method) and in rabbits, and their monospecificity was demonstrated by a modified Western blot immunodetection technique.     

THE DEVELOPMENT OF MICROBODIES AND PEROXISOMAL ENZYMES IN GREENING BEAN LEAVES

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 µm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 µm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.     

Violaxanthin is an abscisic Acid precursor in water-stressed dark-grown bean leaves

The leaves of dark-grown bean (Phaseolus vulgaris L.) seedlings accumulate considerably lower quantities of xanthophylls and carotenes than do leaves of light-grown seedlings, but they synthesize at least comparable amounts of abscisic acid (ABA) and its metabolites when water stressed. We observed a 1:1 relationship on a molar basis between the reduction in levels of violaxanthin, 9′-cis-neoxanthin, and 9-cis-violaxanthin and the accumulation of ABA, phaseic acid, and dihydrophaseic acid, when leaves from dark-grown plants were stressed for 7 hours. Early in the stress period, reductions in xanthophylls were greater than the accumulation of ABA and its metabolites, suggesting the accumulation of an intermediate which was subsequently converted to ABA. Leaves which were detached, but not stressed, did not accumulate ABA nor were their xanthophyll levels reduced. Leaves from plants that had been sprayed with cycloheximide did not accumulate ABA when stressed, nor were their xanthophyll levels reduced significantly. Incubation of dark-grown stressed leaves in an ¹⁸O₂-containing atmosphere resulted in the synthesis of ABA with levels of ¹⁸O in the carboxyl group that were virtually identical to those observed in light-grown leaves. The results of these experiments indicate that violaxanthin is an ABA precursor in stressed dark-grown leaves, and they are used to suggest several possible pathways from violaxanthin to ABA.     

Influence of Glyphosate on Nitrate Reductase Activity in Soybean (Glycine max)

Experiments were conducted to determine the influence of glyphosate[N-(phosphonomethyl)glycine] on extractable nitrate reductaseactivity during light and dark growth of soybean (Glycine max)seedlings. Glyphosate (5?10^–4 M), applied via root-feedingto three-day-old etiolated seedling, significantly reduced enzymeactivity in roots (48 to 96 h) and leaves (96 h) of seedlingsplaced in the light, but had little effect on enzyme activityin cotyledons compared to enzyme levels in tissues of untreatedseedlings. During dark-growth, nitrate reductase activity increasedwith time in cotyledons of untreated seedlings (activity about85-fold less than in cotyledons of light-grown plants) but muchlower enzyme levels were found in cotyledons of glyphosate-treatedseedlings after 72 and 96 h. In leaves of dark-grown seedlings,glyphosate reduced nitrate reductase levels by 95%. Most inhibitionof extractable enzyme activity occurred in newly developingorgans (leaves and roots) which correlates well with reportsthat glyphosate is rapidly translocated to these sites. However,the fact that glyphosate inhibits growth prior to lowering enzymeactivity levels indicates a secondary effect on nitrate reductase. (Received May 18, 1984; Accepted February 12, 1985)     

Inhibitory Effect of Gabaculine on the Biosynthesis of 5-Aminolevulinate in the Tigrina d12 Mutant of Barley

Six-day-old, dark-grown, seedlings of barley homozygous forthe recessive mutation tigrina d¹² accumulated 5-aminolevulinicacid (ALA) and protochlorophyll (ide) in amounts exceeding thewild type level. Transferring the etiolated mutant to lightresulted in the destruction of pigments and the deteriorationof the ALA forming system. Such deleterious effects did notoccurusing light-grown mutant or etiolated and greened wildtype seedlings. Gabaculine (GAB) at 50 µM inhibited ALAsynthesis by about 85% when etiolated wild type seedlings wereexposed to light. In light-grown leaves of either wild typeor mutant strain, ALA production was also sharply (ca. 75%)inhibited by GAB. During dark incubation, however, the inhibitionof ALA accumulation did not exceed 50% in all types of tissues.The results give further evidence for the operation of the C₅pathway in such seedlings since GAB decreased the biosynthesisof ALA to the same extent in both tigrina d¹² mutant and wildtype of barley. (Received July 2, 1990; Accepted May 7, 1991)     

Action Spectra for the Inhibition of Hypocotyl Growth by Continuous Irradiation in Light and Dark-Grown Sinapis alba L. Seedlings

Action spectra for the inhibition by continuous (24-hour) irradiation of hypocotyl growth in 54-hour-old Sinapis alba L. seedlings were measured using seedlings which had had four different pretreatments. These seedlings were either (a) dark-grown with a high total phytochrome level, (b) dark-grown with a low total phytochrome level, (c) light-grown with chlorophyll, or (d) light-grown with no chlorophyll [treated with 4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)-3(2H) -pyridazinone (San 9789)].