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.     

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.     

Effects of freezing and hardening on the sulfhydryl groups of protein fractions from cabbage leaves

Disc electrophoresis was used to separate water soluble proteins from hardy, non-hardy, and frost killed cabbage (Brassica oleracea var. capitata) leaves. Amidoschwarz staining failed to reveal any new bands as a result of hardening although the relative amounts of proteins in individual bands changed. Sulfhydryl groups in the protein bands were stained with 2,2-dihydroxy-6,6-dinaphthyl disulfide and labeled with ¹⁴C p-chloromercuribenzoate. Significant decreases in the sulfhydryl content of the total water soluble protein were found during hardening and as a result of frost death. The decrease during hardening was paralleled by a significant increase in the water soluble protein. There was a significant increase in the sulfhydryl content per unit high molecular weight protein but a decrease in the sulfhydryl content per total protein as a result of frost death. This was interpreted as evidence for intermolecular disulfide bond formation during freezing.     

Growth regulator induced movement of photosynthetic products into fruits of ;black corinth' grapes

The effect of exogenous growth regulators on movement of assimilates into flowers and young fruits of `Black Corinth' grapes was studied. Clusters were treated with growth regulator and after 0.5 hr to 5 days the leaves above the clusters were exposed to ¹⁴CO₂. Control shoots received ¹⁴CO₂ but no growth regulator. At harvest, counting and radioautographic techniques were used to ascertain amount and distribution of activity in clusters. Clusters were dipped in 4-CPA (4-chlorophenoxyacetic acid), GA₃ (gibberellic acid), or BA (benzyladenine). All berries were heavier than controls within 3 days. Total counts in the fruits were increased by 4-CPA, and the distribution of radioactivity among the sugar, organic acid, and amino acid fractions was usually altered by all treatments. In a time series experiment, within 6 hr after treatment of fruits with GA₃ there was almost an 8-fold increase in total counts relative to the control. After 12 hr there was about a 9-fold and 6-fold increase in counts in tartaric and malic acids, respectively, and in γ-aminobutyric acid, pipecolic acid, and valine increases of 56, 150, and 330%. Radioactivity in fructose was increased 70% in gibberellin-treated clusters over the controls. After 96 hr there were only about 1000 cmp per g fr wt in controls, but there were about 31,000 cpm counts in treated clusters. Treatment of clusters with gibberellin attracted less assimilates into the fruits when shoots had also been sprayed with gibberellin. Dipping portions of clusters in gibberellin increased the movement of ¹⁴C assimilates into the treated portions. Hormonal control of mobilization is discussed.     

Control of senescence in rumex leaf discs by gibberellic Acid

The kinetics of chlorophyll and protein decomposition and the effect of gibberellic acid (GA) were examined in senescing leaf discs of Rumex crispus and R. obtusifolius. Loss of Rumex total chlorophyll proceeds at a slow rate for about 2 days followed by a period of rapid logarithmic decline. Chlorophyll b is lost at a slightly faster rate than chlorophyll a during senescence in discs as well as in situ. GA causes a complete cessation of net chlorophyll and protein degradation for several days in Rumex, in contrast to the incomplete senescence inhibition generally observed with cytokinins. GA is fully effective even when added at the middle of the logarithmic phase of chlorophyll loss. Senescence inhibition by GA is apparently gradually reversed upon GA removal. The cytokinins, kinetin and 6-benzylaminopurine, were also effective in Rumex leaf discs, indicating that the senescence retarding effect was not restricted to the gibberellins.     

Regulation of senescence in bean leaf discs by light and chemical growth regulators

The senescence of excised discs of primary leaves of Phaseolus vulgaris, L., var. Red Kidney was followed by measuring the net breakdown of protein and chlorophyll. The chemical growth regulators indoleacetic acid, 2,4-dichlorophenoxy-acetic acid, gibberellic acid, kinetin, and 6-benzylaminopurine were relatively ineffective in retarding senescence in this tissue. White light, on the other hand, was very effective in senescence retardation. The response to light did not have the characteristics of a low energy (phytochrome) response and was blocked by concentrations of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which inhibited photosynthesis in the leaf discs. The light-induced retardation of senescence was concluded to be dependent on photosynthesis.     

Inhibition of in Vitro Pollen Tube Growth by Isolated S-Glycoproteins of Nicotiana alata

Pollen from three S-genotypes of Nicotiana alata was grown in vitro in the presence of S-glycoproteins isolated from styles of the same three genotypes. Pollen germination was not affected by the presence of the S-glycoproteins, but pollen tube growth of all genotypes was inhibited. S2 pollen was preferentially inhibited by the S2-glycoprotein and S3 pollen by the S3-glycoprotein. The S6-glycoprotein preferentially inhibited growth of both S2 and S6 pollen over S3 pollen. Heat treatment dramatically increased the inhibitory activity of the S-glycoproteins as inhibitors both of pollen germination and tube growth; after heat treatment, S-allele specificity of pollen tube inhibition was not detected.     

Effects of the developmental state of the tissue on the competence for flower bud regeneration in pedicel explants of tobacco

The competence of pedicel explants of tobacco (Nicotiana tabacum L. cv Samsun) to regenerate flower buds in response to auxin was manipulated by preincubating excised tissues in the absence of auxin. When exposed to 1 micromolar 1-naphthaleneacetic acid, these tissues formed fewer buds than controls that were not preincubated. The number of buds eventually formed correlated with the 1-naphthaleneacetic acid concentration in the tissue 6 hours after the start of hormone application. The internal concentrations in pretreated explants were lower than in tissues that were not pretreated due to diminished uptake per milligram fresh weight and increased hormone conjugation. The change in the developmental state induced by auxin deprivation had a dual effect on bud regeneration: (a) the pretreatment caused fewer buds to be formed at any 1-naphthaleneacetic acid concentration tested, and (b) a higher auxin concentration in the medium was required to get a maximum bud number on precultured explants. An increase of the 1-naphthaleneacetic acid concentration in the medium led to an elevated hormone level in freshly cut as well as in preincubated tissues. It was concluded that the developmental state of the tissue directly affects the maximum number of buds that can be regenerated. Apart from that there is an indirect effect exerted via modulation of the ratio between external and internal auxin concentration. The change in this ratio can be compensated for by an adjustment of the auxin concentration in the medium.