Transformation of androstane derivatives by Spirodela oligorrhiza

Spirodela oligorrhiza (duckweed) is capable of transforming some steroids of the androstane series. Hydrolysis of the acetates of testosterone and of 3β-hydroxyandrost-5-en-17-one by this species yielded the corresponding alcohols. Further transformation of testosterone and reduction of androst-4-ene-3,17-dione indicated the interconversions of the hydroxyl-ketone function on C-17 and reduction of the Δ⁴-double bond to the trans-A/B system. Only a trace amount of 3β-hydroxyandrost-5-en-17-one underwent further transformations.     

Nitrogen metabolism of Spirodela oligorrhiza

Summary Wheras ammonium inhibited almost completely utilization of nitrate (NO₃) by Spirodela oligorrhiza, asparagine, aspartate, glutamine, and glutamate had only a sparing effect. Asparagine and glutamine were used more rapidly than NO₃; aspartate and glutamate at about the same rate. These differences in utilization of NO₃ were not determined by differences in the levels of nitrate reductase.     

Nitrogen metabolism of Spirodela oligorrhiza

Summary Spirodela oligorrhiza grown in axenic culture was able to use either ammonium, nitrate, or nitrite as sole source of nitrogen, although the morphology of the plants was affected. Plants utilizing ammonium contained higher levels of NH₄, arginine, asparagine and glutamine than did those utilizing NO₃, whereas concentrations of other amino acids were similar.The utilization of NH₄ inhibited that of NO₃ by inhibiting, at least partially, NO₃ uptake, and by inhibiting almost completely the reduction of NO₃ to NO₂. NO₂ also inhibited the utilization of NO₃. NH₄ and NO₂ were taken up and assimilated simultaneously when they were supplied together in the medium.     

Nitrate Reductase Independent Stimulation of Seed Germination in Sisymbrium officinale L. (Hedge Mustard) by Light and Nitrate

After a 72 h preincubation in darkness at 15 °C seed germinationof Sisymbrium officinale L. (hedge mustard) at 24 °C wasstimulated by a combination of red light and nitrate. In thepresence of nitrate the seeds escaped from the inhibiting effectof far-red irradiation with an escape time of approx. 8 h. Afterred light, the exposure of seeds to nitrate could be delayedfor 3 h without affecting maximal germination. Prolonged delayresulted in a decrease of the germination response. The possibilitythat nitrate reduction was involved in the stimulation of germinationwas studied by pre-incubating seeds for 72 h in nitrate andsubsequently transferring them to water and irradiating withred light. During the first 8 h period after the red irradiationin which induction of germination occurred, total nitrate levels(endogenous + leachate) remained constant, indicating an absenceof nitrate reductase activity. During the next 8 h visible germinationstarted and total nitrate levels declined, suggesting inductionof nitrate reduction. It is concluded that nitrate reductiondocs not play a role in the induction of germination. The conclusionwas supported by the lack of inhibition of seed germinationby sodium chlorate and sodium tungstate in spite of an inhibitionof nitrate reduction of 80 and 100%, respectively. The contrastsbetween our results and hypotheses concerning the mechanismof action of nitrate in seed germination are discussed Sisymbrium officinale L., hedge mustard, germination, light, nitrate, nitrate reductase     

The nitrogen metabolism of Spirodela oligorrhiza

Summary Nitrate reductase (NO₃R) in Spirodela oligorrhiza has a specific requirement for NAD, and no activity could be detected when reduced NADP was supplied as a cofactor. NO₃R and nitrite reductase (NO₂R) are adaptive enzymes, being present only when the plant is supplied with NO₃ or NO₂. There is significant correlation between the concentration of NO₃ in plants, and the levels of NO₃R that they contain. When plants not containing NO₃R or NO₂R are supplied with NO₃, there was a rapid increase in the levels of the two enzymes and in the concentration of NO₃ in the plants. These increases were retarded but not prevented by NH₄ in the medium. When NO₃-grown plants were depleted of N, there was a rapid decrease in the levels of NO₃R and NO₂R.The utilization of NH₄ prevented the assimilation of NO₃ even by plants that contained high levels of NO₃R and NO₂R. It was therefore concluded that NH₄, or the processes or products of its assimilation must inhibit the activity of NO₃R. NH₄, arginine, asparagine or glutamine, tested separately or together, had little effect on the in vitro activity of NO₃R.     

Effect of Ammonium, Sucrose and Light on the Regulation of Nitrate Reductase Level in Pisum sativum

In shoot apices of 7-day-old dark-grown peas the addition of ammonium along with the inducer nitrate resulted in a more than two-fold increase in nitrate reductase activity. Individual amino acids, amides and amino-acid mixture could not replace the ammonium effect. Ammonium also stimulated NADH-glutamate dehydrogenase but not glucose-6-phosphate dehydrogenase. Sucrose caused a marked stimulation of nitrate reductase induction and showed synergistic effect with light. In presence of cordycepin and cycloheximide, induction of nitrate reductase was inhibited more if ammonium or sucrose was supplied along with the inducer. With actinomycin D, α-amanitin or chloramphenicol, no differential inhibition took place in presence of ammonium. The inhibition of enzyme activity by chloramphenicol and 3-(3,4-dichlorophenyl)-l,dimethyl urea was completely relieved by sucrose. Incorporation of ¹⁴C-lysine was markedly stimulated by sucrose, but was not affected by ammonium. The effect of sucrose and light on ¹⁴C-lysine incorporation was additive. Cordycepin and cycloheximide did not have any differential effect on ¹⁴C-lysine incorporation in the presence of ammonium as well as sucrose. The inhibition of ¹⁴C-lysine incorporation caused by chloramphenicol was relieved by sucrose. Sucrose also caused a marked increase in ³H-uridine incorporation but ammonium had no effect. Actinomycin D and cordycepin blocked the sucrose dependent increase in ³H-uridine incorporation. The results suggest that ammonium mediated stimulation may depend on a regulatory protein(s) synthesized in response to ammonium, whereas sucrose acts mainly by an overall increase in RNA and protein synthesis. The effect of light does not seem to be dependent on photosynthetic light reactions.     

Cytokinin control of growth of Spirodela oligorrhiza in darkness

Summary Continuous heterotrophic growth of Spirodela oligorrhiza cultures following transfer to darkness requires cytokinins, or periodic brief treatment with red light. In the absence of cytokinins or red light growth ceases after 2–3 days. However, growth resumes spontaneously after 3–4 weeks in darkness to produce etiolated plants. The growth rate of these etiolated plants is not stimulated by kinetin.Although the kinetin concentration in treated plants reaches a plateau 30–60 min after adding kinetin to dormant plants in darkness new fronds do not appear for 24 h. Dormant colonies treated with kinetin in darkness for only 6–12 h subsequently grow in darkness at the same rate as plants treated with kinetin for 1, 2 or 3 days. Treatments which inhibit growth in the light, for example cold, chloramphenicol or actidione, eliminate the requirement for cytokinin and allow subsequent growth in darkness. The results suggest that a growth inhibitor may be present but ineffective in Spirodela growing in the light. The inhibitor is active in darkness but slowly decays. Kinetin appears to inactivate the inhibitor in darkness.     

Response of Spirodela oligorrhiza to Phosphorus Deficiency

The duckweed Spirodela oligorrhiza, growing in sterile defined nutrient media, was used to study some responses of plants to phosphorus deficiency. On a phosphate-deficient medium, growth of Spirodela soon slowed and eventually ceased. Older leaves became chlorotic, but newly formed leaves were dark green and contained much anthocyanin. The photosynthesis rate fell gradually, roots elongated, and chloroplasts became filled with starch.     

硝酸盐对硝酸还原酶活性的诱导及硝酸还原酶基因的克隆

硝酸盐在植物体内的积累过多已成为影响蔬菜品质并影响人类健康的重要因素。硝酸还原酶（NR）是硝酸盐代谢中的关键酶,提高其活性有利于硝酸盐的降解。为了解植物不同组织中NR的活性,用活体测定法检测了经50mmol/L的KNO₃诱导不同时间后的油菜、豌豆和番茄幼苗根茎叶中NR活性,同时为了明确外源诱导剂浓度与植物体内NR活性的关系,检测了经不同浓度KNO₃诱导2h后的矮脚黄、抗热605、小白菜和番茄叶片中的NRA。结果表明,不同植物组织NR活性有很大差异,叶中NR活性较高,根其次,茎最低;不同植物的NR活性随诱导时间呈不同的变化趋势,相同植物不同组织的NR活性变化趋势相似;不同植物叶片NRA为最高时KNO₃浓度不同。用30mmol/L的KNO3诱导番茄苗2h后,从番茄根和叶中提取总RNA,用RT-PCR方法获得NR cDNA,全长2736bp,编码911个氨基酸。为进一步利用该基因提高植物对硝酸盐的降解能力打下基础。     

Light and Nitrate Requirements for Induction of Nitrate Reductase Activity in Hordeum vulgare

The importance of light to the induction of nitrate reductase activity in barley (Hordeum vulgare L.) was studied. Activity in etiolated leaves in darkness stayed at a low endogenous level even while large amounts of nitrate were actively accumulated. Light was required for any increase in activity, though the requirement may be satisfied to a limited extent before nitrate is available. Nitrate reductase activity was induced in the dark in green leaves which had not previously had nitrate but were supplied nitrate at the beginning of the dark period. If the nitrate then made available was sufficient, nitrate reductase activity increased until the effect of the previous light treatment was exhausted. Activity then decreased even though nitrate uptake continued. Upon returning the leaves to light, enzymatic activity increased again, as expected. Nitrate uptake was eliminated as an experimental variable by giving dark-grown plants nitrate, then detaching the leaves for induction studies. Under these conditions light saturation occurred between 3600 and 7700 lux at exemplary periods of illumination. At intensities of 3600 lux and above, activity increased sharply after a 6-hour lag period. As light intensity was decreased below 3600 lux the lag period became longer. Thus, when sufficient nitrate was available, the extent of induction of nitrate reductase activity was regulated by light.     

Increase in Nitrate Reductase Activity with Ammonium Chloride in Bacillus licheniformis by Shaking Culture

In shaking culture, nitrate reductase activity in the cell-free extracts of Bacillus licheniformis increased with the addition of NH₄Cl to the medium containing NaNO₃ as a single nitrogen source, where amounts of nitrogen sources were sufficient for cell growth. This increase of nitrate reductase activity therefore suggests that the activity is not for nitrate assimilation but for other physiological functions containing a dissimilatory nitrate reduction.     

Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza

Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size.     

Induction of high affinity phosphate transporter in the duckweed Spirodela oligorrhiza

Duckweed plants (Spirodela oligorrhiza) grown under phosphate (Pi)-deficient conditions (- P plants) exhibited more than 50-fold higher Pi uptake activity than plants grown under Pi-sufficient conditions (+ P plants). The Pi uptake activity of - P plants measured using (32)Pi was significantly inhibited by carbonylcyanide m-chlorophenylhydrazone, indicating that Pi uptake is energized by the electrochemical proton gradient across the plasma membrane (PM). When Pi uptake was examined at various concentrations of Pi, more active uptake of Pi was observed in - P plants than in + P plants, irrespective of the Pi concentrations. An immunoblot analysis of the PM proteins using antiserum against the conserved sequence of the high-affinity Pi transporter recognized the occurrence and large accumulation of a novel protein band at 48 kDa in - P plants. The protein was almost completely extracted with chloroform-methanol (2:1, v/v), but only a trace amount of the protein was detected in + P plants. Immunohistochemical studies of plant roots using the same antiserum demonstrated a large accumulation of high-affinity Pi transporters at the outermost cortical cells of - P plants, but not of + P plants. When an immunoblot analysis of PM proteins was performed using antiserum against the PM H(+)-ATPase, a positive band of about 96 kDa was detected in both plants with a similar signal intensity. Furthermore, ATP-hydrolytic and ATP-dependent H(+)-transporting activities of PM H(+)-ATPase in - P plants were not higher than those in + P plants. However, kinetic analyses showed that the PM H(+)-ATPase in - P plants had a lower K(m) value and a higher coupling efficiency between ATP hydrolysis and H(+) pumping than the corresponding values in + P plants. These results suggest that the significant stimulation of Pi uptake in - P plants may be due mainly to the induction and accumulation of the high-affinity Pi transporter in the PM, and that the electrochemical proton gradient across the PM may be generated by the high-ATP-affinity and energy-efficient H(+) pump in - P plants. This would facilitate the acquisition of Pi in S. oligorrhiza under Pi-depleted conditions.     

Increase in Nitrate Reductase Activity in Bean Leaves by Light Involves a Regulator Protein

Nitrate reductase activity, assayed either in vivo or in vitro was considerably higher in bean (Phaseolus vulgaris L.) leaves from 7-day-old light grown seedlings than those from dark grown, both in the absence as well as presence of nitrate. Cytochrome c reductase activity was however similar in both regimes, while peroxidase was lower in light than in dark. The light stimulated increase in nitrate reductase activity in leaf segments from dark grown seedlings was inhibited by cycloheximide, DNP, chloramphenicol, and sodium tungstate and was unaffected by lincomycin and DCMU. Under similar conditions, the increase in total chlorophyll was inhibited completely by cycloheximide and DNP, partially by chloramphenicol and lincomycin, and was unaffected by tungstate and DCMU. A supply of 1~5 mm reduced glutathione increased enzyme activity in the dark and also to some extent in light. The substrate induction of enzyme activity started after a lag of one hr in light or dark and continued for either 5 hr in the dark or 8 hr in light. Two proteinaceous inhibitors (Factors I and II) of nitrate reductase were isolated by ammonium sulfate precipitation and Sephadex gel filtration. The amount of Factor I was higher in the dark than in light. The amount and activity of Factor II was however, almost equal in light and dark. The inhibition of enzyme activity by these inhibitors increased with their concentration. It is proposed that light increases nitrate reductase activity by decreasing the amount of a nitrate reductase inhibitor.