Inhibition of chlorophyll biosynthesis by mercury in excised etiolated maize leaf segments during greening: effect of 2-oxoglutarate

Mercury (0.01-1.0 mM) inhibited chlorophyll formation in greening maize leaf segments. However, supplementing incubation medium with 2-oxoglutarate, maintained substantially higher level of chlorophyll in absence of metal after an initial period of 8 hr. On preincubation of leaf segments with HgCl2, per cent inhibition of chlorophyll synthesis by metal was same in the presence and absence of 2-oxoglutarate. Supply of 2-oxoglutarate (0.1-10.0 mM) exerted concentration dependent effect on chlorophyll formation in absence or presence of metal. Increase in delta-amino levulinic acid dehydratase as well as NADH-glutamate synthase activity and decrease in NADH-glutamate dehydrogenase activity by 2-oxoglutarate in the presence of Hg suggested that glutamate for delta-amino levulinic acid synthesis could be made available from NH4+ assimilation via., glutamine synthetase/glutamate synthase pathway during mercury toxicity.     

Effect of cadmium on chlorophyll biosynthesis and enzymes of nitrogen assimilation in greening maize leaf segments: role of 2-oxoglutarate

Supply of cadmium chloride (0.5 mM) inhibited chlorophyll formation in greening maize leaf segments, while lower concentration of Cd (0.01 mM) slightly enhanced it. Inclusion of 2-oxoglutarate (2-OG, 0.1-10 mM) in the incubation mixture increased chlorophyll content in the absence as well as presence of Cd. Substantial inhibition of chlorophyll formation by Cd was observed at longer treatment both in the absence and presence of 2-OG. When the tissue was pre-incubated with 2-OG or Cd, the inhibition (%) of chlorophyll formation by Cd was lowered in the presence of 2-OG. Treatment with Cd inhibited ALAD activity and ALA formation and the inhibition (%) of ALA formation by Cd was strongly reduced in the presence of 2-OG. Glutamate dehydrogenase (GDH) activity was increased by the supply of Cd both in the absence as well as presence of 2-OG. In the presence of 2-OG, Cd supply significantly increased glutamate synthase (GOGAT) activity and reduced inhibition (%) of glutamine synthetase (GS) activity. The results suggested the involvement of the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway of ammonia assimilation to provide the precursor, glutamate, for ALA synthesis under Cd toxicity and 2-OG supplementation.     

Regulation of ethylene biosynthesis during senescence of oat leaf segments

The evolution of endogenous ethylene, the conversion of 1-aminocylopropane-1-car-boxylic acid (ACC) to ethylene and the amounts of ACC (free and conjugated) have been followed during the senescence of oat ( Avena sativa L. cv. Victory) leaf segments. During the first three days of incubation of leaf segments in darkness, endogenous ethylene evolution and ACC-dependent ethylene production displayed a close relationship, both showing an increase followed by a decrease to the basal rate. However, unlike ethylene production, the level of ACC increased during the five days of incubation in the dark without any decline. It is concluded that ACC synthesis does not limit ethylene production, at least in the last stages of leaf senescence when ethylene production markedly decreased. The level of conjugated ACC increased and reached a plateau already at the first day of incubation. Yet, at the progressive stages of senescence, when the level af ACC gradually increased, no further conjugation of ACC could be detected. Thus, conjugation of ACC cannot account for ethylene drop at the last stages of oat leaf senescence.     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

Selective inhibition of chlorophyll biosynthesis by nicotinamide

Rhodobacter sphaeroides grown in the presence of nicotinamide excreted bacteriochlorophyll precursors, 2,4-divinyl protochlorophyllide (DV-Pchlide) and a small amount of 2-monovinyl protochlorophyllide (MV-Pchlide). Accumulation of these pigments indicates that nicotinamide inhibited the bacteriochlorophyll biosynthetic pathway site-specifically between DV-Pchlide and MV-Pchlide. This phenomenon is also observed in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114. Among 12 nicotinamide derivatives and isomers tested, only nicotinamide was effective, indicating that in addition to the completeness of the pyridine ring skeleton at positions 1 to 3, the carboxylic acid amide group is essential for this inhibition. The technique described in this report permits the simple preparation of large quantities of DV-Pchlide.     

Enzymes of chlorophyll biosynthesis

The enzymes responsible for chlorophyll biosynthesis in plants, algae and cyanobacteria are identified and described, with emphasis on their protein composition and structure, required cofactors, physical and catalytic properties, protein-protein interactions and allosteric modulation of activity. Properties and features of the pathway that enable it to operate in a coordinated way while using unstable and light-sensitive intermediates in potentially hostile biochemical environments are discussed. The evolutionary relationships and possible origins of the chlorophyll biosynthetic enzymes are also discussed.     

Light-dependent potassium uptake by Pisum sativum leaf fragments

A net K⁺ influx into chopped pea leaves bathed in 5 mM KCl,0.26 M sucrose and illuminated with 4000 lux amounted to about7.5 µmoles/g fresh weight-hr, while essentially no netflux occurred in the dark. This light-dependent K⁺ uptake waslinear with time for nearly 2 hr and continuously increasedas the light intensity was raised to 9000 lux. Over half ofthe K⁺ uptake was balanced by H⁺ release into the bathing solution,possibly by a mechanism in which bicarbonate was the anion accompanyingK⁺. The replacement of Cl^– by HCO₃^– increased thelight-dependent K⁺ uptake to 56 µmoles/g fresh weight-hr.About 23% of the light-dependent K⁺ uptake in 5 mM KCl was accompaniedby a Cl^– uptake. This net Cl^– influx was less sensitiveto the uncoupler tri-Fl-CCP and more sensitive to DCMU in thebathing solution than was the K⁺ uptake. The remaining net K⁺influx into pea leaf fragments was balanced by effluxes of sodium(accounting for 5%), magnesium (8%) and calcium (1%). (Received March 31, 1969; )     

Recent advances in chlorophyll biosynthesis

The importance of chlorophyll (Chl) to the process of photosynthesis is obvious, and there is clear evidence that the regulation of Chl biosynthesis has a significant role in the regulation of assembly of the photosynthetic apparatus. The understanding of Chl biosynthesis has rapidly advanced in recent years. The identification of genetic loci associated with each of the biochemical steps has been accompanied by a greater appreciation of the role of Chl biosynthesis intermediates in intracellular signaling. The purpose of this review is to provide a source of information for all the steps in Chl and bacteriochlorophyll a biosynthesis, with an emphasis on steps that are believed to be key regulation points.     

Recent advances in chlorophyll biosynthesis

The importance of chlorophyll (Chl) to the process of photosynthesis is obvious, and there is clear evidence that the regulation of Chl biosynthesis has a significant role in the regulation of assembly of the photosynthetic apparatus. The understanding of Chl biosynthesis has rapidly advanced in recent years. The identification of genetic loci associated with each of the biochemical steps has been accompanied by a greater appreciation of the role of Chl biosynthesis intermediates in intracellular signaling. The purpose of this review is to provide a source of information for all the steps in Chl and bacteriochlorophyll a biosynthesis, with an emphasis on steps that are believed to be key regulation points.     

Inhibition of 5-amino levulinic acid dehydratase activity by arsenic in excised etiolated maize leaf segments during greening

In vivo as well as in vitro supply of sodium arsenate inhibited the 5-Amino levulinic acid dehydratase (5-aminolevulinate-hydrolyase EC 4.2.1.24, ALAD) activity in excised etiolated maize leaf segments during greening. The percent inhibition of enzyme activity by arsenate (As) was reduced by the supply of KNO3, but it was increased by the glutamine and GSH. Various inhibitors, such as, chloramphenicol, cycloheximide and LA, decreased the % inhibition of enzyme activity by As. The % inhibition of enzyme activity was also reduced by in vivo supply of DTNB. The enzyme activity was reduced substantially by in vitro inclusion of LA, both in the absence and presence of As. In vitro inclusion of DTNB and GSH inhibited the enzyme activity extracted from leaf segments treated without arsenate (-As enzyme) and caused respectively no effect and stimulatory effect on arsenate treated enzyme (+As enzyme). Increasing concentration of ALA during assay increased the activity of -As enzyme and +As enzyme to different extent, but double reciprocal plots for both the enzymes were biphasic and yielded distinct S0.5 values for the two enzymes (-As enzyme, 40 micromol/L and +As enzyme, 145 micromol/L) at lower concentration range of ALA only. It is suggested that As inhibits ALAD activity in greening maize leaf segments by affecting its thiol groups and/or binding of ALA to the enzyme.     

Degradation of ribulose-1,5-bisphosphate carboxylase and chlorophyll in senescing barley leaf segments triggered by jasmonic acid methylester, and counteraction by cytokinin

Barley ( Hordeum vulgare L. cv. Salome) primary leaf segments responded to the application of a putative plant growth regulator, ± jasmonic acid methylester (JA-Me). with accelerated senescence, as indicated by the loss of chlorophyll and the rapid decrease in activity and immunoreactive protein content of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39). The senescence-promoting action of JA-Me differed in light and in darkness; e.g. the initial rates of chlorophyll and RuBP carboxylase breakdown were markedly higher in light than in darkness in the presence of 4.10⁻⁵ M JA-Me. Cytokinin (benzyladenine, 4.10⁻⁵ M ) stopped the loss of chlorophyll and RuBP carboxylase during senescence; however, the rapid drop induced by JA-Me in the early phase of leaf segment senescence could not be prevented by concomitant or previous addition of BA. On the other hand, BA added 24 h after JA-Me application resulted in a recovery of chlorophyll and RuBP carboxylase at the later stages, indicating a possible rapid inactivation of JA-Me in the tissues. The activities of a number of other chloroplastic and cytosolic enzymes were not significantly altered in JA-Me-treated leaf segments compared with controls floated on water. Time-dependent chlorophyll decrease in isolated chloroplasts did not change upon JA-Me addition to the isolated organelles. It is suggested that JA-Me acts on chloroplast senescence by promoting cytoplasic events which eventually bring about the degradation of chloroplast constituents.     

Studies of chlorophyll biosynthesis in Russia

History of the studies of chlorophyll biosynthesis by Russian and Byelorussian scientists starting from those by Kliment Timiriazeff (also spelled as Timiriazev) and Nikolay ( also spelled as Nikolai) Monteverde (late 19th century) to the present time are summarized here. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inhibition of Cuticular Lipid Biosynthesis in Pisum sativum by Thiocarbamates

Treatment of slices of young pea leaves (Pisum sativum) with μM solutions of α-chlorallyl diethyldithiocarbamate, dichloroallyl diisopropylthiocarbamate, or S-ethyldipropylthiocarbamate resulted in inhibition of incorporation of [1-¹⁴C]acetate into C₃₁ alkane and C₃₁ secondary alcohol, very little effect on the synthesis of C₂₆ and C₂₈ fatty alcohols, and an accumulation of ¹⁴C in shorter chain cuticular lipids, particularly C₂₂ acid. Higher concentrations of the thiocarbamates caused inhibition of synthesis of C₂₆ and C₂₈ fatty alcohols and an accumulation of label in C₂₂ acid. Further increase in thiocarbamate concentration resulted in inhibition of C₂₂ acid synthesis also. The three thiocarbamates at μM concentration also inhibited incorporation of [1-¹⁴C]stearic acid specifically into C₃₁ alkane and C₃₁ secondary alcohol. These results suggest that thiocarbamates reduce cuticular lipid formation by a concentration-dependent inhibition of the various chain-elongating enzyme systems.     

Inhibition of chlorophyll synthesis by kinetin in Cucumis cotyledons

Kinetin is shown to inhibit the synthesis of chlorophyll inlight while causing expansion of isolated cotyledons of Cucumis.The inhibitory effect is more marked at higher concentrationsof kinetin. It has been suggested that specific kinins may beinvolved in different tissues for stimulating chlorophyll synthesis. (Received August 14, 1969; )     

Redox regulation of chlorophyll biosynthesis

Chlorophyll captures and redirects light-energy and is thus essential for photosynthetic organisms. The demand for chlorophyll differs throughout the day and night and in response to changing light conditions. Moreover, the chlorophyll biosynthesis pathway is up to certain points shared between the different tetrapyrroles; chlorophyll, heme, siroheme and phytochromobilin, for which the cell has different requirements at different time points. Combined with the phototoxic properties of tetrapyrroles which, if not properly protected, can lead to formation of reactive oxygen species (ROS), the need for a strict regulation of the chlorophyll biosynthetic pathway is obvious. Here we describe the current knowledge on regulation of chlorophyll biosynthesis in plants by the chloroplast redox state with emphasis on the Mg-chelatase situated at the branch point between the heme and the chlorophyll pathway. We discuss the proposed role of the Mg-chelatase as a key regulator of the tetrapyrrole pathway by its effect on enzymes both up- and downstream in the pathway and we specifically describe how redox state might regulate the Mg-branch. Finally, we propose that a recently identified NADPH-dependent thioredoxin reductase (NTRC) could be involved in redox regulation or protection of chlorophyll biosynthetic enzymes and describe the possible modes of action by this enzyme.     

Inhibition of IAA-induced elongation in Avena coleoptile segments by lead: a physiological and an electron microscopic study.

A high resolution growth measuring apparatus was used to demonstrate the inhibition of auxin-induced cell elongation in oat coleoptile segments (Avena sativa L. var Holden) by lead at concentrations ranging from 2 x 10-6 M to 2 x 10-3 M. The inhibition was immediate, having no measurable lag period. Electron micrographs of lead-treated and control segments revealed that in the treated material, lead became localized as electron-dense granules in the cell walls and in vesicles associated with dictyosomes. These granules were found to be lead hydroxide phosphate by electron diffraction techniques. The possible significance of this localization and identification with regard to phosphatase activity is discussed.     

Age-dependent response of maize leaf segments to cadmium treatment: effect on chlorophyll fluorescence and phytochelatin accumulation

The relationship between the age of leaf tissue and response of the photosynthetic apparatus and phytochelatin accumulation to Cd treatment was studied. Studies were carried out with seedlings of Zea mays L. cv. Hidosil grown in the presence of 100-200 mumol/L Cd for 14 days under low light conditions. The third leaf was divided into 3 segments of equal length differing in the stage of tissue maturity and used for measurements of chlorophyll content, chlorophyll fluorescence, glutathione and phytochelatin content and Cd accumulation. A close relationship between the age of leaf tissue and response of the photosynthetic apparatus to Cd was shown. Cadmium (200 mumol/L) reduced photochemical processes more in older than younger leaf segments as seen in the Chl fluorescence parameters Fv/F0, and t1/2, while the chlorophyll fluorescence decrease ratio (Rfd) was inhibited more strongly in younger ones. Fv/Fm was slightly affected. Cd-induced enhancement of GSH content was correlated with higher phytochelatin accumulation to a greater extent in younger than in older leaf segments. Phytochelatin level corresponded to changes of photochemical processes in older leaves. The peptide thiol:Cd molar ratio for the phytochelatins varied depending on Cd concentration and age of leaf segments. The protective role of phytochelatins for the photosynthetic apparatus is discussed.     

Inhibition of aldosterone biosynthesis by staurosporine

Staurosporine (STS) is a very potent broad-range kinase inhibitor, and its antiproliferative properties made it a lead compound for protein kinase C (PKC) inhibitors with therapeutic potential. Because STS also causes hypotension, we investigated in this study whether it directly interferes with the terminal steps of aldosterone biosynthesis; these are catalysed by a mitochondrial steroid hydroxylase system consisting of adrenodoxin reductase, adrenodoxin, and the cytochrome P450 enzyme hCYP11B2 (aldosterone synthase). Here we demonstrate that nanomolar concentrations of STS significantly reduced aldosterone synthase activity in transiently transfected COS-1 cells and in stably transfected V79MZh11B2 cells (IC50 = 11 nM). However, STS did not inhibit bovine aldosterone synthase in a reconstituted steroid hydroxylation assay. In transiently transfected COS-1 cells, the protein level of adrenodoxin (but not that of adrenodoxin reductase or of hCYP11B2) was significantly reduced after treatment with 2 nM STS. Finally, we show that STS treatment (1 microg/day) of mice reduced their aldosterone/renin ratio by almost 50% (p = 0.015). To the best of our knowledge, this is the first report of a direct in vivo effect of STS on the renin-angiotensin-aldosterone system. We conclude (i) that the hypotensive effect of staurosporine is at least partly due to inhibition of aldosterone biosynthesis via adrenodoxin depletion, and (ii) that aldosterone biosynthesis can be regulated in vivo at the level of adrenodoxin availability.     

Effects and interactions of gibberellic acid and cytokinins on the retention of chlorophyll and phosphate in barley leaf segments

In leaf segments of barley ( Hordeum vulgare L. cv. Mozoncillo), the cytokinin specificity for retarding the loss of phosphate is different from that of retarding the loss of chlorophyll. Some cytokinins require the simultaneous application of gibberellins to delay the loss of phosphate. Although both chlorophyll and phosphate losses occur in senescence, they are apparently controlled by cytokinins through different mechanisms.