Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : III. Evidence for Functional Heterogeneity of the ALA Pool

Gabaculine and 4-amino-5-hexynoic acid (AHA) up to 3.0 millimolar concentration strongly inhibited 5-aminolevulinic acid (ALA) synthesis in developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts, while they hardly affected protochlorophyllide (Pchlide) synthesis. Exogenous protoheme up to 1.0 micromolar had a similar effect. Exogenous glutathione also exhibited a strong inhibitory effect on ALA synthesis in organello but hardly inhibited Pchlide synthesis. Pchlide synthesis in organello was highly sensitive to inhibition by levulinic acid, both in the presence and in the absence of gabaculine, indicating that the Pchlide was indeed formed from precursor(s) before the ALA dehydratase step. The synthesis of Pchlide in the presence of saturating concentrations of glutamate was stimulated by exogenous ALA, confirming that Pchlide synthesis was limited at the formation of ALA. The gabaculine inhibition of ALA accumulation occurred whether levulinic acid or 4,6-dioxohepatonic acid was used in the ALA assay system. ALA overproduction was also observed in the absence of added glutamate and was noticeable after 10-minute incubation. These observations suggest that although Pchlide synthesis in organello is limited by ALA formation, it does not utilize all the ALA that is made in the in organello assay system. Gabaculine, AHA, and probably also protoheme, inhibit preferentially the formation of that portion of ALA that is not destined for Pchlide. A model proposing a heterogenous ALA pool is described.     

Regulation of 5-aminolevulinic Acid synthesis in developing chloroplasts : I. Effect of light/dark treatments in vivo and in organello

Intact chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons regenerated protochlorophyllide (Pchlide) in the dark with added cofactors from either exogenous glutamate or endogenous substrates. No other intermediates of the chlorophyll biosynthetic pathway accumulated. When inhibitors of 5-aminolevulinic acid (ALA) dehydratase were added, the Pchlide that failed to form was replaced by an excessive amount of ALA. When greening seedlings were returned to the dark, ALA-synthesizing activity in the isolated chloroplasts decreased dramatically and recovered if the dark-treated seedlings were again exposed to continuous white light prior to chloroplast isolation. Both the decline and the recovery of ALA-synthesizing activity were complete in approximately 50 minutes. Changes in chloroplast structure during in vivo light to dark and dark to light transitions (as evidenced by electron microscopy) were much slower. Exposing isolated chloroplasts from dark-treated seedlings to short white flashes before incubation transformed nearly all the endogenous Pchlide, but hardly stimulated ALA synthesis, suggesting that Pchlide does not act as a feed-back inhibitor on ALA synthesis. Chloroplasts isolated from dark-treated tissue did not form Pchlide from glutamate when incubated in the dark with added cofactors; moreover, the endogenous Pchlide did not turn over in organello. However, these chloroplasts did synthesize Pchlide from added ALA at the normal rate and synthesized ALA from glutamate at a reduced, but still significant, rate. Mg chelation was not affected by in vivo dark treatment.     

Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : II. Regulation of ALA-Synthesizing Capacity by Phytochrome

When dark-grown cucumber (Cucumis sativus L.) seedlings previously exposed to white light for 20 hours were returned to darkness, the ability of isolated chloroplasts to synthesize 5-aminolevulinic acid dropped by approximately 70% within 1 hour. The seedlings were then exposed to light, and the synthetic ability of the isolated chloroplasts was determined. Restoration of the synthetic capacity was promoted by continuous white or red light of moderate intensity. Intermittent red light was also effective. Blue and far-red light did not restore the synthetic capability. Blue light given after a red pulse did not enhance the effect of the red light. Far-red light given immediately after each red pulse prevented the stimulation due to intermittent red light. Restoration of the biosynthetic activity by in vivo light treatments was inhibited by cycloheximide indicating the requirement for translation on 80 S ribosomes for the in vivo light response. These findings suggest that the majority of the plastidic 5-aminolevulinic acid synthesis is under phytochrome regulation.     

Inhibition of phytochrome synthesis by gabaculine

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a transaminase inhibitor, also inhibits chlorophyll formation in plants, and the effect of this compound can be counteracted by 5-aminolevulinic acid (ALA) (Flint, personal communication, 1984). Since it is probable that ALA also serves as a precursor to phytochrome, the effects of gabaculine on phytochrome synthesis in developing etiolated seedlings were examined using in vivo spectrophotometry. Preemergence treatment with gabaculine was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis, perhaps due to preexisting phytochrome in the seed. Foliar treatment of etiolated pea seedlings prior to light-induced destruction of phytochrome inhibited subsequent phytochrome resynthesis in the dark. These results suggest that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein.     

Characterisation of abscisic acid inhibition of stomatal opening in isolated epidermal strips

Abscisic acid (ABA) inhibited the light-induced opening of stomata in isolated epidermal strips of Commelina benghalensis. It did not alter stomatal closure in the dark. The ABA-induced inhibition in light was released under conditions conducive for cyclic photophosphorylation and remarkably reversed by ATP in the presence of pyruvate. Cyclic photophosphorylation rates of isolated guard cell chloroplasts were significantly reduced by ABA. It is proposed that the direct effect of ABA on stomatal opening was mediated in two ways: (1) by inhibition of cyclic photophosphorylation activities of guard cell chloroplasts and (2) by blocking organic acid formation in guard cells.     

A re-examination of 5-aminolevulinic acid synthesis by isolated intact,developing chloroplasts: The O2 requirement in the light

Synthesis of 5-aminolevulinic acid (ALA) in organello was re-examined with developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var. Beit Alpha) cotyledons. In the dark, ALA accumulated in the presence of ATP, reducing power (NADPH and glucose-6-phosphate), glutamate and levulinic acid (or 4,6-dioxoheptanoic acid).Under continuous illumination there was no requirement for added ATP and reducing power, unless DCMU was added or O₂ was removed, indicating that ATP and reducing power could be supplied endogenously by photosynthesis in the presence of O₂. No mitochondrial involvement could be demonstrated in this system. Under anaerobic conditions in the light oxaloacetic acid (OAA) could replace O₂ and permit a high accumulation of ALA. The fact that OAA could replace O₂ suggests that an acceptor of non-cyclic electron flow may be required to provide ATP or some other cofactor of ALA synthesis. The phosphorylation uncoupler, 2,4-dinitrophenol, inhibited ALA synthesis. Light-dependent ALA in air was strongly inhibited by methylene blue (MB) and NaN₃, but only very slightly by KCN.     

A Low Molecular Weight Polypeptide Which Accumulates upon Inhibition of Porphyrin Biosynthesis in Maize

Levulinic acid, an inhibitor of porphyrin biosynthesis, causes marked accumulation of a low molecular weight polypeptide in greening maize (Zea mays L.) leaves. Additional compounds which interfere with porphyrin synthesis (e.g. aminooxyacetate, iron-chelators, 4,6-dioxoheptanoic acid) had a similar effect. The polypeptide accumulated in the cytosol and could not be detected in the plastid stroma. Its molecular weight was estimated as 4800 daltons by electrophoresis in sodium dodecyl sulfate-acrylamide gels containing urea and glycerol. The accumulation of the polypeptide did not result from inhibition of chlorophyll or protoheme syntheses. Compounds which caused its accumulation markedly reduced the activity of nitrite reductase. It is suggested that the accumulation is caused by inhibition of siroheme synthesis which interferes with the formation of nitrite or sulfite reductase.     

Natural Inhibitor for Volatile C6-Aldehyde Formation from C18-Unsaturated Fatty Acids

The homogenate of tea seed cotyledons contained an inhibitor for C₆-aldehyde formation from linoleic acid and linolenic acid by isolated tea chloroplasts. Seed homogenates of other plants, such as soybean, kidney bean, cucumber, Japanese radish and rice, also contained the inhibitor for C₆-aldehyde formation. The inhibitor from tea seed and cucumber seed inhibited C₆-aldehyde formation by the homogenate of cucumber hypocotyl. Hydroperoxides of linoleic acid detected were reduced when the tea seed inhibitor was added to the reaction mixture, but the enzyme activities of lipoxygenase and hydroperoxide lyase were not inhibited. This means that the inhibitor is a decomposer of fatty acid hydroperoxides as an intermediate of C₆-aldehyde formation. The tea seed inhibitor was formed during the seed ripening and it was stable during the seed germination. These findings obtained here suggest that the inhibitor is widely present in plant seeds and inhibits C₆-aldehyde formation by a variety of plant tissues.     

Inhibition of oxygen evolution in chloroplasts by ferricyanide

Preincubation of chloroplasts from pea leaves (Pisum sativum L. cv. Kelvedon) with 0.5 millimolar ferricyanide in the dark, caused a parallel inhibition of the rate of rise of the variable fluorescence and the rate of electron transport. Both reactions were inhibited to a similar extent by varying the time of preincubation, the concentration of ferricyanide during preincubation, and by raising the concentration of salts in the preincubation medium. Ferricyanide treatment of Tris-washed chloroplasts did not inhibit electron transport from the Photosystem II (PSII) electron donor 1,5-diphenylcarbazide to methylviologen. The inhibition of the variable fluorescence rise and of NADP reduction (caused by ferricyanide pretreatment) was bypassed by addition of the PSII electron donor couple hydroquinone/ascorbate. It was concluded that preincubation of chloroplasts with ferricyanide in the dark inhibited electron transport between water and PSII.     

Arginine modifiers as energy transfer inhibitors in photophosphorylation

Photophosphorylation by spinach chloroplasts is inhibited after they have been incubated in the dark with either phenylglyoxal or butanedione. Inhibition by phenylglyoxal is strongest when N-ethylmorpholine is the buffer used during the incubation; that by butanedione requires the presence of borate as buffer. The inhibitions are not reversed by simply washing out the inhibitor, suggesting that a covalent modification of one or more arginine residues is responsible. This is supported by the reversibility of the butanedione inhibition if both the inhibitor and borate buffer are removed. ATPase of the chloroplasts, and of extracted protein, is inhibited, whether activated by trypsin or by heating. This indicates that arginine residues of the coupling factor are the probable major site(s) for attack by these modifiers, leading to the observed inhibitions.     

Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.     

Energy charge and emergence of the coleoptile and radicle at varying oxygen levels in Echinochloa crus-galli

The role of protein synthesis in senescence and in the inhibition of senescence by light and kinetin was studied in barley ( Hordeum vulgare L. cv. Hassan) leaves with different inhibitors of protein synthesis. A comparison of the actions of D- and L-chloramphenicol was made to compensate for the effects of D-chloramphenicol not mediated by inhibition of protein synthesis. The involvement of phytochrome was also studied. The results suggest that: 1) cytoplasmic protein synthesis is required for senescence in the light and in the dark; 2) chloroplasts, in the dark, synthesize protein which accelerates senescence; 3) kinetin inhibits the synthesis by chloroplasts of senescence-accelerating protein; 4) light changes the type of protein synthesized by chloroplasts from those accelerating to those retarding senescence; and 5) lightretar-dation of senescence is mediated by phytochrome and, probably, by photophos-phorylation.     

Light-controlled sugar-starch interconversion in epidermal chloroplasts of light-grown cucumber hypocotyl sections and its relationship to cell elongation

Sugar-starch interconversion in epidermal chloroplasts of light-grown cucumber hypocotyl sections as a regulatory mechanism of the osmotic potential of the cell was studied in relation to cell elongation. The presence of chloroplasts in epidermal cells was confirmed by electron microscopy, and also the chloroplasts were shown to act as the site of sucrose-starch interconversion. Chloroplast starch formation was induced by light, which was more distinct in the presence of sucrose (50 mM). The starch formation was microscopically detectable even at 1 hr incubation in the light with sucrose. On the other hand, no starch formation was observed in the dark both in the presence and absence of sucrose. Red light was effective, but not blue light. A photosynthetic inhibitor, 3-(4-chlorophenyl)-1:1-dimethylurea, also inhibited starch formation. Thus, epidermal chloroplast starch formation was induced under conditions where cell elongation is small and the osmotic potential (Ψ ₀) of the epidermal cell is high. The sugar quantity (free sugar and reducing sugar) as the osmotica of the cell was larger in the dark than in the light, whereas the quantity of starch was greater in the light than in the dark. It is assumed from these results that one of the regulatory mechanisms of the osmotic potential of the epidermal cells in sugar-starch interconversion which occurs in epidermal chloroplasts.     

Investigation of the Mechanism of Action of a Chlorosis-Inducing Toxin Produced by Pseudomonas phaseolicola

A toxin that induced chlorotic haloes (typifying haloblight disease) on primary leaves of Phaseolus vulgaris L. (var. Canadian Wonder) was partially purified from culture filtrates of the causative agent Pseudomonas phaseolicola (Burkh.) Dowson. This material was used to investigate chlorosis induction. Haloes could only be induced in those bean leaves that were expanding and synthesizing chlorophyll (Chl); the toxin, therefore, does not promote Chl breakdown. Chl, carotene, and xanthophyll synthesis were inhibited in sections of greening barley (Hordeum vulgare L.) leaves, irrespective of the irradiance level. In parallel experiments, the toxin decreased the level of 5-aminolevulinic acid by amounts sufficient to account for toxin-inhibition of Chl synthesis. Electron microscopy revealed no difference between the transformation of etioplasts into chloroplasts in toxin-treated and control tissue, despite a 60% reduction in Chl in the former. The incorporation of [¹⁴C]acetate into lipid by greening barley leaf sections and by isolated Pisum sativum chloroplasts in the light and the dark was inhibited about 60% by the toxin. The distribution of radioactivity among the spectra of acyl residues was the same in the control and toxin-treated material. It is suggested that the toxin interferes with an early process common to the synthesis of different lipids, including Chl.     

5-Aminolevulinic Acid Induced Photodynamic Reactions in Thylakoid Membranes of Cucumber (Cucumis sativus L.) Cotyledons

The cucumber (Cucumis sativus L.) plants were sprayed with 20 mM 5-aminolevulinic acid or distilled water (control) and incubated in dark for 14 hr. The thylakoid membranes prepared from the intact chloroplasts, isolated from the above plants in dark, were illuminated with low light intensity (100 W/m2) for 30 min. Due 10 photodynamic reactions, the photochemical function of photosystem II was damaged by 50% in treated thylakoids whereas it was only slightly (8%) affected in control thylakoids. The photosystem I was, however, not affected. The exogenous electron donors, MnCl2, diphenyl carbazide and NH2OH failed to restore the photosystem II activity suggesting that the photodynamic damage had taken place very close to photosystem II reaction center. Singlet oxygen scavenger, histidine, could protect the photosystem II activity while superoxide radical scavengers, superoxide dismutase and 1, 2-dihydroxybenzene-3, 5-disulphonic acid disodium salt, and hydroxyl radical scavenger, formate, failed to protect the same.     

Inhibitory effect of p-nitrothiophenol in the light on the Photosystem II activity of spinach chloroplasts

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH's caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiophenol to causing the specific inhibition of Photosystem II.     

Die Wirkung von Chloramphencol und Streptomycin auf die Lamellarproteine der Chloroplasten von Vicia faba

Vicia faba plants sprayed with chloramphenicol (0.1%) or streptomycin (0.5%) produce chlorotic chloroplasts with a modified pattern of lamellar proteins. The antibiotics were found to affect the protein patterns by specific inhibition of at least two lamellar proteins. The proteins whose synthesis was found to be inhibited by the antibiotics are evidently produced by the 70S ribosomes of chloroplasts.     

Chemical modification studies of chloroplast membranes. Water oxidation inhibition by diazonium-benzenesulfonic acid

The water-soluble chemical modifier, diazonium benzene-sulfonic acid, significantly inhibited photosystem II-dependent water oxidation (oxygen evolution) when the compound was reacted with chloroplast membranes in the light but not in the dark. The photochemistry of photosystem II was not affected by the diazonium treatment, shown by complete restoration of photosystem II-dependent electron flow from the alternate electron donor diphenylcarbazide.Paralleling the inhibition of oxygen evolution the illuminated chloroplasts bound significantly more diazonium reagent than did chloroplasts treated in the dark. Both the inhibition of oxygen evolution and the increased binding of the diazonium to the membranes were dependent on photosystem II electron flux, which could not be replaced by photosystem I cyclic electron flow. A dark base to acid or acid to base transition resulted in a similar inhibition of water oxidation and increased diazonium binding.The results suggest a membrane conformational change associated with photosystem II electron flow that exposes otherwise buried diazo reactive groups at the external grana membrane surface.