Chloroplast development in 4-thiouridine-cultured radish seedlings III. Photochemical activities in isolated plastids

Accumulation of chlorophyll, development of photosystem I andII activities and contents of chloroplastic components wereinvestigated in greening radish seedlings germinated and grownwith 4-thiouridine (4SU). The development of photosystem I activityprior to that of photosystem II was observed also in the 4SU-culturedgreening radish cotyledons in which chlorophyll accumulationwas inhibited up to 60–80% of that of the control. Photochemicalactivities expressed on a plastid protein basis decreased withthe increase of 4SU in the culture medium. In contrast to ferredoxinand ferredoxin-NADP reductase, which were present in significantamounts in the treated cotyledons, chloroplastic cytochromes(f, b₅₅₉ and b₆ decreased in the plastids from 4SU-culturedcotyledons. These results suggest that 4SU interferes in partwith protein synthesis in plastids and thereby with chloroplastdevelopment. (Received December 4, 1979; )     

Changes in some enzymes of microbodies and plastid development in excised radish cotyledons: effect of narciclasine

Narciclasine (NCS), isolated from mucilage of Narcissus bulb, showed inhibitory effects on growth and plastid development of excised radish cotyledons. NCS (0.1 mumol/L) started to show inhibitory effects on isocitrate lyase and hydroxypyruvate reductase activities after 24 h incubation in light. When NCS concentration was increased to 10 mumol/L, the activities of both enzymes are completely inhibited. From ultrastructural studies, NCS markedly prevented the degradation of protein bodies and lipid bodies, as well as chloroplast formation of excised radish cotyledons. There was only little degradation of protein and lipid bodies, and almost no chloroplast formation in the excised radish cotyledon treated with 1 mumol/L NCS. Therefore, our results provide clear evidence that NCS inhibited the transition of glyoxysomes and peroxisomes, and chloroplast development.     

Inhibition by mevinolin of plant growth, sterol formation and pigment accumulation

To obtain information on the importance of a functional mevalonate synthesis for plant growth and development, we investigated the effect of mevinolin, a highly specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A reductase (the mevalonate-producing enzyme) on growth, sterol accumulation and pigment formation of radish seedlings (Raphanus sativus L. cv. Saxa Treib) and in part also wheat seedlings (Triticum aestivum L. cv. Kolibri). Mevinolin applied during germination inhibits root elongation and development of lateral roots in etiolated and light-grown radish seedlings. This effect cannot be overcome by exogenous GA₃, but by addition of mevalonic acid, the product of the internally inhibited reaction. This emphazises the specifity of the mevinolin effect and indicates that the biosynthesis of mevalonic acid is a mandatory requirement for root growth. In light-grown radish seedlings mevinolin also affects hypocotyl length-growth and inhibits sterol accumulation, but has little effect on the chlorophyll and carotenoid accumulation in the chloroplasts of the cotyledons. This indicates the possible presence of an independent mevalonate synthesizing pathway within the plastids and suggests a low transport rate of mevinolin from the radish roots to the cotyledons. When mevinolin is directly applied to the leaves at higher concentrations, it also reduces the light-induced chlorophyll and carotenoid accumulation as has been shown with etiolated primary leaves of wheat. This inhibition is age-dependent and proceeds to a higher extent in older than in younger etiolated leaf tissue. From our results we conclude that plastids possess an independent HMG-CoA reductase. In the cotyledons of radish, mevinolin seems to induce a senescence retardation and sun-type growth response, as has been evaluated by measuring the fast and slow chlorophyll fluorescence induction kinetics (Kautsky effect). These responses may be due to inhibitor-induced changes in the intracellular phytohormone balance.     

Fatty-acid metabolism in senescing and regreening soybean cotyledons

Changes in fatty-acid metabolism were studied in soybean (Glycine max Merr.) cotyledons during senescence as well as in cotyledons which had been caused to regreen by removal of the epicotyl from the seedling. The activities of the enzymes acetyl-CoA synthetase (EC 6.2.1.1) and fatty-acid synthetase in plastids isolated from the cotyledons decreased during senescence but increased in response to regreening. These changes in enzyme activities followed the same pattern as changes in the quantities of chlorophyll and polyunsaturated fatty acids in this tissue. The in-vivo incorporation of [¹⁴C]acetate into total fatty acids in the senescing and regreening cotyledons did not vary markedly with age. In addition, the quantity of label in fatty acids did not decrease for as much as 60 h after the removal of the substrate. During this 60-h period however, there was substantial redistribution of the label among the individual fatty acids. While the labelling pattern of the individual fatty acids did not vary significantly with respect to age in the senescing cotyledons, there was a substantial increase in the synthesis of labelled polyunsaturated fatty acids in the regreening tissue. Thus, the incorporation of [¹⁴C]acetate into fatty acids did not reflect the changes in the quantities of the individual fatty acids in senescing tissue as well as they did in regreening tissue.     

The capacity of plastids from developing pea cotyledons to synthesise acetyl CoA

In order to determine whether the enzymes required to convert triose phosphate to acetyl CoA were present in pea (Pisum sativum L.) seed plastids, a rapid, mechanical technique was used to isolate plastids from developing cotyledons. The plastids were intact and the extraplastidial contamination was low. The following glycolytic enzymes, though predominantly cytosolic, were found to be present in plastids: glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), and pyruvate kinase(EC 2.7.1.40). Evidence is presented which indicates that plastids also contained low activities of enolase (EC 4.2.1.11) and phosphoglycerate mutase (EC 2.7.5.3). Pyruvate dehydrogenase, although predominantly mitochondrial, was also present in plastids. The plastidial activities of the above enzymes were high enough to account for the rate of lipid synthesis observed in vivo.Abbreviations FPLC fast protein liquid chromatography - PPi pyrophosphate     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings V. Protein Synthesis in Vivo

Incorporation of ¹⁴C-amino acids into proteins in radish cotyledonswas suppressed by 4-thiouridine (4SU) culture. The inhibitoryeffect of 4SU was similar to that of chloramphenicol. 4SU culturedid not reduce the content of ferredoxin (Fd) and the labelinginto Fd significantly, but it did decrease the content and thesynthesis of ribulose bisphosphate carboxylase (RuBPCase). Thesynthesis of thylakoid chlorophyll-proteins I and II also wasinhibited by 4SU culture. In 4SU-cultured seedlings, the ratioof labeling into the large and small subunits of RuBPCase andthat into the two chlorophyll-proteins were the same as thosein the controls grown without 4SU. (Received September 29, 1980; Accepted January 27, 1981)     

Accumulation of Vacuolar H+-Pyrophosphatase and H+-ATPase during Reformation of the Central Vacuole in Germinating Pumpkin Seeds

Protein storage vacuoles were examined for the induction of H+-pyrophosphatase (H+-PPase), H+-ATPase, and a membrane integral protein of 23 kD after seed germination. Membranes of protein storage vacuoles were prepared from dry seeds and etiolated cotyledons of pumpkin (Cucurbita sp.). Membrane vesicles from etiolated cotyledons had ATP- and pyrophosphate-dependent H+-transport activities. H+-ATPase activity was sensitive to nitrate and bafilomycin, and H+-PPase activity was stimulated by potassium ion and inhibited by dicyclohexylcarbodiimide. The activities of both enzymes increased after seed germination. On immunoblot analysis, the 73-kD polypeptide of H+-PPase and the two major subunits, 68 and 57 kD, of vacuolar H+-ATPase were detected in the vacuolar membranes of cotyledons, and the levels of the subunits of enzymes increased parallel to those of enzyme activities. Small amounts of the subunits of the enzymes were detected in dry cotyledons. Immunocytochemical analysis of the cotyledonous cells with anti-H+-PPase showed the close association of H+-PPase to the membranes of protein storage vacuoles. In endosperms of castor bean (Ricinus communis), both enzymes and their subunits increased after germination. Furthermore, the vacuolar membranes from etiolated cotyledons of pumpkin had a polypeptide that cross-reacted with antibody against a 23-kD membrane protein of radish vacuole, VM23, but the membranes of dry cotyledons did not. The results from this study suggest that H+-ATPase, H+-PPase, and VM23 are expressed and accumulated in the membranes of protein storage vacuoles after seed germination. Overall, the findings indicate that the membranes of protein storage vacuoles are transformed into those of central vacuoles during the growth of seedlings.     

Intraplastidic Localization of the Enzymes That Convert delta-Aminolevulinic Acid to Protoporphyrin IX in Etiolated Cucumber Cotyledons

The intraplastidic localization of the enzymes that catalyze the conversion of δ-aminolevulinic acid (ALA) to protoporphyrin IX (Proto) is a controversial issue. While some researchers assign a stromal location for these enzymes, others favor a membranebound one. Etiochloroplasts were isolated from etiolated cucumber cotyledons (Cucumis sativus, L.) by differential centrifugation and were purified further by Percoll density gradient centrifugation. Purified plastids were highly intact, and contamination by other subcellular organelles was reduced five- to ninefold in comparison to crude plastid preparations. Most of the ALA to Proto conversion activity was found in the plastids. On a unit protein basis, the ALA to Proto conversion activity of isolated mitochondria was about 2% that of the purified plastids, and could be accounted for by contamination of the mitochondrial preparation by plastids. Lysis of the purified plastids by osmotic shock followed by high speed centrifugation, yielded two subplastidic fractions: a soluble clear stromal fraction and a pelleted yellowish one. The stromal fraction contained about 11% of the plastidic ALA to Proto conversion activity while the membrane fraction contained the remaining 89%. The stromal ALA to Proto conversion activity was in the range of stroma contamination by subplastidic membrane material. Complete solubilization of the ALA to Proto activity was achieved by high speed shearing and cavitation, in the absence of detergents. Solubilization of the ALA to Proto conversion activity was accompanied by release of about 30% of the membrane-bound protochlorophyllide. It is proposed that the enzymes that convert ALA to Proto are loosely associated with the plastid membranes and may be solubilized without the use of detergents. It is not clear at this stage whether the enzymes are associated with the outer or inner plastid membranes and whether they form a multienzyme complex or not.     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings XI. Inhibition of RNA Synthesis by 4-Thiouridine Nucleotides during Germination of Radish Seeds

The effects of 4-thiouridine and its metabolites on RNA synthesisin radish (Raphanus sativus) cotyledons were investigated. The4-thiouridine nucleotides, 4-thioUTP and 4-thioUDP, were foundto inhibit DNA-dependent RNA polymerase activities of isolatednuclei and of chloroplasts from radish cotyledons. These inhibitorsappeared to compete with UTP for its binding to RNA polymerase.Neither 4-thiouridine nor 4-thioUMP inhibited RNA polymeraseactivities. Reduced RNA accumulation accompanied by the inhibition of protochlorophyllideaccumulation was observed in cotyledons of dark-grown radishseedlings germinated and grown with 4-thiouridine. On the otherhand, 4-thiouridine had no effect on chloroplast developmentin the control seedlings germinated and grown without 4-thiouridine.These results suggest that the inhibition of chloroplast developmentby 4-thiouridine may in part be due to the inhibition of RNAsynthesis by 4-thiouridine nucleotide during germination, resultingin inhibition of etioplast development. (Received December 9, 1985; Accepted June 26, 1986)     

Mechanism of Benzyladenine-Induced Stimulation of the Synthesis of 5-Aminolevulinic Acid in Greening Cucumber Cotyledons: Benzyladenine Increases Levels of Plastid tRNAGlu

The mechanism of the stimulatory effect of a cytokinin, namely,benzyladenine (BA), on the synthesis of 5-aminolevulinic acid(ALA) in cucumber cotyledons was studied. The rate of synthesisof ALA by plastids isolated from BA-treated cotyledons was twicethat by plastids from untreated controls. Western blot analysisof stromal proteins showed that BA did not affect the levelof glutamyl-tRNA synthetase or of glutamate l-semialdehyde (GSA)aminotransferase. Analysis of free amino acids revealed thatBA did not increase the level of glutamate in the stroma. However,the amount of total plastidic RNA was doubled in BA-treatedcotyledons. Northern blot analysis showed that the level ofplastid tRNA^Glu was increased by treatment with BA to the sameextent as that of another plastid tRNA, reflecting an increasein total plastidic RNA. The rate of formation of glutamyl-tRNAwas also doubled in plastids from BA-treated cotyledons. Theresults indicate that stimulation of the synthesis of ALA byBA is due to an increased level of tRNA^Glu in plastids. (Received June 6, 1993; Accepted November 26, 1993)     

Light-dependent development of single cell C4 photosynthesis in cotyledons of Borszczowia aralocaspica (Chenopodiaceae) during transformation from a storage to a photosynthetic organ

BACKGROUND AND AIMS: Previous work has shown that Borszczowia aralocaspica (Chenopodiaceae) accomplishes C4 photosynthesis in a unique, polarized single-cell system in leaves. Mature cotyledons have the same structure as leaves, with chlorenchyma cells having biochemical polarization of dimorphic chloroplasts and C4 functions at opposite ends of the cell. KEY RESULTS: Development of the single-celled C4 syndrome in cotyledons was characterized. In mature seeds, all cell layers are already present in the cotyledons, which contain mostly lipids and little starch. The incipient chlorenchyma cells have a few plastids towards the centre of the cell. Eight days after germination and growth in the dark, small plastids are evenly distributed around the periphery of the expanding cells. Immunolocalization studies show slight labelling of Rubisco in plastids in seeds, including chlorenchyma, hypodermal and water storage, but not epidermal, cells. After imbibition and 8 d of growth in the dark labelling for Rubisco progressively increased, being most prominent in chlorenchyma cells. There was no immunolabelling for the plastid C4 enzyme pyruvate, Pi dikinase under these conditions. Cotyledons developing in light show formation of chlorenchyma tissue, induction of the cytosolic enzyme phosphoenolpyruvate carboxylase and development of dimorphic chloroplasts at opposite ends of the cells. Proximal chloroplasts have well-developed grana, store starch and contain Rubisco; those located distally have reduced grana, lack starch and contain pyruvate, Pi dikinase. CONCLUSIONS: The results show cotyledons developing in the dark have a single structural plastid type which expresses Rubisco, while light induces formation of dimorphic chloroplasts from the single plastid pool, synthesis of C4 enzymes, and biochemical and structural polarization leading to the single-cell C4 syndrome.     

Formation of Tyrosine Phenol-lyase by Bacteria

Formation of protochlorophyllide and ribulose-1,5-diphosphate carboxylase, both of which were constituents proper to chloroplast, was inhibited in the radish cotyledons grown with 4-thiouridine (0.5 mm), though the growth of the seedlings, activities of a cytoplasmic enzyme glucose-6-phosphate dehydrogenase and of a mitochondrial enzyme glutamate dehydrogenase in the cotyledones were not affected. Photo-induced formation of chlorophyll pigments and of chloroplastic ribosomal RNAs in the 4-thiouridine treated cotyledones were depressed as compared with controls. Interestingly, the effect of 4-thiouridine, however, disappeared with continuous illumination. Based upon these results, specific development of the chloroplast is discussed.     

NCS对离体萝卜子叶微体中酶活性及细胞超微结构的影响

研究了天然生物活性物质narciclasine(NCS)对离体萝卜子叶光下生长发育期间叶绿素含量变化、异柠檬酸裂解酶及羟基丙酮酸还原酶活性的影响;并对萝卜子叶细胞的超微结构变化进行了观察。结果表明,NCS明显抑制光下培养的离体萝卜子叶叶绿素含量及鲜重增加,对异柠檬酸裂解酶及羟基丙酮酸还原酶活性也显示出明显的抑制作用。电镜观察显示,NCS对蛋白体及脂质体的降解、叶绿体的发育也表现出强烈的抑制效应。NCS的各种抑制作用均随其浓度的增加而增加,而且高浓度的NCS(10^-5mol／L)基本上完全阻止了离体萝卜子叶的光下生长及其转绿。     

Effect of benzyladenine on the development of plastids and microbodies in excised watermelon cotyledons

Excised watermelon (Citrullus vulgaris Schrad.) cotyledons were grown in the dark in the presence of 0.1 mM benzyladenine (BA). Under these conditions reserve breakdown and organelle differentiation progress very slowly. Treatment with BA accelerates, breakdown of reserves and stimulates development of organelles. Electron micrographs of cells from treated cotyledons show a larger number of plastids with a more developed inner membrane system. The levels of plastid pigments and enzymes are increased while starch content is reduced. Glyoxysomal enzyme levels are increased by BA during the first three days of development and their decline is accelerated thereafter. Also the activity of hydroxypyruvate reductase (EC 1.1.1.81.), a peroxisomal enzyme, is increased, but this increase is not followed by a decay phase. In water controls, hydroxypyruvate reductase bands together with glyoxysomal enzymes after equilibrium centrifugation in a sucrose gradient. In treated cotyledons the equilibrium position of glyoxysomal enzymes is uchanged while that of hydroxypyruvate reductase is shifted to a lower density.Abbreviations BA benzyladenine - RuDP ribulose-1,5-diphosphate - HPR hydroxypyruvate reductase     

The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants

1. Possible mechanisms regulating the activities of three enzymes involved in nitrate assimilation, nitrate reductase, nitrite reductase and glutamate dehydrogenase, were studied in radish cotyledons. 2. Nitrate-reductase and nitrite-reductase activities are low in nitrogen-deficient cotyledons, and are induced by their substrates. 3. Glutamate dehydrogenase is present regardless of the nitrogen status, and the enzyme can be increased only slightly by long-term growth on ammonia. 4. Although nitrate is the best inducer of nitrate reductase, lower levels of induction are also obtained with nitrite and ammonia. The experiments did not distinguish between direct or indirect induction by these two molecules. 5. Nitrite reductase is induced by nitrite and only indirectly by nitrate. 6. The induction of both nitrate reductase and nitrite reductase is prevented by the inhibitors actinomycin D, puromycin and cycloheximide, indicating a requirement for the synthesis of RNA and protein. 7. The decay of nitrate reductase, determined after inhibition of protein synthesis, is slower than the synthesis of the enzyme. Nitrite reductase is much more stable than nitrate reductase. 8. The synthesis of nitrate reductase is not repressed by ammonia, but is repressed by growth on a nitrite medium. 9. There is no inhibition of nitrate reductase, nitrite reductase or glutamate dehydrogenase by the normal end products of assimilation, but cyanate is a fairly specific inhibitor of nitrate reductase.     

Localization of carbamoylphosphate synthetase and aspartate carbamoyltransferase in chloroplasts

The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-grown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSase, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed.     

Changes in soluble carbohydrates and related enzymes induced by low temperature during early developmental stages of quinoa (Chenopodium quinoa) seedlings

Low temperature represents one of the principal limitations in species distribution and crop productivity. Responses to chilling include the accumulation of simple carbohydrates and changes in enzymes involved in their metabolism. Soluble carbohydrate levels and invertase, sucrose synthase (SS), sucrose-6-phosphate synthase (SPS) and alpha-amylase activities were analysed in cotyledons and embryonic axes of quinoa seedlings grown at 5 degrees C and 25 degrees C in the dark. Significant differences in enzyme activities and carbohydrate levels were observed. Sucrose content in cotyledons was found to be similar in both treatments, while in embryonic axes there were differences. Invertase activity was the most sensitive to temperature in both organs; however, SS and SPS activities appear to be less stress-sensitive. Results suggest that 1) metabolism in germinating perispermic seeds would be different from endospermic seeds, 2) sucrose futile cycles would be operating in cotyledons, but not in embryonic axes of quinoa seedlings under our experimental conditions, 3) low temperature might induce different regulatory mechanisms on invertase, SS and SPS enzymes in both cotyledons and embryonic axes of quinoa seedlings, and 4) low temperature rather than water uptake would be mainly responsible for the changes observed in carbohydrate and related enzyme activities.     

Enzymes of starch and sugar phosphate metabolism in achlorophyllous ribosome-deficient plastids from high-temperature-grown rye leaves

Several enzymes of non–photosynthetic sugar phosphate and starch metabolism were measured in gradient–purified chloroplasts from normal rye leaves ( Secale cereale L. cv. Halo) grown at 22°C and in the non-photosynthetic plastids isolated from 70S ribosome-deficient rye leaves grown at a non–permissive elevated temperature of 32°C. Activities of the enzymes phosphoglycerate kinase (EC 2.7.2.3), hexokinase (EC 2.7.1.1), phosphoglucose isomerase (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate de-hydrogenase (EC 1.1.1.46), ADPglucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), and phosphorylase (EC 2.4.1.1) were present in ribosome-deficient plastids from 32°C-grown leaves indicating a cytoplasmic origin of the plastid-specific forms of these enzymes. While the photosynthetic marker enzyme NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) was considerably diminished, both the specific activities and the total activities per leaf of the plastid-specific forms of hexokinase, phosphoglucose isomerase and phosphoglucomutase were markedly increased in the ribosome–deficient plastids, relative to normal chloroplasts. The results demonstrate that after elimination of functional protein synthesis in the chloroplasts the supply of chloroplast–specific enzymes by the cytoplasm is not generally suppressed as observed for many enzymes and proteins involved in photosynthesis, but may even be increased in accord with changed metabolic demands.