Inhibition studies on acetyl group incorporation into chloroplast fatty acids

Abstract. Whilst L-acetylcarnitine acted as a substrate for fatty acid synthesis by isolated pea leaf chloroplasts, D-acetylcarnitine did not. This result, together with those obtained using the inhibitors D-carnitine and deoxycarnitine, indicated that L-acetylcarnitine was not being hydrolysed to free acetate prior to incorporation into chloroplast fatty acids. Seventy-five per cent and 66% inhibitions of L-acetylcarnitine incorporation into fatty acids, brought about by adding equimolar quantities of D-carnitine and deoxycarnitine, respectively, were suggestive of competitive inhibition at two points: an integral membrane translocator in the chloroplast envelope: and the carnitine acetyltransferase enzyme of the chloroplast stroma, which converts L-acetylcarnitine to acetyl CoA. Isotope competition experiments between acetate and L-acetylcarnitine confirmed that L-acetylcarnitine was the preferred substrate for pea chloroplast fatty acid synthesis.     

Characterization of a proton pump from pea stem microsomes

Abstract The present work deals with the characterization of an ATP-dependent proton translocation monitored by the ΔpH probe acridine orange. The ATP-dependent proton translocation has an optimum activity at pH 6.5 and is substrate specific for ATP. It is stimulated by Cl⁻, HCO₃⁻ and Br⁻, but is insensitive to several monovalent cations. Divalent cations (Mg²⁺ or Mn²⁺) are required for proton translocation, while in the presence of Ca²⁺ no uptake is observed. NO₃⁻, NO₂⁻ and citrate strongly inhibit proton uptake. On the contrary, F⁻, SO₄²⁻, malate, pyruvate, succinate, oxalate and acetate have no inhibitory effect. Proton uptake is stimulated by valinomycin and unaffected by molybdate. Two thiols, dithioerythritol and dithiothreitol, are able partially to prevent the FCCP-abolished proton uptake or partially restore the ATP-dependent proton translocation in FCCP-collapsed vesicles. It is suggested that pea stem microsomes possess an electrogenic ATPase, acting as a proton pump, which, on the basis of its characteristics, can be tentatively associated with membranes of tonoplast origin.     

The role of Coenzyme A in lipid synthesis by a particulate fraction from germinating peas

The effect of CoA on fatty acid synthesis by the microsomal fraction from germinating pea (Pisum sativum) was examined. Increasing concentrations of CoA progressively decreased total fatty acid synthesis from [¹⁴C]malonyl-CoA. However, the synthesis of very long chain fatty acids was relatively unaffected so that their proportion in the reaction products increased. Other CoA-esters also decreased total fatty acid synthesis while increasing the relative accumulation of radioactivity in very long chain fatty acids. The addition of CoA also altered the distribution of newly synthesized fatty acids in different lipid fractions. Complex lipid labelling was relatively increased while that of acyl-acyl carrier proteins was decreased. Very long chain fatty acids accumulated in lipids rather than thioesters. The role of CoA in controlling fatty acid synthesis in the pea microsomal fraction is discussed.     

Inhibition of chloroplast development by tentoxin

Light-dependent chloroplast development in detached pea shoots was measured in terms of chlorophyll synthesis and the synthesis of Fraction 1 protein. Both synthetic processes were inhibited more than 90% by the fungal metabolite, tentoxin (1 or 10 μg/ml). These results place Pisum sativum in the class of tentoxin-sensitive higher plants. Tentoxin, actinomycin D, lincomycin, D-threo-chloramphenicol and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were compared in their ability to inhibit RNA and protein synthesis by isolated pea chloroplasts. Energy for the synthetic reactions was supplied either by light or by added ATP. Only CCCP gave the same pattern of inhibition as tentoxin, i.e. inhibition of both RNA and protein synthesis in the light-driven system but no inhibition in the ATP-driven system. It is concluded that chloroplast developmental processes are inhibited by tentoxin through the inhibition of photophosphorylation.     

Jasmonic Acid is Induced in a Biphasic Manner in Response of Pea Seedlings to Wounding

The role of jasmonic acid （JA） in plant wounding response has been demonstrated. However, the source of JA in wound signaling remains unclear. In the present study, pea seedlings were used as material to investigate the systemic induction of JA and the activation of lipoxygenase （LOX）-dependent octadecanoid pathway upon wounding. The results showed that endogenous JA could induce two peaks in the wounded leaves and the stalks, while only one peak in the systemic leaves. LOX activity and its protein amount were also induced and the stimulation mainly occurred in the late phase, while one peak of induction was present after pretreatment with JA. Applied nordihydroguaiaretic acid （NDGA）, an inhibitor of LOX activity, only inhibited the induction of JA in the late phase, and the resistance of pea was impaired. Furthermore, 13（S）- hydroperoxy-9（Z）, 11 （E）-octadecadienoic acid （13（S）-H（P）ODE） was confirmed to be the main product of LOX throughout the experimental time. in addition, immunocytochemical analysis also revealed the occurrence of JA biosynthesis and transport upon wounding. These results demonstrated that wound-induced JA in wounded leaves resulted from its biosynthesis and conversion from its conjugates, while in systemic leaves resulted from its transport and biosynthesis; and proved that the LOX pathway was vital to the wound-induced defense response involved in JA biosynthesis.     

Ascorbic acid effect on the onset of cell proliferation in pea root

The ability of ascorbic acid to induce cell proliferation of non-cycling cells was investigated in quiescent embryo root of Pisum sativum L. cv. Lincoln, as well as in the active plantlet root meristem, where a minor portion of the cells is non-proliferating. Quiescent embryo cells speeded up the G₀–G₁ transition during germination in the presence of ascorbic acid. In addition, proliferating cells present in the root tip of 3-day-old plantlets, arrested at the G₁/S boundary by hydroxyurea, resumed the cycle earlier than the control, when treated with ascorbic acid. In contrast, ascorbic acid was unable to induce the proliferation of non-cycling cells present in the active meristem. Therefore, these data suggest that the ability of ascorbic acid lo induce cell proliferation depends on the physiological status of the cell. In particular the data indicate that ascorbic acid is involved in cell proliferation as a factor necessary to enable already competent cells to progress through the cell cycle phases, but not as a factor able to induce non-competent cells to overcome proliferation arrest.     

Cytokinin oxidase/dehydrogenase activity as a tool in gibberellic acid/cytokinin cross talk

Changes in endogenous cytokinin (CK) content and cytokinin oxidase/dehydrogenase activity (CKX) in response to gibberellic acid (GA₃) in two pea cultivars with different life span were assessed. The control leaves of cv. Scinado, which developed faster, had higher initial cytokinin content and lower CKX activity, while opposite trend was observed in cv. Manuela with longer life span. Increased CKX and decreased CK content were detected in leaves of cv. Scinado after treatments with 0.5, 1 and 5 μM GA₃. Changes in CK content and CKX activity in GA₃-treated cv. Manuela leaves were reciprocal to those in cv. Scinado. CK content and CKX activity in roots were not significantly influenced by the application of GA₃. The slight repression of CKX activity in some of the root samples was accompanied by increased isopentenyladenine and isopentenyladenine riboside content. Obtained results suggest that CKX was responsible for the changes in endogenous cytokinin pool in GA₃-treated plants and most probably this enzyme represents an important link in GA/cytokinin cross talk.     

Re-examination of the localization of Mg-chelatase within the chloroplast

In a plastid-free assay, Mg-chelatase from pea ( Pisum sativum L. cv. Spring) and cucumber ( Cucumis sativus L. cv. Sumter) chloroplasts is inhibited to equal extents by the mercurial reagents. p -chloromercuribenzoate (PCMB) and p -chloromercuribenzene sulfonate (PCMBS). However, in intact chloroplasts PCMB inhibits Mg-chelatase fourfold more strongly than does PCMBS. Since PCMBS cannot penetrate membranes as readily as PCMB, Mg-chelatase may be localized interior to the inner chloroplast envelope. When Mg-chelatase is assayed with photosynthetically generated ATP, the presence of an external ATP trap does not inhibit activity, suggesting that the enzyme is not located in the interenvelope space. None of the components of Mg-chelatase are integral membrane proteins: Mg-chelatase activity is readily solubilized by washing the total chloroplast membranes in buffers of low MgCl₂ content. This precludes localization by purifying individual thylakoid and envelope membranes which requires low MgCl₂ concentrations.     

Catabolism of indole-3-acetic acid in chloroplast fractions from light-grown Pisum sativum L. seedlings

Abstract The catabolism of indole-3-acetic acid was investigated in chloroplast preparations and a crude enzyme fraction derived from chloroplasts of Pisum sativum seedlings. Data obtained with both systems indicate that indole-3-acetic acid undergoes decarboxylative oxidation in pea chloroplast preparations. An enhanced rate of decarboxylation of [1′-¹C]indole-3-acetic acid was obtained when chloroplast preparations were incubated in the light rather than in darkness. Results from control experiments discounted the possibility of this being due to light-induced breakdown of indole-3-acetic acid. High performance liquid chromatography analysis of [2′-¹⁴C]indole-3-acetic acid-fed incubates showed that indole-3-methanol was the major catabolite in both the chloroplast and the crude enzyme preparations. The identification of this reaction product was confirmed by gas chromatography-mass spectrometry when [²H₅]indole-3-methanol was detected in a purified extract derived from the incubation of an enzyme preparation with ³²H₅]indole-3-acetic acid.     

A temperature-sensitive nodulation mutant (sym 5) of Pisum sativum L.

Abstract. In peas ( Pisum sativum L.) homozygous for sym 5, nodulation has an unusual temperature dependence. These sym 5 mutants nodulate poorly at a root temperature of 20°C but nodulate better at 12°C. By lowering the root temperature of the sym 5 mutants from a lightroom temperature of 20/15°C to a constant 12°C, 8d after planting, the number of nodules can be further increased. A cool period (12°C) as short as 6h, early in the infection process, is sufficient to significantly increase nodulation of plants otherwise growing at 20/15°C. This temperature-sensitivity of nodulation is not due to a temperature induced change of a sym 5-related, 66-kD peptide but may involve accumulation of a gas in the rhizosphere.     

Roots of Pisum sativum L. exhibit hydrotropism in response to a water potential gradient in vermiculite

In the present study, root hydrotropism in an agravitropic mutant of Pisum sativum L. grown in vermiculite with a steep water potential gradient was examined. When wet and dry vermiculite were placed side by side, water diffused from the wet (-0.04 MPa) to the dry (-1.2 MPa) and a steep water potential gradient became apparent in the dry vermiculite close to the boundary between the two. The extent and location of the gradient remained stable between the fourth and sixth day after filling a box with vermiculite, and the steepest gradient (approx. 0.02 MPa mm-1) was found in the initially dry vermiculite between 60 and 80 mm from the boundary. When seedlings with 25-35 mm long roots were planted in the initially dry vermiculite near where the gradient had been established, each of the main roots elongated toward the wet vermiculite, i.e. toward the high water potential. Control roots elongated without curvature in both the wet and the dry vermiculite, in which no water potential gradient was detectable. These results show that pea roots respond to the water potential gradient around them and elongate towards the higher water potential. Therefore, positive hydrotropism occurs in vermiculite just as it does in air. Hydrotropism in soil may be significant when a steep water potential gradient is apparent, such as when drip irrigation is applied.     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Jasmonic Acid is Induced in a Biphasic Manner in Response of Pea Seedlings to Wounding

The role of jasmonic acid (JA) in plant wounding response has been demonstrated. However, the source of JA in wound signaling remains unclear. In the present study, pea seedlings were used as material to investigate the systemic induction of JA and the activation of lipoxygenase (LOX)-dependent octadecanoid pathway upon wounding. The results showed that endogenous JA could induce two peaks in the wounded leaves and the stalks, while only one peak in the systemic leaves.LOX activity and its protein amount were also induced and the stimulation mainly occurred in the late phase, while one peak of induction was present after pretreatment with JA. Applied nordihydroguaiaretic acid (NDGA), an inhibitor of LOX activity, only inhibited the induction of JA in the late phase, and the resistance of pea was impaired. Furthermore, 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid (13(S)-H(P)ODE) was confirmed to be the main product of LOX throughout the experimental time. In addition, immunocytochemical analysis also revealed the occurrence of JA biosynthesis and transport upon wounding. These results demonstrated that wound-induced JA in wounded leaves resulted from Its biosynthesis and conversion from its conjugates, while in systemic leaves resulted from its transport and biosynthesis; and proved that the LOX pathway was vital to the wound-induced defense response involved in JA biosynthesis.     

Rapid decarboxylation of the products of dark fixation of CO2 in roots of pisum and Plantago

Seedlings of Pisum sativum and excised roots of Plantago major and P. lanceolata were given, in the dark, a pulse of ¹⁴CO₂ in air followed by a chase in ¹²CO₂ in air. A very substantial proportion of the ¹⁴C fixed into organic compounds in the pulse was lost from the tissues in the chase. The activity of NAD malic enzyme in extracts of roots of all three species exceeded their rate of respiration. Azide, 2-n-butylmalonate, and salicylhydroxamic acid each inhibited CO₂ fixation by excised roots of pea. The first two compounds inhibited respiratory gas exchange, but the third stimulated it. Arguments are presented for the widespread diversion of phosphoenolpyruvate from glycolysis to oxaloacetate and thence to malate in the cytosol followed by transport of the malate into the mitochondria for conversion to pyruvate via NAD malic enzyme. No differences, in the above respects, were found between the two species of Plantago.     

Effect of number and configuration of fruits, photon flux and age on the growth and dry matter distribution of fruits of Pisum sativum L.

Abstract. In experiments with Pisum sativum cv. Sleaford Orbiter in a controlled environment, the effect of fruit number and position, photon flux density and developmental stage on fruit growth was studied. During early development (up to 22 d from anthesis) growth of the first fruit was unaffected by the presence of one or two additional fruits irrespective of their position. When grown to maturity in competition with fruits at the same node a small decrease in weight of this fruit was observed. Where plants retained a full complement (20-30) of fruits the growth of the first fruit was markedly decreased at all stages of development (6-40 d). In all instances where competition was observed, the pericarp was more affected than the seeds. This was particularly so when photon flux was decreased 18-22 d from anthesis compared with a decrease at an earlier stage. Partition of dry matter between fruits showed a progressively increasing allocation to the later-formed fruits with time for all treatments. The actual proportions allocated to different fruits were not changed by the number of competing fruits. Decreasing photon flux by more than two-thirds decreased fruit growth rates but had little effect on dry matter partitioning in most cases, although where all fruits were retained, there was a tendency for fruits at the lower reproductive nodes to be less affected. These findings are discussed in relation to known sources of assimilate for fruits, assimilate transport and sink demand. It is suggested that partition of dry matter between fruits can be estimated on the basis of fruit size and the developmental trend in relative growth rate of fruits grown in the absence of competition for assimilate from other fruits.     

Neutral growth inhibitors in light-grown dwarf pea shoots –separation, identification and growth regulation

To correlate endogenous growth inhibitors of dwarf pea to its dwarfism a thorough search of growth inhibitors was made in the neutral fraction of an acetone extract from the shoots of light-grown seedlings of Pisum sativum L. cvs Progress No 9 and Alaska. From both cultivars six inhibitors were separated and named A-1, A-2, A-3, B-1, B-2 and B-3 based on their order of elution from the silica gel column. Their contents as determined by cress root bioassay were: in cv. Progress 0.40, 16.5, 6.36, 1.02, 0.11 and 0.10, and in cv. Alaska 0.33, 2.35, 3.51, 0.95, 0.10 and 0.09 cress units (g fresh weight)⁻¹. Their contributions to growth regulation of the relevant pea plants were estimated as the products of the above-stated contents times the ratios of the specific activities of each standard sample in the cress roots and the relevant pea cultivars, and they were; in cv. Progress A-2, 5.44 and A-3, 2.10, and in cv. Alaska A-2, 0.68 and A-3, 0.88, those of A-2 and A-3 constituting more than 90% of the total contribution of the six inhibitors in either cultivar. The great differences in the content and contribution to growth regulation of A-2 and A-3 between the two cultivars suggest that the higher contents of and greater responsiveness to the two inhibitors in cv. Progress may be causes of dwarfism of this cultivar. A-l and A-3 were spectroscopically identified with pisatin and a mixture of cis, trans– , and trans, trans xanthoxins.     

Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6- dinitroanilino)-N-methylpropionamide

Selective effects of lincomysin and cycloheximide in detached shoots of Pisum sativum on the synthesis of photosystem I and II proteins, and a chloroplast membrane protein of molecular weight 32000, confirm results obtained from studies of protein synthesis by isolated chloroplasts. A model is proposed in which one role of chloroplast ribosomes is to synthesize membrane proteins required for the immobilization of chloroplast components, such as photosystem I protein, which are synthesized by cytoplasmic ribosomes. 2-(4-Methyl-2,6-dinitroanilino)-N-methylpropionamide rapidly inhibits the synthesis of both the large and small subunits of Fraction I protein in greening detached pea shoots. This observation can be reconciled with the site of synthesis of the large subunit being in the chloroplast by a model which proposes that the small subunit is a positive initiation factor for the synthesis or translation of the messenger RNA for the large subunit.     

Interaction of ethylene with indole-3-acetic acid in regulation of rooting in pea cuttings

Cuttings of pea (Pisum sativum L. cv Marma) were treated with 1-aminocyclopropane-l-carboxylic acid (ACC). This treatment caused increased ethylene production and reduction of root formation. The effect of 0.1 mM ACC on the level of endogenous indole-3-acetic acid (IAA) in the rooting zone and in the shoot apex was analyzed by gas chromatography-single ion monitoring mass spectrometry or by high pressure liquid chromatography with fluorimetric detection (HPLC). Concentrations of indole-3-acetylaspartic acid (IAAsp) in the stem bases were also determined using HPLC. The ACC treatment had little effect on the IAA level in the base measured after 24 h, but caused a considerable decrease during the 3 following days. IAAsp increased in the base on days 1, 2 and 3 and then declined. The build up of IAAsp in the base was not affected by ACC during the first two days of the treatment, but later this conjugate decreased more rapidly than in controls. No effect of the ACC treatment was found on the level of IAA in the apex. IAA (1 µM) applied to the cuttings during 24 h reduced the number of roots formed. The possibility that IAA-induced ethylene is involved in this response was investigated.Our results support earlier evidence that the inhibitory effect of ethylene on rooting in pea cuttings is due to decreased IAA levels in the rooting zone. The inhibitory effect of applied IAA is obtained if the internal IAA level is maintained high during the first 24 h, whereas stimulation of rooting occurs if the internal IAA level remains high during an extended period of time. Our results do not support the suggestion that ethylene mediates the inhibitory effect of applied IAA.     

The influence of high levels of brief or prolonged supplementary far-red illumination during growth on the photosynthetic characteristics, composition and morphology of Pisum sativum chloroplasts

Abstract. Peas were grown in controlled environments (12h white fluorescent light. ∼47 μmol photons m^-2 s 1/12 dark, 25 °C), using (1) 15-min far-red illumination at the end of each photoperiod (brief FR) to simulate the increase in the far-red/red ratio near the end of the day, and (2) high levels of supplementary far-red light (red:far-red ratio=0.04) during the entire photoperiod (long-term FR) to simulate extreme shade conditions under a plant canopy. Brief FR illumination led to marked morphological effects attributable to phytochrome regulation, namely, an increase in internodal length, but a decrease in leaflet area, chloroplast size and chlorophyll content per chloroplast compared with the control. Significantly, brief FR illumination had little or no effect on the amounts of the major chloroplast components (ribulose 1.5-biphosphate carboxylase, adenosine triphosphate synthase, cytochrome b/f complex and Photosystem II) relative to chlorophyll or Photosystem I, and the leaf photosynthetic capacities per unit chlorophyll were similar. In contrast, supplementing high levels of far-red light during the entire photoperiod not only led to the phytochrome effects above, but there was also a marked increase in leaf photosynthetic capacity per unit chlorophyll. due to increased amounts of the major chloroplast components relative to chlorophyll or Photosystem I. We hypothesize that supplementary far-red light, absorbed by Photosystem I, induced an increase in the major chloroplast components by a photosynthetic feedback mechanism. In fully greened leaves, we propose that the two photosystems themselves, rather than phytochrome, may be the predominent sensors of light quantity in triggering modulations of the stoichiometries of chloroplast components, which in turn lead to varying photosynthetic capacities.     

ATP-dependent proton uptake inhibited by Cercospora beticola toxin in pea stem microsomal vesicles

Abstract. The effect of Cercospora beticola toxin (CBT) on ATP-dependent and nigericin-induced proton translocation, monitored by acridine orange uptake in pea stem microsomal vesicles, was studied. CBT inhibits ATP-dependent proton translocation, but not the nigericin-induced H⁺/K⁺ exchange. The inhibitory effect is dependent on CBT concentration, time of preincubation with CBT and protein concentration of the vesicle suspension.
The previously observed effects of CBT on membrane transport phenomena, in the light of the present results, are in agreement with the hypothesis that the primary effect of the toxin is exerted on an ATPase of plasmalemma and/or tonoplast, acting as a proton pump.