Lipid biosynthesis by isolated plastids from greening pea, Pisum sativum

Isolated etioplasts from 8-day-old dark-grown pea seedlings incorporated [1-(14)C]acetate into lipid at a relatively low rate. Plastids from seedlings that had been illuminated for at least 2 hr showed an enhanced incorporation provided the plastids were illuminated during incubation with the labeled acetate. Dark incubation or the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased the acetate-incorporating activity of the developing chloroplasts to the level observed with etioplasts. Light had a marked effect on the type of fatty acid into which acetate was incorporated by the developing chloroplasts. Unsaturated fatty acids (mostly oleic acid) accounted for 60-80% of the incorporated label if the plastids were illuminated, but in the dark or in the presence of DCMU the unsaturated acids accounted for only 0-15% of the label incorporated into lipid. The effect of ATP on incorporation was dependent on the maturity of the chloroplasts; mature pea chloroplasts were inhibited by ATP, whereas in developing plastids there was a slight stimulation by ATP. Inhibition of acetate incorporation into lipid by DCMU appears to be due to inhibition of noncyclic phosphorylation. Incorporation was restored by reduced 2,3,5,6-tetramethylphenylenediamine, which restored phosphorylation, but not by reduced N,N,N',N'-tetramethylphenylenediamine.     

Effects of the selective herbicide fluazifop on fatty acid synthesis in pea (Pisum sativum) and barley (Hordeum vulgare).

Concentrations of fluazifop-butyl sprayed on intact plants caused large decreases in the incorporation of radioactivity from [1-14C]acetate into lipids of barley (Hordeum vulgare) leaves and stems, but did not affect leaves or stems of pea (Pisum sativum). Labelling of all acyl lipids, but not pigments, was reduced. The effects of the active acid form, fluazifop, were also determined in leaf pieces and chloroplasts. Concentrations of (R,S)-fluazifop up to 100 microM had no affect upon quality or quantity of fatty acids produced from [1-14C]acetate in pea. In barley, however, 100 microM-(R,S)-fluazifop caused 89% (leaf) or 100% (chloroplasts) inhibition in labelling of fatty acids from [1-14C]acetate. Lower concentrations of fluazifop (less than 25 microM) caused incomplete inhibition and significant decreases in the proportion of C18 fatty acids synthesized, particularly by isolated chloroplasts. Synthesis of fatty acids from [2-14C]malonate was also inhibited (59%) in barley leaf tissue by 100 microM-(R,S)-fluazifop. The labelling pattern of products showed that elongation reactions were unaffected by the herbicide, but synthesis de novo was specifically diminished. By using resolved stereoisomers, it was found that the (R) isomer was the form which inhibited fatty acid synthesis, a finding that is in agreement with its herbicidal activity. These results suggest that inhibition of fatty acid synthesis de novo forms the basis for the selective mode of action of fluazifop.     

Additive and synergistic effects of kinetin and ethrel on germination, thermodormany, and polyribosome formation in lettuce seeds

The inhibition of germination of Grand Rapids lettuce (Lactuca sativa L.) seeds at 35 C was removed to a marked extent by kinetin and 2-chloroethylphosphonic acid (ethrel). When both compounds were used together, an additive effect was observed. A synergistic effect was, however, noted when ethrel promoted the kinetin reversal of abscisic acid inhibition of seed germination (light- as well as gibberellic acid-, induced). Both kinetin and ethrel increased the total ribosomal material and the percentage of polyribosomes in lettuce seeds imbibed in the light for 24 hours. A combination of the two compounds showed a synergism in polyribosome formation only at high ethrel concentration. The inability of ethrel to reverse abscisic acid inhibition indicates that kinetin action cannot always be substituted by ethrel. The possible mechanisms involved in the enhanced response by a combination of kinetin and ethrel are discussed.     

生菜种子低温处理后耐冻性和脂肪酸合成代谢的关系研究

以生菜(Lactuca sativa)种子为研究对象,通过不同时间的吸水处理分析其含水量变化,再通过程序降温处理,分析不同含水量种子发芽率的差异,以及脂肪酸合成有关基因(FAD2、FAD3、PPT、ELOVL)和冷调节基因ICE1的表达。结果表明,种子含水量随吸水时间增加而升高。程序降温至同样的低温冷冻条件下(-20℃、-22℃),吸水时间小于6 h的种子发芽率较高,而吸水8 h以上的种子发芽率显著降低。种子吸水8 h含水量处于饱和状态,在此状态下种子对低温较为敏感,说明含水量对种子耐冻性有影响。冷冻处理后生菜种子基因表达检测结果表明,脂肪酸去饱和酶基因(FAD2、FAD3)、蛋白质棕榈酰基硫脂酶相关基因(PPT）、长链脂肪酸延伸酶相关基因(ELOVL)的表达水平均随着种子含水量增加呈上升趋势,吸胀10 h的种子表达量最高,此时种子由于高含水量所受冷冻伤害最大。基因ICE1在冷冻处理种子中的表达也随着吸水时间增加而升高,在吸水10 h时种子中表达量到最高水平。综上,种子含水量越高,所受冷冻伤害越大。但种子在低温条件下具有一定的抗冷反应,可通过相关基因的过表达调控合成更多不饱和脂肪酸、抗冻蛋白等提高含水种子耐冻性。     

L-acetylcarnitine, a substrate for chloroplast fatty acid synthesis

Abstract. H¹⁴CO₃ was not incorporated into fatty acids by isolated pea leaf chloroplasts, which, therefore, do not possess a self-contained pathway for the synthesis of fatty acids from early intermediates of the Calvin cycle. Citrate, pyruvate, acetate and L-acetylcarnitine were all shown to act as sources of acetyl groups for fatty acid synthesis by pea leaf chloroplasts. L-acetylcarnitine was the best substrate, being incorporated into fatty acids at rates that were at least five-fold higher than those achieved with the other substrates. Citrate was incorporated into fatty acids at the lowest rate, followed by pyruvate, with acetate being incorporated at the second highest rate of all. When the isolated chloroplasts were ruptured, an inhibition of L-acetylcarnitine incorporation into fatty acids was noted, whilst acetate incorporation remained unaffected. L-acetylcarnitine also increased the ratio of monoenoic: saturated fatty acids synthesized, compared with a 1:1 ratio observed when citrate, pyruvate and acetate were supplied as substrates. It is suggested that L-carnitine and carnitine acyltransferases play a central role in plant acyl CoA metabolism by facilitating the transfer of activated acyl groups across membranes (acyl CoA barriers).     

Lipase-induced alterations of fatty acid synthesis by subcellular fractions from germinating pea (Pisum sativum L.).

1. The effect of exogenous lipases on fatty acid synthesis from [14C]malonyl-CoA by the microsomal and soluble fractions from germinating peas was studied. 2. Addition of phospholipase A2 or the lipase from Rhizopus arrhizus had no effect on total fatty acid synthesis by the soluble fraction but caused severe inhibition of that by the microsomal fraction. 3. The addition of enzymes with phospholipase activity particularly inhibited the microsomal stearate elongase. 4. Control studies indicated that the phospholipase-induced inhibition of fatty acid synthesis was due to the location of fatty acid synthetase, palmitate elongase and stearate elongase on the outside of the microsomal vesicles. 5. Experiments with a trypsin-like proteinase showed that approximately half the microsomal fatty acid synthesis was resistant to proteolysis. 6. Although addition of exogenous phospholipases had no effect on total fatty acid synthesis by the soluble fraction, it did increase alpha-hydroxylation of newly-formed palmitate and stearate. 7. The results provide further evidence for differences between the soluble and particulate fatty acid synthetase and palmitate elongase activities of germinating pea.     

Sidedness studies of thylakoid phosphatidylglycerol in higher plants.

The transmembrane distribution of phosphatidylglycerol was determined in thylakoids from barley (Hordeum vulgare), lettuce (Lactuca sativa) and pea (Pisum sativum) chloroplasts. Phospholipase A2 and phospholipase D digestion and chemical-labelling methods were used. Phosphatidylglycerol was preferentially localized in the outer (stromal) leaflet. The proportion of the phospholipid in this leaflet ranged from about 66% in pea to about 75% for barley and lettuce thylakoids. One of the main fatty acids, trans-delta 3-hexadecenoic acid, was exclusively located in the outer leaflet in all three plant types. The data are discussed in relation to suggested roles for phosphatidylglycerol in thylakoid function.     

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.     

Site of action of inhibitors of carbon dioxide assimilation by whole lettuce chloroplasts

The sites of action of several compounds, reported to inhibit CO(2) fixation by chloroplast preparations were located by developing assays in lettuce chloroplasts to test their effect on partial reactions of the carbon cycle and on carbonic anhydrase. The results indicated that: d, l-glyceral-dehyde and 5'-AMP inhibit phosphoribulose kinase or isomerase. 3-Phosphoglyceric acid and 6-phosphogluconate inhibit ribulose diphosphate carboxylase. Azide, Mg(2+), and nitrite inhibit the activity of carbonic anhydrase of lettuce chloroplasts and light-dependent CO(2) fixation by intact chloroplasts with similar sensitivities. None of these inhibited CO(2) fixation in ruptured chloroplasts. It is suggested that the inhibition by azide, nitrite, and magnesium ions of CO(2) fixation by intact chloroplasts is due to their inhibition of the activity of carbonic anhydrase.     

The influence of endogenous acyl-acyl carrier protein concentrations on fatty acid compositions of chloroplast glycerolipids.

The concentrations of long-chain acyl-acyl carrier proteins (acyl-ACP) occurring during fatty acid synthesis from [1-14C]acetate within chloroplasts isolated from spinach, pea, and amaranthus leaves were manipulated by making minor changes to a basal incubation medium containing sn-glycerol 3-phosphate (G3P). Pools of oleoyl-, stearoyl-, and palmitoyl-ACP were compared with those of the corresponding 1-acyl glycerol 3-phosphates to determine how endogenous acyl-ACP concentrations affected the fatty acid compositions of chloroplast glycerolipids. The 1-acyl G3P synthesized by isolated chloroplasts contained more palmitate than would be expected for the precursor of thylakoid phosphatidylglycerol in the different plant species. However, treatments which increased ratios of oleoyl- to palmitoyl-ACP by about 50% increased synthesis of sn-1-oleoyl G3P to the extent anticipated from known fatty acid compositions of the different phosphatidylglycerols. Since stearate constituted 70-73% of the acyl-ACP and 48-51% of the 1-acyl-G3P pool of spinach and pea chloroplasts incubated in the presence of cyanide, it is transferred to G3P much more efficiently in situ than would be predicted from competition studies using mixtures of acyl donors and purified acyltransferases. Increasing concentrations of G3P in incubation media from 0.1 to 2 mM had relatively little effect on the amounts and proportions of acyl-ACPs but forced the synthesis of palmitoyl-G3P and, ultimately, disaturated glycerolipid. It is concluded that the chloroplast G3P acyltransferases are primarily responsible for determining the fatty acid compositions of procaryotic glycerolipids in plants, but that acyl-ACP concentrations may play a more important role than would be anticipated from the kinetics of the purified enzyme. However, those kinetics may be quite complex; allosteric effectors may influence the affinities of the enzyme for oleoyl-ACP and for G3P.     

Inhibition of photophosphorylation by kaempferol

Kaempferol, a naturally occurring flavonol, inhibited coupled electron transport and both cyclic and noncyclic photophosphorylation in isolated pea (Pisum sativum) chloroplasts. Over a concentration range which gave marked inhibition of ATP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids. It is suggested that kaempferol acts as an energy transfer inhibitor.     

Synergism of ammonium and palmitic acid in uncoupling of electron transfer and ATP synthesis in chloroplasts

Uncoupling by ammonium of electron transfer and ATP synthesis during linear transfer of electrons from water to photosystem 1 acceptors was studied in pea chloroplasts. It was shown that 40 μM palmitic acid decreased several-fold the ammonium concentrations necessary for 50% inhibition of ATP synthesis. The protonophore carbonyl cyanide m-chlorophenylhydrazone has no such property. The enhancement by palmitate of ammonium-induced uncoupling is accompanied by acceleration of basal electron transfer and decrease in the photoinduced uptake of hydrogen ions (H⁺). In the absence of ammonium, palmitate has no effect on basal transport and stimulates uptake of hydrogen ions. This means that in the case of combined action of palmitate and ammonium an additional leakage of H⁺ takes place, resulting in dissipation of the pH gradient. Synergic action of two metabolites, free fatty acid and ammonium, is supposed to provide for functioning of a system of mild regulation of energy coupling processes in native plant cell chloroplasts. Possible mechanisms of synergism are discussed.     

The effect of growth retardants on phytochrome-induced lettuce seed germination

Experiments were carried out to explore the involvement of the plant hormone gibberellin (GA) in the light-induced germination of lettuce seeds. Three growth retardants known to be inhibitors of GA biosynthesis were tested for their effect on red-light-induced germination. Chlormequat chloride (CCC) and AMO-1618 had no effect, but ancymidol was strongly inhibitory. Moreover, the inhibition caused by ancymidol was completely overcome by GA₃. CCC and AMO-1618 inhibit the formation ofent-kaurene, while ancymidol blocks the oxidation ofent-kaurene toent-kaurenoic acid. Ancymidol also was found to inhibit GA-induced dark germination of lettuce seeds, and this inhibition was partially reversed by higher levels of GA. Therefore, the results suggest two possibilities for the relationship between phytochrome and GA in this system: first, the rate-limiting step in the germination of light-sensitive lettuce seeds, that which is regulated by phytochrome, is the oxidation ofent-kaurene toent-kaurenoic acid. Alternatively, red-light treatment may result in the release of active GAlike substances which, in turn, induce germination. In either case the results presented here support the view that phytochrome exerts its effect on lettuce seed germination by means of GA rather than via an independent pathway.     

Pea chloroplast carnitine acetyltransferase

The purpose of this study was to resolve the controversy as to whether or not chloroplasts possess the enzyme carnitine acetyltransferase (CAT) and whether the activity of this enzyme is sufficient to support previously reported rates of fatty acid synthesis from acetylcarnitine. CAT catalyses the freely reversible reaction: carnitine + short-chain acylCoA <--> short-chain acylcarnitine + CoASH. CAT activity was detected in thc chloroplasts of Pisum sativum L. With membrane-impermeable acetyl CoA as a substrate. activity was only detected in ruptured chloroplasts and not with intact chloroplasts, indicating that the enzyme was located on the stromal side of the envelope. In crude preparations, CAT could only be detected using a sensitive radioenzymatic assay due to competing reactions from other enzymes using acetyl CoA and large amounts of ultraviolet-absorbing materials. After partial purification of the enzyme, CAT was detected in both the forward and reverse directions using spectrophotometric assays. Rates of 100 nmol of product formed per minute per milligram of protein were obtained, which is sufficient to support reported fatty acid synthesis rates from acetylcarnitine. Chloroplastic CAT showed optimal activity at pH 8.5 and had a high substrate specificity, handling C2-C4 acyl CoAs only. We believe that CAT has been satisfactorily demonstrated in pea chloroplasts.     

Differential inactivation of seed exudate stimulation of Pythium ultimum sporangium germination by Enterobacter cloacae influences biological control efficacy on different plant species

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.     

Abscisic Acid Inhibition of Kinetin Nucleotide Formation in Germinating Lettuce Seeds

Imbibing ‘Grand Rapids’ lettuce (Lactuca saliva L.) seeds take up ¹⁴C-kinetin, and metabolize this cytokinin to the 5′-nucleotide. The identity of the labeled nucleotide in seed extracts was verified by Sephadex LH-20 column chromatography, paper and thin layer chromatography, and high voltage paper electrophoresis. Incubations with kinetin in the presence of abscisic acid lead to an apparent specific inhibition of kinetin nucleotide formation. ABA has no effect on kinetin uptake, and does not inhibit kinetin nucleotide synthesis in vitro by a cell-free preparation from lettuce seeds. Additionally, ABA does not inhibit adenylate synthesis from exogenously supplied adenine. These results represent a specific cytokinin-ABA interaction, which might play a significant role in the hormonal regulation of lettuce seed germination.     

Osservazioni Sull'effetto Inibente di Elevate Pressioni Osmotiche su Diversi Tipi di Crescita

Abstract

On the inhibition of seeds germination and of growth by cell enlargement by the osmotic pressure of the medium. — The mechanism of inhibition by osmotic pressure (O.P.) of the medium on growth and respiration of germinating wheat, castor bean and lettuce sèeds and of etiolated pea internode segments was investigated.

The following results were obtained:

1 - External osmotic pressure (up to 0.3 M) of various substances such as mannitol, urea, glucose, NaCl, was shown to inhibit the germination and growth of lettuce, wheat and castor bean seeds.

2 - a) A remarkable decrease of the development of respiration during the first 48 h of germination was demonstrated in embryos of wheat seeds germinated and maintained in mannitol solutions at concentration from 0,2 to 0,3 M.

b) A slight but reproducible inhibition of óxygen uptake by O.P. was also observed in embryos isolated from wheat seeds germinated in water for 24 and 34 h and transported respectively in water or into 0,2 M mannitol solutions.

This is interpreted as indicating that high external O.P. inhibits both the respiratory metabolism and the development with time of enzyme systems supporting respiration.

3 - Mannitol solutions (0,2–0,3 M) inhibited completely growth by cell enlargement in pea internode sections, while they did not at all affect oxygen uptake and protein synthesis ( ¹⁴ C - leucine incorporation). The stimulatory effect of auxin on pea elongation was almost completely suppressed by mannitol, whereas the hormone stimulation of respiration remained unchanged.

These data are interpreted as indicating that in tissues, presenting an advanced differentiation, high external O.P. inhibits growth by a direct physico-chemical mechanism; while the inhibitory effect in embrional tissues seems to comprehend, besides this direct effect, a complicated metabolic component, apparently influencing protein synthesis.     

Final step of phosphatidic Acid synthesis in pea chloroplasts occurs in the inner envelope membrane

The second enzyme of phosphatidic acid synthesis from glycerol-3-phosphate, 1-acylglycerophospate acyltransferase, was localized to the inner envelope membrane of pea chloroplasts. The activity of this enzyme was measured by both a coupled enzyme assay and a direct enzyme assay. Using the coupled enzyme assay, phosphatidic acid phosphatase was also localized to the inner envelope membrane, although this enzyme has very low activity in pea chloroplasts. The addition of UDP-galactose to unfractionated pea chloroplast envelope preparations did not result in significant conversion of newly synthesized diacylglycerol to monogalactosyldiacylglycerol. Thus, the envelope synthesized phosphatidic acid may not be involved in galactolipid synthesis in pea chloroplasts.     

Fatty acid synthetase from chloroplasts of soybean cotyledons: ACP activation and CoA inhibition

Fatty acid synthetase from chloroplasts of soybean cotyledons was activated by preincubation with acyl carrier protein and dithiothreitol. The synthetase reaction had a 3–10 min lag which was not eliminated by the preincubation. Acetyl-CoA and malonyl-CoA had no effect on the activation. Fatty acid synthetase from spinach chloroplasts was neither activated by preincubation nor had a lag. The variability of the activity of the soybean enzyme with preincubation suggested that the fatty acid synthetase was present in two forms and that the acyl carrier protein caused conversion to the active form. This fatty acid synthetase and the same synthetase from spinach chloroplasts were inhibited by CoA. The type of inhibition by CoA in soybean was competitive with respect to malonyl-CoA and the Ki was 80μM.     

Inhibition of lettuce seed germination and seedling growth by antimetabolites of nucleic acids,and reversal by nucleic acid precursors and gibberellic acid

Summary Germination of White Paris lettuce seeds is inhibited by 2-thiouracil up to 24 hours. This inhibition is reversed by RNA precursors only. Seedling growth of lettuce is inhibited by 2-thiouracil and 5-fluorouracil; and white the effect of 2-thiouracil is counteracted by RNA precursors, inhibition due to 5-fluorouracil is not reversed significantly by any nucleic acid precursors. Possibly 2-thiouracil controls germination and seedling growth by interfering with RNA synthesis, while the effect of 5-fluorouracil is non-specific.In the presence of gibberellic acid, 5-fluorouracil and 2-thiouracil are relatively ineffective in causing inhibition of hypocotyl growth. Mechanism of gibberellic acid action remains obscure.