Chloroplast Content Research Articles

Trypsin inhibition of electron transport

1. 1. A relaxation spectrophotometer was employed to measure the effects of trypsin treatment on electron transport in both cyclic and non-cyclic chloroplast reactions. The parameters measured were electron flow rate through P700 (flux) and the time constant for dark reduction of P700. 2. 2. In the reduction of methyl viologen by the ascorbate-2,6-dichlorophenol-indophenol (DCIP) donor couple, there was no effect of trypsin on P700 flux or on the time constant for dark reduction of P700. In the phenazine methosulfate (PMS) cyclic system, trypsin had either a slightly stimulatory or slightly inhibitory effect on the P700 flux, depending on the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU): either effect being marginal compared to trypsin effects on Photosystem II. With both ferricyanide and methyl viologen reduction from water, trypsin treament gave a first order decline in P700 flux: which matched the trypsin-induced decline in electron transport with the water to DCIP system, measured by dye reduction. This implies that Photosystem II is inhibited. The inhibition of Photosystem II was up to 90% with a 6–10-min trypsin treatment. This result is consistent with the concept of Photosystem I (P700) being in series with Photosystem II in the electron transfer sequence. 3. 3. Cyclic phosphorylation was severely inhibited (85%) by trypsin treatment which had a somewhat stimulatory effect on P700 flux, indicating uncoupling. Non-cyclic phosphorylation was uncoupled as well as electron flow being inhibited since the P 2e ratio decreased more rapidly as a function of trypsin incubation time than inhibition of electron flow. The two effects, uncoupling and non-cyclic electron flow inhibition, are separate actions of trypsin. It is probably that the uncoupling action of trypsin is due to attack on the coupling factor protein, known to be exposed on the outer surface of thylakoids. 4. 4. Trypsin treatment caused an increase in the rate constant, k d, for the dark H + efflux, resulting in a decreased steady state level of proton accumulation. The increased proton efflux and the inhibition of phosphorylation are consistent with an uncoupling effect on trypsin. 5. 5. Trypsin treatment did not reduce the manganese content of chloroplasts: as reported by others, Tris washing did remove about 30% of the chloroplast manganese. 6. 6. Electron micrographs of both negatively stained and thin-sectioned preparations showed that, under these conditions, trypsin does not cause a general breakdown of chloroplast lamellae. Inhibition by trypsin must therefore result from attacks on a few specific sites. 7. 7. Both System II inhibition and uncoupling occur rapidly when trypsin treatment is carried out in dilute buffer, a condition which leads to thylakoid unstacking, but both are prevented by the presence of 0.3 M sucrose and 0.1 M KCl, a condition that helps maintain stacked thylakoids. Evidently vulnerability to trypsin requires separation of thylakoids. 8. 8. Since trypsin does not appear to disrupt thylakoids nor prevent their normal aggregation in high sucrose-salt medium and since the trypsin molecule is probably impermeable, it is probable that the site(s) of trypsin attack in System II are exposed on the outer thylakoid surface.

Gibberellic Acid Induced Changes in the Ultrastructure of Chloroplasts and the Content of Chlorophyll in Leaves of Dwarf Maize (Zea mays L.)

Summary Water culture experiments with a complete nutrient solution were conducted to investigate the effect of GA 3 -treatment on the ultrastructure and the pigment content in the 3rd leaf blade of dwarf maize. Tall maize was also included for comparison. It was found that dwarfism in maize was nearly without effect on the ultrastructure and the pigment content of chloroplasts. GA 3 induced an absolute decrease in the total chlorophyll content in the leaves of dwarf plants. The ultrastructure of both mesophyll and bundle sheath chloroplasts of GA 3 -treated dwarf plants was markedly changed. In mesophyll chloroplasts, the grana and stroma thylakoids were swollen and showed a certain degree of desorientation. A great number of osmiophilic granules were scattered between the swollen thylakoids. The chloroplasts in the bundle sheath cells differed in their sensitivity to GA 3 . Some of them were deformed and quite irregular in shape and showed a great number of osmiophilic granules and large vacuoles. Other chloroplasts exhibited well-developed small grana and small starch grains. Chloroplasts with normal structure were also found. The observed effects of GA 3 -treatment on the ultrastructure of plastids are not primary effects of the growth substances. The interrelationships between GA 3 -treatment, structure of chloroplasts and calcium content will be discussed.

Decrease in Chloroplast Content of Barley Leaves infected with Powdery Mildew

POWDERY mildew fungi (the Erysiphales) cause many important plant diseases. These fungi are obligate parasites, as they attack only living host tissue and cannot yet be cultured axenically. They exhibit extreme host specific parasitism and the biochemical mechanisms which determine host resistance or susceptibility are still obscure. Because of their basic metabolic importance, people have sought to determine whether alterations occur in nucleic acids after infection. Millerd and Scott1 reported that infection of susceptible barley leaves with powdery mildew resulted in increased amounts of RNA in host tissue and four days after inoculation there was approximately 20% more RNA/μg DNA than in non-infected leaves. In contrast, no change was observed in the amount of RNA after inoculation of resistant barley varieties. Malca et al.2 determined the base composition of RNA in susceptible mildew-infected barley leaves at various stages of disease development. They found a relative increase in guanylic acid and larger amounts of all nucleotides, reflecting the higher RNA content of mildewed leaves. Plumb et al.3 also reported a slight increase in RNA in susceptible wheat leaves infected with powdery mildew.

Die unterschiedliche Synthese der lipophilen Plastidenchinone in Sonnen- und Schattenblättern von Fagus silvatica L. / The Unequal Synthesis of the Lipophilic Plastidquinones in Sun- and Shade Leaves of Fagus silvatica L.

The synthesis of plastidquinones was studied from leaf development until autumnal leaf degeneration in sun and shade leaves of Fagus silvatica and compared with chlorophyll and carotinoid synthesis. 1. The lipoquinone content of Fagus chloroplasts increases steadily with increasing age of leaf tissue. This lipoquinone accumulation takes place in sun and shade leaves, it is however much more expressed in sun leaves. On a chlorophyll or a leaf square (100 cm2) basis the latter possess a several times (3 to 9 x) higher lipoquinone content than shade leaves. 2. The augmentation of the plastidquinone level in both leaf types is mainly due to the synthesis of the reduced benzoquinone forms, plastohydroquinone 45 and the chromanol a-tocopherol. The larger part of the reduced lipoquinones represent excess amounts which are deposited extrathylakoidal in the osmiophilic plastoglobuli of the plastid stroma. 3. The oxidized benzoquinones, plastoquinone 45 and α-tocoquinone, as well as the naphthoquinone vitamin K1 (phylloquinone) are present in much lower concentration. Their synthesis parallels that of chlorophylls and carotinoids. They are formed either not in excess (vitamin K1) or in very low excess amounts (α-tocoquinone, plastoquinone) . These oxidized quinone forms are in turn preferably located together with chlorophylls and carotinoids in the photochemically active thylakoids. 4. The production of excess plastidquinones (mainly reduced benzoquinones) starts very early during leaf development. The main synthesis phase begins after the end of chlorophyll and thylakoid synthesis and continues in senescent Fagus leaves during the breakdown of chlorophyll and thylakoids until the early yellow stage of sun and shade leaves. In senescent leaves the level of the oxidized benzoquinones, plastoquinone and α-tocoquinone, increases slowly by oxidation of plastohydroquinone and α-tocopherol, respectively. 5. The differences between sun and shade leaves in the total and relative amounts of the various lipid classes such as chlorophylls, carotinoids, benzoquinones and the naphthoquinone K1 (100 cm2 leaf square, dry weight or chlorophyll a as reference system) are not yet significant in May, several days after leaf unfolding, but get established thereafter. 6. By August the benzoquinone content of sun leaves reaches even that of chlorophyll a, whereas in shade leaves the lipophilic benzoquinones make up only 20 to 30% of the chlorophyll a content. Shade leaves exhibit a benzoquinone level which is little lower than that of carotinoids. Sun leaves, in contrast, contain a benzoquinone content which is by ca. 4 times higher than the carotinoid content. The ratio benzoquinones to vitamin K1 amounts 60 - 70 in shade leaves and 100 - 120; n sun leaves. In addition the two lipophilic redox systems plastoquinone/plastohydroquinone 45 and α-tocoquinone/α-tocopherol are present in sun leaves to a higher extent in the reduced state (84% and 96%) than in shade leaves (60% and 90% respectively). 7. From the results it is assumed, that the steady increase of the benzoquinone level with increasing age of plant tissue may be correlated with thylakoid turn-over. The possibility, that the enormous benzoquinone accumulation in sun leaves is mainly caused by higher light intensities in connection with a reduced protein synthesis is discussed.

The Effect of 6‐Benzylaminopurine on the Starch Metabolism of Stellaria media

AbstractThe effect of 6‐benzylaminopurine (BAP) was studied by measuring the amylase activity of leaf extracts and the starch content of chloroplasts in Stellaria media‐plants cultivated for five days in solutions containing different quantities of BAP.Small amounts of BAP decreased amylase activity in weak‐growing plants. In strong‐growing ones the effect was variable. However, in all experiments very high amount of this growth regulator caused a great increase in amylase activity which was accompanied by a relatively high concentration of starch in the chloroplasts. The induction of amylase activity was thus not high enough to diminish the starch content. This is supposed to be due to the lack of a sink organ since the growth of roots and shoots was greatly inhibited in concentrated BAP‐solutions (10‐5M or more). In these cases the plants usually died during the first week.In some cases small amounts of BAP increased the growth of S. media, and high concentrations inhibited it causing characteristic morphological changes, such as creeping growth pattern, small, thick leaves and the turning up of the leaf margins.Benzylaminopurine also caused increased resistance to aging, especially in low concentrations (10–7–10–6M).

Inhibitory site of carbonyl cyanide m-chlorophenylhydrazone in the electron transfer system of the chloroplasts

The inhibitory effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the electron transport system of chloroplasts was investigated. At concentrations higher than 1 · 10 −7 M, CCCP acted as an inhibitor of the Hill reaction. Two different processes of CCCP inhibition were discernible; the one which was accomplished very rapidly and independently of light, and the other which developed slowly and only in light. Both processes of inhibition were not reversed by washing the inhibited chloroplasts. The rapid type of inhibition in the dark was observed not only in the Hill reaction in normal (untreated) chloroplasts, but also in ascorbate plus hydroquinonesupported NADP + photoreduction and manganese-supported 2,6-dichlorophenol-indophenol (DCIP) photoreduction in heated chloroplasts, whereas NADP + photoreduction supported by reduced DCIP was not inhibited. CCCP inactivated the Hill reaction by affecting the quantum efficiency of the reaction without influencing the rate constant of the dark reaction. There was no loss in the manganese content in chloroplasts caused by incubation with CCCP in the dark. The variable fluorescence in chloroplasts was decreased by CCCP at concentrations higher than 1 · 10 −7 M, with concomitant elimination of the induction phenomenon. However, on addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to the CCCP-inhibited chloroplasts there occurred a recovery of the induction phase of fluorescence. The pool size of the electron acceptor of photosystem II decreased with increasing concentrations of CCCP, but the pool size of the primary electron acceptor determined in the presence of DCMU was not affected by the poison. It is postulated that the site of CCCP inhibition is located between the endogenous redox substance (Y 1) which receives electrons from artificial electron donors such as ascorbate, and the primary electron donor of photosystem II (Y 2).

Effects of temperature on accumulation of starch or lipid in chloroplasts of grapevine

The amount of starch accumulated in chloroplasts of grapevine leaves fell, with increasing temperature, from 23% of dry weight at 18/13° C (day/night) to 1% at 35/30° C, whereas total lipids in the leaf rose from 6 to 16% of dry weight. Preparations viewed by electron microscopy had large starch granules in the chloroplasts at 18/13° C, and large lipid droplets at 35/30° C. At 35/30° C chlorophyll content of chloroplasts was high and grana prominent, suggesting that the results were due to selective effects of temperature on metabolic pathways.

The plastocyanin content of chloroplasts from some higher plants estimated by a sensitive enzymatic assay

1. A sensitive enzymatic assay of plastocyanin is described. The method is based on the stimulation of cytochrome c photo-oxidation in detergent-treated chloroplasts. Under appropriate conditions the rate of reaction is proportional to the amount of plastocyanin present and as little as 0.5 natom plastocyanin-copper may be determined in chloroplast samples. 2. The method is specific for plastocyanin when applied to sonic extracts of pea chloroplasts. 3. The plastocyanin content of chloroplasts from various higher plants varied from 2.0 to 9.0 natoms copper per mg chlorophyll whereas cytochrome f was present in quantities 2–5 times less (on a haem basis). 4. The total copper content of chloroplasts from most plants tested was in excess of the plastocyanin. In the case of barley chloroplasts the total copper was equal to the plastocyanin copper. We conclude that plastocyanin is likely to be the only copper compound involved in photosynthetic electron transport.

Starch Accumulation Associated with Growth Reduction at Low Temperatures in a Tropical Plant

Growth of Digitaria decumbens is severely reduced by night temperatures of 10 degrees C or below. Ultra-structure of leaves and chemical analyses show a high starch content in chloroplasts of plants illuminated and kept at a temperature of 30 degrees C. This starch disappears after a period in the dark at 30 degrees C, but it remains if the temperature during the dark period is 10 degrees C. The inhibition or slowing of starch translocation out of chloroplasts appears to account for reduced photo-synthesis and growth at low night temperatures.

Sites of function of manganese within photosystem II. Roles in O 2 evolution and system II

The Mn content of spinach chloroplasts has been decreased by growth deficiency, extraction and by ageing at 35°. We studied the effect of subnormal Mn content upon the chloroplasts capacity to evolve O 2 and to photooxidize electron donors other than water via Photosystem II. We observed the following: 1. 1. In fresh chloroplasts ascorbate and other reducing agents, if present in sufficient concentration, fully replace water as the System II oxidant and can sustain maximum rates of 1000–1200 equiv/chlorophyll per h. 2. 2. None of the studied donors proved entirely specific for System II; to a variable extent all could react with the oxidant of System I. We therefore considered only the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-(DCMU)-sensitive fraction of the observed rates as pertinent. 3. 3. Normal fresh chloroplasts contained 3 Mn 200 chlorophylls II and showed flash yields of approx. 1 O 2 1600 chlorophylls. This indicates that each System II trapping and O 2-evolving center contains three Mn atoms. 4. 4. O 2 evolution capacity is abolished when about 2 3 of the total Mn pool is removed by way of Tris or hydroxylamine extraction, i.e. upon removal of two of the three Mn atoms normally present per reaction center. Between the limits of 1 Mn per trap and 3 Mn per trap O 2 evolution capacity is linear with Mn content. 5. 5. Mn removal affects the rates of O 2 evolution in strong light and in weak light (quantum yield) in the same fashion. This indicates that complete O 2 reaction centers are inactivated. 6. 6. With Mn removal the capacity for donor (ascorbate or p-phenylenediamine) photooxidation in strong light declines in a very similar fashion as the O 2 evolving capacity. However, after removal of 2 3 of the Mn pool (by Tris or hydroxylamine extraction) 15–20% of the maximum rate remains (100–250 equiv/chlorophyll per h) as previously noticed by other workers. Secondly, the rate in weak light (quantum yield) of these photooxidations remains unaffected by Mn removal. This shows that for donor photooxidation the larger of the two Mn pools is not essential. 7. 7. Complete removal of Mn ( < 1 Mn 4000 chlorophylls) led to 90–95% loss of donor photooxidation in strong light. 8. 8. Removal of 2 3 of the Mn left a low fluorescence yield (variable fraction = 0) which could be fully restored by adding DCMU. After complete removal of Mn ( < 1 Mn 4000 chlorophylls) DCMU enhanced the yield of the variable fluorescence to only 1 2 the maximum level but the full maximum could be restored by chemical reduction. This indicates that fluorescence quencher of System II, Q, is not affected by Mn removal. 9. 9. Of the three Mn associated with each trapping center, one is linked more closely to the center than the other two. While all three are essential for O 2 evolution, artificial donors can enter with various rate constants at several loci on the oxidant side of System II.

Ion and water transport in limonium. V. The ionic status of chloroplasts in the leaf of Limonium vulgare in relation to the activity of the salt glands

A freeze-drying and non-aqueous extraction technique has been used to record the salt content of chloroplasts in leaf segments of Limonium vulgare. Changes in the level of K +, Na + and Cl −were observed when leaf segments were treated with NaCl or KCl solutions which induce pumping of the salt glands. The effects of treatments designed to inhibit glandular activity are also recorded. Net accumulation of ions into chloroplasts is shown to be a process in vivo. The results provide evidence for a direct effect of glandular activity on cytoplasmic ion levels and they provide intracelluar evidence for the induction of glandular activity after a certain lag period.

The effect of nitrate and nitrite on oxygen evolution and carbon-dioxide assimilation and the reduction of nitrate and nitrite by intact chloroplasts

Intact chloroplasts isolated from spinach reduced NO3 (-) and NO2 (-) in the light without the addition of either co-factors or added enzymes. The maximum rate observed, however, for the reduction of NO3 (-) was approximately 3μMoles hr(-1) mg(-1) (chlorophyll) and for NO2 (-) 6 μMoles hr(-1) mg(-1) (chlorophyll). These rates were consistent with the enzyme content of whole chloroplasts, but much lower than those found in whole leaf extracts.The addition of both NO3 (-) and NO2 (-) in low concentrations resulted in transient increases in both O2 evolution and CO2 fixation. The increases in oxygen evolution were not consistent in amount and bore no relation to the amount of substrate reduced. Similar transients were observed in a number of experiments when NaCl or NH4Cl were added.The addition of NO2 (-) at concentrations of 10(-4) M and above resulted in marked inhibition of both O2 evolution and CO2 fixation. NO2 (-) appears to inhibit by blocking the reduction of NADP. NO3 (-) at similar concentrations had no such effect.An increase in the soluble amino nitrogen content of the chloroplasts was observed when NO3 (-) or NO2 (-) was reduced. There was, however, no increase in the incorporation of (14)C from (14)CO2 into amino acids under these conditions. Even with the addition of ammonia the amount of (14)C incorporated into the amino acids was not changed from less than 5% of the total (14)C fixed. We conclude that while intact chloroplasts do have the ability to reduce both NO3 (-) and NO2 (-) at low rates, they do not synthesize appreciable amounts of amino acid directly, and this fact must be considered when formulating any pathways for nitrogen metabolism during photosynthesis.

Überschüssige Plastidenchinone in Blättern

The synthesis of plastidquinones was studied during the development through the degeneration of Ficus leaves and compared with chlorophyll and carotenoid synthesis. 1. The plastidquinone content of Ficus chloroplasts increases steadily with increasing age of leaf tissue. The augmentation of the plastidquinone level is mainly due to the synthesis of the reduced quinone forms plastoquinol 45 (Plastohydrochinon 45) and a-tocopherol, the chromanol of α-tocoquinone. In several years old leaves the concentration (moles) of plastoquinol 45 and α-tocopherol reaches or exceeds that of chlorophyll a. 2. The naphthoquinone vitamin K1 (phylloquinone) as well as the oxidized quinone forms (α-tocoquinone and plastoquinone 45) seem not to be produced in excess amounts. The levels of the latter increase, however, in older and senescent leaves, apparently by oxidation of some α-tocopherol and plastoquinol 45 respectively. 3. The production of excess plastidquinones starts very early during leaf development. The main synthesis phase begins after the end of chlorophyll and thylakoid synthesis and continues in senescent Ficus leaves during the breakdown of chlorophyll until the early yellow stage. 4. The young pale green leaves still rolled up within the buds, possess a quinone and carotenoid composition which is quite different from that of fully expanded leaves. It resembles that of shortly illuminated etiolated seedlings. 5. The carotenoid level of older fully grown Ficus leaves slowly increases, mainly by synthesis of lutein. These additional carotenoids represent excess amounts, which are deposited together with the excess plastidquinones in the plastoglobuli of the plastid stroma. 6. There is no indication for a re-incorporation of excess plastidquinones in the general chloroplast metabolism. Thus the excess plastidquinones, accumulated in the osmiophilic plastoglobuli, may be regarded as secondary plant products.

The effect of mineral deficiency and leaf age on the nitrogen and chlorophyll content of spinach chloroplasts

The nitrogen and chlorophyll content of chloroplasts from spinach plants varies with leaf age and the nutritional status of the plant. Both chlorophyll and nitrogen content of the chloroplasts increase with leaf age when plants are grown on full nutrient. Chloroplasts from manganese- and iron-deficient plants contain less chlorophyll but similar amounts of nitrogen to chloroplasts from plants grown on full nutrient. Chloroplasts from sulphur- and potassium-deficient plants contain lower amounts of both chlorophyll and nitrogen than control plants.

Light-induced changes in the ionic content of chloroplasts in Pisum sativum

1. 1. The content of chloroplasts isolated in two minutes from pea plants in the light or dark was ascertained both via osmotic responses in sucrose solutions interpreted by the Boyle-Van 't Hoff relation and from direct determination of the K +, Na +, Mg 2+, Ca 2+ and Cl − associated with the chloroplasts. 2. 2. From packed-volume and Coulter-counter results, the chloroplasts contained about 3.52 μmoles of osmotically active particles per mg chlorophyll when isolated from plants in the dark and only 2.39 μmoles per mg chlorophyll when isolated from plants in the light. Likewise, the total amount per mg chlorophyll of the five ions determined was about 32% lower for chloroplasts from plants in the light compared with the dark. 3. 3. The concentration of ions in the osmotically responding space was calculated using the chloroplast volume measured at the same osmolality as the cell sap (about 288 mosmolal). Chloroplasts both from plants in the light and the dark contained about 99 mM K +, 92 mM Cl −, 16 mM Mg 2+, 15 mM Ca 2+ and 10 mM Na +. 4. 4. Based on an extension of the conventional Boyle-Van 't Hoff relation using irreversible thermodynamics, the reflection coefficient of the chloroplast limiting membranes for internal solutes was greater than 0.7. Sucrose was essentially excluded from these chloroplasts. 5. 5. The light-induced decrease of K + in the chloroplasts and its reversal when the plants were placed in the dark had half-times of about 4 min. This decrease occurring in vivo was 75% inhibited by p-trifluoromethoxycarbonylcyanide phenylhydrazone and was completely abolished by nigericin, indicating that the light-induced K + efflux from chloroplasts required either ATP or a high-energy intermediate created by photosynthetic electron flow.

Nicotinamide cofactors of intact chloroplasts isolated on a sucrose density gradient

1. 1. The potassium content of chloroplasts expressed as a potassium-chlorophyll ratio was adopted as a new criterion of chloroplast integrity. 2. 2. Chloroplast fractions from pea seedlings were prepared using a sucrose density gradient and found to contain not less than 80% intact chloroplasts and no recognisable cellular components as contaminants. 3. 3. NADP +, NADPH, NAD + and NADH assays on these preparations are reported, and compared with published values. 4. 4. The light-induced reduction of endogenous NADP + and NAD + was followed and similarly the reduction of exogenous NADP +. A NADPH peak occurred during the initial 30-sec illumination, in agreement with data published for illumination of the whole leaf. 5. 5. The observations indicate that these chloroplasts are freely permeable to NADP + and may accumulate it against a concentration gradient. There is also some evidence to indicate that conversion of exogenous NADP + into NAD(H) occurs on illumination.

Accumulation and Disintegration of Starch in the Chloroplasts of Stellaria media Grown under Constant Illumination

AbstractThe persent paper contains a study un the starch content of the chloroplasts of the leaves of Stellaria media, performed by hsitochemical methods. Three leaves were collected at short intervals, fixed and cut in 10 μ sections, and stained according to the PAS‐procedure. The starch content of single chloroplasts was measured by a microscope photometer. The chemical composition of the stainable material was demonstrated by enzyme specific experiments.At the end of a 12 hours' dark period the chloroplasts contained only traces of starch. Light caused the starch accumulation to begin and after a time chloroplasts seemed to be filled with starch. Subsequently, however, a sudden decrease in the starch content of the chloroplasts took place, in spite of constant illumination. A rather high level of starch content was restored in a few hours.The author puts forward some ideas about the nature of the factors causing the transitory disintegration of starch in illuminated leaves. The induction or the activation of certain enzymes seems to be the most probable explanalion.

Release of free fatty acids and loss of hill activity by aging spinach chloroplasts.

The free fatty acid content of spinach chloroplasts, isolated at pH 5.8 to 8.0, has been found to vary between 3.1 and 5.5% of the total chloroplast fatty acids. When chloroplasts were incubated at room temperature for 2 hours, the free fatty acids increased by 42% and the Hill activity decreased by 70%. After 2 hours of incubation at 37 degrees , the free fatty acids increased about 3-fold and the Hill activity decreased to almost 0. The addition of crystalline bovine serum albumin largely prevented the loss of Hill activity at room temperature and at 5 degrees , but had little effect during incubation at 37 degrees . Both the release of free fatty acids and the loss of Hill activity were pH dependent. The losses were the least during incubation at pH 5.8 and the greatest during incubation at pH 8.0. The major free fatty acids released at pH 5.8 were saturated, while those released at pH 7.0 or 8.0 were mainly the unsaturated acids, alpha-linolenic acid and hexadecatrienoic acid.

Effect of benzimidazole and kinetin on the nicotinamide nucleotide content of senescing wheat leaves.

Assays of nicotinamide nucleotides were made on detached leaves of wheat, Triticum dicoccum var. Khapli, immediately after excision and at the end of a 16-h photoperiod and 8-h dark period at 1, 3, and 5 days on leaves floated on water, and on benzimidazole, kinetin, or both. The total nicotinamide nucleotide content of 25 nmoles per gram fresh weight was increased by treatments with these kinins. A diurnal rhythm was observed in all treatments involving an increase of NADP(H) and a decrease of NAD(H) during the photoperiod, whereas the opposite was the case in darkness. The ratio NAD(H)/NADP(H) was about equimolar after excision and decreased to a mean value of 0.4 in all treatments after the first photoperiod. Thereafter, despite fluctuations in light and darkness and increased synthesis of NADPH in the treatments, the ratio increased to a mean value of 1.5 after 5 days, with no significant difference between treatments. The transformations in light can be explained by the combined operation of NAD kinase, NADH transhydrogenase, photosynthetic electron transfer forming NADPH, and biosynthetic reductive reactions restoring NADP.Assays were also made on chloroplasts isolated in nonaqueous media from leaves treated similarly but under continuous illumination. The NAD content of chloroplasts was 11.2 nmoles per μmole chlorophyll as compared to 3.14 for NADP. The oxidized nucleotides predominated in chloroplasts. Chloroplasts from leaves floated on water had lost all their NADP after 6 days, whereas those treated with benzimidazole or kinetin increased or maintained their level of NADP. The evidence is discussed with reference to the mechanism of action of benzimidazole and kinetin in delaying the onset of visible and metabolic changes that occur during the senescence of wheat leaves.

Manganese and copper content of chloroplasts and cytoplasts

Employing catalytic methods, the Mn- and Cu-content was determined in different cell fractions (chloroplasts, cytoplasts) of freeze-dried leaves ofValerianella olitoria isolated with a new dry method byBehrens et al.1. Cu was found essentially in the fractions with high chloroplast content; Mn was found mainly in the fractions with low chloroplast content.