Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed.

1. [14C]Oleoyl-CoA was metabolized rapidly and essentially completely by microsomal preparations from developing safflower (Carthamus tinctorius) cotyledons, and most of the [14C]oleate was incorporated into 3-sn-phosphatidylcholine. 2. In aerobic reaction mixtures containing NADH2 the [14C]oleate in 3-sn-phosphatidylcholine was converted into [14C]linoleate without any change in the specific radioactivity of the lipid. Over a 60 min incubation period the extent of conversion of [14C]oleoyl phosphatidylcholine into [14C]linoleoyl phosphatidylcholine was generally greater than 60%. The rate of desaturation of endogenous [14C]oleoyl phosphatidylcholine labelled from [14C]oleoyl-CoA was much greater that of exogenous [14C]dioleoyl phosphatidylcholine the specific radioactivity of the oleoyl moiety of the lipid remained constant, indicating that labelled and unlabelled oleate were desaturated at the same rate. On this assumption an initial rate of desaturation of about 15 nmol of oleate desaturated/min per mumol of 3-sn-phosphatidylcholine was estimated. 4. [14C]Oleate esterified at positions 1 and 2 of both endogenous and exogenous 3-sn-phosphatidylcholine was desaturated. 5. Attempts to demonstrate the presence of an oleoyl-CoA desaturase in safflower microsomal fractions by the appearance of linoleoyl-CoA in reaction mixtures were inconclusive.     

Evidence for the reversibility of the acyl-CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver.

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.     

The interconversion of diacylglycerol and phosphatidylcholine during triacylglycerol production in microsomal preparations of developing cotyledons of safflower (Carthamus tinctorius L.).

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalyse the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. Under these conditions the radioactive glycerol in sn-glycerol 3-phosphate accumulates in phosphatidic acid, phosphatidylcholine, diacyl- and tri-acylglycerol. The incorporation of glycerol into phosphatidylcholine is via diacylglycerol and probably involves a cholinephosphotransferase. The results show that the glycerol moiety and the acyl components in phosphatidylcholine exchange with the diacylglycerol during the biosynthesis of diacylglycerol from phosphatidic acid. The continuous reversible transfer of diacylglycerol with phosphatidylcholine, which operates during active triacylglycerol synthesis, will control in part the polyunsaturated-fatty-acid quality of the final seed oil.     

Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons.

The major cytochrome in microsomal membrane preparations from developing seeds of safflower (Carthamus tinctorius, var High Linoleate), has a reduced-minus-oxidized difference spectrum characteristic of a b-type cytochrome, and was identified from its midpoint-potential (E'7.2) value as cytochrome b5. Cytochromes P-450 and P-420 were also present. The cytochrome b5 content of microsomal preparations from a number of oilseed species was found to be in the order of 200-300 pmol/mg of protein. The cytochrome b5 was reduced in the membrane preparations by NADH, demonstrating the presence of an NADH: cytochrome b5 reductase; NADPH was a less effective donor. Microsomal membranes catalysed the NAD(P)H-dependent conversion of radioactive oleate into linoleate, indicating acyl-CoA: lysophosphatidylcholine acyltransferase and 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) activity. Desaturation of oleate to linoleate was unaffected by CO, but inhibited by CN-. The addition of oleoyl-CoA to the NADH-reduced membranes resulted in the CN(-)-sensitive partial re-oxidation of cytochrome b5, indicating that electrons from NADH were transferred to the site of desaturation via this cytochrome. The delta 12-desaturase in safflower, therefore, is CN(-)-sensitive and appears to require cytochrome b5 and NADH: cytochrome b5 reductase for activity.     

Triacylglycerols are synthesised and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L.) seeds

Microsomal membrane preparations from the immature cotyledons of safflower (Carthamus tinctorius) catalysed the interconversion of the neutral lipids, mono-, di-, and triacylglycerol. Membranes were incubated with neutral lipid substrates, ¹⁴C-labelled either in the acyl or glycerol moiety, and the incorporation of radioactivity into other complex lipids determined. It was clear that diacylglycerol gave rise to triacylglycerol and monoacylglycerol as well as phosphatidylcholine. Radioactivity from added [¹⁴C] triacylglycerol was to a small extent transferred to diacylglycerol whereas added [¹⁴C] monoacylglycerol was rapidly converted to diacylglycerols and triacylglycerols. The formation of triacylglycerol from diacylglycerol occurred in the absence of acyl-CoA and hence did not involve diacylglycerol acyltransferase (DAGAT) activity. Monoacylglycerol was not esterified by direct acylation from acyl-CoA. We propose that these reactions were catalyzed by a diacylglycerol: diacylglycerol transacylase which yielded triacylglycerol and monoacylglycerol, the reaction being freely reversible. The specific activity of the transacylase was some 25% of the diacylglycerol acyltransferase activity and, hence, during the net accumulation of oil, substantial newly formed triacylglycerol equilibrated with the diacylglycerol pool. In its turn the diacylglycerol rapidly interconverted with phosphatidylcholine, the major complex lipid substrate for Δ12 desaturation. Hence, the oleate from triacylglycerols entering phosphatidylcholine via this route could be further desaturated to linoleate. A model is presented which reconciles these observations with our current understanding of fatty acid desaturation in phosphatidylcholine and oil assembly in oleaceous seeds. Received: 8 November 1996 / Accepted: 5 February 1997     

The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied ¹⁴C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [¹⁴C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [¹⁴C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [¹⁴C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [¹⁴C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.Abbreviations FW fresh weight - TLC thin-layer chromatography     

EMS-induced cytomictic variability in safflower (Carthamus tinctorius L.)

Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3h, 5h and 7h) of 0.5 % Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future.     

Improvements in rooting regenerated safflower (Carthamus tinctorius L.) shoots

A continuing obstacle for regenerating safflower (Carthamus tinctorius L.) plants from cultured explants or callus has been a reliable method for rooting shoots. For shoots directly regenerated from primary explants, 76% of shoots rooted after a 7-d exposure to 10 mg/1 indole-3-butyric acid. Auxin source, concentration or exposure time did not greatly affect root formation or morphology, but strongly affected callus production. Shoots infected with Agrobacterium rhizogenes produced massive numbers of fibrous roots, but shoots did not elongate or survive transfer to soil. Shoot hyperhydricity symptoms were reduced by including 1 g/1 activated charcoal in rooting media. The optimal protocol for inducing root formation consisted of a 7-d exposure to 10 mg/l indole-3-butyric acid in root induction media, followed by incubation in media containing 15 g/l sucrose and 1 g/1 activated charcoal for 21 d.Abbreviations IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA anaphthalene acetic acid - POP 2,3,5-trichloro--phenoxypropionic acid     

不同产地红花的挥发油成分

不同产地红花的挥发油成分郭美丽张汉明张芝玉苏中武（第二军医大学药学院,上海２００４３３）Ｔｈｅｃｏｎｓｔｉｔｕｅｎｔｓｏｆｅｓｅｎｔｉａｌｏｉｌｆｒｏｍｓａｆｆｌｏｗｅｒ（ＣａｒｔｈａｍｕｓｔｉｎｃｔｏｒｉｕｓＬ．）ｉｎｄｉｆｆｅｒｅｎｔｌｏｃａｌｉ...     

Association-dissociation and denaturation-renaturation of high-molecular-weight protein: carmin from safflower seed (Carthamus tinctorius L.) in alkaline solution

The effect of alkalinepH on the association, dissociation, and denaturation of carmin, the high-molecular-weight protein from safflower seed was investigated in thepH range 7–12, using various biophysical techniques. The results indicate that the multimeric protein carmin dissociates atpH 8.0 where denaturation has not set in. The association-dissociation of the protein can be represented schematically as 11S 7S 4S 2S. AbovepH 10, the protein undergoes simultaneous dissociation and denaturation. The denaturation process appears to be complete at pH 12.5. The protein undergoes conformational change and covalent modifications and cleavage during the denaturation process. A reversibility study shows that the process of dissociation is reversible to a large extent, whereas denaturation appears to be irreversible. These results are discussed in terms of association-dissociation, denaturation and alkaline-catalyzed covalent modifications and cleavage of seed proteins.     

Penconazole alleviates salt-induced damage in safflower (Carthamus tinctorius L.) plants

It has been shown that penconazole (PEN) acts as an endogenous signal molecule responsible for inducing stress tolerance in plants. The effect of PEN (15?mg?l^–1) and sodium chloride (0, 100, and 200 mM NaCl) on some biochemical and molecular responses of safflower was studied. Results revealed that chlorophylls and total soluble protein contents decreased under salinity, however total carotenoid, anthocyanin, flavonoid, and carbohydrate contents increased as well as SOS1 and NHX1 genes expression. The exogenous PEN had a positive effect on chlorophylls, carotenoid, anthocyanin, flavonoid, soluble protein and carbohydrate contents. In addition, RT-qPCR analysis showed that the exogenous PEN induced expression of SOS1 and NHX1 genes in both salt-treated and untreated plants. Our data indicate that PEN helps safflower plants to better cope with salt stress. The results can provide new insights to better realizing the responsible mechanisms to regulate salinity resistance in safflower. PEN can be considered in order to ameliorate salinity effects, due to the low price and their availability.     

Inheritance of flower color and spininess in safflower (Carthamus tinctorius L.)

Safflower (Carthamus tinctorius L.) flowers are used for coloring and flavoring food and also as fresh-cut and dried flowers. The most important characteristics which contribute to the ornamental value of safflower are flower color and spinelessness. The objective of this study was to determine the inheritance mode and the number of genes controlling spininess and flower color in some Iranian genotypes of safflower. The results indicated that the existence of spines on the leaves and bracts of safflower is controlled by a single dominant gene in which the spiny phenotype was completely dominant to spineless. In some crosses, flower color was controlled by two epistatic loci each with two alleles, resulting in a ratio of 13:3 in the segregating F2 population for plants with orange and yellow flowers. Also, other mechanisms of genetic control, such as duplicate dominance and duplicate recessive types of epistasis, were observed for flower color in other crosses that led to ratios of 7:9 and 15:1 for plants with orange and yellow flowers, respectively. The results suggest that for ornamental use or in the food dying industry, genotypes with orange or yellow flowers and without spines on the leaves and bracts can be produced.     

Estimates of broad-sense heritability for seed yield and yield components of safflower (Carthamus tinctorius L.)

This study was carried out to estimate the broad-sense heritability for seed yield and some yield components of safflower (Carthamus tinctorius L.) cultivars. The experimental design was a randomized complete block design with three replications in the 2004 growing season in the Middle Black Sea Region conditions of Turkey. Three safflower cultivars (5-154, Din?er and Yenice) were grown at five locations (Bafra, Ladik, Suluova, Gümü?hacik?y and Osmancik). The heritability for seed yield, plant height, first branch height, number of branch, head diameter, number of seed per head, 1000-seed weight and oil content were estimates as 35%, 93%, 99%, 45%, 21%, 69%, 81% and 59%, respectively. It was found that first branch height was the least affected trait over environments and followed plant height, thousand seed weight and number of seed per head. On the other hand, head diameter, seed yield, number of branch and oil content were the most affected traits versus environmental conditions. The first branch height, plant height and 1000-seed weight could be used to succeed in selection in early generation.     

The procaryotic nature of the fatty acid synthetase of developing Carthamus tinctorius L. (safflower) seeds

All component activities involved in the synthesis of fatty acid were detected in crude extracts of developing safflower seeds. The crude extracts were fractionated into three portions by polyethylene glycol (0–5, 5–15, and 15% supernatant). Acetyl-CoA:acyl carrier protein (ACP) transacylase was precipitated about 66% by 5% polyethylene glycol. β-Ketoacyl-ACP reductase and enoyl-ACP reductase I were completely recovered in the 5–15% fraction. β-Ketoacyl-ACP synthetase and enoyl-ACP reductase II were in the 15% supernatant fraction. Malonyl-CoA:ACP transacylase and β-hydroxyacyl-ACP dehydrase were distributed into both fractions of 5–15 and 15% supernatant. When the 5–15% fraction was gel-filtrated on Sephadex G-200 column, β-hydroxyacyl-ACP dehydrase and malonyl-CoA:ACP transacylase were clearly separated from other enzymes, but β-Ketoacyl-ACP reductase and enoyl-ACP reductase I overlapped. However, by hydroxyapatite chromatography, these two reductases were clearly separated. Properties of each enzyme were examined with the samples fractionated by polyethylene glycol. β-Ketoacyl-ACP reductase preferably utilized NADPH (K_m = 16 μM) as hydrogen donor. The K_m for acetoacetyl-ACP was 9 μm. β-Hydroxyacyl-ACP dehydrase had a K_m of 12 μm for crotonyl-ACP. Enoyl-ACP reductase had two forms, I and II, and these two reductases differed from each other as follows: (a) separation by polyethylene glycol (15%) fractionation; (b) the optimum pH; (c) the hydrogen donor specificity; (d) the substrate specificity. From these results, it is concluded that the FAS system of developing safflower seeds was nonassociated and similar to the procaryotic type of Escherichia coli.     

The biosynthesis of triacylglycerols in microsomal preparations of developing cotyledons of sunflower (Helianthus annuus L.)

The synthesis of triacylglycerols was investigated in microsomes (microsomal fractions) prepared from the developing cotyledons of sunflower (Helianthus annuus). Particular emphasis was placed on the mechanisms involved in controlling the C18- unsaturated-fatty-acid content of the oils. We have demonstrated that the microsomes were capable of: the transfer of oleate from acyl-CoA to position 2 of sn-phosphatidylcholine for its subsequent desaturation and the return of the polyunsaturated products to the acyl-CoA pool by further acyl exchange; the acylation of sn-glycerol 3-phosphate with acyl-CoA to yield phosphatidic acid, which was further utilized in diacyl- and tri-acylglycerol synthesis; and (3) the equilibrium of a diacylglycerol pool with phosphatidylcholine. The acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine coupled to the equilibration of diacylglycerol and phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C18 polyunsaturated fatty acids for triacylglycerol production. Similar reactions were found to operate in another oilseed plant, safflower (Carthamus tinctorius L.). On the other hand, the microsomes of avocado (Persea americana) mesocarp, which synthesize triacylglycerol via the Kennedy [(1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940] pathway, were deficient in acyl exchange and the diacylglycerol in equilibrium phosphatidylcholine interconversion. The results provide a working model that helps to explain the relationship between C18- unsaturated-fatty-acid synthesis and triacylglycerol production in oilseeds.     

Restriction endonuclease cleavage site map of safflower (Carthamus tinctorius L.) chloroplast DNA

Summary The restriction endonucleases SalI, PstI, KpnI and HindIII have been used to construct a physical map of safflower (Carthamus tinctorius L.) chloroplast DNA. This was accomplished by hybridizing Southern blots of single and double digested chloroplast DNA with ³²P-dCTP nick-translated SalI, KpnI and HindIII probes which were individually isolated from agarose gels. The chloroplast DNA was found to be circular and to contain approximately 151 kbp. In common with many other higher plant chloroplast DNAs a sequence of about 25 kbp is repeated in an inverted orientation. The small and large single copy regions separating the two repeated segments contain about 20 kbp and 81 kbp, respectively. The rRNA structural genes were also mapped by Southern blot hybridization and are co-linear with several other plant species.     

Effect of acidicpH on the protein carmin from safflower seed (Carthamus tinctorius)

The effect of a decrease inpH on the structural integrity of carmin has been monitored by a variety of biophysical techniques. The protein undergoes initial dissociation up topH 3.5–4.0 without any significant denaturation. Below thispH the process of dissociation and denaturation appears to be simultaneous. Further, in thepH range of 2.5–1.6 the protein reassociates to probably a different polymer resulting from possibly, an entropically driven hydrophobic interaction. The process of dissociation appears to be reversible to a large extent. The process of denaturation appears to be governed by the kinetic path that the denatured protein molecule follows either by a sudden decrease inpH or through a gradual decrease inpH. These results are interpreted while keeping in view the oligomeric and globular structure of carmin at neutralpH. The results would help in understanding of structure-function relationship of the protein and its role in hydrogen ion bindingin vivo.     

Generation and analysis of drought stressed subtracted expressed sequence tags from safflower (Carthamus tinctorius L.)

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of?~1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78?%) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22?%) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study.     

An analysis of association of components of yield and oil in safflower (Carthamus tinctorius L.)

Summary Inter-relationships of various component characters with yield and oil content were analysed using 215 entries of safflower from India and U.S.A. Correlation of capsule number per plant and capsule weight with yield per plant was pronounced and they showed large direct effects on yield. All other components influenced seed yield mainly through these two components. Seed size had little effect on yield while seed number exerted a positive influence. The proportion of hull expressed as per cent of the whole seed revealed a highly significant and inverse relationship with oil content and was mainly responsible for the observed variability in oil content in the material. Although negative association was indicated between seed size and oil content, it was observed to be due to the indirect effect of hull content and not due to direct effect of seed size. Interestingly, yield per plant and its major components, number of capsules and capsule weight, revealed a negligible relationship with oil content. Both direct as well as indirect effects of hull percent and yield per plant were responsible for the favourable effect of seed number on oil content. The correlation of plant height, days to first flowering and total crop growth period with yield and oil content was either negligible or low, offering scope for developing early maturing and dwarf varieties with high yield and oil content. Spine index showed a non-significant association with yield and oil content. Capsule number, capsule weight and hull per cent were observed to be the most important components in breeding for higher yield and oil content.     

Experiments on enzymatic acylation of sn-glycerol 3-phosphate with enzyme preparations from pea and spinach leaves

Summary Optimal reaction conditions were investigated for acylation of sn-[U-¹⁴C]glycerol 3-phosphate in cell-free systems from leaves of Pisum sativum L. and Spinacia oleracea L. With palmitoyl-CoA as acyl donor the major product formed was monoacyl glycerol phosphate. In pea seedlings enzymatic activity was found to be dependent on the age of shoots going through a maximum 12 days after planting. In such plants the highest enzymatic activity is found in leaves and not in the shoot apex. Also in leaves activity varies according to the age of these organs. In leaves of maximal activity the highest total and specific activity was found in soluble proteins from chloroplasts. In older pea and spinach leaves higher activities were observed in membranes from chloroplasts and microsomes.