Δ1-Pyrroline-5-carboxylate: The product of proline dehydrogenase from Cucurbita moschata cotyledons

The product of oxidation of proline by pumpkin proline dehydrogenase reacted with o-aminobenzaldehyde to give a yellow compound that had an absorption spectrum similar to that obtained from chemically synthesized Δ¹-pyrroline-5-carboxylate. The product of the proline dehydrogenase reaction and synthetic Δ¹-pyrroline-5-carboxylate had identical R_f values. Both authentic Δ¹-pyrroline-5-carboxylate and the product of the enzyme gave a pink colour with acid ninhydrin on paper chromatograms and both had identical elution patterns on Dowex 50(H⁺) columns. Neither synthetic Δ¹-pyrroline-5-carboxylate nor the product of proline-dehydrogenase produced γ-amino butyrate with hydrogen peroxide.     

Epiphytic Occurrence of Azorhizobium caulinodans and Other Rhizobia on Host and Nonhost Legumes

A large population of Azorhizobium caulinodans was present on Sesbania rostrata; up to 5 × 10⁻⁵ cm⁻² were found on leaves and fewer were found on flowers. Although A. caulinodans was also present on the leaves of Sesbania aculeata (nonhost), the populations were much smaller than that observed on S. rostrata. The population of S. aculeata rhizobia on host leaves was less than 30 cm⁻², and their presence on host flowers was sporadic. Aeschynomene afraspera and Aeschynomene aspera rhizobia, which are profusely stem nodulating, were found on the leaves of host and nonhost plants and on the flowers of host plants, but, Aeschynomene pratensis and Aeschynomene sensitiva rhizobia were not found on the leaves and flowers of host plants.     

Synthesis and biological evaluation of novel pyrazolopyrimidines derivatives as anticancer and anti-5-lipoxygenase agents

A novel series of 6-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones 3a-h were synthesized in a single step via condensation of carboxamide 2 with some aromatic aldehydes (presence of iodine). Treatment of aminopyrazole 1a with acetic anhydride afforded pyrazolopyrimidines 4 which on treatment with ethyl chloroacetate in refluxing dry DMF furnished a single product identified as ethyl 2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl) acetate 5. On the other hand, esterification of compound 6 with different alcohol, led to the formation of new esters linked pyrazolo[3,4-d]pyrimidinones hybrids 7a-f. The reaction of compound 2 with 3-propargyl bromide gave the compound 8 used as a dipolarophile to access to triazoles (4- and 5-regioisomers (9a-e) and (10a-e), respectively) via the 1,3-dipoar cycloaddition reaction. Finally, condensation reaction of aminopyrazole 1b with α-cyanocinnamonitiles gave the new pyrazolo[1,5-a]pyrimidine-3,6-dicarbonitriles 11a-e. Structures of compounds were established on the basis of ¹H/¹³C NMR and ESI-HRMS. Compounds were screened for their cytotoxic (HCT-116 and MCF-7) and 5-lipoxygenase inhibition activities. The structure-activity relationship (SAR) was discussed.     

Ivermectin-derived leishmanicidal compounds

In the present study a family of macrocyclic and acyclic analogues as well as seco-analogues of avermectins were prepared from commercial Ivermectin (IVM) and their antileishmanial activity assayed against axenic promastigote and intracellular amastigote forms of Leishmania amazonensis. Contrarily to the filaricidal activity, the leishmanicidal potentiality of avermectin analogues does not appear to depend on the integrity of the non-conjugated Δ^3,4-hexahydrobenzofuran moiety. Conjugated Δ^2,3-IVM or its corresponding conjugated secoester show higher anti-leishmania activity than the parent compound. Surprisingly, the diglycosylated northern sub-unit exhibits the same anti-amastigote potentiality as the southern hexahydrobenzofuran. As expected for compounds derived from the widely used Ivermectin antibiotic, little toxicity has been noticed for most of the novel analogues prepared.     

Potential difference responses to secretory K+, Cl− and Na+ changes in secreting and resting states of frog stomach

The effects of changes in secretory concentrations of K⁺, Cl⁻ and Na⁺ on transmembrane potential difference (PD) and resistance were compared for secreting fundus and resting fundus of Rana pipiens. In the resting fundus experiments histamine was present, and SCN and omeprazole gave similar results. Increase of K⁺ from 4 to 80 mM, decrease of Cl⁻ from 160 to 16 mM and decrease of Na⁺ from 156 to 15.6 mM gave, respectively, 10 min after the change, in the secreting fundus ΔPD = 7.6, 10.0 and −2.2 mV and in the resting fundus ΔPD = 4.3, 14.4 and 0 mV. With cimetidine and no histamine, increase of K⁺ from 4 to 80 mM gave a ΔPD which decreased to near zero after exposure to cimetidine for at least 30 min. For the same K⁺ change, replacement of cimetidine with SCN or omeprazole and without histamine maintained ΔPD near zero and subsequent addition of histamine with inhibitor present gave a ΔPD of about 12 mV. The change in ΔPD was attributed to histamine increasing the secretory membrane area, which results in an increase in K⁺ conductance. Increase in ΔPD in the resting fundus compared to the secreting fundus for a decrease from 160 to 16 mM Cl⁻ may be due to relatively little Cl⁻ entering the lumina from cells in the resting fundus, which would result in a greater change of the ratio intracellular Cl⁻/luminal Cl⁻ in the resting fundus than in the secreting fundus for the decrease in Cl⁻ studied.     

Chloroplastic ATP synthase optimizes the trade-off between photosynthetic CO2 assimilation and photoprotection during leaf maturation

In the present study, we studied the role of chloroplastic ATP synthase in photosynthetic regulation during leaf maturation. We measured gas exchange, chlorophyll fluorescence, P700 redox state, and the electrochromic shift signal in mature and immature leaves. Under high light, the immature leaves displayed high levels of non-photochemical quenching (NPQ) and P700 oxidation ratio, and higher values for proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes but lower values for the activity of chloroplastic ATP synthase (g_H⁺) than the mature leaves. Furthermore, g_H⁺ was significantly and positively correlated with CO₂ assimilation rate and linear electron flow (LEF), but negatively correlated with pmf and ΔpH. ΔpH was significantly correlated with LEF and the P700 oxidation ratio. These results indicated that g_H⁺ was regulated to match photosynthetic capacity during leaf maturation, and the formation of pmf and ΔpH was predominantly regulated by the alterations in g_H⁺. In the immature leaves, the high steady-state ΔpH increased lumen acidification, which, in turn, stimulated photoprotection for the photosynthetic apparatus via NPQ induction and photosynthetic control. Our results highlighted the importance of chloroplastic ATP synthase in optimizing the trade-off between CO₂ assimilation and photoprotection during leaf maturation.     

Light Restriction Delays Leaf Senescence in Winter Oilseed Rape (Brassica napus L.)

Oilseed rape (Brassica napus L.) is a crop with a complex aerial architecture that can cause self-shading leading to a vertical light gradient over the foliage. Mutual shading between neighboring plants at a high sowing density also results in an alteration of photosynthetically active radiation (PAR) absorption by lower leaves. The aim of this study was to analyze the impact that light restriction on lower leaves has on shoot architecture, biomass production and allocation, nitrogen (N) fluxes, and progression of sequential senescence. Field-grown plants were collected at the end of the vegetative rest period and grown in hydroponic conditions until pod maturity. A shading treatment corresponding to a 43.4 % reduction of PAR was applied at the early flowering stage. N uptake and fluxes of N allocation and remobilization were determined by supplying K¹⁵NO₃ in the nutrient solution. Photosynthesis and expression of SAG12 and Cab genes (indicators of leaf senescence progression) were also analyzed on different leaf ranks. The results showed that shading enhanced leaf development on the main stem and ramifications to optimize light capture. The expression pattern of the SAG12/Cab molecular indicator suggested a delay in leaf senescence that allowed leaf life span to be extended resulting in a more efficient leaf compound remobilization, with lower N residual contents in fallen leaves under shading. N uptake increased and N remobilization fluxes were enhanced from source organs (leaves and stem) toward sink organs (flowers). Profuse branching and late senescing varieties would be of interest for further selection programs under high sowing densities.     

Distribution of charged flavones and caffeylshikimic acid in Palmae

Identification of the phenolic constituents in flowers of nine palm species has revealed that charged C-glycosylflavones and caffeylshikimic acid are characteristically present. Flavonol glycosides are also common; the 3-glucosides, 3-rutinosides and 3,4′-diglucosides of quercetin and isorhamnetin and the 7-glucoside and 3,7-diglucoside of quercetin are all variously present. Tricin 7-glucoside, luteolin 7-rutinoside and several unchanged C-glycosylflavones were also detected. Male flowers of Phoenix canariensis differ from female flowers in having flavonol glycosides. As expected, in most species studied, flavonoid patterns in the flowers vary considerably from those found in the leaves.     

Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum

The aim of this work was to establish the pathways of carbohydrate oxidation used in the dark by leaves of Pisum sativum and Triticum aestivum. Segments of young and mature leaves of pea released the carbons of glucose-[¹⁴C] as ¹⁴CO₂ in the order 3,4 > 1 > 2 > 6 whereas in segments of young and mature leaves of wheat the order was 3,4 > 1 > 6 > 2. The detailed labelling of the constituents of mature leaves of wheat by glucose-[1-¹⁴C], -[2-¹⁴C], -[3,4-¹⁴C], and -[6-¹⁴C] was determined and showed that the high yield of CO₂ from C-6 relative to that from C-2 was due to release of C-6 during pentan synthesis. Estimates were made of the maximum catalytic activities of phosphofructokinase and glucose-6-phosphate dehydrogenase in pea and wheat leaves of three ages. The results of all the above investigations strongly indicate that both pea and wheat leaves in the dark oxidize carbohydrate via glycolysis and the pentose phosphate pathway with the latter accounting for no more than a third of the total. No evidence was obtained of any major change in the relative activities of the two pathways during the development of either type of leaf.     

Modifications at C-3 and C-4 of 2-amino-2-deoxy-d-glucose

Modifications at C-3 and C-4 of 2-amino-2-deoxy-d-glucose have been developed. A 3,4-double bond was introduced into benzyl 2-acetamido-2-deoxy-3,4-di-O-Methylsulfonyl-α-d-glucopyranoside by treatment with NaI and Zn. Epoxidation of the double bond with m-chloroperoxybenzoic acid gave an exo-epoxide exclusively. The stereochemistry of the epoxidation product has been confirmed by an alternative synthesis. An analysis of the ¹H-n.m.r. spectra indicates that both the 3,4-unsaturated derivatives and the epoxide exist in the °H₁ (d) conformation. Nucleophilic reagents (F^?, I^?) opened the 3,4-epoxide to give 4-substituted derivatives having the d-gulo configuration. Thus, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-iodo-α-d-gulopyranose and 2-acetamido-1,3,6-tri-O-acetyl-3,4-dideoxy-4-fluoro-α-d-gulopyranose have been synthesized. Reduction of the double bond in the key intermediate and deprotection gave 2-acetamido-2,3,4-trideoxy-d-glucopyranose. Some of the derivatives were active as inhibitors of growth of mouse, mammary adenocarcinoma cells in culture.     

Distribution of different forms of sterols in three cellular subfractions of Calendula officinalis leaves

From Calendula officinalis leaves three cellular subtractions (mitochondrial, Golgi membranes and microsomal) were obtained and enzymatically characterized. The contents of Δ⁰, Δ⁵, Δ⁷, Δ^{5, 22} sterols, as well as those of 24-methylenecholesterol and clerosterol, in the free and bound in the form of esters, glucosides and acylated glucosides were determined in these fractions. The results revealed the predominance of free sterols in the microsomal fraction, of esters in the mitochondrial fraction and of steryl glucosides and acylated glucosides in the Golgi fraction.     

Assessment of the Environmental Fate of the Biological Control Agent of Fire Blight, Pseudomonas fluorescens EPS62e, on Apple by Culture and Real-Time PCR Methods

The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 10² cells g (fresh weight)⁻¹ and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 10⁷ to 10⁸ cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.     

Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74-related enzyme of oxylipin biosynthesis in brown algae

Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [¹⁸O₂]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.     

Nitrate reductase activity (NRA) in Asphodelus aestivus Brot. (Liliaceae): distribution among organs,seasonal variation and differences among populations

Nitrate reductase activity (NRA) in different compartments (leaves, inflorescence stalks, flowers and tuberous roots) of Asphodelus aestivus Brot. (Liliaceae) and actual mineral nitrogen (NO₃⁻-N and NH₄⁺-N) in soil surrounding the roots were investigated over one year. Although the highest NRA was found in the leaves, the other plant compartments, such as flowers and tuberous roots, also have nitrate assimilation capacity. High nitrate assimilation capacity under suitable conditions is considered to be a good strategy for development and dominance of this species in Mediterranean environments. There was a seasonal variation in nitrate assimilation in leaves and actual NO₃⁻-N content of soils. Depending on actual nitrate content of soils, nitrate assimilation increased in winter.     

Lipoxygenase pathway in brown algae: The biosynthesis of novel oxylipins ‘ectocarpins’ by hydroperoxide bicyclase CYP5164A3 of Ectocarpus siliculosus

The sequence encoding the CYP5164A3 of the brown alga Ectocarpus siliculosus (Stramenopiles, SAR) was heterologously expressed in E. coli cells. The resulting recombinant CYP74 clan-related protein CYP5164A3 possessed a selective activity towards the α-linolenic acid 13(S)-hydroperoxide (13-HPOTE) and eicosapentaenoic acid 15(S)-hydroperoxide (15-HPEPE). The major products were the heterobicyclic oxylipins. For instance, the 13-HPOTE was converted into plasmodiophorols A, B, and C formed at about 14:3:2 ratio. Plasmodiophorols A-C have been recently described as the products of enzyme hydroperoxide bicyclase CYP50918A1 of cercozoan Plasmodiophora brassicae (Rhizaria, SAR). Furthermore, an unknown compound 1 was detected. Purified product 1 (Me) was identified as a novel substituted 3-propenyl-6-oxabicyclo[3.1.0]hexane based on its MS and NMR spectral data. Conversion of 15-HPEPE by CYP5164A3 resulted in products 7 and 8, analogous to plasmodiophorols A and B. This work uncovered the CYP5164A3 as the first hydroperoxide bicyclase in brown algae. Apparently, this enzyme plays a crucial role in the biosynthesis of heterobicyclic oxylipins like hybridalactone, ecklonilactones, and related natural products, widespread in brown algae.     

Enzymic formation of carbonyls from linoleic acid in leaves of Phaseolus vulgaris

Homogenization of Phaseolus vulgaris leaves at acid pH results in the evolution of hexanal, cis-3- and trans-2-hexenal. With cell-free extracts of leaves, linoleic and linolenic acids are enzymically converted to their hydroperoxides (predominantly the 13-hydroperoxide isomers) and to hexanal or hexenal respectively. Activity was highest in young, dark-green leaves and was stimulated by Triton X-100. Oleic acid is not a substrate for these reactions. Both 9- and 13-hydroperoxides were cleaved to carbonyl fragments and are proposed as intermediates in the formation of volatile aldehydes and non-volatile ω-oxoacids in P. vulgaris leaves. Properties of the enzyme systems are described.     

Essential oils of five Tanacetum vulgare genotypes

The essential oils of five Tanacetum vulgare genotypes were investigated and their main components identified. Artemisia alcohol, γ-campholenol, davanone, lyratol, lyratyl acetate and 4-thujen-2α-yl acetate have not been reported before as constituents of Tanacetum vulgare. This is the first time that γ-campholenol has been isolated from a natural source. 4-Thujen-2α-yl acetate is a novel compound.     

Source/Sink relations of abscising and nonabscising soybean flowers

The partitioning of recently fixed ¹⁴C to setting and abscising flowers within the axillary raceme of `Clark' isoline E1t soybeans (Glycine max L. Merr) was examined as a function of time after anthesis of individual flowers. In such racemes, the first four flowers showed a 17% abscission while the next four flowers showed 47% abscission.

Source/sink relations of flowers I-IV (normally setting) were compared to those of flowers V-VIII (normally abscising) by pulse labeling source leaves with ¹⁴CO₂ and determining the radioactivity of individual flowers after a 4-hour chase period. The relative specific activity (RSA;% disintegrations per minute per% dry weight), sink strength (% disintegrations per minute), and its components, sink size (milligrams dry weight) and sink intensity (% disintegrations per milligram dry weight) were then calculated as a function of days after anthesis.

Sink intensity (i.e. the competitive ability to accumulate photoassimilate per unit mass) was very high prior to anthesis of both setting and abscising flowers. Sink intensity then became very low for the first 3 days following anthesis after which it recovered in normally setting flowers, but failed to recover in normally abscising flowers. It is concluded that soybean reproductive abscission is determined at or very near the day of anthesis.

     

A MORPHOGENETIC STUDY OF LANCEOLATE,A LEAF-SHAPE MUTANT IN THE TOMATO

Mathan , D. S., and J. A. Jenkins . (U. California, Berkeley.) A morphogenetic study of lanceolate, a leaf-shape mutant in the tomato . Amer. Jour. Bot. 49(5): 504–514. Illus. 1962.—The single-gene mutant, lanceolate (La/La⁺), which has simple, entire leaves rather than the oddpinnately compound leaves of the normal tomato (Lycopersicon esculentum), differs from normal (La⁺/La⁺) in many characters which can be related to fewer and larger cells in meristematic regions. The homozygous lanceolate (La/La) is sometimes lethal, but is usually expressed as reduced, which consists of a hypocotyl without cotyledons, or occasionally either by modified, in which there is a single fused cotyledon and a bud, or by narrow, in which the bud produces a shoot with simple leaves even smaller than those of lanceolate and a tendril-like inflorescence without flowers. In an attempt to overcome the effect produced by the lanceolate gene, the mutant was treated with a number of substances. Embryos without cotyledons cultured in White's medium plus adenine or tyrosine or both gave a higher frequency of narrow plants. With added gibberellic acid, no narrow plants developed from embryos without cotyledons. When tyrosine was sprayed on young narrow plants, they developed larger leaves and morphologically normal but sterile flowers. On the other hand, gibberellic acid sprayed on young lanceolate plants altered their development in the direction of narrow.     

The development of photophosphorylation and photosynthesis in greening bean leaves

Photophosphorylation and oxygen evolution were measured in 8-day-old dark-grown bean leaves (Phaseolus vulgaris) after various times of greening in far red light and in white light. The sequence of development was the same for both greening regimes, but the processes were much more rapid in white light. The capacity for photophosphorylation, as assayed by the firefly luciferase assay, appeared after 12 hours in far red light. At this stage and for times up to 24 hours, photophosphorylation was not inhibited by 10⁻⁵m 3-(3,4-dichlorophenyl)-1,1-dimethylurea. At 24 hours, the capacity for oxygen evolution appeared and photophosphorylation became partially inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations which inhibited oxygen evolution. In white light photophosphorylation appeared after 15 minutes, and oxygen evolution at one hour. Photophosphorylation became partially sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea when oxygen evolution appeared. Carbonylcyanide m-chlorophenyl-hydrazone inhibited photophosphorylation and photosynthesis at low concentrations, 10⁻⁵m, with immature leaves, but the leaves developed resistance to carbonylcyanide m-chlorophenyl-hydrazone as they greened.