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Gas chromatographic, mass spectrometric and stable carbon isotopic investigations of organic residues of plant oils and animal fats employed as illuminants in archaeological lamps from Egypt.

Man's use of illuminants in lamps or as torches to extend the working day and range of environments accessible to him would have been a major technological advance in human civilisation. The most obvious evidence for this in the archaeological record comes from pottery and stone vessels showing sooting due to the use of a wick in conjunction with a lipid-based fuel or illuminant. A wide range of potential fuels would have been exploited depending upon availability and burning requirements. Reported herein are the results of chemical investigations of a number of lamps recovered from excavations of the site of Qasr Ibrim, Egypt. Gas chromatographic, mass spectrometric and stable carbon isotopic analyses of both free (solvent extractable) and 'bound'(released from solvent extracted pottery by base treatment) lipids have revealed a wide range of saturated fatty acids, hydroxy fatty acids and alpha, omega-dicarboxylic acids. Examination of the distributions of compounds and comparisons with the fatty acid compositions of modern plant oils have allowed a range of fats and oils to be recognised. Specific illuminants identified include Brassicaceae (Cruciferae) seed oil (most likely radish oil, Raphanus sativus), castor oil (from Ricinus communis), animal fat, with less diagnostic distributions and delta(13)C values being consistent with low stearic acid plant oils, such as linseed (Linum usitatissimum) or sesame (Sesamum indicum) oils. The identifications of the various oils and fats are supported by parallel investigations of illuminant residues produced by burning various oils in replica pottery lamps. The findings are entirely consistent with the classical writers including Strabo, Pliny and Theophrastrus.

Archaeology↗

Gene flow by pollen into small populations: Data from experimental and natural stands of wild radish.

Gene flow can have an especially strong impact on the evolution of small populations. However, empirical studies on the actual rates and patterns of gene flow into small populations are few. Thus, we sought to measure gene flow into small populations of wild radish. Raphanus sativus. We found significant differences in gene flow receipt among experimental populations and within those populations over a season. A maximum-likelihood estimate revealed that almost all of the gene flow into these synthetic populations had its origin in relatively distant (>650 m), large natural populations rather than the proximal (255-400 m), small synthetic populations. We also estimated rates of interpopulation mating from simple paternity analysis of progeny produced by seven small (ca. 50 plants) natural populations. Again, we found significant heterogeneity in gene flow receipt. Although these populations varied 10-fold in their range of isolation distances (100-1000 m), gene flow rates did not vary with distance. The magnitude of gene flow rates estimated in all but one population was great enough for gene flow to play an important role in the evolution of these small populations.

Journal Article↗

Spatial pattern of cdc2 expression in relation to meristem activity and cell proliferation during plant development.

The p34 protein kinase encoded by the cdc2 gene is a key component of the eukaryotic cell cycle required for the G1- to S-phase transition and entry into mitosis. To study the regulation of plant meristem activity and cell proliferation, we have examined the tissue-specific accumulation of cdc2 transcripts in Arabidopsis thaliana and the related crucifer radish (Raphanus sativus) by in situ hybridization using A. thaliana cdc2 cDNA sequences as a probe. cdc2 transcripts accumulated in leaf primordia and within the vegetative shoot apical meristem. During flower development, high levels of expression were observed in meristems, in the basal regions of developing organs, in the developing vasculature, and associated with rib meristems elaborated late in the development of some floral organs. In root tips, cdc2 transcripts accumulated in the meristematic region and adjacent daughter cells but were not detected in the quiescent center. There was strong hybridization throughout the pericycle, and a further localized accumulation of cdc2 transcripts was observed in the initial stages of the activation of a new meristem at sites of lateral root development. We conclude that cdc2 expression is a critical factor in the regulation of meristem activity and establishment of proliferative competence.

Base Sequence↗

Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes.

Hs-AFP1, an antifungal plant defensin from seed of the plant Heuchera sanguinea, was radioactively labeled using t-butoxycarbonyl-[35S]L-methionine N-hydroxysuccinimidyl ester, resulting in a 35S-labeled peptide with unaltered antifungal activity. [35S]Hs-AFP1 was used to assess binding on living hyphae of the fungus Neurospora crassa. Binding of [35S]Hs-AFP1 was found to be competitive, reversible, and saturable with an apparent Kd of 29 nM and a Bmax of 1.4 pmol/mg protein. [35S]Hs-AFP1 also bound specifically and reversibly to microsomal membranes derived from N. crassa hyphae with a Kd of 27 nM and a Bmax of 102 pmol/mg protein. The similarity in Kd value between binding sites on hyphae and microsomes indicates that Hs-AFP1 binding sites reside on the plasma membrane. Binding of [35S]Hs-AFP1 to both hyphae and microsomal membranes could be competed to some extent by four different structurally related plant defensins but not by various structurally unrelated antimicrobial peptides. In addition, an inactive single amino acid substitution variant of the antifungal plant defensin Rs-AFP2 from Raphanus sativus seed was also unable to displace [35S]Hs-AFP1 from its binding sites, whereas Rs-AFP2 itself was able to compete with [35S]Hs-AFP1.

Antifungal Agents↗

Defensins from insects and plants interact with fungal glucosylceramides.

Growth of the yeast species Candida albicans and Pichia pastoris is inhibited by RsAFP2, a plant defensin isolated from radish seed (Raphanus sativus), at micromolar concentrations. In contrast, gcs-deletion mutants of both yeast species are resistant toward RsAFP2. GCS genes encode UDP-glucose:ceramide glucosyltransferases, which catalyze the final step in the biosynthesis of the membrane lipid glucosylceramide. In an enzyme-linked immunosorbent assay-based binding assay, RsAFP2 was found to interact with glucosylceramides isolated from P. pastoris but not with soybean nor human glucosylceramides. Furthermore, the P. pastoris parental strain is sensitive toward RsAFP2-induced membrane permeabilization, whereas the corresponding gcs-deletion mutant is highly resistant to RsAFP2-mediated membrane permeabilization. A model for the mode of action of RsAFP2 is presented in which all of these findings are linked. Similarly to RsAFP2, heliomicin, a defensin-like peptide from the insect Heliothis virescens, is active on C. albicans and P. pastoris parental strains but displays no activity on the gcs-deletion mutants of both yeast species. Furthermore, heliomicin interacts with glucosylceramides isolated from P. pastoris and soybean but not with human glucosylceramides. These data indicate that structurally homologous anti-fungal peptides present in species from different eukaryotic kingdoms interact with the same target in the fungal plasma membrane, namely glucosylceramides, and as such support the hypothesis that defensins from plants and insects have evolved from a single precursor.

Animals↗

Radical scavenging and iron-chelating activities of some greens used as traditional dishes in Mediterranean diet.

This study aimed at evaluating the antioxidative activity of nine different families of greens. Raphanus raphanistrum (wild radish), Anchusa azurea (bugloss), Daucus carota (wild carrot), Sonchus oleraceus (sowthistle), Papaver rhoeas (corn poppy), Malva sylvestris (blue mallow), Foeniculum vulgare (fennel), Cichorium intybus (chicory) and Salicornia europaea (jointed glasswort) are native to the Mediterranean and are commonly consumed as a salad or an ingredient in some recipes. The antioxidative activities, including the radical scavenging effects, inhibition of hydrogen peroxide (H(2)O(2)), and Fe(2+)-chelating activity, were studied. All samples showed antioxidant activity as a radical scavenger in the experiment using the DPPH* radical. The ratio between the slopes of the kinetic model was used to compare antioxidant efficiency of different greens. Greens also possessed antioxidative activity toward H(2)O(2). Especially, greens exhibited a marked scavenging effect on H(2)O(2) at 0.2 g/ml concentration. The Fe(2+) ion-chelating activities of the samples except jointed glasswort were greater than 70%. The antioxidant activity of samples with different methods based on the inhibition of different reactions could not be compared. The current dietary guidelines include recommendations for an increase in the consumption of plant foods. Greens should provide an optimal supply of antioxidant substances in the diet.

Antioxidants↗

Polyribosomes and cisternal accumulations in root cells of radish.

The zone of root hair formation of seedling radish roots, Raphanus sativus L., was studied by phase-contrast and electron microscopy. Localized dilations of the endoplasmic reticulum, which contained a moderately dense proteinaceous material, were found to be a common component of the cytoplasm in cells of the epidermis and cortex. The surfaces of these dilations were covered with polyribosomes in discrete coils commonly composed of 15 to 17 ribosomes. The function of these structures and the fate of the material accumulated in them are unknown. Their similarity to structures described in some types of animal cells is discussed.

Endoplasmic Reticulum↗

Rescuing activity of galactoglycerolipids from cellular lesions induced by 5-aminolevulinic acid.

An anti-oxygen radical reagent of a bacterial metabolite, M874 monogalactoglycerolipid (di-O-12-methyl-tetradecanoyl-3-O-beta-D-galactopyranosyl-sn-glycerol ), was tested for its ability to protect two organisms against cellular lesions induced by 5-aminolevulinic acid (ALA) and light. In Corynebacterium flavescens ATCC 10340, extracellular uroporphyrin and coproporphyrin were the main porphyrin products. Although less than 2 mM ALA increased porphyrin synthesis, ALA levels above 3 mM inhibited the synthesis. Depending on the light intensity, the amount of porphyrin decreased and ALA-induced cytotoxicity increased. The lesion was more severe in the case of coproporphyrin than uroporphyrin. The porphyrin lesion produced in low intensity light (300 lx) was considerably reduced by 100 microM M874 glycolipid, although the reduction in intense light (3,000 lx) was restricted to a lower level. Similar results were obtained with radish (Raphanus sativus). The ALA concentration that inhibited porphyrin synthesis and stem growth was similar to that seen with C. flavescens. Although the exogenous addition of M874 glycolipid to the radish did not prevent ALA-induced cellular injury, the co-culture of radish and a glycolipid producing bacterium (Microbacterium sp. M874) resulted in a significant prevention of cellular injury. This was true only under enforced adhesion conditions through the action of a polysaccharide flocculant H12. Some species of monogalactoglycerolipids were found in Corynebacterium and radish that showed prominent oxygen radical-protecting activities similar to that of M874 glycolipid. These monogalactoglycerolipids might function in vivo as agents to prevent ALA-induced cytological lesions, although the concentrations were low in Corynebacterium and radish.

Aminolevulinic Acid↗

Amino acid sequences of ferredoxin isoproteins from radish roots.

Three ferredoxin isoproteins (R-Fd A, R-Fd B-1, and R-Fd B-2) were purified from white roots of radish (Raphanus sativus L. var. acantiformis cultivar Miyashige) and two isoproteins (L-Fd A and L-Fd B) from leaves. The amino acid sequences of three of them (L-Fd A, R-Fd B-1, and R-Fd B-2) were determined and compared with one another and with those of other higher plant ferredoxins so far studied. L-Fd A and R-Fd B-1 had heterogeneities at four and two amino acid sites, respectively. Two isoprotein (R-Fd B-1 and R-Fd B-2) were deduced to be expressed only in root tissue on the basis of sequence studies and amino acid compositions of all isoferredoxins isolated from the radish plant. The root ferredoxins sequenced in this study were similar to each other, but quite different from other higher plant ferredoxins, all of which were isolated from leaf tissue. The coupling activities of these ferredoxin isoproteins were measured in the NADP+-photoreduction system of radish chloroplasts and glutamate synthase [EC 1.4.7.1] systems isolated from radish leaf and root tissues. No distinctive physiological characteristics were observed among these isoferredoxins.

Amino Acid Sequence↗

Molecular cloning, water channel activity and tissue specific expression of two isoforms of radish vacuolar aquaporin.

A major membrane intrinsic protein (VM23) in vacuoles of radish (Raphanus) tap root was investigated. The cDNAs for two isoforms of VM23, gamma- and delta-VM23, encode polypeptides of 253 and 248 amino acids, respectively. gamma- and delta-VM23 correspond to the gamma- and delta-TIP (tonoplast intrinsic protein) of Arabidopsis. The deduced amino acid sequences of the two VM23 isoforms were 60% identical. The amino-terminal sequence of gamma-VM23 showed agreement with the direct sequence of the purified VM23, suggesting that gamma-VM23 is the most abundant molecule among the VM23 isoforms. When mRNAs of gamma- and delta-VM23 were injected into Xenopus oocytes, the osmotic water permeability of oocytes increased 6-fold (60 to 200 microns s-1) of the control oocytes. The transcripts of both isoforms were detected in a high level in growing hypocotyls and young leaves, but delta-VM23 was not detected in seedling roots. Light illumination enhanced the transcription of two genes of VM23 in cotyledons and roots but suppressed their expression in hypocotyls the growth of which was inhibited by light. These findings suggest that the expression of VM23 is tightly related to cell elongation.

Amino Acid Sequence↗

Water-soluble chlorophyll protein in Brassicaceae plants is a stress-induced chlorophyll-binding protein.

Two kinds of water-soluble chlorophyll (Chl) proteins (WSCPs) have been found, e.g., a WSCP from Chenopodium, Atriplex, Polygonum, and Amaranthus species (class I) and that from Brassica, Raphanus, and Lepidium species (class II). Classes I and II WSCPs differ mainly in their photoconvertiblity. Class I WSCPs show a light-induced absorption change, whereas Class II WSCPs do not. The molecular and functional properties of Class I WSCP are largely uncertain. On the other hand, recent studies on the adaptation of plants to osmotic stress revealed the participation of drought-stress induced proteins with molecular masses of 20-22 kDa possessing a sequence similarity with class II WSCPs. This mini review focuses on the molecular signature of class II WSCPs. The physiological function of class II WSCPs has not been clarified either, but, their water-solubility, low Chl content, and stress-inducibility suggested little contribution to photosynthesis. Several molecular properties predicting its physiological role are as follows. The WSCP tetramer, may have only one or no Chl molecules in each subunit. All WSCPs possess a motif for Künitz-type proteinase inhibitor family in their sequence. WSCP is induced by drought- and heat-stresses suggesting its protective role during stress conditions. Monomeric recombinant apo-WSCP is able to remove Chls from the thylakoid membrane in aqueous solution and form into a tetramer. Brassica-WSCP contains a signal sequence targeted to endoplasmic reticulum. The highly conserved, C-terminal region is missing in the mature WSCP. Possible functions of class II WSCPs in plant tissues are discussed.

Adaptation, Physiological↗

Predicting variability in biological control of a plant-pathogen system using stochastic models.

A stochastic model for the dynamics of a plant-pathogen interaction is developed and fitted to observations of the fungal pathogen Rhizoctonia solani (Kühn) in radish (Raphanus sativus L.), in both the presence and absence of the antagonistic fungus Trichoderma viride (Pers ex Gray). The model incorporates parameters for primary and secondary infection mechanisms and for characterizing the time-varying susceptibility of the host population. A parameter likelihood is developed and used to fit the model to data from microcosm experiments. It is shown that the stochastic model accounts well for observed variability both within and between treatments. Moreover, it enables us to describe the time evolution of the probability distribution for the variability among replicate epidemics in terms of the underlying epidemiological parameters for primary and secondary infection and decay in susceptibility. Consideration of profile likelihoods for each parameter provides strong evidence that T. viride mainly affects primary infection. By using the stochastic model to study the dependence of the probability distribution of disease levels on the primary infection rate we are therefore able to predict the effectiveness of a widely used biological control agent.

Models, Biological↗

Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus.

Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species.

Defective Viruses↗

Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins.

We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.

Amino Acid Sequence↗

Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase.

A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage.

2S Albumins, Plant↗

Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings).

The effects of calmodulin and of controlled trypsin treatments on the activity of the Ca2+ pump were investigated in plasma membrane purified from radish (Raphanus sativus L.) seedlings. Treatment of the plasma membrane with ethylenediaminetetra-acetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Ca2+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Ca2+ pump activity was maximal at low free Ca2+ (2-5 [mu]M) and decreased with the increase of free Ca2+ concentration. The Ca2+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the Ca2+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free Ca2+ concentration in the assay medium. Moreover, the Ca2+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.

Journal Article↗

Controlled Proteolysis Mimics the Effect of Fusicoccin on the Plasma Membrane H+-ATPase.

We analyzed the effects of controlled treatments with trypsin of plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings on the activity of the PM H+-ATPase, and we compared them with those of fusicoccin (FC). Mild treatments of the PM with trypsin, which led to a decrease of the molecular mass of the peptide of about 10 kD, markedly increased the H+-ATPase activity. The effect strongly increased with the increase of pH of the assay medium from 6.1 to 7.5, so the pH optimum of the enzyme activity shifted from 6.8 in untreated PM to 7.1 in trypsin-treated PM. The proteolytic treatment activated only the portion of PM H+-ATPase activity that is stable to preincubation in assay medium in the absence of ATP and determined a strong increase of Vmax and a less marked decrease of the apparent Km for Mg-ATP. All of these effects were very similar to those determined by FC, which activated the PM H+-ATPase without promoting its proteolytic cleavage. FC did not further activate the H+-ATPase activity of trypsin-treated PM under conditions in which the FC receptor was protected from the attack of trypsin. Conversely, trypsin treatment had little effect on the PM H+-ATPase preactivated with FC. Moreover, the activity of the PM H+-ATPase preactivated with FC was not further activated by Iysolecithin. These results indicate that the modification of the PM H+-ATPase of higher plants triggered by the FC-receptor complex hinders the inhibitory interaction of the regulatory C-terminal domain with the active site.

Journal Article↗

Comparative Studies on Ferredoxin-NADP+ Oxidoreductase Isoenzymes Derived from Different Organs by Antibodies Specific for the Radish Root- and Leaf-Enzymes.

Determination of the prosthetic group and titration of sulfhydryl group of ferredoxin-NADP+ oxidoreductase (FNR) from roots of radish (Raphanus sativus var acanthiformis cv Miyashige) confirmed its similarity to leaf-FNR. Antisera directed against radish root-FNR and leaf-FNR distinguished the enzyme forms from roots and leaves of radish as well as other flowering plants. The FNR isoenzymes showed organ-specific distributions. In horsetail (Equisetum arvense L.) and cultured liverwort cells (Marchantia polymorpha), at least two FNR isoenzymes were distinguished by the antisera. FNR from Chlorella vulgaris reacted only with the anti-root-FNR antiserum. FNR from a cyanobacterium, Spirulina spp., failed to react with either antiserum.

Journal Article↗