Simple sequence repeat-based assessment of genetic relationships among Prunus rootstocks

Ten SSR loci, previously developed for Prunus, were analyzed to examine genetic relationships among 23 rootstock candidates for sweet and sour cherries, of the species P. avium, P. cerasus, P. mahaleb, and P. angustifolia. Five genotypes of P. laurocerasus, not used as rootstock, were included in the molecular analysis. The number of alleles per locus ranged from 8 to 12, with a mean of 9, while the number of microsatellite genotypes varied from 8 to 17, indicating that the SSRs were highly informative. The degree of heterozygosity (0.61) was high. Clustering analysis resulted in two main clusters. The first cluster was divided into two subclusters; the first subcluster consisted of P. avium and P. cerasus, and the second subcluster consisted of P. laurocerasus. The second cluster was divided into two subclusters. The first subcluster consisted of P. mahaleb genotypes and the second consisted of P. angustifolia genotypes. The reference rootstocks also clustered with their associated botanical species. Unweighted pair-group method with arithmetic mean analysis demonstrated that P. laurocerasus genotypes had less genetic variation and that P. avium genotypes were more closely related to P. cerasus. The SSR-based phylogeny was generally consistent with Prunus taxonomy information, suggesting the applicability of SSR analysis for genotyping and phylogenetic studies in the genus Prunus.     

Xylem cavitation in the leaf of Prunus laurocerasus and its impact on leaf hydraulics

This paper reports how water stress correlates with changes in hydraulic conductivity of stems, leaf midrib, and whole leaves of Prunus laurocerasus. Water stress caused cavitation-induced dysfunction in vessels of P. laurocerasus. Cavitation was detected acoustically by counts of ultrasonic acoustic emissions and by the loss of hydraulic conductivity measured by a vacuum chamber method. Stems and midribs were approximately equally vulnerable to cavitations. Although midribs suffered a 70% loss of hydraulic conductance at leaf water potentials of -1.5 MPa, there was less than a 10% loss of hydraulic conductance in whole leaves. Cutting and sealing the midrib 20 mm from the leaf base caused only a 30% loss of conduction of the whole leaf. A high-pressure flow meter was used to measure conductance of whole leaves and as the leaf was progressively cut back from tip to base. These data were fitted to a model of hydraulic conductance of leaves that explained the above results, i.e. redundancy in hydraulic pathways whereby water can flow around embolized regions in the leaf, makes whole leaves relatively insensitive to significant changes in conductance of the midrib. The onset of cavitation events in P. laurocerasus leaves correlated with the onset of stomatal closure as found recently in studies of other species in our laboratory.     

Variation in seed dormancy and germination among populations of Silybum marianum (Asteraceae)

Milk thistle (Silybum marianum) is a medicinal plant; however, lack of consistency in past dormancy studies has hindered propagation of this species from seeds. We tested the germination responses of freshly harvested and after-ripened (stored for 2 and 7 months; 25°C at 50% relative humidity) seeds from three populations (P1, P2 and P3) in Iran at varying constant or alternating temperatures, with or without GA₃ and in light and continuous darkness. No germination occurred in freshly harvested seeds incubated at any condition without GA₃ application, indicating that all the seeds were dormant. Seeds from P1 and P2, which developed under relatively dry, warm conditions, germinated over a wider range of temperatures after 2 months of dry storage, indicating type 6 of non-deep physiological dormancy (PD). Seeds from P3, which developed under relatively wet, cool conditions, incubated at constant temperatures (especially on GA₃), exhibited an increase in maximum temperature for germination, indicating type 1 of non-deep PD. Light improved germination of after-ripened seeds, and GA₃ application substituted for the light requirement for germination. This is the first report that environmental conditions during seed development may be correlated with differences in the type of non-deep PD. We conclude that milk thistle seeds are positively photoblastic and photodormant and the germination responses of after-ripened seeds from different populations are different under darkness. Therefore, the impacts of genetic differences and maternal effects on the induction of dormancy during seed development should be considered in attempts to domesticate this medicinal plant.     

Isocitrate lyase: artifacts and multiple enzyme forms

Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.     

Steady-state kinetic analysis of isocitrate lyase from Lupinus seeds: considerations on a possible catalytic mechanism of isocitrate lyase from plants

Isocitrate lyase catalyzes the reversible cleavage of isocitrate into glyoxylate and succinate. The kinetic mechanism of bacterial isocitrate lyase has been reported to be ordered uni-bi. Moreover, it has been proposed that isocitrate lyase in higher plants may be switched on and off by a succinylation/desuccinylation mechanism. Similarly to bacterial citrate lyase, in which an acetylation/deacetylation mechanism is operative, succinylation might also play a role in the catalytic mechanism of plant isocitrate lyase. We have investigated the kinetic mechanism of isocitrate lyase from Lupinus seeds. The results reported in this paper show that the system follows a preferentially ordered uni-bi pathway in which the succinate is released first. On the basis of our results and some other recently reported data, we conclude that it is unlikely that bacterial and plant isocitrate lyases have different catalytic mechanisms.     

Effect of light on the development of glyoxysomal functions in the cotyledons of mustard (Sinapis alba L.) seedlings

The degradation of storage fat in the cotyledons of mustard seedlings is unaffected by phytochrome and photosynthesis (irradiation with continuous red or far-red light from sowing of the seeds) although light imposes a strong constraint on the translocation of organic matter from the cotyledons into the seedling axis. Likewise, the development and disappearance of glyoxysomal enzyme activities (isocitrate lyase, malate synthase, citrate synthase) takes place independently of light. It is concluded that the mobilization of storage fat (fatcarbohydrate transformation) is independent of photomorphogenesis. The surplus of carbohydrate produced from fat in the light seems to be converted to starch grains in the plastids, which function as a secondary storage pool in the cotyledons.Abbreviations CS citrate synthase - ICL isocitrate lyase - MS malate synthase     

Sphingolipid-to-glycerophospholipid conversion in SPL-null cells implies the existence of an alternative isozyme

Sphingosine-1-phosphate (S1P) lyase catalyzes the cleavage of the bioactive lipid molecule S1P to phosphoethanolamine and hexadecenal, both of which are utilized as glycerophospholipid precursors. Until now, only one gene, SPL, has been identified as encoding a S1P lyase. In the present study, SPL-null F9 cells were able to convert radiolabeled dihydrosphingosine to glycerophospholipids, albeit at much lower efficiency than parent cells. Lysates prepared from the SPL-null cells exhibited weak but significant dihydrosphingosine-1-phosphate lyase activity in vitro. These results provide evidence of the existence of an alternative S1P lyase.     

Molecular cloning of the cDNA coding for the (R)-(+)-mandelonitrile lyase of Prunus amygdalus: temporal and spatial expression patterns in flowers and mature seeds

A gene highly expressed in the floral organs of almond (Prunus amygdalus Batsch), and coding for the cyanogenic enzyme (R)-(+)-mandelonitrile lyase (EC 4.1.2.10), has been identified and the full-length cDNA sequenced. The temporal expression pattern in maturing seeds and during floral development was analyzed by RNA blot, and the highest mRNA levels were detected in floral tissues. The spatial mRNA accumulation pattern in almond flower buds was also analyzed by in-situ hybridization. The mRNA levels were compared during seed maturation and floral development in fruit and floral samples from cultivars classified as homozygous or heterozygous for the sweet-almond trait or homozygous for the bitter trait. No correlation was found between these characteristics and levels of mandelonitrile lyase mRNA, suggesting that the presence of this protein is not the limiting factor in the production of hydrogen cyanide. Received: 3 December 1997 / Accepted: 18 April 1998     

Albumins, glyoxysomal enzymes and globulins in dry seeds of cucumis sativus: qualitative and quantitative analysis.

1) Albumins and globulins were prepared from dry seeds of cucumbers (Cucumis sativus) by differential extraction. The globulin fraction was analyzed by gel electrophoresis under denaturing conditions in the presence and absence of mercaptoethanol. The subunit (Mr = 54000) of the tetramer (Mr = 240000) was shown to be composed of two different peptides. Microheterogeneity rendered the exact interpretation of the analysis difficult. 2) Glyoxysomal proteins were already present in dry seeds: malate synthase, isocitrate lyase, citrate synthase, malate dehydrogenase, catalase and crotonase could be detected unequivocally. It was demonstrated that the enzymatic and immunological properties of malate synthase and isocitrate lyase were not distinguishable from that of enzymes assigned to glyoxysomes of fully developed cotyledons. 3) Homogenates prepared from seeds by cautious cell disintegration were subjected to sucrose density gradient centrifugation and yielded microbody and protein body fractions, among other things.     

Isolation and spectroscopic characterization of a recombinant bell pepper hydroperoxide lyase

Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense. HPO lyase from bell pepper has been identified as a heme protein which shares 40% homology with allene oxide synthase, a cytochrome P450 (CYP74A). HPO lyase of immature bell pepper fruits was expressed in Escherichia coli and the enzyme was purified and characterized by spectroscopic techniques. The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands. The various complexes were characterized by using UV-visible absorption and electron paramagnetic resonance spectroscopy. The spectroscopic data demonstrated that the isolated recombinant HPO lyase has a pentacoordinate, high-spin heme with thiolate ligation. Addition of the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation. The striking similarities between both the ferric and ferrous HPO lyase-NO complexes with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features.     

Synthesis and biochemical evaluation of a range of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P-450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. Here, we report the synthesis and biochemical evaluation of a range of benzyl imidazole-based compounds which have been targeted against the two components of this enzyme, that is, 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are good inhibitors, with N-4-iodobenzyl imidazole (5) (IC50=10.06 microM against 17alpha-OHase and IC50=1.58 microM against lyase) showing equipotent activity against lyase compared to the standard compound, ketoconazole (KTZ) (IC50=3.76+/-0.01 microM against 17alpha-OHase and IC50=1.66+/-0.15 microM against lyase). Furthermore, the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha).     

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C9 aldehydes from HPL action.     

Physiological and enzymatic activity of pepper seeds (Capsicum annuum) during priming

Pepper ( Capsicum annuum L. cv. Keystone Resistance Giant 3) seeds were monitored during priming to determine if seed treatments which accelerate the rate of germination could be correlated with specific physiological changes within the seeds. Pepper seeds primed with −0.90 and −1.35 MPa NaCl solutions at 23°C for 18 days did not completely equilibrate with the osmotic potential of the priming solution. Seed respiratory rates indicated that priming extends the lag phase of germination following imbibition. Soluble protein levels increased 115% in primed seeds, and the uptake and incorporation of [¹⁴C(U)] labelled amino acids into the acid insoluble fraction increased throughout the priming treatments. Alcohol dehydrogenase (EC 1.1.1.1, anaerobic metabolism), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44, pentose phosphate pathway) activities remained stable throughout the priming treatment, but were higher after 6 days. than the water-imbibed controls. Aldolase (EC 4.2.1.1. glycolysis) and isocitrate lyase (EC 4.1.3.1, glyoxylate cycle) activities increased with imbibition and were 61 and 56% (respectively) higher in primed seeds as compared to the water-imbibed controls after 12 days. Treatment with the −0.90 MPa NaCl solution was more effective than the −1.35 MPa solution in improving the rate of germination, yet there were no significant differences between the protein concentrations or enzyme activities of the two priming treatments. However, the incorporation of labelled amino acids into pepper seeds was significantly higher in the −0.90 MPa priming treatment.     

The induction of enzyme activity in the endosperm of germinating castor-bean seeds.

Endosperm extracts were prepared at various times during germination from intact castor-bean seeds and from seeds from which the embryos had been removed. The sterilized seeds were incubated either on solid water agar or on agar containing 0.3 mM-gibberellic acid. 2. Isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase had very low activities in the mature seeds, but increased 44-fold and 27-fold respectively during germination. In contrast, the extracts of mature seeds had considerable acid and alkaline lipase activity and this only increased two- to three-fold during the incubation period. 3. Incubation of the seeds with gibberellic acid accelerated the rate of appearance of isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase. It also increased the total activity attained. However, the application of hormone had, in comparison, little effect on the development of lipase activity. 4. The removal of the embryo had little influence on the development of enzyme activity in the endosperm tissue; only with isocitrate lyase was a decrease in activity observed in the absence of the embryo.     

Comparative Physiology of Dimethyl Sulfide Production by Dimethylsulfoniopropionate Lyase in Pseudomonas doudoroffii and Alcaligenes sp. Strain M3A

Dimethylsulfoniopropionate (DMSP) lyase enzymatically cleaves DMSP, an algal metabolite, to produce acrylate, a proton, and dimethyl sulfide (DMS), the most abundant volatile sulfur compound emitted from oceans. The physiology of DMS production by DMSP lyase was studied in vivo in an Alcaligenes-like organism, strain M3A, a salt marsh bacterial isolate, and in a marine strain, Pseudomonas doudoroffii. Enzymes from both strains were induced at optimum rates by 1 mM DMSP and vigorous aeration. P. doudoroffii was very sensitive to continued aeration and lost activity rapidly; the enzyme was more stable when aeration ceased. In addition to DMSP, acrylate and several of its analogs acted as inducers of DMSP lyase in Alcaligenes sp. strain M3A but not in P. doudoroffii. Turnover of DMSP by P. doudoroffii was enhanced by 3.5% NaCl or seawater, whereas the Alcaligenes sp. strain M3A enzyme was not salt dependent and salt did not greatly affect its activity. The pH profile showed two peaks of DMSP lyase activity (6.5 and 8.8) for Alcaligenes sp. strain M3A and a single peak at pH 8 for P. doudoroffii. Enzyme activity in both organisms was inhibited by methyl-3-mercaptopropionate and homocysteine. Cyanide, azide and p-chloromercuribenzoate inhibited only the P. doudoroffii DMSP lyase. The apparent K(infm) values for DMSP for cell cultures of Alcaligenes sp. strain M3A and P. doudoroffii were ca. 2 mM and <20 (mu)M, respectively. The differences in the physiology of DMSP metabolism in these two bacterial isolates may enable them to exist in diverse ecological niches.     

Sphingosine-1-phosphate lyase SPL is an endoplasmic reticulum-resident, integral membrane protein with the pyridoxal 5'-phosphate binding domain exposed to the cytosol

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that functions as a bioactive lipid molecule. S1P is degraded either by S1P lyase or by S1P phosphohydrolase. The gene encoding mammalian S1P lyase, SPL, has been identified. Here, we characterize the SPL protein in its expression, localization, and topology. The expression levels of the SPL protein correlated well with the dihydrosphingosine-1-phosphate (DHS1P) lyase activity in most tissues. However, liver and heart exhibited high DHS1P lyase activities compared to their SPL protein levels. The SPL mRNA expression was temporally regulated during mouse embryonal development. Immunofluorescence microscopy demonstrated that SPL is localized at the endoplasmic reticulum. Proteinase K digestion studies revealed that the large hydrophilic domain, containing the active site, faces the cytosol. This active site orientation is opposite to that of S1P phosphohydrolase, indicating that the degradation of S1P by two S1P-degrading enzymes occurs in spatially separated sides of the endoplasmic reticulum.     

Increases in 1,5-anhydroglucitol levels in germinating amaranth seeds and in ripening banana

To examine whether 1,5-anhydroglucitol (AG) is derived from starch degradation in plant tissues, we colorimetrically measured AG contents of germinating amaranth seeds and ripening banana pulp. In both cases, as starch degradation proceeded, AG levels were significantly increased, but were 1,700-5,000 times lower than those of total soluble carbohydrates. alpha-1,4-Glucan lyase activity, which is measured by the 1,5-anhydrofructose (AF) liberated from non-reducing glucose residues of starch or glycogen, was too low to be detected in amaranth or banana by the 3,5-dinitrosalicylic acid method. On the other hand, AF reductase, which reduces AF to AG, was detected in germinating amaranth seeds and banana pulp. Thus, the increases in AG levels are conceived to be derived from starch breakdown, although further investigation is needed to answer whether the starch degradation pathway via alpha-1,4-glucan lyase/AF reductase exists in plant tissues.     

Increases in 1,5-Anhydroglucitol Levels in Germinating Amaranth Seeds and in Ripening Banana

To examine whether 1,5-anhydroglucitol (AG) is derived from starch degradation in plant tissues, we colorimetrically measured AG contents of germinating amaranth seeds and ripening banana pulp. In both cases, as starch degradation proceeded, AG levels were significantly increased, but were 1,700-5,000 times lower than those of total soluble carbohydrates. α-1,4-Glucan lyase activity, which is measured by the 1,5- anhydrofructose (AF) liberated from non-reducing glucose residues of starch or glycogen, was too low to be detected in amaranth or banana by the 3,5-dinitrosalicylic acid method. On the other hand, AF reductase, which reduces AF to AG, was detected in germinating amaranth seeds and banana pulp. Thus, the increases in AG levels are conceived to be derived from starch breakdown, although further investigation is needed to answer whether the starch degradation pathway via α-1,4-glucan lyase/AF reductase exists in plant tissues.     

The C-S lyases of higher plants: preparation and properties of homogeneous alliin lyase from garlic (Allium sativum)

Alliin lyase from garlic (Allium sativum) has been purified to homogeneity. The purification procedure involves the use of affinity chromatography on concanavalin A-Sepharose 4B. Addition of polyvinylpolypyrrolidone to the homogenizing medium greatly improves the specific activity of the extract. The enzyme is a glycoprotein as seen by its ability to bind to concanavalin A-Sepharose 4B and by its positive periodic acid-Schiff base stain. It has a carbohydrate content of 5.5%. Km values for this enzyme were estimated to be 5.7 mM for S-ethyl-L-cysteine sulfoxide and 3.3 mM for S-allyl-L-cysteine sulfoxide. The molecular weight of this garlic enzyme, as determined by gel filtration, was found to be 85,000; the molecule consists of two equal subunits of Mr 42,000. The amino acid content was found to be similar to that reported previously for onion alliin lyase, although there is twice as much tryptophan in the garlic alliin lyase as in the onion enzyme. By both chemical and spectral methods the enzyme was found to have two molecules of pyridoxal 5-phosphate per enzyme molecule, suggesting one per subunit. There are significant differences in the nature of these findings from those previously reported from this laboratory for the onion enzyme. Studies are in progress to compare further the alliin lyases from garlic and onion.     

Purification and Characterization of a Novel (R)-Mandelonitrile Lyase from the Fern Phlebodium aureum

Using high-performance liquid chromatography and nuclear magnetic resonance we identified vicianin as the cyanogenic compound of Phlebodium aureum. The (R)-hydroxynitrile lyase involved during cyanogenesis in the catabolism of the aglycon ([R]-mandelonitrile) was purified to apparent homogeneity. The purified holoenzyme is a homomultimer with subunits of Mr = 20,000. At least three isoforms of the enzyme exist. In contrast to other hydroxynitrile lyases, mandelonitrile lyase (MDL) from P. aureum was not inhibited by sulfhydryl- or hydroxyl-modifying reagents, suggesting a different catalytic mechanism. The enzyme is active over a broad temperature range, with maximum activity between 35 and 50[deg]C, and a pH optimum at 6.5. In contrast to (R)-MDLs isolated from several species of the Rosaceae family, (R)-MDL from P. aureum is not a flavoprotein. The substrate specificity was investigated using immobilized enzyme and diisopropyl ether as solvent. The addition of cyanide to aromatic and heterocyclic carbonyls is catalyzed by this (R)-MDL, whereas aliphatic carbonyls are poorly converted.