Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.     

In vitro response as a reflection of genomic diversity in long-term cultures of Musa

Summary Ten commercial cultivars of Musa representing five different types of genomic constitutions were studied for in vitro multiplication through meristem culture. In addition, the effects of various genomic constitutions at different ploidy levels on growth and meristem proliferation in long-term cultures were analysed statistically. Plantlets were readily obtained by culturing the excised meristems on MS semisolid medium supplemented with IAA, IBA and BAP at various concentrations. The regenerative potential of all cultivars of Musa, irrespective of their genomes, remained unaffected in long-term culture, even after 28–30 months. The genomic influence on both the nature and rate of proliferative growth was evident. Statistical analysis revealed that the rates of meristem proliferation between different cultivars of the same passage and between different passages of the same cultivar were significantly different. Those cultivars having only an A genome showed a low rate of meristem proliferation, while under the same culture conditions, cultivars having one or two B genomes in addition to the A exhibited a very high rate.     

Genetic variation in natural populations of the cereal cyst nematode (Heterodera avenae Woll.) submitted to resistant and susceptible cultivars of cereals

The purpose of the present work was to study the genetic characteristics of cereal cyst nematode (Heterodera avenae) populations re-established after the long-term use of resistant oat cultivars in field conditions. Population features were analyzed through fitness components and variation in enzymatic polymorphism (esterase and malate dehydrogenase loci). The longest (6 year) use of the same resistance genes (oat cv Panema) at high frequency (Rotation IB) led to the selection of a resistance-breaking pathotype and to a decrease in viability which suggested either a founder effect or a lower reproductive potential for the new pathotype. Analysis of esterase allelic frequencies led to the conclusions that: (1) the genetic constitution of this pathotype was different from the reference population maintained on the susceptible host (oat cv Peniarth), and (2) that the esterase locus may develop a disequilibrium linkage with loci involved in virulence. Random mating was recorded at the whole-field level but not always at the single-plant level, suggesting that cultivation practices such as annual ploughing could play a major role in homogenizing subpopulations developed in the vicinity of a plant. These phenomena showed that the long-term use of highly effective resistance could provok marked genetic modifications in populations. These risks should be taken into account when deciding strategies for optimal use of resistance genes in nematode management programs.

---

     

Extent of genetic variability of endosperm esterases in Triticum aestivum L. 2n=6x=42

Summary Genetic variability of endosperm esterase has been studied in 42 cultivars of Triticum aestivum L. 2n=6x=42. Different techniques, including sequential electrophoresis and electrofocusing, have been used with various substrates and esterase inhibitors. The electrophoretic patterns in each cultivar are described. Chromosomal location using the nullitetrasomic and ditelosomic lines of Chinese Spring was carried out in order to relate and/or locate the esterase genes to specific chromosomes. Most of the esterase isozymes located were in the long arm of the chromosomes of the homoeology group 3; but we have found six located in the short arms, five of them in the chromosome 3AS and one in the 3DS. This location increases the number of esterase genes described, because no esterase genes had been described so far in short arms of chromosomes of the homoeology group 3. The genetic control is discussed and, according to our results, between 12 and 15 loci, organized in five compound loci, control the endosperm esterases in wheat. Also one modifier gene modifying the mobility of two esterase bands and present in all the cultivars studied is postulated.This work was supported by a personal grant (L. Rebordinos) from the P.F.P.I. and by an institutional grant from the C.A.I.C.Y.T. (PB85-0153)     

Soluble Proteins, Esterases and Superoxide Dismutase in Stem Tissue of Pepper Plants in Relation to Age-related Resistance to Phytophthora capsici

Patterns of soluble proteins and isoenzymes of esterase and superoxide dismutase were investigated in healthy and infected stems of two pepper cultivars resistant and susceptible to Phytophthora capsici. By the use of two-dimensional SDS-polyacrylamide gel electrophoresis, it was possible to compare precisely the cultivars Hanbyul and Kingkun susceptible or resistant to P. capsici with respect to their protein patterns. The two-dimensional electrophoresis identified three proteins (25—27 kD) from the healthy stem extracts of Kingkun, which were absent in Hanbyul. Some particular proteins appeared in pepper stems of both cultivars at later developmental stage of plants, suggesting their role in the expression of age-related resistance. Some proteins which were not detectable in the healthy stem extracts also existed in large amounts in the diseased ones. By contrast, other proteins present in the healthy stems disappeared from the diseased stems. Some esterase isoenzymes appeared in the two cultivars only at late developmental stage. Other esterase isoenzymes were produced only in the diseased stems. There were no differences in the patterns of superoxide dismutases between the cultivars and also between developmental stages. Large activities of several superoxide dismutases were detected in the diseased stems.     

Protein complex and esterase isoenzyme patterns ofAllium sativum L. cultivars and clones-regenerants

Protein complex patterns of cloves and esterase isoenzyme patterns of apical buds of cloves were studied with Czechoslovak virus-free cultivars ofAllium sativum L. and the wild speciesA. longicuspis Regel, Similarly, four clones-regenerants obtained using explant culture techniques from A.sativum L. cv. Bzenecky paličák (two somaclones and two clones derived from plants regenerated from meristem cultures treatedin vitro with colchicine) differing in their ploidy, morphology, and yields were studied. Immunophoreograms of protein complexes of theA. sativum L. cultivars under investigation differed from one another in the number and mobility of protein fractions in both the cathodic and anodic regions and thus these cultivars can be distinguished. On the basis of esterase isoenzyme patterns, the Czechoslovak cultivars of A. sativum L. can be arranged into three groups - bolting winter cultivars with broad leaves, non-bolting winter cultivars with broad leaves, and non-bolting spring cultivars with narrow leaves. All the clones-regenerants showed the same protein complex and esterase isoenzyme patterns as their original cultivar.A. longicuspis Regel markedly differed in its protein complex and esterase isoenzyme patterns from all the other genotypes studied.Received May 17, 1989: accepted January 5,1990     

Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization

The lipA gene encoding a thermostable esterase was cloned from Thermoanaerobacter tengcongensis and overexpressed in Escherichia coli. The recombinant esterase, with a molecular mass of approx. 43 kDa determined by SDS-PAGE, was purified to homogeneity through Sephadex G-100 gel filtration. The purified enzyme actively hydrolyzed tributyrin but not olive oil. Maximum activity was observed on p-nitrophenyl (NP)-propionate (C3) and p-NP-butyrate (C4), with little activity towards p-NP-palmitate (C16). The esterase was optimally active at 70 °C (over 15 min) and at pH 9. It is highly thermostable, with a residual activity greater than 80% after incubation at 50 °C for more than 10 h. The activity was not inhibited by 5 mM EDTA and PMSF, indicating the esterase is not a metalloenzyme and may contain a specific structure around the catalytic serine residue. In addition, it was stable for 1 h at 37 °C in 1% CHAPS and Triton X-100 but not stable in 1% Tween 20 or SDS.     

Identification of Flax and Linseed Cultivars by Isozyme Markers

A set of 28 fibre flax and linseed cultivars differing in plant morphology and technological parameters were analysed by isozyme markers in five ontogenetic phases. Relatively high isozyme polymorphism was observed using polyacrylamide gel electrophoresis. Altogether 18 isozyme systems produced 145 different bands; 66 of them (45.52 %) have been found to be polymorphic. The highest level of polymorphism was found in acid phosphatase and esterase, polymorphism was detected in aconitase, diaphorase, glutamate dehydrogenase, peroxidase and superoxide dismutase as well. The highest number of unique isozymic spectra (cultivar × enzyme × ontogenetic phase) was detected in the phase of shoot with removed cotyledons. Electrophoretic analysis of all polymorphic isozymes enabled to distinguish 20 cultivars (71 %) in the screened cultivar set.     

Genetic variability of leaf esterases in Triticum aestivum L. 2n=6x=42

Summary Starch gel electrophoresis with two different buffer systems and several substrates and inhibitors have been used to study the electrophoretic variability of esterases in leaves of cultivars of Triticum aestivum. Each one of the buffer systems showed different levels of variability, according to the electrophoretic patterns. At the same time green and etiolated leaves showed different patterns in each buffer system. The variability was dependent upon the developmental stage of the leaves. According to the results from chromosomal location, the genes controlling esterases in green leaves were located in homoeology group 3, while the genes controlling esterases in etiolated leaves were in homoeology group 6. But both esterase isozymes showed a similar electrophoretic migration and a similar reponse to substrates and inhibitors. The possible origin of both sets of genes due to an interchromosomal duplication is discussed.     

Influence of phosphorus level on VA Mycorrhizal colonization and growth of cowpea cultivars

Vesicular-arbuscular mycorrhizal (VAM) associations often vary according to the abundance of available soil phosphorus (P). Therefore, understanding the response of crop plants to colonization by VAM fungi necessitates the study of the response of colonized and noncolonized plants, from a range of cultivars, to differing levels of P. Cowpea is grown throughout the world, often on impoverished soils in which it can benefit from formation of mycorrhizae. The present study was conducted to determine the response of four cultivars of cowpea (Vigna unguiculata (L.) Walp.), varying in nitrogen fixation capacity, to inoculation withGlomus fasciculatum at four levels of added P in the rooting medium. In a greenhouse experiment, four cowpea cultivars, Mississippi Silver, Brown Crowder, Six Week Browneye and MI 35, were grown with and without the mycorrhizal fungus at four levels of added P, 0, 10, 20 and 30 ppm. Root colonization (%) was negatively correlated with P content of the growth medium and shoot P concentration. Intraspecific variability was shown for shoot dry weight and leaf area in response to inoculation withG. fasciculatum at different P levels. The range of P required in the growth medium which allowed benefit fromG. fasciculatum was identified for individual cultivars using shoot dry weight and leaf area, and collectively across cultivars for other parameters.     

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

Summary Immature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as aged callus. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.Florida Agricultural Experiment Station Journal Series No. R-00494     

Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6

Esterase 6 fromDrosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directedin vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for β-naphthyl esters andp-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb’s free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.     

Effects of growth regulators and incubation period on in vitro regeneration of adventitious shoots from gerbera petioles

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pseudogamous embryogenesis and the degree of parthenogenesis inAllium tuberosum

Summary Early stages of egg embryony and antipodal embryony in ovules from both pollinated and unpollinated flowers ofAllium tuberosum, a diplosporous apomict, were observed. Whether flowers were pollinated or not, autonomous egg and antipodal embryonies occurred at nearly equal frequencies and progressed almost synchronously for several days. An electrophoretic analysis of endosperm esterase demonstrated the fertilization of polar nuclei. It was thus confirmed thatA. tuberosum is a pseudogamous apomict. The degree of parthenogenesis, calculated as the percentage of ovules in which egg cells showed autonomous embryonic development, ranged between 62% and 94% among six cultivars and was nearly equal to the degree of diplospory previously reported. The present results show that most unreduced egg cells and egg-like antipodal cells ofA. tuberosum have the ability to start embryogenesis independently of pollination.     

Screening ofExiguobacterium acetylicum from soil samples showing enantioselective and alkalotolerant esterase activity

About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed hgh apparent enantioselectivity (E _app>100) for (R)-2PB-O-res and were identified asExiguobacterium acetylicum. The JH13 strain showed high esterase activity onp-nitrophenyl acetate (pNPA), but showed low lipase activity onp-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.     

Isozyme Polymorphism of Esterases in the Genus Lanistes (Mollusca: Prosobranchiata) and Genetic Analysis of Populations

Esterases from the digestive gland of the snails Lanistes carinatusand Lanistes boltenicollected from four Egyptian governorates were extracted and analyzed using starch gel electrophoresis and five substrates. Twelve esterase bands were detected in both Lanistes species. The esterase bands were distributed in three main zones, which could be classified as acetylesterases, carboxylesterases, and cholinesterases. Depending on the substrate specificity, inhibition properties, and relative mobility of esterase bands, the three zones of esterase activity could be traced to eight genetic loci. Locality-specific loci were found. Inter- and intrapopulation variations are discussed. There is an absence of equilibria at all esterase loci in all populations studied, and a high proportion of genetic diversity in different esterase loci. The absence of interspecific variations proves that Lanistessnails in Egypt belong to one species.     

HPLC separation of enterobactin and linear 2,3-dihydroxybenzoylserine derivatives: a study on mutants ofEscherichia coli defective in regulation (fur), esterase (fes) and transport (fepA)

Reversed-phase HPLC separation of enterobactin and its 2,3-dihydroxybenzoylserine derivatives was used for a comparative analysis of mutants of Escherichia coli, defective in the regulation of enterobactin biosynthesis (fur), enterobactin transport (fepA) and enterobactin esterase (fes). A complete separation of all 2,3-dihydroxybenzoylserine compounds was achieved: the monomer (DHBS), the linear dimer (DHBS)₂ and trimer (DHBS)₃, the cyclic trimer, enterobactin, as well as 2,3-dihydroxybenzoic acid. The production of all these compounds was followed after ethylacetate extraction from acidified culture fluids. Enterobactin was found to be the predominant product in all mutant strains. The mutant strains behaved differently with regard to the breakdown products. All degradation products, such as DHBS, (DHBS)₂ and (DHBS)₃, were detected in the overproducing fur mutant where both transport and esterase are still functioning, while only the monomer, DHBS, was detected in the fepA mutant and no degradation was found in the esterase-deficient fes mutant. From the pattern of breakdown products it may be inferred that the esterase acts in two different ways, depending on whether transport is functioning or not. Thus, esterolytic cleavage of ferric enterobactin after entering the cells results in a mixture of all three hydrolysis products, i.e. DHBS, (DHBS)₂ and (DHBS)₃, while cleavage of iron-free enterobactin subsequent to its biosynthesis yields only the monomer. Thus, the results of quantitative HPLC analysis of enterobactin and its breakdown products show that different enterobactin esterase products arise, depending on whether iron is bound to enterobactin or not.     

Genetics of esterase isoenzymes in Malus

Summary Three main zones of esterase activity (EST-I, EST-III, EST-IV) identified in leaf extracts of cultivated apple and Malus species were determined by the genes EST-1, EST-3 and EST-4, respectively. In addition to earlier reported alleles of EST-1 (a, b) three further bands c, d and f were identified in the EST-I zone of which c was found to be determined by an allele, c. Two alleles, a, b, and a null allele were found for both the genes EST-3 and EST-4. Differences in allelic frequency were observed between cultivars, rootstocks and Malus species. Allele EST-1a was rare amongst the rootstocks. The examination of Malus species and derivatives showed a geographical relationship. Allele EST-1c was confined to species of Asian origin, and EST-1d was confined to American species.     

Esterase D and carbonic anhydrase 2 in a natural population ofCebus apella from Brazil

The electrophoretic patterns of esterase D (ESD; E.C.3.1.1.1) and carbonic anhydrase 2 (CA 2, E.C.4.2.1.1) were studied in 147 specimens ofCebus apella. Three phenotypes were detected at the esterase D system,ESD ¹ allele showing a frequency of 44%, markedly different from those observed in Old World monkeys. CA2 also proved to be polymorphic, with three alleles being detected at the following frequencies:CA2 ¹, 98%;CA2 ² andCA2 ³, both 1%. The CA2 activity was absent in newborn animals and in fetuses.     

Production and characterization of esterase and lipase from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis>

The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h⁻¹ and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l⁻¹ using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature, pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal values for growth rate and productions of esterase and lipase: 0.086 h⁻¹ (at 42.5°C, pH 7.4, and 3.6 mol l⁻¹ NaCl), 2.3 U l⁻¹ (at 50°C, pH 7.5, and 4.3 mol l⁻¹ NaCl), and 0.58 U l⁻¹ (at 50°C, pH 7.6, and 4.5 mol l⁻¹ NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l⁻¹ NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained 50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the first report evidencing the synthesis of esterase and lipase by H. marismortui.