Esterase Isozymes Research Articles

Genetic analysis of disease resistance to all strains of BaYMV in a Chinese barley landrace, Mokusekko 3

A Chinese landrace of barley, Mokusekko 3, is unique in being completely resistant against all strains of barley yellow mosaic virus (BaYMV). The present investigation revealed that the resistance of Mokusekko 3 is governed by two recessive genes. As one of the resistance genes was known to be tightly linked with alleles at the Est complex locus, consisting of the Est1, Est2 and Est4 loci for esterase isozymes, each of the resistance genes could be separated by means of marker-assisted selection using an isozyme allelic combination as a marker. One of the resistance genes, ym1, is linked to K (hooded lemma) and gl3 (glossy leaf 3) with recombination values of 25.3% and 9.7% respectively, and these three genes are located in the order K-gl3-ym1 on chromosome 4. Another newly designated resistance gene, ym5, is linked to alleles at the Est complex locus and cu2 (curly growth 2), with recombination values of 1.9% and 19.5% respectively, in the order cu2-Est-ym5 from proximal to distal on the long arm of chromosome 3. The complete resistance of Mokusekko 3 is caused by combining two resistance genes, ym1 and ym5. However, almost all the “resistant” cultivars derived from crosses with Mokusekko 3 are susceptible to the recently detected strain BaYMV-III in Japan, since they contain only one resistance gene, ym5. Marker-assisted selection to combine resistance genes into a cultivar is discussed for the breeding of stabilizing resistance to BaYMV.

Differential expression of esterase and MDH isozymes during in vitro culture in maize (Zea mays L.)

Isozyme patterns of esterase and malate dehydrogenase were analyzed at different stages of in vitro culture of immature embryos and glumes of Zea mays L. viz. explant, callus formation, root formation and shoot formation. Significant changes in isoenzyme patterns of esterase and MDH were observed besides the appearance of specific and new isozymes. Specific fast migrating isozymes were noted in differentiating calli of embryo and glume calli which were absent at other stages suggesting a possible association of these isozyme patterns with in vitro differentiation.

Immunological Detection of Greenbug (Schizaphis graminum) Esterases Associated with Resistance to Organophosphate Insecticides

Organophosphate resistance in the greenbug,Schizaphis graminum,has been at least partially attributed to two distinct patterns of nonspecific esterase isozymes (Type I and II). Antiserum raised against a purified Type II esterase was specific for this enzyme and did not cross-react with the Type I esterase, indicating that the two greenbug esterases are immunologically distinct. The antiserum reacted identically with the Type II esterase in both resistant and susceptible insects based on both immunoblots and quantitative immunoelectrophoresis despite the much higher activity of this isozyme associated with the resistant strain. These results indicate that the isozyme is immunologically indistinguishable in resistant and susceptible insects and that the resistance appears to involve a modification of the enzyme rather than overexpression. These results contrast with those obtained for the Type I esterase, which gave a strong reaction with an antiserum against the E4 esterase associated with resistance in the green peach aphid,Myzus persicae.Western blot analysis indicated that the Type I esterase is present at much higher levels than in the susceptible insects and is likely to involve a mechanism resulting in overexpression of the enzyme.

Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCR‐RFLP analyses

SUMMARYThe early development of indigenous and introduced Scots pine (Pinus sylvestris L.) ectomycorrhiza in natural forest humus was examined using molecular fingerprinting techniques. Non‐mycorrhizal or mycorrhizal (Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) Fr.) seedlings were placed in transparent two‐dimensional microcosms on a thin layer of sieved humus to allow observation of ectomycorrhizal development and fungal growth. Twelve ectomycorrhizal morphotypes, mainly based on colour, gross morphology and outer mantle structure, were identified over a 3‐month period. No successional trends in mycorrhiza formation were observed. For further characterization, individual ectomycorrhiza representing the different morphotypes were subjected to analyses of esterase (EST) isozyme profiles and RFLPs of the internal transcribed spacer (ITS) from fungal rRNA genes following amplification using PCR. All 10 morphotypes analysed displayed different esterase isozyme profiles, and the characteristic s. bovinus species diagnostic band (S. b. EST8) was detected in two white morphotypes. Ten Pink and Black ectomycorrhiza were all separated into one and three groups, respectively. A fast‐running plant‐specific polymorphic locus (Z) was also confirmed in most ectomycorrhizal morphotypes. Diagnostic species‐specific (S. bovinus and Pink) EST bands were detected in intact external mycelium colonizing soil. Successful amplification of the ITS from individual ectomycorrhiza, of eight different morphotypes, was found to be mainly influenced by the DNA template concentration. Different RFLPs (Hinf I, Mbo I and Hha I) of the ITS placed the white morphotypes into two groups, one corresponding to S. bovinus, in agreement with the esterase fingerprinting. By contrast, Pink and Beige morphotypes displayed different EST profiles but nearly identical RFLP fingerprints. The ITS amplification success rate of standardized DNA template concentrations from 10 individual Pink and Black ectomycorrhiza was 90 and 50%, respectively. In all successful amplifications, the black morphotype yielded the smallest ITS fragment, similar to earlier reports for Cenococcum geophilum, that gave identical RFLPs. The ecological significance of the observed mycorrhizal diversity and combined application of these two identification methods is discussed.

The assessment of genetic variation in Spanish primitive cultivars of barley, Hordeum vulgare L., by a combination of isozymes and hordeins

Five isozyme and endosperm reserve protein systems were analysed using electrophoretic techniques in order to investigate the genetic diversity of 222 accessions of Spanish, local varieties of barley, Hordeum vulgare L., maintained at the Centro de Conservacion de Recursos Fitogeneeticos of the I.N.I.A. (Alcala de Henares, Spain). The esterase (EST) isozymes provided more information than did the other systems analysed, showing a total of 14 variable markers. The cathodic peroxidases (CPX) and acid phosphatases (ACPH) were also polymorphic. Malate dehydrogenase (MDH) and glutamate oxaloacetate transaminase (GOT) were monomorphic. The hordeins showed patterns of up to 15 bands, the majority of which were very useful in distinguishing genotypes. 17.2% of accessions showed a uniform genotype, 29.8% showed practically identical genotypes and 53% showed mixtures of different genotypes. It is noteworthy that the use of only two systems (EST and hordeins) and the analysis of only six loci (Est-1, Est-2, Est-4, Hor-1, Hor-2 and Hor-3) is sufficient to reveal the genetic diversity of the collection.

Characterization of the esterase isozymes ofIps typographus (coleoptera, scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I50 of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.

Comparison of different staining methods for analysing esterase (EST) isozyme of fungi and plants

EST isozymes are one of the frequently used biochemical markers in genetic analysis of fungi, and the staining is an important process in electrophoresis analysis of studying Est. The effects were compared among different stain recipes for Est of 3 kinds of fungi-Lentinusedodes,Pleurotus sapidus, Phellinus igriarius and 2 kinds ofplants-Populus sp andBrassica chinensis. Of the four kinds of Est staining recipes tested, the recipe α-acetic acid-naphther showed the best effect. and followed by β-aceticacid-naphther, semicontent α-aceticacid-naphther and α+β-aceticacid-nathpher.

Genetic relatedness of North American populations of Tomicus piniperda (Coleoptera:Scolytidae).

We used DNA fingerprinting by random amplified polymorphic (RAPD) DNA and electrophoretic characterization of esterase isozymes to investigate the genetic relatedness of North American populations of the exotic bark beetle Tomicus piniperda (L.). Cluster analyses of genetic distances among populations identified the Illinois population as an outlier population with mean genetic distances to other populations averaging 0.895 (where complete dissimilarity = 2), compared with genetic distance averages of 0.595 among populations excluding Illinois. When genetic distance means and geographical distance between populations were compared, the results suggested that T. piniperda populations in the United States were established separately in Illinois near Lake Michigan and in Ohio along Lake Erie. Molecular markers indicated that insects derived from the 2 founder groups were interbreeding in contiguous regions in western Indiana.

Identification of the root-knot nematode, Meloidogyne incognita, using monoclonal antibodies raised to non-specific esterases

The esterase isozyme from Meloidogyne incognitawith pI ∼4.95 was purified using a Rotofor electrophoresis system. The purified enzyme was then used to raise monoclonal antibodies. Hybridoma cell lines producing antibodies were cloned and screened against the purified esterase in an enzyme-linked immunosorbent assay (ELISA). A selected monoclonal antibody was then tested by western blot analysis of crude soluble protein extracts of all the major species of Meloidogyneseparated by native and SDS-PAGE electrophoresis. Although the antibody was cross-reactive in western blots of denatured proteins in the presence of SDS and in ELISA, it was not cross-reactive in western blots of native protein. The results demonstrate that a monoclonal antibody to a diagnostic non-specific esterase enables M. incognitato be discriminated from M. javanicawithout the need to separate the esterases by electrophoresis first. This is an approach which could be adopted in a number of situations where organisms can be identified by a diagnostic protein which has already been identified by electrophoresis.

Expression of isoenzymes altered by both Glomus intraradices colonization and formononetin application in corn ( Zea mays L.) roots

We searched for and found alterations in isoenzymes that were characteristic of the establishment of the Zea mays-Glomus intraradices symbiosis. Isoenzyme activities for NAD-dependent malate dehydrogenase, esterase, and total peroxidase were examined in corn ( Zea mays L. cv. Great Lakes-hybrid 582) roots or mycorrhizas during the first 3 weeks of growth. Soil was either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith or was left uninoculated. Plants were also grown in the presence or absence of formononetin. At 2 weeks of growth, there was an 80% relative increase in colonization in plants that received both formononetin and Glomus as compared to plants inoculated with the mycorrhizal fungus alone. One of the malate dehydrogenase isozyme was differentially expressed in VAM roots at 3 weeks. The activity of this isozyme was further enhanced in the presence of formononetin. At 2 and 3 weeks, four esterase isozymes showed higher activity in mycorrhizas than in non-mycorrhizal roots. Formononetin enhanced the activities of these four esterases following fungal colonization. At 2 weeks of growth, mycorrhizal roots had the highest total peroxidase activity compared to formononetin-treated or control roots. In the presence of formononetin, peroxidase activity was reduced below the amounts of untreated controls. According to these results, the differential expression of certain malate dehydrogenase and esterase isozymes, and the change in total peroxidase activity indicate very early physiological alterations in roots during the establishment of a VAM symbiosis. Furthermore, by examining the shifts in isoenzyme activity, the effect of exogenously-applied formononetin on root colonization could be detected at the earliest stages of the Zea-Glomus symbiosis.

Proteins and esterase isozymes during developmental stages of organophosphate resistant strains in Anopheles stephensi liston

The proteins and esterase isozymes from Anopheles stephensi were analysed by polyacrylamide gel electrophoresis. Changes in proteins and esterase isozyme patterns during developmental stages were studied. A few specific protein bands were present especially in the third instar larval stage. In most strains, eggs showed only one esterase band. During the larval development many bands were gradually formed and most anodal bands were lost upon pupation. The adults showed differences in intensity and mobility exhibiting sexual dimorphism. The protein and esterase isozyme patterns of organophosphate (fenthion, methyl parathion and malathion) resistant and susceptible strains during developmental stages were compared. The zymogram and relative mobility showed variation in the number of intensity of proteins and esterase bands in the resistant strains as compared to the susceptible strains.

Variation of leaf esterases in some Ethiopian tetraploid wheat landraces

Esterase isozymes of tetraploid wheat landraces originating from four localities in the central highlands of Ethiopia were studied using the horizontal starch gel electrophoresis. The landraces studied consisted of 60 Triticum turgidum L., one T. dicoccon Schrank, one T. polonicum L. and an improved local cultivar. One introduced durum cultivar was also included for comparison.

Electrophoretic Evaluation of Esterase Isozymes from Turfgrass Seed Blends and Mixtures

Turfgrass seeds are commonly blended to improve performance. However, once physically blended, no fast, reliable technique is available to identify the cultivars and the proportion of each cultivar in the blend. The objective of this study was to isolate specific species and cultivars in seed blends or mixtures through examination of esterase banding patterns on electrophoretic gels, and to compare band intensities unique to each cultivar or species when blended in increasing or decreasing proportions. Two cultivars of Kentucky bluegrass (Poa pratensis L.) were blended by weight in proportions ranging from 0 to 100%. Protein extracted, separated on isoelectric focusing gels (pH gradient 3 to 9) and stained for esterase activity. Mixtures of fine fescue (Festuca spp.) species only and mixtures of both fine fescue species and Kentucky bluegrass cultivars were prepared in the same manner. Gels were visually analyzed for the presence of characteristic bands unique to each cultivar or species and for intensities of characteristic bands when cultivar proportions were varied within a seed blend or mixture. The results indicated that visual discriminations could be made between cultivars and species in turfgrass blends or mixtures and that bands unique to a cultivar or species varied in intensity with their concentration in a blend or mixture. These findings have importance to seed companies and consumers interested in monitoring the composition of seed blends and mixtures subsequent to physical mixing.

Differences in esterase isozymes between Panonychus citri populations infesting citrus and Osmanthus

Gene frequencies were investigated in the α-Est1 locus between Japanese populations of Panonychus citri occurring on some fruit trees and on the garden trees, Osmanthus trees and Ilex crenata. A new allele, A3, was found in the α-Est1 of populations collected on Osmanthus trees. Populations on I. crenata, Citrus unshiu and Pyrus serotina had one or both A1 and A2 alleles. However, the populations on Osmanthus trees had only the A3 allele and did not vary geographically.

Genomic relationships between Dasypyrum villosum (L.) Candargy and D. hordeaceum (Cosson et Durieu) Candargy

The origin and genomic constitution of the tetraploid perennial species Dasypyrum hordeaceum (2n = 4x = 28) and its phylogenetic relationships with the annual diploid Dasypyrum villosum (2n = 2x = 14) have been investigated by comparing the two genomes using different methods. There is no apparent homology between the conventional or Giemsa C-banded karyotypes of the two Dasypyrum species, nor can the karyotype of D. hordeaceum be split up into two similar sets. Polymorphism within several chromosome pairs was observed in both karyotypes. Cytophotometric determinations of the Feulgen-DNA absorptions showed that the genome size of D. hordeaceum was twice as large as that of D. villosum. Both the cross D. villosum x D. hordeaceum (crossability rate 12.1%) and the reciprocal cross (crossability rate 50.7%) produced plump seeds. Only those from the former cross germinated, producing sterile plants with a phenotype that was intermediate between those of the parents. In these hybrids (2n = 21), an average of 13.77 chromosomes per cell paired at meiotic metaphase I. Trivalents were only rarely observed. Through dot-blot hybridizations, a highly repeated DNA sequence of D. villosum was found not to be represented in the genome of D. hordeaceum. By contrast, very similar restriction patterns were observed when a low-repeated DNA sequence or different single-copy sequences of D. villosum or two sequences in the plastidial DNA of rice were hybridized to Southern blots of the genomic DNAs of the two Dasypyrum species digested with different restriction endonucleases. By analyzing glutamic-oxaloacetic-transaminase, superoxide dismutase, alcohol dehydrogenase, and esterase isozyme systems, it was shown that both Dasypyrum species shared the same phenotypes, which differed from those found in hexaploid wheat. In situ hybridizations using DNA sequences encoding gliadins showed that these genes were located close to the centromere of three pairs of D. villosum chromosomes and that they had the same locations in six pairs of D. hordeaceum chromosomes. We conclude that the autoploid origin of D. hordeaceum from D. villosum, which cannot be defended on the basis of chromosomal traits, is suggested by the other findings obtained by comparing the two genomes. Key words : Dasypyrum hordeaceum, Dasypyrum villosum, phylogenetic relationships.

イネ寄生およびマコモ寄生ニカメイガの交尾時刻の違いについて

The Rice Stem Borer, Chilo suppressalis (WALKER), is a serious pest of rice plants in Asia. In Japan, host plants of are mainly rice plants (Oryza sativa) and the water-oat (Zizania latifolia). Our previous study indicated significant differences in insecticide susceptibility and esterase isozyme patterns between the rice-feeding and water-oat-feeding strains. In the present study, the mating time of the two strains was investigated in the laboratory. The mating time was significantly different. The peak of mating time of the rice-feeder was 5h earlier than that of the water-oat-feeder, suggesting that the two strains are reproductively isolated.

Intraspecific somatic and sexual hybridisation between dihaploid lines of Solanum tuberosum L.: evaluation of morphological traits and resistance to late blight Phytophthora infestans (Mont.) de Bary in the foliage

Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.

Characteristics of Cold-Preserved Embryogenic Suspension Cells in Fennel, Foeniculum vulgare Miller

An embryogenic suspension cell line of fennel, Foeniculum vulgare Miller, directly induced from hypocotyl, was developed. The suspension cells were cold-preserved at 4 °C for up to 12 weeks and then cultured at 25 °C for 2 weeks. The packed cell volume of the cultured cells decreased as the cold-preservation period became longer. The cells, with a size of 32-82μm and having embryogenic potential, diminished suddenly with the passage of the cold-preservation period. The cells cold-preserved for 2 to 6 weeks were capable of forming normal somatic embryos that were identical to those obtained from the cells without cold-preservation. The resulting somatic embryos regenerated to develop into normal plantlets when cultured in hormone-free Murashige and Skoog medium under illumination. Esterase isozyme analysis on polyacrylamide gel electrophoresis revealed the appearance of different bands in the embryos treated with 4-week and 6-week cold-preservation, in contrast to the embryos from the cells without cold-preservation. Anethole was detected in the methanol extracts of embryos induced from the cold-preserved cells at the same level as that of embryos induced from the cells without cold-preservation.

Purification and characterization of an esterase isozyme from insecticide resistant and susceptible strains of german cockroach, Blattella germanica (L.)

The most active forms of esterases (E5, E6 and E7) from the German cockroach, Blattella germanica (L.) were purified from resistant and susceptible strains. About 45–155 fold purification with a 11–16% of total esterase recovery was achieved after different column chromatography and preparative gel electrophoresis. Elution profiles of resistant and susceptible strains were similar, but esterase E6 activity was higher in the resistant strains. Kinetic analyses indicate no differences in K m values between the resistant and susceptible strains. However V max was significantly higher in resistant strains. Inhibition of esterase activity by paraoxon, chlorpyrifos and propoxur did not suggest any structural differences in esterase E6 between strains. From these results we suggest that insecticide resistance in German cockroach is due to the increased production of E6 esterase. The role of E6 may be sequestration of toxic molecules rather than hydrolysis.

Changes of peroxidase, esterase isozyme activities and some cell inclusions in regenerated vascular tissues after girdling inBroussonetia papyrifera (L.) Vent

The isozyme zymogram of peroxidase and esterase, and some cell inclusion contents were changed with the differentiation of regenerated vascular stem tissues after girdling inBroussnetia papyrifera (L.). Vent. Presence or disappearance of some peroxidase and esterase isozyme bands was related to wounding. Some isoperoxidase bands disappeared at the time of vascular tissue formation, but some esterase isozyme bands appeared in phloem or cambial regions as sieve-like elements or mature xylem were formed. The inclusion grains progressively disappeared with the formation of callus and initiation of vascular meristems. The cell inclusions reappeared during the formation of regenrated vascular tissues. Histochemical study indicated that the inclusion grains could be a complex compound of a protein mass encircling polysaccharide in the center with a proteinous nucleus.