supernatant was carefully collected using a 100-μL pipette and stored at –80 °C until isoelectric focusing (IEF). Gel preparation. A 0.5-mm thick gel was used, which required a total volume of 25 mL of gel solution (6.25 mL acrylamide/bis solution
Mason T. MacDonald, Rajasekaran R. Lada, A. Robin Robinson, and Jeff Hoyle
L. Mark Lagrimini, Jill Vaughn, John Finer, Karen Klotz, and Patrick Rubaihayo
Tomato plants (Lycopersicon esculentum cv. OH 7814) were transformed via Agrobacterium tumefaciens with a chimeric tobacco anionic peroxidase (EC 188.8.131.52) gene joined to the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants obtained by selection on kanamycin were found to have more than five times the total leaf peroxidase activity of control plants. Transformed tomato plants chronically wilted upon reaching sexual maturity. Two independently selected transformants were self-fertilized, and progeny were obtained that were homozygous for the foreign gene. Isoelectric focusing gels stained for peroxidase activity revealed a new tomato leaf peroxidase isoenzyme with a pI of 3.75, which is similar to that seen in Nicotiana sylvestris L. Mature tomato fruit were found to have up to 1600-fold higher peroxidase activity in transformants expressing the tobacco anionic peroxidase (TobAnPOD) than control plants. Tissue blots showed the tobacco enzyme evenly distributed throughout the tomato fruit tissue. Progeny plants possessing the tobacco peroxidase gene (now homozygous) showed stunting, and fruit size was reduced by >80%. However, fruit set was normal and the rate of ripening was not altered from control plants. Fruit from transformed plants were found to have normal pigmentation, but the soluble solids concentration was 400% higher than in control tomato fruit. This result was predicted from the peroxidase-induced water stress. Possible roles for the tobacco anionic peroxidase in growth, development, and stress resistance are discussed.
Hongwen Huang, Fenny Dane, and Joseph D. Norton
Allozyme polymorphism in chestnut (Castanea) species was investigated using isoelectric focusing in thin-layer polyacrylamide slab gels. Genetic analysis of the progenies of intraspecific crosses and interspecific F2s and backcrosses (BC1s) allowed the verification of 11 polymorphic isozyme loci from 11 enzyme systems. The following loci were defined: Acp, Adh, Est-1, Est-2, Est-5, Me, Prx-1, Prx-2, Prx-3, Skd-3, and Skd-4. All polymorphic loci behaved as single-locus Mendelian genes. Skd showed unique species specificity. Skd-1 and Skd-2 were unique to the American chestnut (C. dentata Borkh.) and the European chestnut (C. sativa Mill.), whereas Skd-3 and Skd-4 were unique to the Chinese chestnut (C. mollissima Bl.) and the Japanese chestnut (C. crenata Sieb.). Linkage analysis revealed linkage for three pairs of loci: Skd-3/Skd-4, Est-1/Est-2, and Est-5/Prx-1. The single-tree progeny method was used successfully for isozyme genetic analysis. Forty-seven chestnut cultivars in six chestnut species were characterized using 12 isozyme loci and can be unambiguously identified by 12 multi-locus genotypes. The interspecific and geographic relationships among species were also discussed.
Hongwen Huang, Desmond R. Layne, and R. Neal Peterson
The utility of isozyme phenotypes for identifying and determining genetic variation in pawpaw cultivars was studied using isoelectric focusing in thin-layer polyacrylamide gels. Based on a sample of 32 clones (cultivars and advanced selections) and 23 enzyme systems, 7 enzymes were found to be polymorphic, involving 9 polymorphic loci [acid phosphatase (ACP), dihydrolipoamide dehydrogenase (DDH), malic enzyme (ME), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), peroxidase (PRX), and shikimate dehydrogenase (SKD)]. Altogether these 9 loci and 32 clones yielded 28 multi-locus isozymic phenotypes useful for cultivar identification; 24 of the 32 clones were uniquely identified. The allozyme variation in these clones has the average of other long-lived woody perennials of widespread geographic range in temperate regions with insect-pollinated outcrossing breeding systems, secondary asexual reproduction, and animal-dispersed seed. Genetic differentiation among these pawpaw clones, measured by Nei's distance, D, was substantial: 496 pairwise comparisons of genetic distance among the 32 clones indicated that they differed on average of D = 0.068 ± 0.04 and ranged from 0 to 0.188. Cluster analysis (UPGMA) produced a most likely division of the 32 clones into 7 groups; however, these groups did not conform to known pedigree relations. Additional polymorphic enzymes are needed for accurate allozyme-based genetic discrimination.
Hongwen Huang, Desmond R. Layne, and Don E. Riemenschneider
As a new National Clonal Germplasm Repository for Asimina species at Kentucky State University (KSU), of major concern to us is the genetic variation within our germplasm collection. The present study investigated the extent of genetic diversity for the pawpaw germplasm in our collection and the geographical pattern of genetic diversity among populations using isozyme markers. Allozyme diversity was high in Asimina triloba (L.) Dunal (Annonaceae) collected from all nine different states, as is typical for temperate woody perennial, widespread and outcrossing plant species. Averaged across populations, mean number of alleles per locus (A), percent polymorphic loci (P), effective number of alleles per locus (Ae), and expected heterozygosity (He) were 1.54, 43.5, 1.209, and 0.172, respectively. Significant deviations from Hardy-Weinberg equilibrium were found in nine populations at an average of 4.8 loci. Observed heterozygosity was higher than expected. Partitioning of genetic diversity showed that 88.2% resided within populations. The proportion of genetic diversity among populations (Gst = 0.118; FST = 0.085) was either lower than or within the range of those species with similar ecological and life-history traits. The mean genetic identity among populations was high (I = 0.988). An analysis using UPGMA clustered most populations as one major group, with the southernmost (Georgia) and the westernmost (Illinois) populations readily separated from the main group. The relationships discovered by principal component analysis (PCA) were similar to those revealed by UPGMA. In addition, PCA separated the northernmost population (New York) from the major group. Sampling strategies for future germplasm collection of A. triloba are also discussed.
T. Gregory McCollum, Hamed Doostdar, M. Bausher, Richard T. Mayer, and Roy E. McDonald
Polygalacturonase-inhibiting proteins (PGIPs) are believed to be one component of plants inherent defense mechanisms against fungal pathogens. We have purified a PGIP from mature grapefruit (Citrus paradisi cv. Marsh) flavedo using ammonium sulfate precipitation, preparative isoelectric focusing and ion exchange chromatography. Two peaks of PGIP activity were separated by isoelectric focusing, one at pH 6–7 and one at pH 9–10. The basic protein was more abundant than the neutral protein and was selected for further purification. The basic protein binds to S Sepharose at pH 6.1 and has an apparent Mr of ≈43,000 based on SDS-PAGE analysis. The protein is glycosylated as revealed by binding to ConA sepharose and is serologically similar to PGIPs from bean hypocotyl and pear fruit. Two dimensional PAGE analysis revealed the presence of two bands of similar Mr but with slightly different pIs (≈9.0–9.5). The N-terminal amino acid sequence of grapefruit PGIP shows high homology with PGIPs from fruit of other species and with a cDNA clone of PGIP that was isolated from a Citrus sinensis cv. Hamlin expression library. Grapefruit PGIP inhibits polygalacturonases from Aspergillus niger, and the citrus pathogen Penicillium italicum. We are interested in the role of PGIP in resistance of citrus fruit to postharvest decay fungi.
William C. Mitchell and Gojko Jelenkovic
The NAD-dependent and NADP-dependent alcohol dehydrogenase activities of strawberries (Fragaria xananassa Duch.) were found to have broad substrate specificities including those alcohols and aldehydes responsible for strawberry aroma and flavor either directly or through their ester products. NAD-dependent activities were greatest against short-chained alcohols, whereas the NADP-dependent activities were most active against aromatic and terpene alcohols. Differences were seen in substrate specificity between receptacle and achene alcohol dehydrogenase activities. Alcohol dehydrogenase activities were found to be developmentally regulated in receptacle tissue and increased during the period of fruit maturation and ripening. Isoelectric focusing of NAD-dependent ADH activities showed that several isozymes of this enzyme exist, that they differ between receptacle and achene tissues, and that they vary among specific genotypes. Our results suggest that NAD- and NADP-dependent ADH activities are integral components of flavor and fragrance volatile production in ripening strawberries.
Nicole A. Cardwell and Gary L. McDaniel
The pathogenesis-related protein, chitinase, is implicated in the resistance mechanisms involved in dogwood anthracnose, which is caused by Discula destructiva. Chitinase isozymes were isolated from Discula-inoculated Cornus mas, a highly resistant species, and from inoculated C. florida, a highly susceptible species. Chitinase activity was identified in C. mas on days 2-12 following inoculation, but was expressed only on day 8 following inoculation in C. florida. Both dogwood species expressed a constitutive chitinase level in noninoculated control leaves, but Discula-inoculated leaves of C. mas contained three chitinase isozymes, whereas C. florida leaves expressed only two. Molecular masses of isozymes were 21, 32, and 35 kDa for C. mas and 21 and 35 kDa for C. florida. Isoelectric focusing demonstrated three chitinase isozyme isoelectric points for C. mas (pI = 5.6, 6.8, and 8.9), but only two for C. florida (pI = 5.6 and 6.8). These differences in synthesized isozymes and rate of accumulation suggest that chitinase may have a role in the defense of dogwood against D. destructiva infection.
Attila Hegedüs, Zoltán Szabó, József Nyéki, Júlia Halász, and Andrzej Pedryc
The most commercially grown peach [Prunus persica (L.) Batsch.] cultivars do not require cross-pollination for reasonable fruit set; however, self-incompatibility is a well-known feature within the Prunoideae subfamily. Isoelectric focusing and native polyacrylamide gel electrophoresis of S-ribonucleases; PCR analyses of S-RNase and S-haplotype-specific F-box genes as well as DNA sequencing were carried out to survey the self-(in)compatibility allele pool and to uncover the nature of self-compatibility in peach. From 25 cultivars and hybrids with considerable diversity in phenotype and origin, only two S-haplotypes were detected. Allele identity could be checked by exact length determination of the PCR-amplified fragments and/or partial sequencing of the peach S 1-, S 2-, and Prunus davidiana (Carr.) Franch. S 1-RNases. S-RNases of peach were detected to possess ribonuclease activity, and a single nucleotide polymorphism in the S 1-RNase was shown, which represents a synonymous substitution and does not change the amino acid present at the position in the protein. A 700-bp fragment of the peach SFB gene was PCR-amplified, which is similar to the fragment size of functional Prunus L. SFBs. All data obtained in this study may support the contribution of genes outside the S-locus to the self-compatible phenotype of peaches.
T.G. McCollum and R.E. McDonald
Grapefruit (Citrus paradisi) flavedo is a rich source of peroxidase (POD) (EC 184.108.40.206). Changes in POD have been related to senesence and environmental stress in a variety of plant tissues. However, due to the large number of POD isoenzymes as well as the broad substrate specificity, measurement of POD activity in crude extracts is of limited value for gaining an understanding of the role of POD in vivo. We have begun to purify and characterize POD isoenzymes from grapefruit flavedo. HPLC gel permeation chromatography reveals 2 peaks of POD activity with apparent MW of 66 kD and 30 kD. Native PAGE (8% bis-acrylamide, pH 8.8) followed by activity staining indicates that the PODs differ in Pi; the 30 kD POD migrates anodally, whereas the 66 kD POD does not migrate. Isoelectric focusing has been used to separate flavedo PODs into acid (Pi ca 4.0) and basic (Pi > 8.5) forms. Treatment of grapefruit with ethylene (2 ppm 72 hours) induces a basic POD not present in freshly-harvested fruit or in nonethylene-treated controls.