Three soluble invertase isoforms from Lilium longiflorum flower buds that had been separated by DEAE-Sephacel chromatography were purified to near homogeneity by further chromatography on hydroxylapetite, Con-A sepharose, phenyl agarose, and Sephacryl S-200 gel filtration. Nondenaturing polyacrylamide gel electrophoresis (PAGE) gave a single band in all three invertases that corresponded to a band of invertase activity in a duplicate gel. The SDS-PAGE of the purified invertase I resulted in a single band with apparent relative molecular mass of 78 kDa. Invertase II and III were resolved to a similar polypeptide pattern by SDS-PAGE with three bands of 54, 52, and 24 kDa. Antiserum of tomato acid invertase cross-reacted with all three invertase protein bands. Antiserum of wheat coleoptile acid invertase cross-reacted only with 54 and 52 kDa bands of invertase II and III but did not recognize invertase I protein. Con-A peroxidase was bound to invertase I protein and all three protein bands of invertase II and III, suggesting that all proteins were glycosylated. Invertase I protein could be completely deglycosylated by incubating with peptide-N-glycosidase F to result in a peptide of 75 kDa. Invertase II and III were partially deglycosylated by peptide-N-glycosidase F resulting proteins bands of 53, 51, 50, and 22 kDa.
Anil P. Ranwala and William B. Miller
Rajeev Arora, Michael Wisniewski, and Lisa J. Rowland
Seasonal changes in cold tolerance and proteins were studied in the leaves of sibling deciduous and evergreen peach [Prunus persica (L.) Batsch]. Freezing tolerance [defined as the subzero temperature at which 50% injury occurred (LT50)] was assessed using electrolyte leakage. Proteins were separated by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. Electroblots were probed with anti-dehydrin and anti-19-kD peach bark storage protein (BSP) antibodies. Leaf LT50 decreased successively from -5.8 °C on 18 Aug. to -10.3 °C in the evergreen genotype and from -7.0 °C to -15.0 °C in the deciduous genotype by 14 Oct. Protein profiles and immunoblots indicated the accumulation of a 60- and 30-kD protein during cold acclimation in the leaves of deciduous trees; however, levels of these proteins did not change significantly in the evergreen trees. Immunoblots indicate that the 60-kD protein is a dehydrin-like protein. Gel-electrophoresis and immunoblots also indicated that the 19-kD BSP progressively disappeared from summer through fall in leaves of deciduous peach but accumulated to a high level in bark tissues. A similar inverse relationship was not evident in evergreen peach.
Mark J. Gatschet, Charles M. Taliaferro, Jeffrey A. Anderson, David R. Porter, and Michael P. Anderson
Cold acclimation (CA) of `Midiron' and `Tifgreen' turf bermudagrasses (Cynodon dactylon L. Pers. × C. transvaalensis Burtt-Davy) induced tolerance to lower freezing temperatures and altered protein synthesis in crowns. LT50 (lethal temperature for 50% of plants) values were lowered ≈5C after 4 weeks in controlled-environment chambers under CA [8/2C (day/night) cycles with a 10-hour photoperiod] vs. non-CA (28/24C) conditions. LT50 values for `Midiron' plants decreased from -6.5 to -11.3C after CA and from -3.6 to -8.5C for `Tifgreen'. Proteins synthesized by isolated crowns were radiolabeled in vivo for 16 hours with 35 S-methionine and 35 S-cysteine. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography revealed increased synthesis of several cold-regulated (COR) proteins in CA crowns of both cultivars. Synthesis of intermediate molecular weight (MW) (32 to 37 kDa) and low-MW (20 to 26 kDa) COR proteins was greater in `Midiron' than `Tifgreen' crowns.
I.E. Yates, E.A. Carter, T.A. Wilkins, and B.W. Wood
Polypeptides from pecan [Carya illinoensis (Wangenh.) C. .Koch] leaves were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. Pecan leaf protein profiles were similar irrespective of cultivar (Desirable and Stuart), leaflet position, reproductive status of the allied shoot, or seasonal leaf age relative to fruit development. The large subunit of ribulose l,5-bisphosphate carboxylase and the majority of the other polypeptides were consistently present. However, the most striking change in the polypeptide composition was the seasonal decline of a polypeptide with an approximate molecular mass of 24.5 kDa. This leaf polypeptide was present in leaves collected in June and July, coinciding with the periods of initial fruit elongation and rapid increase in fruit volume. A detectable decrease occurred by mid-August, when kernel development was initiated. Changes in the abundance of this polypeptide relative to other polypeptides were observed over two growing seasons. Cells of young leaves collected early in the growing season contained more ribosomes and starch granules, but fewer vesicles and smaller electron-dense osmophilic granules than old leaves collected late in the growing season.
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.
M.V. Bhaskara Reddy, Alain Asselin, and Joseph Arul
We have investigated the relationship between chitosan treatments and maceration of potato tissue by macerating enzymes secreted by Erwinia carotovora causal agent of soft rot of potato. Erwinia isolated from potato showing soft rot symptoms was used for inoculation. The bacteria secreted a wide spectrum of enzymes that degraded potato cell walls. Polygalacturonase (PG), pectate lyase (PL), pectinmethylesterase (PME), cellulase, xylanase, and protease showed the highest activity in potato tissue inoculated with the pathogen. Accordingly increased maceration and cell death were observed. On the other hand, in chitosan-treated tissue and challenged with the pathogen, significant decrease in enzymatic activity and tissue maceration were observed, more so with increasing chitosan concentration. This observation confirmed that chitosan interfered with multiplication and pathogenic powers of the bacteria, thereby improving cell texture and viability. Crude extracts obtained from treatments were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess pectinase activity. The electophoretic profiles showed significant lytic zone of pectin degradation in the control, which decreased with increase in chitosan concentrations. No lytic zone was observed at 8 mg·ml–1 chitosan concentration and was comparable to intact activity in untreated potato tissue. Pectic enzyme reaction products were analyzed to see the action pattern of pectinases in the crude extracts. Cellulose choromatographic profiles revealed monomers and dimers of polygalacturonic acid up to 6 mg·ml–1 chitosan concentrations. The results suggest that chitosan significantly inhibits bacterial growth and the production of macerating enzymes by the pathogen and thus chitosan can be a potential anti-bacterial agent.
V.M. Russo and C. Biles
Cucumber (Cucumis sativus L.) seed require soil temperature to be around 20C for efficient germination. This hinders early planting in cool soils. This study was conducted to determine how germinating seed of cucumber cultivars Earlipik 14 and Arkansas Little Leaf at 13.9, 15.6, and 20C in the dark affected protein formation. Seed were removed from moist chambers at 0, 12, 24, 48, 72, 84, 96, 120, and 168 h. Germination, defined as the radicle being at least 5 mm long, was determined at each time. Germinated and ungerminated seed was prepared for polyacrylamide gel electrophoresis. At 20C, 90% to 100% of seed had germinated by 48 h. At 15.6C, 20% to 50% of seed germinated by 168 h, and at 13.9C, ≈2% of seed had germinated by 168 h. For seed incubated at 20C, concentrations of proteins at 70.1 kDa decreased, while those at 37.4, 43.4, and 50 kDa increased after 24 h, which corresponded to formation and elongation of the majority of radicles. These changes were expressed later for seed germinated at 15.6 and 13.9C. Identification of the proteins is being attempted. The importance of these proteins in germination and early development will be discussed.
Kathryn C. Taylor, Danielle R. Ellis, and Luciano V. Paiva
Zinc in xylem and phloem of the citrus rootstock, rough lemon [Citrus jambhiri (L.)] was associated with a Zn-binding protein, designated citrus vascular Zn-binding protein (CVZBP). The apparent molecular mass of the CVZBP was 19.5 kDa after nondenaturing size exclusion chromatography and 21.8 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion exchange chromatography demonstrated that CVZBP was anionic, requiring 0.43 n NaCl for elution from quaternary aminoethyl Sepharose. Antiserum to the protein cross-reacted more with total protein extracts from leaf midveins than with total protein from the rest of the leaf lamina, further suggesting a vascular location of the Zn-binding protein. Quantitative analysis indicated that ≈2 to 3 mol of Zn were associated with 1 mol of native protein. Binding studies with the partially purified CVZBP demonstrated a capacity to bind several divalent cations: Cd, Ni, Pb, and Zn. Reaction with Ellman's reagent suggested that the protein has significant sulfhydryl group content that may be involved in metal binding. N-terminal sequencing demonstrates identity with papaya latex trypsin inhibitor, sporamin, or other Kunitz soybean proteinase inhibitors.
A. Belaj, I. Trujillo, R. de la Rosa, L. Rallo, and M.J. Giménez
Random amplified polymorphic DNA (RAPD) analysis was performed on the main Mediterranean cultivars of olive (Olea europaea L.) from the Germplasm Bank of the Centro de Investigación y Formación Agraria “Alameda del Obispo” in Cordoba, Spain. One hundred and ninety reproducible amplification fragments were identified using 46 random primers followed by agarose gel electrophoresis. Some 63.2% of the amplification products were polymorphic, with an average of 2.6 RAPD markers obtained for each primer. The combination of polymorphic markers resulted in 244 banding patterns. The high degree of polymorphism detected made identification of all the cultivars (51) possible by combining the RAPD banding patterns of just only four primers: OPA-01, OPK-08, OPX-01, and OPX-03. Cultivar-specific RAPD markers and banding patterns were also found. A dendrogram based on unweighted pair-group method cluster analysis was constructed using a similarity matrix derived from the RAPD amplification products generated by the 46 primers. Three major groups of cultivars could be distinguished by RAPD analysis: 1) cultivars from east and northeast Spain, 2) Turkish, Syrian, and Tunisian cultivars, and 3) the majority of common olive cultivars in Spain. The dendrogram thus showed a good correlation between the banding patterns of olive cultivars and their geographic origin. A higher level of polymorphism was observed when polyacrylamide gel electrophoresis was used to separate the amplification products. Thus, adequate use of RAPD technology offers a valuable tool to distinguish between olive cultivars.
Gregory A. Lang and Joshua Tao
Plant dormancy research has long been stifled by the lack of appropriate biochemical markers to characterize the changing physiological status of dormant vegetative or reproductive buds. Two sets of experiments were conducted in an attempt to identify changes in soluble protein profiles during endodormancy of peach and blueberry reproductive apices. Bud samples from the peach cultivars `La Festival' (low chilling requirement) and `La White' (moderate chilling requirement) were taken every 15 days in the orchard during December and January, extracted for soluble proteins, and analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Outshoots were forced at 25C in a growth chamber to determine the intensity of endodormancy. A further experiment utilized potted `Bluechip' and `Meader' (troth high chilling requirement) blueberry plants given varying periods of cold (4.5C) chamber treatment, followed by forcing at 25C in a growth chamber. Bud samples were taken following cold treatment for extraction and SDS-PAGE. The relationship of the resulting protein profiles to chilling unit accumulation and intensity of endodormancy will be discussed.