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S.S. Nielsen, C.I. Osuala, and W.E. Brandt

Five greenhouse-grown cowpea [Vigna unguiculata (L.) Walp] cultivars were tested in a generalized random complete-block design to determine the effect of early leaf harvest on dry weight and protein concentration of plant parts at maturity. The most recent, fully expanded leaves on each branch from one group of plants were harvested at 5 and 7 weeks after planting. On the other groups of plants, no early leaf harvest was performed. Dry weight and protein concentration (dry weight basis) were determined for leaves, stems, and seeds at maturity and for leaves harvested early. Weight and protein concentration of seeds, leaves, and stems differed significantly between cultivars; protein concentration of leaves harvested at 5 or 7 weeks did not. Dry weight of leaves harvested at 5 vs. 7 weeks did not differ significantly, but leaf protein concentration was significantly higher at 5 weeks compared to 7 weeks. Across all cultivars, early leaf harvest had no significant effect on leaf or stem weight per plant at maturity. However, there was a significant decrease in seed weight when leaves were harvested early. Results suggest that even limited leaf harvest at 5 and 7 weeks has detrimental effects on yield, but not on protein concentration, of cowpea seeds harvested at maturity.

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Kathryn C. Taylor and Danielle R. Elli

A 22-kDa Zn-binding protein (ZBP) was isolated from the phloem tissue and evacuated xylem sap of `Valencia' sweet orange [Citrus sinensis (L.) Osbeck] on rough lemon [C. jambhiri (L.)], as well as Valencia on Rangpur lime [Citrus limonia Osbeck]. Phloem and xylem Zn was associated with the 22 kDa ZBP. The Mr value of this ZBP was estimated to be 19,500 by size exclusion chromatography and 22,800 by SDS-PAGE. This protein was isolated with an isoelectric point of 7.5. Ion exchange chromatography demonstrated that 22-kDa ZBP was highly anionic, requiring 0.43 M NaCl for elution from QAE Sepharose. The 22-kDa ZBP appears unique to citrus, having no cross reaction with protein from several tissues from a range of plant species. Accumulation decreased under Zn-deficient conditions, was enhanced by osmotic stress, and the protein completely disappeared with wounding. Amino acid composition demonstrated that the protein was rich in aspartate, and glutamate; and contained 6 cysteine, and 4 histidine residues. These amino acids may be involved in metal binding. N-terminal amino acid sequencing demonstrated that the 22-kDa ZBP had identity with sporamin A&B precursors, Kunitz-type trypsin inhibitors, and miraculin. It is suggested that the genes that encode these proteins are derived from a common ancestral gene.

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Alan G. Smith and Kenneth J. McNeil

The sporophytic tissue of the anther and, in particular, the tapetum, a cell layer surrounding the pollen sac, is know to be essential for the production of pollen. The isolation and characterization of the gene 92B from tomato that encodes an extracellular glycine rich protein (GRP) has been used to further elucidate the role of the tapetum in pollen development. RNA from the 92B gene accumulates exclusively in the tapetum. Polyclonal antibodies raised against the 92B GRP detect four proteins in stamens with microspores beginning meiosis. In pollen extracts, the antibodies detect a single protein. Expression of the tomato 92B gene in transgenic tobacco indicates that the four protein products are derived from only the 92B gene. The 92B GRP is localized to the tapetum, the callose wall of microspore mother cells, the exine (outer wall) of mature pollen, and orbicules. Orbicules are globular bodies derived from tapetal material that form on the tapetum wall and line the exterior of the pollen sac. Expression of 92B antisense RNA resulted in a significant decrease of 92B RNA and protein levels in transgenic tomatoes. This reduction was correlated with a decrease in pollen germination and an abnormal exine morphology. The function of the 92B protein in pollen development and function will be discussed.

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Michael A. Arnold and Eric Young

Bare-root Malus × domestica Borkh. seedlings were chilled for 0, 600, 1200, or 1800 hours at 5C (CH). Seedlings were then placed with roots and/or shoots in all combinations of 5 and 20C forcing conditions (FC) for up to 21 days. Virtually no growth occurred at 5C FC. When the whole plant was forced at 20C, all measures of root and shoot growth increased in magnitude, occurred earlier and at a faster rate with increasing CH. Thus, roots and shoots responded similarly to chilling. When shoots or roots were subjected to 20C FC, while the other portion of the plant was at 5C, the responses were reduced in magnitude and delayed. However, the overall growth enhancement by chilling was not negated. Root and shoot growth enhancement by chilling appeared to be increased if the other portion of the plant was actively growing also, but not dependent on it. Growth of adventitious shoots on roots (root suckers) was greatly enhanced with increasing CH on plants subjected to 5C shoot and 20C root FC. While total root and shoot bark protein levels on a per-seedling basis were similar, protein concentrations were lower in root bark than in shoot bark. During chilling, total protein per seedling generally increased until just before the time that chilling requirements for vegetative budbreak were satisfied. Protein degradation then began, resulting in lower protein levels through 2300 CH. Rapid protein breakdown (1200 to 1800 CH, roots; 1000 to 1800 CH, shoots) occurred at about the same time that root (1000 to 1800 CH) and shoot (800 to 1800 CH) growth responses to chilling were increasing. Warm FC resulted in increased protein breakdown with increased CH and forcing time.

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Ahmed Mahhou and Frank G. Dennis Jr.

Siberian C peach (Prunus persica L.) seeds were stratified at 5 and 20C. DWs and soluble protein content remained constant regardless of stratification temperature and duration. Seed extracts subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the intensity of nine polypeptides in the cotyledons of seeds held at 5C during weeks 5 through 8, coinciding with an increase in germination capacity. These changes were confined to cotyledons held at 5C, and were observed only when the seeds were able to germinate. The effects of stratification and the imbibition degree on changes in the protein content of seeds of two additional peach biotypes (`Farouki' and `Maloussi') were also evaluated. Germination of fully imbibed seeds at 20C increased steadily as stratification time at 5C increased. Partially imbibed seeds (25 % or 50% of full imbibition) did not germinate regardless of stratification time. However, when these seeds were soaked in water after stratification, their germination paralleled that of fully imbibed seeds. Thus, dormancy was broken, even though the seeds could not germinate. Changes in protein profiles in fully imbibed seeds confirmed those previously reported for Siberian C seeds. Similar changes occurred in cotyledons of partially imbibed seeds during stratification at 5C, but at a slower rate. Those changes were, however, delayed by partial imbibition, whereas germination capacity (ability to germinate when fully imbibed) was not. Changes in cotyledon protein profiles were not affected by removing the embryonic axis before stratification, a result indicating that such changes are not controlled by the axis. Gibberellic acid (GA3 induced 35 % to 40% germination of nonchilled seeds. It hastened the loss of protein band intensity in `Farouki' but not in `Maloussi'. However, GA3-treated seeds germinated before any visible changes occurred in protein profiles. We conclude that the effects of chilling on breaking dormancy are independent of its effects on the protein changes observed in this study.

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Gene Lester

Using an aqueous polymer two-phase [polyethylene glycol (PEG) 3400/dextran T500, 6.2%: 6.2%, w/w] partitioning procedure combined with isopycnic fractionation, plasma membranes derived from muskmelon (Cucumis melo L. var. reticulates Naud.) leaf blades have been isolated and examined for marker enzyme activity, density, and molecular composition. After aqueous polymer partitioning, plasma membranes were centrifuged on a linear sucrose density gradient, and a single band was found at the 31% (w/w) sucrose (1.13 g-cm-3). Identification of plasma membranes was performed by the combination of K+-stimulated ATPase, pH 6.5, vanadate inhibition of ATPase and KNO3-insensitive ATPase activity. Plasma membranes from seedling leaves grown for 5 days at 15C had the highest concentration of total phospholipids, the lowest concentration of proteins, and a total sterol concentration not significantly different from leaves grown at 30C. The total sterol to total phospholipid ratio of the plasma membrane from leaves grown for 5 days at 15C was ≈1:1; from leaves grown for 10 days at 15C or 5 days at 30C the ratio was ≈2:1; and from leaves grown for 10 days at 30C the ratio was ≈3:1. The plasma membrane phospholipid saturated to unsaturated fatty acid ratio from leaves grown for 5 days at 15C was ≈0.8:1.0; from leaves grown for 10 days at 15C or 5 days at 30C the ratio was ≈1.0:1.0; and from leaves grown for 10 days at 30C it was 1.4:1.0.

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Guowei Fang and Rebecca Grumet

Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.

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G.N.M Kumar and N.R. Knowles

The physiological mechanisms leading to a decline in sprout-vigor, root growth potential and apical dominance during long-term aging of potato seed tubers are currently under investigation. Malondialdehyde (MDA) and ethane, products of peroxidative degradation of PUFA increase in seed-tuber tissues with advancing age (from 2 to 32 months of storage). MDA is known to react with free amino acids to produce lipofuscin-like fluorescent compounds (FC), which build-up in aging/senescent tissues of plants and animals. With advancing seed-tuber age, an increase in free amino acids, MDA and FC concentrations was evident. Moreover, high levels of MDA have been shown to reduce protein synthesis in both plant and animal cells. We therefore examined the extent to which seed-tuber age affects protein synthesizing capacity of tuber tissues during sprouting. Tissue disks from 6- and 18-mo-old seed-tubers at various stages of sprouting, were compared for their protein synthesizing ability by monitoring the incorporation of radiolabelled amino acids into TCA precipitable products. The rate of incorporation (dpm mg protein-1 min-1) was 1.8 to 5.4-fold higher in tissue from 6-mo-old, as compared to that from 18-mo-old seed tubers, at similar stages of sprout development. Loss in protein synthesizing ability (possibly due to direct peroxidative damage) may be an important factor contributing to loss of sprout-vigor from aged potato seed-tubers.

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Tridate Khaithong, Brent S. Sipes, and Adelheid R. Kuehnle

Lack of a conclusive evidence of ingestion of plastid components by plant-parasitic nematodes cautions the use of plastid transformation technology for nematode resistance. Nematode-resistant effector proteins generally require ingestion by the nematode to be effective. Transgene-encoded proteins produced in plastids are not known to be exported into the cytoplasm. Disintegration of plant cell organelles after nematode feeding suggests that nematodes possibly ingest plastid components. Proof of ingestion will validate the development of plastid transformation for nematode resistance. Small subunit ribulose-1,5-bisphosphate carboxylase/oxygenase (SSRubisco) protein is prevalent in chloroplasts and thus chosen as a study molecule. The migratory endoparasitic nematodes Pratylenchus penetrans and Radopholus similis were cultured on green carrot callus containing chloroplasts. Total nematode proteins were extracted and subjected to western blot analysis using cross-reactive polyclonal antibody raised against spinach SSRubisco. Positive detection of SSRubisco occurred in protein extracts from nematodes fed on green carrot callus, but not in extracts from nematodes cultured on non-green alfalfa root callus as negative control. These results confirm the ingestion of plastid components of MW 14 kDA by migratory endoparasitic nematodes.

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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.