No description available.

No description available.

No description available.

No description available.

Genes encoding representatives of two subfamilies from the vicilin storage protein family in pea (Pisum sativum L.) have been sequenced and characterised. One, encoding convicilin, shows that this protein differs from vicilin by the insertion of a hydrophilic region near the N-terminus. The transcription start point has been determinedand the pattern of expression in developing seeds elucidated. By theexpression of this gene in tobacco, the specific polypeptide product ofthe gene was identified as a minor component of convicilin , with a lowerMr than the major species . The other gene subfamily investigated wasThat encoding the vicilin 47,000 Mr polypeptide. A gene and a cDNA weresequenced, and the gene found to diverge from the cDNA in the 3" regionof the coding sequence. No product from this divergent gene could beidentified.A member of the legumin gene family (legK) was sequenced and found to be inactive due to a mutation of the start codon. The region of DNAencoding the start codon of this gene was amplified by polymerase chainreaction from a pea line in which the gene was known to be active . Thesequence of this revealed the presence of a normal start codon. Two dimensional protein gels were run with seed extracts from these twolines , and the product of (legK) demonstrated by its occurrence in theline with the functional gene. A method for the extraction and purification of the major pea root protein was established. The protein was shown to have a Mr of 16,000 and not to be susceptible to cleavage by cyanogen bromide. Partial amino acid sequence data was obtained from the purified protein .A differential screen of cDNA from purple and green poddedvarieties of pea was conducted, and differentially expressed cDNAsisolated . The nature of the expression of these cDNAs was studied in thetwo lines and the cause of instability in the purple podded phenotypeinvestigated . A genomic library was constructed from the purple poddedline . Two genes were selected by the differentially expressed cDNAs andtheir DNA sequences determined. A gene encoding a pectinestera- likesequence, and the pod expressed cDNA used to select it , were found to betwo members of a small multigene family in pea. The second gene selectedproved to encode a protein containing two distinct domains; the N-terminal region being of a repetitive proline-rich nature and the C-terminal region being hydrophobic and cysteine rich . This gene waspresent as a single copy in the pea genome and its expression appearedto be linked to pigmentation.

published_or_final_version / Botany / Doctoral / Doctor of Philosophy

No description available.

No description available.

Research was undertaken to optimize propagation of the southern China native Camptotheca acuminata Decaisne, source of the medicinal compound camptothecin that is used in the treatment of multiple forms of cancer and other diseases. The study focused on cutting propagation, micropropagation, and seed storage. Softwood cuttings of C. acuminata rooted readily in intermittent mist (4 sec on every 6 min.) in coarse vermiculite when treated with K-IBA (indolebutyric acid, potassium salt) quick dips ranging from 4.14 mM to 37.3 mM, with a 29.0 mM quick dip (5 sec.) promoting 82% rooting with little foliar damage. Actively growing shoot tip explants were tissue cultured on media containing Murashige and Skoog, Gamborg's B5, and Woody Plant Medium (WPM) salts in factorial combinations with BA (benzyladenine). WPM containing 4.44 μM BA promoted excellent shoot proliferation; microcuttings were rooted, acclimated, and grown in the greenhouse. Seeds stored in polyethylene bags in a refrigerator (4°C) or freezer (-20°C) maintained good germination (81% and 80%, respectively) while seeds stored at room temperature (25°C) in polyethylene bags lost germination ability quickly (58%) after one year of storage. C. acuminata is readily adaptable to modern nursery techniques for either vegetative or seed propagation.

Understanding the mechanisms plants utilize for seed storage protein (SSP) synthesis, transport and deposition have the potential rewards of enabling high yields of modified or foreign proteins. Hayashi et al. (1999) indicated that the machinery devoted to the synthesis of protein storage vacuoles in cotyledon cells can be induced in vegetative tissue by the constitutive expression of a pumpkin 2S albumin phosphinothricin-acetyl-transferase gene fusion (pumpkin 2S-PAT) resulting in the biogenesis of precursor-accumulating (PAC) vesicles in Arabidopsis leaves. This discovery was the impetus behind the work described which sought to examine this phenomenon further by ectopically evoking SSP trafficking and vesicle biogenesis machinery in leaves. With the aim of elucidating the mechanisms necessary to evoke PAC vesicle biogenesis, a suite of constructs including the pumpkin 2S-PAT and analogous napin-PAT and napin-GFP variants were synthesized. Analysis of these transgenes in Arabidopsis revealed that the pumpkin 2S albumin has a capacity unique from napin peptides to result in fusion protein accumulation. Further, the truncated pumpkin 2S albumin peptide and the pumpkin 2S albumin C-terminus were found to direct deposition to vesicles; however, the C-terminus alone was not enough to direct deposition to vesicles unless combined with a significantly shortened napin peptide. An increased ER protein throughput was correlated to trafficking of the fusion protein by Golgi-independent mechanisms resulting in stable accumulation of the unprocessed protein whereas less ER throughput indicated passage through the Golgi-dependent pathway resulting in accumulation of a processed variant. At the level of gene expression, as examined by a microarray study, both inducible and constitutive ectopic expression of pumpkin 2S-PAT resulted in substantial perturbations of the endomembrane system affecting protein folding, flowering time and ER-associated biosynthetic functions which indicated that modulation of flowering time and photoperiodism are highly dependent on protein trafficking and vacuolar biogenesis mechanisms and that high ER protein throughput occurs at the expense of biosynthesis and cessation of ER functioning.