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.

A thesis submitted by Melinda Jane Mayer, B.Sc.(Bristol) in accordance with the requirements of the University of Durham for the degree of Doctor of Philosophy. Department of Biological Sciences, August 1993.Two cDNA libraries were constructed from desiccating pea cotyledons. Differential screening of the libraries with cDNA from an earlier developmental stage (physiological maturity) demonstrated that the abundant message population during dehydration shows some noticeable differences to the message populations present before desiccation. Clones hybridising to a polyubiquitin probe were isolated from a cDNA library. These clones were identified as messages for the two types of ubiquitin extension proteins (with 52 and 79 residue tails), already characterised in other species as being involved in ribosome biogenesis. The pea ubiquitin extension tail amino acid sequences showed considerable homology to tails from other plants, animals, yeast and protozoa, including a nuclear localisation site and a putative zinc-binding nucleic acid binding domain, the positions of which are conserved within the tail sequences. Sequencing of a second polyubiquitin cDNA from pea leaf demonstrated that pea contains a ubiquitin multigene family of at least four members. The expression of several genes associated with plant response to stress and two abundant seed messages (Leg A and J) was examined in developing and dehydrating cotyledons and axes. This confirmed conspicuous variations in the message levels of the genes examined as the cotyledons aged, with different members of the ubiquitin and legumin multigene families showing differential expression with age. It was also demonstrated that the expression pattern of certain messages in the cotyledons was different to that in the axes and other seed tissues. This was confirmed by an analysis of total and albumin protein fractions in cotyledons and axes. The effect on specific message and protein levels of premature desiccation treatments indicated that the temporal expression of several seed genes is related to the state of hydration of the seed, artificial desiccation leading to premature maturation. Seed storage protein message and protein levels were especially increased by premature desiccation. Legumin seed storage protein messages were also shown to be responsive to exogenous ABA applied to immature cotyledons during the seed filling stage. However, the other stress-related messages examined in pea (ubiquitin and a pea putative metallothionein) were not responsive to exogenous ABA at this developmental stage.

Amylase activities and patterns were compared in extracts from mature seeds and from different parts of seedlings at various stages of seed germination of various varieties of different legume species. Apart from soyabean, all varieties had low levels of amylase activities in extracts from mature seeds, while the former had a remarkably high level. Amylase activities and the number of bands increased during seed germination and showed time and tissue specifity. The extracts from cotyledons had the highest activities and the largest number of bands as compared to that of the various parts of embryonic axes. Amylase activities and patterns had time specific changes during the various stages of seed development of the 7 varieties of different legume species studied. All the different varieties showed changes in zymogram patterns and decreased amylase activity during seed development, except soyabean where amylase activity remained high at all stages. The different components of amylase were characterised in pea (var. Feltham First). One band of a -amylase activity, restricted to the cotyledons, was present in the middle stages of seed development only, then reappeared at the third day of seed germination. The embryonic axes β -amylase activity decreased as the seed developed, to reach zero level in mature seeds, and reappearred during seed germination. The reverse sequence occurred for the cotyledonary β -amylase. The pea a -amylase was partially purified using ethanol precipitation, glycogen complex and hydroxyapatite column chromatography. The purified protein had three major bands with few faint bands on the SDS polyacrylamide gel. The embryonic axes β -amylase of pea was partially purified using the conventional method, ammonium sulphate precipitation, ion exchange chromatography and gel filtration. The purified protein contained two thick bands and many faint bands on the SDS polyacrylamide gel. The zymogram patterns of phosphorylase, EST, GDH, GOT, LDH, ADH and MDH were investigated during seed development of various varieties of different legume species. The most noticeable changes as the seeds developed were the decrease in activities and number of bands of EST in pea and soyabean, shift in GDH activities between isoenzyme forms in pea (var. English Wonder), increase in GDH activities and number of bands in dwarf French bean and soyabean and increase in activities and number of bands of MDH in pea (var. Feltham First).

xiv, 173 leaves : graphs, tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1979) from the Dept. of Agronomy, University of Adelaide

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Apr. 9, 2009). PDF text: xiv, 282 p. : ill. (some col.) ; 10 Mb. UMI publication number: AAT 3339125. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed May 5, 2009). PDF text: xi, 146 p. : ill. (some col.) ; 8 Mb. UMI publication number: AAT 3341869. Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Jeffrey C. Mosley. Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on September 18, 2008) Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1952. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 322-323).

Thesis (M.S.)--University of Wisconsin--Madison, 1953. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 41-43).