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Protein bodies within the endosperm of castor bean (Ricinus communis L. cv. Hale) seeds arise from numerous small vacuoles which progressively become filled with storage protein, of which the crystalloid proteins make up approximately 70%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the crystalloids are a family of at least four proteins which reduce to two complementary groups after 2-mercaptoethanol treatment. The matrix, which comprises the remainder, has two major components, the soluble albumins and the lectins. The lectins are the only glycoproteins within the mature protein body. Both cytochemical staining and SDS-PAGE indicate that the synthesis of the crystalloid and the majority of matrix proteins begins some 20 days after pollination. Additionally, the crystalloid proteins are synthesized concurrently, whereas there is temporal variation in the synthesis of matrix proteins.

In this study we examined the effect of heat stress (up to 6 hours at 43 degrees C) on the biosynthesis and transport of phytohemagglutinin (PHA) in cotyledons of developing seeds of the common bean, Phaseolus vulgaris. Heat stress resulted in a decrease of total protein synthesis and an enhancement of the synthesis of heat shock proteins and PHA. Pulse chase experiments showed that a considerable proportion of the newly synthesized PHA was present in the endoplasmic reticulum (ER)/Golgi fraction and did not readily chase-out. Analysis with endoglycosidase H showed that the oligosaccharide sidechains of PHA were almost entirely in the high mannose configuration, indicating that most of the newly synthesized PHA was in the ER. However, some of the PHA became fucosylated at 43 degrees C, indicating fucosyltransferase activity. That the biosynthesis and secretion of fucosyl-containing cell wall polymers proceeded normally at 43 degrees C provided evidence that certain Golgi functions (i.e. transport to the cell wall) remained unaffected by heat stress. The ER obtained from these heat stress cotyledons had a greater density (1.16 g. cm(-3) at 43 degrees C instead of 1.14 g.cm(-3) at 22 degrees C) in sucrose gradients. Ultrastructural observations showed that the width of the lumen of the ER cisternae had increased from 20 nanometers at 22 degrees C to 60 to 80 nanometers at 43 degrees C; the lumen was filled with electrondense material presumed to be protein. The experiments are interpreted as evidence that heat stress imposes a block in the transport of PHA out of the ER. Whether heat stress affects the ER itself or alters the conformation of PHA, thereby preventing its transport, is not clear.

During seed development, myo-inositol (Ins) hexakisphosphate or phytic acid (PA) is stored in the form of phytin with mineral cations, and is mobilized following germination, releasing these nutrients that are required for seedling growth. Outside its role in seeds, PA and other phosphoylated Ins derivatives play critical roles in biological processes in many eukaryotes. PA also has negative influences on nutrition in both non-ruminant animals and humans due to its lack of digestibility. There have been two parallel PA biosynthetic pathways proposed, yet, the pathway is still poorly understood in terms of its regulation and enzymes involved. Here, genes encoding enzymes putatively implicated in castor bean PA biosynthetic pathways were identified in the genome and expression followed. Isolated castor bean embryos have the ability to resynthesize PA following germination if exogenous phosphate is available. It was found that the genes purported to act in PA synthesis were constitutively expressed in the embryos regardless of the availability of phosphate. Castor bean Ins 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1), catalyzing the final reaction in PA biosynthesis, regardless of pathway, was chosen for further study. Even though only one copy of the RcIPK1 gene was present in the genome, numerous RNA variants were found, most likely due to alternative splicing events. These variants encoded six closely related protein isoforms based on in silico analysis. Functional analyses using yeast mutant strains lacking the IPK1 gene revealed that only three of the mRNA variants could rescue the temperature-sensitive growth phenotype, and it was demonstrated by HPLC analysis of Ins phosphates that their ability to complement the missing yeast IPK1 enzyme was associated with enzyme activity. Only these three isoforms possessed conserved motif III and motif IV important for IPK1 catalytic activity.

This thesis investigates endosperm programmed cell death (PCD) in 'Solanum lycopersicum' L. (tomato) seeds following germination. The expression pattern of a cysteine proteinase-encoding gene, ' SlCysEP', and accumulation of the enzyme in imbibed whole seeds prior to, and following, germination are consistent with that of an enzyme involved in mobilization of the cell corpse following PCD. Further, immuno-gold labelling revealed that the enzyme localizes to a PCD-associated, endoplasmic reticulum-derived organelle, the ricinosome. Overexpression of a fluorescent protein-tagged SlCysEP construct in cultured tobacco cells induces formation of ricinosome-like structures (RLs). Removal of the cysteine proteinase-domain from the construct prevents the formation of RLs suggesting that this domain may be necessary for formation of ricinosomes 'in vivo'. The hormonal control of endosperm PCD was also investigated. This study confirms a previous report that gibberellic acid (GA) promotes endosperm PCD, and demonstrates that abscisic acid antagonizes the action of GA thereby preventing PCD. Limited evidence indicates that the C14 family of cysteine proteinases, in particular the metacaspases, participate in the regulation of some instances of PCD in plants. The latter portion of this thesis reports the cloning and initial characterization of a type II tomato metacaspase gene, 'SlMCA2 '. Transcript analysis of 'SlMCA2' indicate that it is not involved in endosperm PCD, but that it may be involved in instances of PCD in the developing seedling, and perhaps in dying tissues of the anther.

Ranunculus asiaticus' is a horticultural dicot that is able to tolerate long-term desiccation during its annual life cycle. The annual cycle of desiccation and resurrection of 'R. asiaticus' is correlated with the presence of a putative storage protein (Ra14) and stress-related proteins (dehydrins and smHSPs) in its underground storage organs, the tuberous roots. The 14 kDa protein (Ra14) as a major root protein fluctuates seasonally and increases upon drying. It is a glycoprotein and has an amphipathic character. A 492 bp full-length Ra14 cDNA sequence and a 615 bp genomic sequence were obtained by 3'-RACE and 5'-RACE PCR using oligonucleotide sequences based on the terminal amino acids of the 14 kDa mature protein. A 500 bp probe based on the Ra14 genomic sequence detected at least 4 homologs in 'R. asiaticus ' using Southern blots. The 14 kDa protein was localized in the storage vacuoles in 'R. asiaticus' roots using an antibody raised against it. SDS-PAGE analysis of total proteins extracted from different organs of ' R. asiaticus' revealed that the 14 kDa protein is specifically expressed and synthesized in tuberous roots but not in above-ground vegetative organs such as leaves and stems, and its homolog was only detected in 'Anemone ' corms, a desiccation-tolerant organ. Immunoblots indicated that dehydrins (LEAII D-11 family) are present in desiccated roots and their expression declined upon rehydration. A 477 bp full-length cDNA sequence of Dhn17.5 encoding a 17.5 kDa dehydrin and a 543 bp full-length cDNA sequence of Dhn20 encoding a 20 kDa dehydrin were obtained by degenerate PCR, 3'-RACE and 5'-RACE PCR. The expression of Dhn20 was induced by dehydration and ABA but not by salt. As with dehydrins, smHSPs were also detected by immunoblots in the tuberous roots during desiccation, and their expression was induced by dehydration, ABA and salt. Gel filtration chromatography showed that smHSPs may act as molecular chaperons which are able to bind to other molecules during desiccation to protect them from damage. A possible interaction between the 14 kDa protein and smHSPs was determined using S-tagged 14 kDa protein and total proteins extracted from desiccated roots, but the gel filtration chromatography showed that there is no interaction between the two 'in vivo'.

Peroxisome biogenesis is complex, involving a diverse array of intracellular pathways and mechanisms that mediate their biogenesis and cellular functions. Relevant to our understanding of peroxisome biogenesis is the utilization of peroxisomal membranes for viral genome replication as observed in plant cells infected by several members of the 'Tombusviridae' family of positive-strand RNA viruses; Tomato Bushy Stunt Virus (TBSV), for instance, usurps an array of host factors that facilitate the transformation of peroxisomes into peroxisomal multivesicular bodies (PMVB) the sites of viral RNA replication. In this study, pMVB topology and biogenesis was investigated using transmission electron and epitluorescence microscopy of tobacco and wildtype or mutant budding yeast that were transformed with TBSV replicase proteins and a defective interfering viral RNA. Overall, the results suggest that host-virus interactions specifically associated with Endosomal Sorting Complex Required for Transport (ESCRT) and lipid metabolism are involved in TBSV replication and pMVB biogenesis.

The seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing (galacto)mannan polymers. These are present in the lateral endosperm, but there is little or no cell wall thickening in the micropylar endosperm, except in date seeds. In this species, a ring of thin cells is visible in the micropylar endosperm and surrounding an operculum which is pushed open by the expanding radicle to complete germination. The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion. The activity of endo-[beta]-mannanase, the major enzyme responsible for cell wall weakening, increases only after emergence of the radicle, and hence plays no role in germination. The gene for the transcription factor 'GAMYB,' a component of the GA transduction pathway which responds to the hormone, was studied during tomato seed germination. Two identified 'GAMYB'-like genes show 83% amino acid similarity within their binding domain to barley, rice and 'Arabidospsis GAMYB' and 'GAMYB'-like genes. In 'gib-1' mutant (non-GA producing) and wild-type seeds; expression increases during germination and declines thereafter. While ' LeGAMYBL1' transcripts are present in 'gib-1' mutant seeds imbibed in water and they are upregulated by GA during germination. However, dissected 'gib-1' embryos germinate when imbibed in either water or GA, with almost no difference in the amount of its mRNAs during this event. This is indicative that neither GA nor expression of the ' LeGAMYBL1' gene is necessary for embryo germination, but that they are involved in the germination of the intact seed. The germination-inhibiting hormone ABA does not affect expression of this gene in the intact seed. Information on the genes and transcripts for endo-[beta]-mannanase in tomato is clarified, particularly with reference to 'LeMAN3,' which was previously known only as a gene sequence, and 'LeMAN5' was identified as being 'LeMAN2.' The 'Le'GAMYBL1 protein binds to the promoter region of 'LeMAN2' and may affect the expression of the latter at the post-transcriptional level. Overexpression lines of 'LeGAMYBL1' in the 'gib-1' plants exhibit a phenotype of compact dark-green leaves.

A variety of cysteine proteinases are implicated in plant programmed cell death, although their function in this process is not completely understood. Here, the cDNAs of two novel cysteine proteinases termed 'SLCysEP' and 'SLCYSPRO' were cloned and found to be members of the C1A superfamily. SLCysEP is somewhat unique as it contains a C-terminal KDEL motif that mediates retrieval of peptides to the ER. Both 'SLCysEP' and 'SLCYSPRO' were found to be single copy genes containing three introns each. Recombinant proteins expressed in 'E. coli' showed 'in vitro' proteolytic activity characteristic of C1A proteinases. Recombinant SLCysEP underwent self-hydrolysis when subjected to an acidic pH; recombinant SLCYSPRO did not. During stamen development, sporophytic cells surrounding the locules and within the stomium undergo programmed cell death, as do endospermic cells of the seed following germination. The ' SLCysEP' transcript and protein were detected at high levels in stamens of pre-dehiscent flower buds but not in any other floral whorl, and the ' SLCYSPRO' transcript and protein were detected in the endosperm of germinating seeds. SLCysEP was localized strictly to the stomium and the middle layer and endothecial cells surrounding the locules of pre-dehiscent anthers, and immunogold labeling with anti-SLCysEP antibodies, coupled with electron microscopy, localized the protein to endoplasmic reticulum-derived precursor protease vesicles.

Using 'Vicia faba' as a model system to study programmed cell death (PCD) in plants, the objectives of this thesis were to study the ultrastructure of pith cells undergoing PCD, and to characterize the genomic sequence of 'vfcyspro' by identifying the 5' end of the transcript unit, the presence of introns, and the promoter region. Hollow pith formation in 'Vicia faba' was found to be ultrastructurally similar to aerenchyma formation in 'Zea mays' root cortex, suggesting that PCD events are common among plant organs and species. Molecular characterization of 'vfcyspro' identified the transcriptional start site to be 50 bp upstream of the translational start codon, determined the presence of three introns within the genomic sequence, and sequenced and cloned the upstream regulatory region of 'vfcyspro '. This promoter region, which was able to drive expression of green fluorescent protein in protoplasts, included several motifs that implicate its involvement in PCD.

The root of dandelion ('Taraxacum officinale' Weber) contains a prominent 18kD protein, which was previously thought to have a storage function. Since the characteristics of proteins can be determined from their sequence and expression of their genes, an isoform of the 18kD protein was purified and its N-terminal amino acid sequence determined. Using this information oligonucleotide primers were constructed and a full-length cDNA sequence obtained using RACE-PCR. The deduced full-length amino acid sequence of the protein showed that it has no signal peptide, it lacks serine, glutamine and tryptophan, has a pI of 5.6, two potential glycosylation and eight potential phosphorylation sites, and has a molecular mass of 16.911 kD. The protein shows high homologies with a group of allergen and intracellular pathogenesis-related proteins with which it shares the common motif GXGGXG. It has two isoforms that are 85% homologous and three gene copies. Although the 18kD protein is present throughout the year exclusively in the dandelion root and subterranean stem, with a small decline in amount in the summer, expression of its gene in these regions is greatly increased in the fall. This expression is affected by environmental perturbations involving temperature change, such as cold shock (20° to 5°C) and heat shock (5° to 20°C). Leaf wounding and defoliation at 5°C also stimulate both gene expression and protein synthesis, but not at 20°C. Thus the proteins may be more involved in a cold response than in having a storage role, and the signal to initiate gene expression may be through a systemic transduction pathway between the leaf and root. Although they share homology to some pathogenesis-related proteins fungal elicitor treatments of root discs do not cause an increase in gene expression.

A cDNA library of germinated V. faba seeds was successfully constructed. From the three largest and overlapping positive clones a cDNA encoding a full length cysteine pre-pro-proteinase was constructed (GenBank access number: U59465). The cDNA contains a full coding region, a 15-base pair $5\sp\prime$-end untranslated region, 334-base $3\sp\prime$-untranslated region and a 19-base poly(A) tail. To my knowledge this the first cDNA coding for a cysteine proteinase constructed from the mRNA of V. faba L. The deduced full length cysteine pre-pro-proteinase was designated as VFCYSPRO. As in other reported cysteine proteinase zymogens, VFCYSPRO contains a N-terminal signal peptide sequence (18 amino acids), a pro-protein region (112 amino acids) and a mature proteinase portion (232 amino acids). The mature protein belongs to the well-characterized papain superfamily although it shares only 37% identity with papain. A 3-D model of the mature enzyme of VFCYSPRO was generated by aligning VFCYSPRO with known protein structures and confirmed this classification. The pro-protein and partial mature enzyme or VFCYSPRO were successfully synthesized in bacterial host E. coli and purified. Antibodies were raised to the fusion proteins and purified to be specific to VFCYSPRO using a simple, inexpensive and reliable method. VFCYSPRO expression is under temporal or developmental control, rather than tissue specific regulation. It is expressed not only in germinated seeds but also in developing leaves, shoots, roots and pod walls. It is not expressed in developing seeds. That both the mRNA and the protein are expressed in the same pattern indicates that the protein content and activity are controlled at the transcriptional level. The phytohormone ethylene significantly promotes the expression of the mRNA for VFCYSPRO, while neither ABA nor GA has a stimulating role. Based on the fact that both VFCYSPRO mRNA and protein are expressed in seeds at post-germinated stages (when most storage proteins, globulins, have been mobilized), in senescing pods, and in the vascular tissues, it is speculated that besides involvement in degradation of storage proteins in seeds, VFCYSPRO plays a role for cell content turnover during programmed cell death.