Protein-bound and free lysine contributed to the total lysine content of maize endosperm, and both of these can be significantly increased by the opaque-2 (o2) mutation. Elongation factor 1A is one of the lysine-rich proteins increased in o2 mutants, and its concentration is highly correlated with the protein-bound lysine content of the endosperm. However, the biological basis of this correlation is unknown. The mechanism by which the free amino acid content, including free lysine, is increased by the o2 mutation is also poorly understood. Understanding the biological basis of these traits could provide new insights for improving maize nutritional quality. A maize genetic linkage map consisting of 83 DNA simple sequence repeat markers was created for two inbred lines (Oh51Ao2 and Oh545o2) that differ in elongation factor 1A and free amino acid content. Quantitative trait locus analysis was performed and identified two significant loci that accounted for 25% of the variance for elongation factor 1A content. One of them was linked with a cluster of 22-kD α-zein genes on the short arm of chromosome 4; the other locus was on the long arm of chromosome 7 and may be linked to the 27-kD γ-zein genes. Quantification of protein and mRNA levels of the major storage proteins suggested that a higher level of α-zein gene expression co-segregates with higher elongation factor 1A content. Furthermore, measurement of protein body size and density predicted a greater protein body surface area (80% higher) in Oh51Ao2 than Oh545o2, and this may partially explain the higher level of elongation factor 1A in Oh51 Ao2 by creating a more extensive cytoskeletal network. Quantitative trait locus analysis of free amino acid content identified four loci accounting for about 46% of the variation for this trait. One locus on the long arm of chromosome 2 is tightly linked to monofunctional aspartate kinase and a bifunctional aspartate kinase-homoserine dehydrogenase gene. Biochemical characterization of these enzymes indicated the aspartate kinase in Oh545o2 is less sensitive to lysine than that in Oh51 Ao2. Consequently, aspartate kinase 2 is the more promising gene involved in this quantitative locus.

This research was conducted to determine the potential for using plants to obtain samples of boron isotopes from groundwater, sod moisture and fracture water. In essence, this work sought to ascertain whether plants can function as in-situ samplers for boron as an environmental isotope. At present, very little is known about the behavior of boron isotopes in plants so this study was designed to reveal any isotopic fractionation that might occur during plant uptake by a specific species under carefully controlled conditions. The relationship between the boron isotope ratios sequestered in the leaves of the species Atriplex canescens and the growth conditions of the plant were investigated using a semi-hydroponic greenhouse experiment. Nutrient boron concentration and solution pH were selected as experimental variables as these parameters span large ranges in nature. In addition, the mechanism through which plants take up anionic nutrients suggests that boron isotope fractionation could occur. The experimental setup was a randomized factorial block design and the plants were provided six different nutrient solutions with pH values ranging from 7.5 to 9.5 and boron concentrations varying from 0.1 mg/L to 10.0 mg/L. Boron concentration in the plant's leaf and stem samples followed expected patterns, with the highest boron amount in the leaves of the plants fed nutrient solution with 10.0 mg/L B. The stern samples of plants fed 0.1 mg/L B contained the least boron. The ratio of boron in plants fed 10.0 mg/L vs. 0.1 mg/L B was far less than the 100:1 ratio of boron in the nutrients, which implies that a component of uptake is actively controlled by the plant. Negative thermal ionization mass spectrometry was employed to analyze the minute amounts of boron extracted from digests of the plant tissues. Statistical tests were utilized to determine that, contrary to the hypothesis, no significant isotopic fractionation occurred during uptake at any treatment pH level. The results of this research indicate that the species Atriplex canescens can provide samples of boron isotopes which closely represent the isotopic signature of the plant's water source.

acase@tulane.edu

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0102. Adviser: Jeffrey D. Palmer. "Title from dissertation home page (viewed Feb. 21, 2007)."

An essential component of development is the accumulation of specific metabolites in a temporal and tissue-specific manner. The growth regulator abscisic acid (ABA), which accumulates at a specific time during seed development, is required for seed maturation and prevents the premature developmental switch from dormancy to germination ABA accumulates differently in two tissues of the seed; levels in the embryo are several-fold higher than in the endosperm and the temporal accumulation of ABA is also different between these tissues. To begin to understand how ABA accumulation is regulated during seed development, the regulation of ABA biosynthesis was investigated. The approach taken was to examine the expression of the biosynthetic enzyme, phytoene desaturase (PDS), which catalyzes a regulated step in ABA synthesis in several other organisms (Bramley, 1985, Sandmann et al., 1989, Hugueney et al., 1992 and Giuliano et al., 1993). Unlike ABA accumulation, PDS transcript and protein levels were higher in the endosperm than in the embryo. The spatial difference in PDS levels did correlate with levels of the pathway intermediate, beta-carotene, suggesting that PDS may control the synthesis of ABA precursors while subsequent enzymes may regulate ABA accumulation. The temporal expression of Pds was also unrelated to ABA accumulation. In the endosperm, transcript levels were initially high and declined during desiccation while protein levels remained high throughout development. In the embryo, transcript levels were low and constant while protein levels declined. There are several maize mutants (viviparous mutants) disrupted in ABA biosynthesis, resulting in decreased levels of ABA and premature germination. Analysis of the Pds allele and transcript in the viviparous-5 mutant showed that the gene contains multiple insertions and deletions, giving rise to a larger transcript. In addition, the 55 kDa PDS protein was not detected in the vp5 mutant by immunoblot analysis, indicating that the vp5 phenotype results from a mutation at the PDS locus. To determine whether the wild type protein encoded by the ABA mutant, vp2, or the pathway intermediate, lycopene, regulate PDS, transcript and protein levels were compared in wild type and mutant (vp2 and vp7, respectively) seeds. The levels of PDS were not significantly different in vp2 or vp7 wild type and mutant seeds, suggesting that neither the VP2 protein nor lycopene regulate PDS at the steady-state transcript or protein level.

This dissertation addresses how small subunit (S) of higher plant Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC4.1.1.39) might influence Rubisco function. Toward this analysis a pea RbcS 3A cDNA expression cassette was introduced into Arabidopsis thaliana Landsberg by Agrobacterium tumefaciens mediated transformation. Analysis of RNA blots and 2-D gels indicate pea RbcS 3A is expressed and the S protein product is transported into Arabidopsis chloroplasts, processed and assembled into a stable chimeric holoenzyme. Incorporation of only one pea S per Arabidopsis Rubisco was sufficient to allow biochemical analyses. Biochemical analyses determined that chimeric enzymes displayed lower carboxylase activity (Vc) than WT Arabidopsis Rubisco coincident and consistent with the amount of pea S present in holoenzymes. Lower Vc is likely the result of reduced carbamylation following activation. Enhancement of Vc following temperature treatment at 42°C is kinetic evidence of increased activity of active sites which not due to differences in carbamylation. Unlike wild-type Rubisco, chimeric enzymes do not display the expected increase in carboxylase activity following activation at 42°C. This indicates S plays a role in allowing increased activity of neighboring L. Thus, both carbamylation and activity are disrupted by the interaction of pea S and Arabidopsis L. Kinetic data and formulae offered here support a structural model whereby S influences activity by allowing S-dependent interaction between L active sites. Also, high-temperature treated Rubisco shows a more pronounced fallover. This suggests that 42°C caused changes within Rubisco which either increase the synthesis of inhibitors or the response to inhibitors. To enhance abundance of pea S relative to Arabidopsis S pea S expressing plants were transformed with oligo-antisense cassettes targeting the 5'UTR and transit peptide of endogenous Arabidopsis RbcS transcripts. These doubly transformed plants were grown on media with 3% sucrose to cause metabolite repression which can further reduce endogenous Arabidopsis RbcS expression. However, both antisense and metabolite repression reduce the amount of pea S relative to Arabidopsis S protein. Genetic crosses between independently transformed plants expressing pea S suggest that expression of different amounts of pea S can be achieved.

One of the major challenges for maize improvement is to enhance the protein quality of the endosperm. We do not know what genes encode the proteins that contribute most to the protein quality, but lysine is the most limiting amino acid. One way to determine the proteins made in the endosperm is by isolating the genes expressed in this tissue and identifying their function. The results of the maize genome project with etiolated seedling and endosperm cDNA libraries support the basis of this strategy, in that the putative functions of a large number of cDNA sequences were identified through sequence similarity comparison and genomic Southern analyses. One of the genes I selected for further analyses is the elongation factor 1 alpha (eEF1A) gene family. eEF1A interacts with many cellular components and is therefore classified as a multifunction protein. In addition, its level is highly correlated with the amount of protein-bound lysine in maize endosperm. Even though many proteins are known to interact with eEF1A, the basis for the relationship between eEF1A and endosperm lysine content is not known. Transcript level and sequence of different members of the maize eEF1A gene family were analyzed in this work. All the eEF1A maize genes examined had GTP, aminoacyl tRNA, eEF1B and actin binding domains. The substitution of glutamic acid for aspartic acid in a region that has been shown to be important for actin binding in eEF1Aa, eEF1Ae and eEF1Af suggest that there might be two groups of eEF1A genes that bind differently to actin. Analysis of transcript levels demonstrated that different members of the eEF1A gene family are expressed at different levels in the tissues examined. The results from the analyses of transcript levels demonstrated that most of the maize eEF1A gene family members are expressed and their mRNAs vary in different tissues and in developing endosperm. Physiological differences were also determined for the two most abundant eEF1As members by the yeast two-hybrid system. This research provides a significant step toward understanding eEF1A functions in maize and why this protein correlates with the lysine content of the endosperm.

Greenhouse and field experiments were conducted to determine how proximity factors and water stress interact to influence competition between purple nutsedge and upland cotton. Purple nutsedge produced more total dry weight than cotton in wet conditions but produced less or similar total dry weight in dry conditions. Cotton's ability to extract water from greater soil depth and maintain a high rate of photosynthesis during water stress enabled it to maintain higher RGR (relative growth rate), LAR (leaf area ratio), and leaf expansion than nutsedge. Absolute growth rate (AGR), initial propagule weight, and early shoot production were important parameters for purple nutsedge competition with cotton. Moisture stress affected the relative importance of intraspecific and interspecific competition between species in both greenhouse and field experiments. intraspecific competition was more important than interspecific competition in determining cotton biomass production in wet conditions but drier conditions further reduced the relative importance of interspecific competition. The results of all experiments indicate that greenhouse addition series competition experiments can be applicable to field conditions provided the experimental design takes into account the biological characteristics of the species being studied. Pot size had a large influence on intra- and interspecific competition between purple nutsedge and cotton. In greenhouse experiments, physiological measurements were initiated one hour after irrigation and repeated every 2 h throughout the day. One hour after the cessation of water stress, the photosynthetic rates of both species increased, but photosynthesis recovered faster in cotton than in purple nutsedge. The faster recovery of photosynthesis in cotton was probably due to the osmotic adjustment that occurred in cotton leaves that protected enzymes and other cellular components during water stress. In additive field experiments, seed cotton yield was reduced because the number of harvestable bolls m⁻¹ was reduced as purple nutsedge density increased. Yield was also reduced by cotton seedling death at the highest nutsedge density. The interference of purple nutsedge with cotton cannot be reduced through water management alone. But, based on the growth characteristics of indeterminate cotton varieties, we suggest that delaying the first post-planting irrigation of some cotton varieties could reduce the competition of purple nutsedge with cotton without affecting final seed cotton yield.

Aphis gossypii Glover (cotton-melon aphid) is a major pest of agriculture worldwide. Cucumis melo L. (melon) possesses monogenic resistance to this aphid, and is a good model for the study of aphid resistance mechanisms in plants. This dissertation presents analyses of the effects of the resistance gene on A. gossypii, and of the gene's effects on biochemical and molecular-genetic properties of melon plants. Nearly isogenic lines (NILs) of melon, either resistant or susceptible to A. gossypii, were compared for their influence on aphid life history traits and feeding behavior. The resistance trait delayed development, increased mortality, and markedly decreased reproduction of aphids confined to leaves of resistant plants. Aphids on resistant plants salivated into phloem sieve elements significantly longer, and were less likely to begin sap ingestion after salivation, suggesting that the resistance factor acts within phloem sieve elements. Biochemical properties of callose synthase were compared between NILs to test the hypothesis that callose deposition plays a role in the resistance mechanism. No differences were detected between resistant and susceptible melon genotypes with respect to callose synthase subunit abundance or in vitro enzyme activity. Sixty-four F₃ families from a melon mapping population were tested for aphid resistance to place the resistance locus on a genetic map of the melon genome. Four molecular markers were found to be linked to the aphid resistance phenotype. The name Agr ( Aphis gossypii resistance) is proposed for this locus. The closest flanking markers were positioned at 4.3 and 7.0 cM from Agr. Evidence suggests Agr might be a member of the nucleotide binding site-leucine-rich repeat (NBS-LRR) family of plant resistance genes, which are known to cluster in plant genomes. Melon genomic DNA sequences homologous to this gene family were isolated to test the hypothesis that Agr is an NBS-LRR homolog. Two of these sequences were tested for genetic linkage to Agr in a population of F₂ plants segregating for the resistance trait. DNA gel blot analysis determined that one sequence, NBS-2, is approximately 2.7 cM distant from Agr, which suggests Agr resides in a cluster of NBS-LRR homologs and could be a member of this gene family.

The goal of this dissertation was to analyze the regulation and function of cytoplasmic class I small heat shock proteins (sHSPs) during seed development in Arabidopsis thaliana. Results show that two class I sHSPs accumulate in late seed maturation, persist in the dry seed and decline rapidly during germination. HSP17.4 accounts for 90% of total class I sHSP in the dry seed. The temporal pattern of sHSP accumulation during seed development suggests that HSP17.4 may help establish seed properties that are acquired during late seed maturation, such as dormancy or desiccation tolerance. Several mutants with reduced seed dormancy were determined to accumulate wild type levels of HSP17.4, however, all desiccation intolerant seeds analyzed had decreased levels of HSP17.4. Thus, HSP17.4 reduction correlates with desiccation intolerance. In total, these data suggest that HSP17.4 is not sufficient for seed dormancy and that it may be necessary for desiccation tolerance. The localization and regulation of HSP17.4 were examined in developing Arabidopsis seeds by transforming plants with hsp17.4 promoter fused to the β-glucuronidase (GUS) gene. HSP17.4::GUS expression was detected in the cotyledons early in seed development and eventually throughout the embryo. Arabidopsis embryos showed a much different pattern of HSP17.4::GUS expression in response to heat indicating distinct mechanisms regulate sHSP transcription during heat shock and during development. To analyze seed specific transcriptional activator regulation of HSP17.4 transcription, HSP17.4::GUS transgenic plants were crossed to seed transcriptional activator mutants. Results showed aberrant localization of HSP17.4::GUS in fus3-3 and lec1-2 seeds and negligible levels in abi3-6. These results strongly implicate AB13 in the transcriptional regulation of HSP17.4. To analyze more specifically HSP17.4 function, transgenic antisense technology was used to suppress hsp17.4 expression to 30--50% of wild type. These lines exhibited a reduced dormancy phenotype as assayed by reduced sensitivity to germination on ABA and by the ability of fresh seed to germinate. These data provide insight into the localization, regulation and function of HSP17.4 during seed maturation. The seed-specific transcriptional activator ABI3 is implicated in controlling hsp17.4 expression during development. Overall, these results demonstrate the importance of HSP17.4 during seed maturation, and establish a role for sHSPs in dormancy.