P-proteins are structurally distinct proteins present in the sieve element-companion cell complexes of phloem tissue. Genomic clones encoding the two major P-proteins, the phloem filament protein (PP1) and the phloem lectin (PP2), were isolated and characterized. To understand mechanisms that control phloem-specific expression of these two genes, approximately 1 kb of the 5' flanking region from PP1 and PP2 genomic clones were fused with the GUS reporter gene and introduced into tobacco plants using Agrobacterium tumefaciens-mediated gene transfer. For both promoters, histochemical staining detected GUS activity specifically in the phloem tissue that was most easily detected in stems followed by midrib, secondary veins, roots, and leaf lamina of transgenic tobacco plants. GUS activity directed by the PP2 promoter was approximately 23 times greater than GUS activity directed by the PPI promoter. A nested set of 5' deletions between nucleotides -1014 and +32 were constructed to localize cis-elements that specify the patterns of PP2 gene expression in the phloem. Deletions within this region revealed that nucleotides -228 to +32 relative to the transcription initiation site contained sufficient information to direct phloem-specific gene expression, while positive regulators of promoter activity appeared to be located upstream of nucleotide -621. Mutation of a conserved 13-bp sequence, TTAAAAGAAGATA, found in the minimal PP2 promoter did not affect reporter gene expression. Sucrose responsive elements were identified in the PP2 promoter that could contribute to increased promoter activity in response to sugar. Finally we initiated studies to construct a phloem-specific promoter that could be induced by wound released compounds such as ethylene. Although not conclusive, our results suggest that it is possible to enhance phloem-specific expression in response to ethylene.

A general paradigm in semi-arid and arid systems is that woody plants with dimorphic root systems will exhibit preferential use of deeper soil water because it represents a more stable source of water than short duration pulses of shallow soil moisture derived from summer rainfall. However, whether this holds across all woody species and whether use of deeper soil water interacts with use of shallow soil water is not determined for many species in different ecosystems. Understanding the amount of water plants derive from groundwater and shallow soil water is critically important to accurate calculations of local and regional water balance. The focus of this research was to determine if dominant woody species in semi-arid riparian ecosystems used shallow soil water and how depth to groundwater and defoliation might affect root proliferation and water uptake. This research found that the functional grouping "phreatophytes" encompasses a variety of responses to environmental variation. Stable isotopic analyses determined that Prosopis velutina Woot. (Velvet mesquite) and Populus fremontii Wats. (Fremont cottonwood) used shallow soil water derived from summer rainfall, and the proportion of shallow soil water was higher at sites with greater depth to groundwater. In contrast Salix gooddingii Ball (Goodding willow) did not use shallow soil water at any location regardless of depth to groundwater. Field experiments using defoliation treatments, to limit carbon assimilation and reduce plant photosynthate pools, confirmed that Prosopis velutina exhibited flexible response in water uptake patterns in response to defoliation. Defoliation, which presumably reduced available photosynthate, increased the reliance of this species on shallow soil water; contrary to predictions that woody species should maintain extensive deep root systems to buffer themselves from seasonal drought. Greenhouse experiments with Populus fremontii and Prosopis velutina also indicated changes in belowground biomass of fine roots, which were associated with changes in water-source use for Populus fremontii, but not for Prosopis velutina. These results imply that in terms of predicting plant response to changes in future climates, or modeling fluxes of water from the soil to the atmosphere that are largely controlled by plant transpiration, intra- and interspecific variability will need to be considered.

K⁺ contributes most to the osmotic pressure in plant cells. Under saline conditions, accumulation of Na⁺ disturbs the ion homeostasis and causes toxicity to the cell. Potassium channels and transporters may mediate Na⁺ entry, yet K⁺ contents must be maintained at a certain level. Regulation of potassium transport under salt stress is important for ion homeostasis and stress tolerance in plants. This dissertation presents analyses of the potassium channels and transporters in the halophyte Mesembryanthemum crystallinum (common ice plant) emphasizing their regulation in the context of ion homeostasis under salinity conditions. Short-term changes of ion content in leaf tissues were measured for salt stressed ice plants. Na⁺ was found to interfere with potassium uptake. Net potassium uptake was reduced at least 50% after a few hours. Potassium contents in the ice plant roots and leaves are known to be reduced after long term salt stress. The cDNAs of three potassium channels MKT1, MKT2 and KMT1, three HAK-type transporters McHAK1-3, and a HKT-type transporter McHKT1 were isolated from the ice plant. McHAK1, McHAK2 and McHKT1 were able to complement a yeast mutant defective in potassium uptake. MKT1 transcripts were mainly detected in roots; MKT2, KMT1 and McHKT1 were expressed primarily in shoots. The McHAK transcripts were found in various tissues. Expression patterns of the channel and transporter transcripts under salt stress were investigated by RNA blot analysis and RT-PCR. In roots, transcripts of the MKT1 channel were reduced significantly after 6 h of stress, while the transcripts of McHAK1, McHAK2 and McHAK3 increased till 24 h of stress. In leaves, McHKT1 was transiently up-regulated within hours of stress and decreased rapidly afterward, but the McHAK1--3 transcripts increased up to 48 h of treatment. The transcripts of KMT1 channel showed transient up-regulation. The tissue-specific expression and coordinated regulation of these genes suggested that upon salt stress, the K⁺ uptake mechanism in the ice plant switches from the MKT1 channel to other systems such as the McHAK and McHKT1 transporters. Physiological relevance of these changes to the salinity adaptive strategy in the ice plant is discussed.

This study was designed to explore correlations between flower and seed characters and the relationship of flower and seed characters to yield. Thirty six lines selected from Composite Cross XXXII material were planted in a randomized block design with four replications. At anthesis, spikes from each plot were collected and fixed in 70% alcohol. At the same time, an equivalent spike was tagged to be harvested at maturity. Measurements of flower characters were made on the preserved spikes and seed. Length, width and weight were obtained on the mature spikes. Flower volume was calculated by multiplying the area of lemma and palea by flower thickness. Correlations indicated that selection for flower characters at anthesis would be an effective selection technique for seed characters.

sHSPs are induced at elevated temperatures in virtually all organisms and they are believed to help the cell survive heat stress, although the mechanism by which this occurs is unclear. Recently sHSPs have been proposed to function as a type of molecular chaperone, acting to prevent irreversible aggregation of other protein substrates. To increase our understanding of the properties of these proteins, I have studied two members of the sHSP family. The first part of the research presented here focuses on the chloroplast-localized sHSP, HSP21, and the second part focuses on the Saccharomyces cerevisiae cytosolic sHSP, HSP26. Plants synthesize several classes of small (15-30 kD monomer) heat shock proteins (sHSPs) in response to heat stress, including the nuclear-encoded chloroplast-localized HSP21. I examined the native structure and phosphorylation of chloroplast HSP21 to understand this protein's basic properties and to compare it to cytosolic sHSPs. Cytosolic sHSPs exist as large oligomers (∼200-800 kD) composed solely or primarily of sHSPs, and phosphorylation of mammalian sHSPs causes oligomer dissociation, which appears to be important for regulation of sHSP function. Initial studies confirmed that, similar to the cytosolic sHSPs, the apparent size of native HSP21 was >200 kD, but in contrast to mammalian cytosolic sHSPs, HSP21 did not dissociate during heat stress. Furthermore, I found no evidence that HSP21 or the plant cytosolic sHSPs are phosphorylated in vivo. A partial HSP21 complex purified from heat stressed Pisum sativum leaves contained no proteins other than HSP21. Mature recombinant pea and Arabidopsis thaliana HSP21 were expressed in E. coli, and purified recombinant Arabidopsis HSP21 assembled into homo-oligomeric complexes with the same apparent molecular weight as HSP21 complexes observed in heat stressed leaf tissue. In total the data indicate that the native, functional form of chloroplast HSP21 is a large, oligomeric complex containing ≥9 HSP21 subunits, and that plant sHSPs are not regulated by phosphorylation-induced dissociation. In the second half of my research I examined the ability of S. cerevisiae cytosolic HSP26 to interact with model protein substrates in vitro. HSP26 was first purified to greater than 95% homogeneity from a yeast strain engineered to overexpress HSP26. The protein behaved as a large homo-oligomer with an estimated size of 625 kD, comprising ≥24 subunits. HSP26 was shown to form stable complexes with model protein substrates and to prevent thermally induced aggregation of the proteins. In addition, HSP26 was able to maintain a thermally inactivated substrate in a conformation which could subsequently be refolded and reactivated in reticulocyte lysate. These characteristics of HSP26 are consistent with sHSPs having a role in protecting other proteins from permanent thermal damage during heat stress.

Although the protective effects of polyols against environmental stress have been demonstrated, mechanisms through which protection is accomplished are unknown. In this dissertation, the potential functions of sugar polyols in osmotic stress protection have been investigated. The function of mannitol as a hydroxyl radical scavenger in plant cells has been tested by using a transgenic plant approach. The presence of mannitol in transgenic plant cells enhanced the hydroxyl radical scavenging capacity and protected phosphoribulokinase from oxidative inactivation. Transgenic plants showed increased resistance to methylviologen (MV)-induced oxidative stress, as documented by increased retention of chlorophyll in transgenic leaf tissue following MV treatment. In addition, mesophyll cells from transgenic plants exhibited higher CO₂ fixation than wild type. It was concluded that mannitol localized in chloroplasts can supplement endogenous radical scavenging mechanisms and reduce oxidative damage of cells by hydroxyl radicals. The role of polyols in osmotic adjustment was evaluated in yeast. A bacterial mannitol-1-phosphate dehydrogenase gene and an apple sorbitol-6-phosphate dehydrogenase gene were introduced into a glycerol-deficient yeast mutant. The presence of sorbitol and mannitol in transformants provided remarkable protection against salt stress. However, this protection was much less than the protection provided by the same concentration of glycerol in the transformants of glycerol-3-P dehydrogenase gene (GPD1). The reduced protection by mannitol and sorbitol suggested that osmotic adjustment by glycerol was either not sufficient for acquisition of salt tolerance or that glycerol had specific functions for which mannitol and sorbitol could not substitute. To understand the role of glycerol in salt tolerance, salt-tolerant suppressor mutants were isolated from the glycerol-deficient mutants. One such suppressor mutant, sr13, partially suppressed the salt-sensitive phenotype of the glycerol-deficient mutant, most likely, due to the double amount of K⁺ accumulated under salt stress. The accumulation of K⁺ and extrusion of Na⁺ in sr13 were not inhibited by a calcineurin inhibitor (FK506), suggesting SR13 may function downstream of the calcineurin signaling pathway or in a separate pathway that regulated ion homeostasis under salt stress.

One-carbon (C₁) metabolism. C₁ metabolism is central to all organisms, because C₁ units have essential roles in biosyntheses of basic materials for living cells, such as protein, nucleic acids, choline and its derivatives. One unique feature of plant C₁ metabolism is that it channels significant C₁ flux from primary carbon metabolism to methylated metabolites. Part I of this dissertation presents functional analysis of plant methyane-THF reductases (MTHFRs) in Arabidopsis and maize, and regulation of the plant C₁ metabolic pathway and glycine betaine (GlyBet) biosynthesis in maize GlyBet near isogenic lines (NILs). Plant MTHFRs were isolated from Arabidopsis and maize and functionally characterized in yeast. Unlike mammalian MTHFRs, the plant enzymes strongly prefer NADH to NADPH and are not inhibited by S-adenosyl-methionine. An NADH dependent MTHFR reaction could be reversible in the cytosol, supported by radiotracer labeling data using [methyl-¹⁴C]methyltetrahydrofolate. Systematic analyses of profiling transcript abundance of plant C₁ genes using microarrays indicated that the regulation of the plant C₁ metabolic pathway may be "fine-tuned", or at a downstream "outlet" point in maize. Comparison of GlyBet NILs through analyses of metabolites, [¹⁴C]formaldehyde labeling and northern blotting of phosphoethanolamine methyltransferase (P-EAMT) indicated that the regulation glycine betaine biosynthesis is at P-EAMT, whose transcript is probably repressed by its product, phospho-choline. Global gene responses to salt stress. To understand how genes respond to salt stress is very important for the genetic improvement of salt tolerance in plants. Part II of this dissertation presents a systematic analysis of global gene responses to salt stress in maize roots, using microarrays, that contained ∼8,000 ESTs. Microarray expression analysis revealed that 916 different ESTs were up- or down-regulated under salt stress (611 up-regulated, 305 down-regulated), representing 11% of ∼8,000 ESTs printed on slides. These up- or down-regulated ESTs were clustered into various subgroups based on repression or induction of the timing, amplitude and duration of their transcripts. Contig analysis assigned 916 up- or down-regulated ESTs into 472 tentative unique genes (51.6%). These responsive genes are involved in a broad range of cellular processes, biochemical pathways and signal transduction cascades etc. The functions of some regulated genes are discussed.

During maize endosperm development, the cell cycle in the majority of cells switches from a mitotic to an endoreduplication cell cycle. This results in cells of varying ploidies within the tissue, and is presumed to be a factor in its rapid growth. Investigating the inheritance of variation in endoreduplication in maize endosperm will begin to elucidate the genetic mechanisms controlling it. It has been hypothesized that retinoblastoma-related proteins (RRBs) negatively regulate the G1/S transition during both mitotic and endoreduplication cell cycles. Testing this hypothesis in both mitotic cells and endoreduplicating endosperm cells will further our understanding of the molecular mechanisms regulating endoreduplication. Flow cytometry was used to assess the variability of endoreduplication in endosperms of maize inbred lines. High levels of endoreduplication were observed in popcorns relative to Midwestern dent corns. To study the genetic regulation of endoreduplication, four inbreds were crossed to B73 and developing endosperms from parental, reciprocal F1, and backcross generations were subjected to flow cytometric analysis. Maternal zygotic effects, often considered a form of parental imprinting, and maternal sporophytic effects were detected. To test the feasibility of introgressing a high endoreduplication phenotype into a Midwestern dent inbred line, a backcross population was generated using B73 as the reciprocal parent and the popcorn Sg18. The heritabilities calculated from an analysis of the backcross population generally agree with the values calculated in the larger crossing experiments. The Wheat Dwarf Virus RepA protein binds RRBs and is predicted to activate the cell cycle. RepA and the maize RRB, ZmRb1, were tested for cell cycle regulatory activity in tobacco BY-2 cells and determined to be an activator and repressor, respectively. The effect of RepA on endoreduplication was evaluated in both mitotically-active maize callus cultures and developing endosperms. Flow cytometric measurements of nuclear ploidy showed that RepA expression was sufficient to convert a mitotic into an endoreduplication cell cycle in calli but had no discernable effect on endopolyploidy in developing endosperm by 18-days after pollination (DAP).

Studies in this thesis concern expression of one class of small HSPs (sHSPs) in field grown desert plant species and the isolation of a new HSP gene encoding an sHSP targeted to plant mitochondria. Expression of class I, cytosolic sHSPs was assessed in three desert species: Screwbean Mesquite, Baja Fairyduster, and Sweet Acacia. Total leaf protein, and if available, flower and pod protein, was extracted from samples and analyzed by SDS-PAGE and Western blotting. Sweet Acacia showed strong sHSP expression in leaves with an apparent diurnal pattern of increased expression in the hotter PM. Screwbean Mesquite pods showed significant sHSP expression, which was not correlated to temperature. The isolation and sequence analysis of a gene encoding a mitochondrion-localized sHSP from Arabidopsis was completed. Comparisons to other plant sHSPs verified it was most similar to other mitochondrial-localized sHSPs from plants.

The measurement of sap flow has been sought after for many years. Various methods have been devised to accomplish this task, one of which is the heat balance method. This method is non-invasive and accurate, but its simplifying assumptions were questionable and needed to be critically examined. This study evaluated the heat balance method and sap flow gauges. The method yielded satisfactory results when compared to the calibration system. The satisfactory results were over a limited range, which exemplified the necessity for the gauges to be calibrated. The heat balance method's simplified heat transfer analysis does not reflect the complexity of the physical situation. Sap flow gauge improvements were suggested.