Magnesium (Mg2+) is an essential mineral nutrient for plants and is the most abundant free divalent cation in plant cells. However, our knowledge of the role of this ion in the plant cell is limited, and the mechanisms of homeostasis and transport of the ion are almost completely unknown. A. Tutone (this laboratory) identified an Arabidopsis thaliana gene by the complementation of a Mg2+-uptake yeast mutant (CM66). This gene, referred to as AtMRS2-11, was expressed as cDNA from a strong yeast promoter and allowed the growth of the CM66 yeast strain on standard media. The conceptually translated AtMRS2-11 protein sequence was used in this study to identify nine additional proteins by sequence homology searches using the BLAST algorithm. The corresponding genes have been cloned from cDNA (A. thaliana ecotype Landsberg erecta) and sequenced. Protein sequence similarity suggests that the family forms a sub-section of the CorA super-family of Mg2+ transport proteins. The mutant yeast used to identify the family initially was also used to show that two family members in addition to AtMRS2-11 were able to complement the Mg2+-dependent growth phenotype. When fused to eGFP, these proteins showed a localisation consistent with some of the protein reaching the yeast cell membrane. The other members of the family were also fused to eGFP and showed a range of localisation patterns within the yeast cell. None of the three AtMRS2 proteins previously able to complement the yeast mutant phenotype did so when fused to eGFP. RNA transcripts from the AtMRS2 family were detected by RT-PCR in organ-scale preparations of total RNA from A. thaliana. Most family members were detected in all of the organs tested. Northern analysis of AtMRS2-11 RNA transcript level showed that the gene was more highly expressed in leaf tissue, but was not affected by decreased levels of Mg2+ in the growth media. The levels of steady state AtMRS2-11 mRNA transcript were elevated two-fold in the light during the diurnal cycle, but no change was detected during light-induced greening of etiolated seedlings. A stable transgenic A. thaliana line expressing the gusA gene from the promoter region of AtMRS2-11 was used to localise the promoter's activity to cells containing chloroplasts. The expression appeared highest in younger cells. VI The AtMRS2-11 protein was predicted to contain a chloroplast targeting peptide. Western analysis demonstrated that AtMRS2-11 was enriched in the total proteins of isolated chloroplasts as compared to extracts from whole plants. The AtMRS2-11:eGFP fusion protein was also detected in chloroplasts by fluorescence microscopy. Flame atomic absorption spectroscopy was used in conjunction with isolated chloroplasts to try to determine the effects of the overaccumulation of the AtMRS2-11 protein in a transgenic A. thaliana plant line (constructed by A. Tutone). A rapid uptake or binding of Mg2+ was seen in chloroplasts isolated from both wild type and transgenic lines, but no differences were observed in either the rate of Mg2+ uptake/binding or the final Mg2+ content.

Magnesium (Mg2+) is an essential mineral nutrient for plants and is the most abundant free divalent cation in plant cells. However, our knowledge of the role of this ion in the plant cell is limited, and the mechanisms of homeostasis and transport of the ion are almost completely unknown. A. Tutone (this laboratory) identified an Arabidopsis thaliana gene by the complementation of a Mg2+-uptake yeast mutant (CM66). This gene, referred to as AtMRS2-11, was expressed as cDNA from a strong yeast promoter and allowed the growth of the CM66 yeast strain on standard media. The conceptually translated AtMRS2-11 protein sequence was used in this study to identify nine additional proteins by sequence homology searches using the BLAST algorithm. The corresponding genes have been cloned from cDNA (A. thaliana ecotype Landsberg erecta) and sequenced. Protein sequence similarity suggests that the family forms a sub-section of the CorA super-family of Mg2+ transport proteins. The mutant yeast used to identify the family initially was also used to show that two family members in addition to AtMRS2-11 were able to complement the Mg2+-dependent growth phenotype. When fused to eGFP, these proteins showed a localisation consistent with some of the protein reaching the yeast cell membrane. The other members of the family were also fused to eGFP and showed a range of localisation patterns within the yeast cell. None of the three AtMRS2 proteins previously able to complement the yeast mutant phenotype did so when fused to eGFP. RNA transcripts from the AtMRS2 family were detected by RT-PCR in organ-scale preparations of total RNA from A. thaliana. Most family members were detected in all of the organs tested. Northern analysis of AtMRS2-11 RNA transcript level showed that the gene was more highly expressed in leaf tissue, but was not affected by decreased levels of Mg2+ in the growth media. The levels of steady state AtMRS2-11 mRNA transcript were elevated two-fold in the light during the diurnal cycle, but no change was detected during light-induced greening of etiolated seedlings. A stable transgenic A. thaliana line expressing the gusA gene from the promoter region of AtMRS2-11 was used to localise the promoter's activity to cells containing chloroplasts. The expression appeared highest in younger cells. VI The AtMRS2-11 protein was predicted to contain a chloroplast targeting peptide. Western analysis demonstrated that AtMRS2-11 was enriched in the total proteins of isolated chloroplasts as compared to extracts from whole plants. The AtMRS2-11:eGFP fusion protein was also detected in chloroplasts by fluorescence microscopy. Flame atomic absorption spectroscopy was used in conjunction with isolated chloroplasts to try to determine the effects of the overaccumulation of the AtMRS2-11 protein in a transgenic A. thaliana plant line (constructed by A. Tutone). A rapid uptake or binding of Mg2+ was seen in chloroplasts isolated from both wild type and transgenic lines, but no differences were observed in either the rate of Mg2+ uptake/binding or the final Mg2+ content.

Magnesium (Mg2+) is an essential mineral nutrient for plants and is the most abundant free divalent cation in plant cells. However, our knowledge of the role of this ion in the plant cell is limited, and the mechanisms of homeostasis and transport of the ion are almost completely unknown. A. Tutone (this laboratory) identified an Arabidopsis thaliana gene by the complementation of a Mg2+-uptake yeast mutant (CM66). This gene, referred to as AtMRS2-11, was expressed as cDNA from a strong yeast promoter and allowed the growth of the CM66 yeast strain on standard media. The conceptually translated AtMRS2-11 protein sequence was used in this study to identify nine additional proteins by sequence homology searches using the BLAST algorithm. The corresponding genes have been cloned from cDNA (A. thaliana ecotype Landsberg erecta) and sequenced. Protein sequence similarity suggests that the family forms a sub-section of the CorA super-family of Mg2+ transport proteins. The mutant yeast used to identify the family initially was also used to show that two family members in addition to AtMRS2-11 were able to complement the Mg2+-dependent growth phenotype. When fused to eGFP, these proteins showed a localisation consistent with some of the protein reaching the yeast cell membrane. The other members of the family were also fused to eGFP and showed a range of localisation patterns within the yeast cell. None of the three AtMRS2 proteins previously able to complement the yeast mutant phenotype did so when fused to eGFP. RNA transcripts from the AtMRS2 family were detected by RT-PCR in organ-scale preparations of total RNA from A. thaliana. Most family members were detected in all of the organs tested. Northern analysis of AtMRS2-11 RNA transcript level showed that the gene was more highly expressed in leaf tissue, but was not affected by decreased levels of Mg2+ in the growth media. The levels of steady state AtMRS2-11 mRNA transcript were elevated two-fold in the light during the diurnal cycle, but no change was detected during light-induced greening of etiolated seedlings. A stable transgenic A. thaliana line expressing the gusA gene from the promoter region of AtMRS2-11 was used to localise the promoter's activity to cells containing chloroplasts. The expression appeared highest in younger cells. VI The AtMRS2-11 protein was predicted to contain a chloroplast targeting peptide. Western analysis demonstrated that AtMRS2-11 was enriched in the total proteins of isolated chloroplasts as compared to extracts from whole plants. The AtMRS2-11:eGFP fusion protein was also detected in chloroplasts by fluorescence microscopy. Flame atomic absorption spectroscopy was used in conjunction with isolated chloroplasts to try to determine the effects of the overaccumulation of the AtMRS2-11 protein in a transgenic A. thaliana plant line (constructed by A. Tutone). A rapid uptake or binding of Mg2+ was seen in chloroplasts isolated from both wild type and transgenic lines, but no differences were observed in either the rate of Mg2+ uptake/binding or the final Mg2+ content.

Magnesium (Mg2+) is an essential mineral nutrient for plants and is the most abundant free divalent cation in plant cells. However, our knowledge of the role of this ion in the plant cell is limited, and the mechanisms of homeostasis and transport of the ion are almost completely unknown. A. Tutone (this laboratory) identified an Arabidopsis thaliana gene by the complementation of a Mg2+-uptake yeast mutant (CM66). This gene, referred to as AtMRS2-11, was expressed as cDNA from a strong yeast promoter and allowed the growth of the CM66 yeast strain on standard media. The conceptually translated AtMRS2-11 protein sequence was used in this study to identify nine additional proteins by sequence homology searches using the BLAST algorithm. The corresponding genes have been cloned from cDNA (A. thaliana ecotype Landsberg erecta) and sequenced. Protein sequence similarity suggests that the family forms a sub-section of the CorA super-family of Mg2+ transport proteins. The mutant yeast used to identify the family initially was also used to show that two family members in addition to AtMRS2-11 were able to complement the Mg2+-dependent growth phenotype. When fused to eGFP, these proteins showed a localisation consistent with some of the protein reaching the yeast cell membrane. The other members of the family were also fused to eGFP and showed a range of localisation patterns within the yeast cell. None of the three AtMRS2 proteins previously able to complement the yeast mutant phenotype did so when fused to eGFP. RNA transcripts from the AtMRS2 family were detected by RT-PCR in organ-scale preparations of total RNA from A. thaliana. Most family members were detected in all of the organs tested. Northern analysis of AtMRS2-11 RNA transcript level showed that the gene was more highly expressed in leaf tissue, but was not affected by decreased levels of Mg2+ in the growth media. The levels of steady state AtMRS2-11 mRNA transcript were elevated two-fold in the light during the diurnal cycle, but no change was detected during light-induced greening of etiolated seedlings. A stable transgenic A. thaliana line expressing the gusA gene from the promoter region of AtMRS2-11 was used to localise the promoter's activity to cells containing chloroplasts. The expression appeared highest in younger cells. VI The AtMRS2-11 protein was predicted to contain a chloroplast targeting peptide. Western analysis demonstrated that AtMRS2-11 was enriched in the total proteins of isolated chloroplasts as compared to extracts from whole plants. The AtMRS2-11:eGFP fusion protein was also detected in chloroplasts by fluorescence microscopy. Flame atomic absorption spectroscopy was used in conjunction with isolated chloroplasts to try to determine the effects of the overaccumulation of the AtMRS2-11 protein in a transgenic A. thaliana plant line (constructed by A. Tutone). A rapid uptake or binding of Mg2+ was seen in chloroplasts isolated from both wild type and transgenic lines, but no differences were observed in either the rate of Mg2+ uptake/binding or the final Mg2+ content.

Magnesium (Mg2+) is an essential mineral nutrient for plants and is the most abundant free divalent cation in plant cells. However, our knowledge of the role of this ion in the plant cell is limited, and the mechanisms of homeostasis and transport of the ion are almost completely unknown. A. Tutone (this laboratory) identified an Arabidopsis thaliana gene by the complementation of a Mg2+-uptake yeast mutant (CM66). This gene, referred to as AtMRS2-11, was expressed as cDNA from a strong yeast promoter and allowed the growth of the CM66 yeast strain on standard media. The conceptually translated AtMRS2-11 protein sequence was used in this study to identify nine additional proteins by sequence homology searches using the BLAST algorithm. The corresponding genes have been cloned from cDNA (A. thaliana ecotype Landsberg erecta) and sequenced. Protein sequence similarity suggests that the family forms a sub-section of the CorA super-family of Mg2+ transport proteins. The mutant yeast used to identify the family initially was also used to show that two family members in addition to AtMRS2-11 were able to complement the Mg2+-dependent growth phenotype. When fused to eGFP, these proteins showed a localisation consistent with some of the protein reaching the yeast cell membrane. The other members of the family were also fused to eGFP and showed a range of localisation patterns within the yeast cell. None of the three AtMRS2 proteins previously able to complement the yeast mutant phenotype did so when fused to eGFP. RNA transcripts from the AtMRS2 family were detected by RT-PCR in organ-scale preparations of total RNA from A. thaliana. Most family members were detected in all of the organs tested. Northern analysis of AtMRS2-11 RNA transcript level showed that the gene was more highly expressed in leaf tissue, but was not affected by decreased levels of Mg2+ in the growth media. The levels of steady state AtMRS2-11 mRNA transcript were elevated two-fold in the light during the diurnal cycle, but no change was detected during light-induced greening of etiolated seedlings. A stable transgenic A. thaliana line expressing the gusA gene from the promoter region of AtMRS2-11 was used to localise the promoter's activity to cells containing chloroplasts. The expression appeared highest in younger cells. VI The AtMRS2-11 protein was predicted to contain a chloroplast targeting peptide. Western analysis demonstrated that AtMRS2-11 was enriched in the total proteins of isolated chloroplasts as compared to extracts from whole plants. The AtMRS2-11:eGFP fusion protein was also detected in chloroplasts by fluorescence microscopy. Flame atomic absorption spectroscopy was used in conjunction with isolated chloroplasts to try to determine the effects of the overaccumulation of the AtMRS2-11 protein in a transgenic A. thaliana plant line (constructed by A. Tutone). A rapid uptake or binding of Mg2+ was seen in chloroplasts isolated from both wild type and transgenic lines, but no differences were observed in either the rate of Mg2+ uptake/binding or the final Mg2+ content.

Master of Science / Department of Biology / Jesse B. Nippert / In 1902, President Theodore Roosevelt signed and enacted the Reclamation Act, which would fundamentally alter the lowland hydrology of the arid southwest over the next century. Flow regulations, groundwater pumping, damming, and river channel changes have led to decreases in water table heights and periodic overbank flooding, and subsequently, increased soil salinity in the arid Southwest. During this period, native riparian tree species have declined significantly and an invasive tree species, Tamarix ramosissima, has increased in abundance and distribution. Increases in soil salinity negatively impact the physiology of native riparian tree species, but the impacts of soil salinity on Tamarix physiology are incompletely known. I studied the impact of increasing soil salinities on the physiology of Tamarix in both field and controlled environments. I first studied the impacts of increasing soil salinities on Tamarix physiology at two semi-arid sites in western Kansas. I concluded that physiological functioning in Tamarix was maintained across a soil salinity gradient from 0 to 14,000 ppm illustrating robust physiological responses. Using cuttings from Tamarix trees at both sites, I subjected plants to higher NaCl concentrations (15,000 and 40,000 ppm). Tamarix physiology was decreased at 15,000 ppm and 40,000 ppm. Tamarix physiological functioning was affected at the induction of treatments, but acclimated over 30-40 days. These results reveal a threshold salinity concentration at which Tamarix physiological functioning decreases, but also illustrate the advantageous halophytic nature of Tamarix in these saline environments. Many arid and semi-arid environments are predicted to become more saline, however, results from both studies suggest that increasing salinity will not be a major barrier for Tamarix persistence and range expansion in these environments.

Master of Science / Department of Agronomy / Tesfaye Tesso / Cold temperature stress is an important abiotic constraint to grain sorghum production in temperate regions. In the United States, low temperature in late spring and early fall has limited sorghum production to a narrow growing period. Deployment of cold tolerance traits may widen this window and hence contribute to increased production. The objectives of this study were (1) to determine the effect of early and mid-season cold temperature stress on growth, phenology and yield components of sorghum, and identify key traits that are most sensitive to cold stress at seedling and flowering stages, and (2) to identify new sources of cold tolerance for use in breeding programs. Series of controlled environment (greenhouse/growth chamber) and field experiments were carried out. Three sorghum genotypes of variable response, Shan Qui Red (tolerant), SRN39 (susceptible) and Pioneer 84G62 (unknown) were subjected to cold (15/13ºC day/night) and normal (25/23ºC day/night) temperature at seedling (Experiment I) and flowering (Experiment II) stages. The genotypes were planted in a greenhouse using a 5L polytainer pots. Each pot consisted of a single plant and each plot was represented by three pots. A split-plot design with three replications was used in both experiments with temperature regimes as main plots and genotypes as sub-plots. Three days after emergence, experiment I plants were moved to the growth chamber and subjected to the designated temperature treatments. For experiment II, the treatments were assigned at heading stage immediately before anthesis had begun. The treatments lasted 10 d in both experiments. Data were collected on seedling characteristics and leaf chlorophyll content in experiment I, days to flowering, maturity, and yield components in both experiments, and anthesis duration in experiment II. For the field experiment, 150 sorghum germplasm collections of potential cold tolerance along with tolerant and susceptible checks were evaluated for emergence and seedling traits under early planting (April 13) at soil temperature of 20.1/13.4 ºC max/min. The normal temperature treatment was applied by planting at regular season (May 26) at soil temperature of 30.0/20.4ºC max/min. Twenty-four genotypes selected based on field emergence and seedling vigor were further screened under controlled environment. Early-season stress significantly reduced leaf chlorophyll content, all seedling traits (height, vigor and dry weight), and also delayed flowering and maturity. But it had no effect on final leaf number, plant height and yield components. Genotypic response to early stress was significant for all traits with the susceptible checks having the lowest score for all seedling traits. Mid-season cold stress prolonged anthesis duration, delayed maturity and highly reduced all yield components. Several genotypes among the 150 had higher seedling vigor and emergence than the tolerant check, Shan Qui Red. In conclusion, reduced seedling vigor as a result of early stress had no effect on final yield provided that stand establishment was not compromised while mid season stress is damaging to yield. The wide genetic variation for the traits indicates the potential for improvement of cold tolerance in sorghum.

Master of Science / Department of Biology / Jesse B. Nippert / The Konza Prairie in northern Kansas, USA contains over 550 vascular plant species; of which, few have been closely studied. These species are adapted to environmental stress as imposed by variable temperature, precipitation, fire, and grazing. Understanding which plant traits relate to drought responses will allow us to both predict drought tolerance and potential future shifts in plant community composition from changes in local climate. Morphological and physiological measurements were taken on 121 species of herbaceous tallgrass prairie plants grown from seed in a growth chamber. Gas exchange measurements including maximum photosynthetic rate, stomatal conductance to water vapor, and intercellular CO[subscript]2 concentration were measured. All plants were exposed to a drought treatment and were monitored daily until stomatal conductance was zero. At this point, critical leaf water potential (Ψ[subscript]crit), an indicator of physiological drought tolerance was assessed. Other measurements include root length, diameter, volume, and mass, leaf area, leaf tissue density, root tissue density, and root to shoot ratio. Traits were compared using pair-wise bivariate analysis and principal component analysis (PCA). A dichotomy was found between dry-adapted plants with thin, dense leaves and roots, high leaf angle, and highly negative Ψ[subscript]crit and hydrophiles which have the opposite profile. A second axis offers more separation based on high photosynthetic rate, high conductance rate, and leaf angle, but fails to provide a distinction between C[subscript]3 and C[subscript]4 species. When tested independently, grasses and forbs both showed drought tolerance strategies similar to the primary analysis. Matching up these axes with long term abundance data suggests that species with drought tolerance traits have increased abundance on Konza, especially in upland habitats. However, traits that relate to drought tolerance mirror relationships with nutrient stress, confounding separation of low water versus low nutrient strategies. My results not only illustrate the utility of morphological and physiological plant traits in classifying drought responses across a range of species, but as functional traits in predicting both drought tolerance in individual species and relative abundance across environmental gradients of water availability.

Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Dale J. Bremer / Jack D. Fry / Researchers and practitioners can use numerous techniques to measure or estimate evapotranspiration (ET) from turfgrass but little is known about how they compare to ET using standard lysimeters. An investigation was conducted to compare measurements of ET from lysimeters (LYS[subscript]E[subscript]T) with ET estimates from the FAO56 Penman-Monteith (PM[subscript]E[subscript]T) and Priestley-Taylor (PT[subscript]E[subscript]T) empirical models, atmometers (AT[subscript]E[subscript]T), eddy covariance (EC[subscript]E[subscript]T), and a canopy stomatal conductance model that estimates transpiration (COND[subscript]T). Methods were compared at the same site during the 2010, 2011, and 2012 growing seasons. Overall, PT[subscript]E[subscript]T and EC[subscript]E[subscript]T were not different from LYS[subscript]E[subscript]T, whereas PM[subscript]E[subscript]T, AT[subscript]E[subscript]T, and COND[subscript]T, increasingly underestimated LYS[subscript]E[subscript]T. Differences exist among ET measurement techniques and one should employ the technique that best fits their situation. An atmometer is an inexpensive tool that can be used to measure turfgrass ET within microclimates, such as those typically found in an urban home lawn. An investigation was conducted to compare AT[subscript]E[subscript]T estimates with PM[subscript]E[subscript]T estimates within a number of lawn microclimates. Home lawns in Manhattan and Wichita, KS, were selected for study during the growing seasons of 2010 and 2011. Open sward AT[subscript]E[subscript]T was 4.73 mm d[superscript]-[superscript]1, whereas PM[subscript]E[subscript]T was 5.48 mm d[superscript]-[superscript]1. Within microclimates, AT[subscript]E[subscript]T was 3.94 mm d[superscript]-[superscript]1 and PM[subscript]E[subscript]T 3.23 mm d[superscript]-[superscript]1. Atmometers can provide practitioners with reliable estimates of PM[subscript]E[subscript]T within microclimates. Zoysiagrass (Zoysia spp.) is a common turfgrass used on home lawns and golf courses. However, poor shade tolerance and cold hardiness have limited its use in the transition zone. A study was conducted to determine changes and differences in growth and physiology among selected Zoysia over a three-year period (2010-2012) in the transition zone. The genotypes were 'Emerald' [Z. japonica × Z. pacifica], 'Zorro' [Z. matrella], 'Meyer' and Chinese Common [Z. japonica], and experimental progeny Exp1 [Z. matrella × Z. japonica], and Exp2 and Exp3 [(Z. japonica × Z. pacifica) × Z. japonica]. 'Zorro' and 'Emerald' experienced winter injury. 'Meyer', Chinese Common, and Exp1 showed poor performance over the three-years. The Exp2 and Exp3 progeny, maintained high percent cover, visual quality, and tiller density, and may provide practitioners more shade-tolerant cultivar choices in the transition zone.

Master of Science / Agronomy / Mithila Jugulam / Common waterhemp (Amaranthus rudis) is a problematic weed species of cropping systems throughout the Midwestern states, including Kansas. Recently, waterhemp populations from Kansas were found to have evolved resistance to the widely used herbicide glyphosate as a result of amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzyme target of glyphosate. The objectives of this research were to 1) perform glyphosate dose-response study and determine the relationship between relative EPSPS genomic copies and EPSPS gene expression in glyphosate-resistant waterhemp, and 2) characterize the genomic configuration and distribution of EPSPS copies using florescence in situ hybridization (FISH) in three glyphosate-resistant waterhemp populations. Waterhemp populations from eastern Kansas were screened with 868 g ae haˉ¹ (field used rate) of glyphosate, and genomic DNA and total RNA was isolated from the survivors to determine the EPSPS genomic copies and EPSPS gene expression relative to the acetolactate synthase (ALS) gene using qPCR. Furthermore, waterhemp specific EPSPS probes were synthesized to perform florescence in situ hybridization (FISH) on these glyphosate-resistant plants. Results of these experiments indicate a positive correlation between level of glyphosate resistance, EPSPS copies, and their expression. As expected, a negative correlation was found between shikimate accumulation and EPSPS copies. Sequencing of the EPSPS gene showed no presence of the proline 106 mutation, which is known to be associated with glyphosate resistance suggesting that an insensitive EPSPS enzyme was not involved in the mechanism of glyphosate resistance. FISH analysis of resistant plants illustrated presence of amplified EPSPS copies on two homologous chromosomes, likely near the centromeric region. . This is the first report demonstrating a positive relationship between EPSPS copies and expressions, as well as chromosome configuration of EPSPS copies in glyphosate- resistant waterhemp from Kansas.