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.

Alternative oxidase (AOX) is a non-energy conserving branch of the mitochondrial electron transport chain (ETC) which has been hypothesized to modulate the level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plant mitochondria. The aim of the research presented herein is to provide direct evidence in support of this hypothesis and to explore the implications of this during plant-pathogen interactions in Nicotiana tabacum. We observed leaf levels of ROS and RNS in wild-type (Wt) tobacco and transgenic tobacco with altered AOX levels and we found that plants lacking AOX have increased levels of both NO and mitochondrial O2 - compared Wt plants. Based on the results we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of electrons from the ETC to O2 or nitrite. We characterized multiple responses of tobacco to different pathovars of the bacterial pathogen Pseudomonas syringae. These included a compatible response associated with necrosis (pv tabaci), an incompatible response that included the hypersensitive response (HR) (pv maculicola) and an incompatible response that induced defenses (pv phaseolicola). We show that the HR is accompanied by an early mitochondrial O2 - burst prior to cell death. Also, we found iii that the appearance of HR and the appearance of the mitochondrial O2 - burst are delayed in transgenic plants lacking AOX. A similar delay is seen in transgenic plants treated with the complex III inhibitor antimycin A. In Wt plants, expression of Aox1a is suppressed during the HR response to pv maculicola despite the accumulation of signaling molecules known to induce Aox1a transcription. Also, MnSOD activity declined during the HR. We suggest that the mitochondrial ROS burst controlled by AOX and MnSOD is an important component for the induction and coordination of the HR during plant-pathogen interactions.

The C3 photosynthetic pathway uses light energy to reduce CO2 to carbohydrates and other organic compounds and is a central component of biological metabolism. In C3 photosynthesis, CO2 assimilation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which reacts with both CO2 and O2. While competitive inhibition of CO2 assimilation by oxygen is suppressed at high CO2 concentrations, O2 inhibition is substantial when CO2 concentration is low and O2 concentration is high; this inhibition is amplified by high temperature and aridity (Sage 2004). Atmospheric CO2 concentration dropped below saturating levels 25-30 million years ago (Tipple & Pagani 2007), and the C4 photosynthetic pathway is hypothesized to have first evolved in warm, low latitude environments around this time (Christin et al. 2008a). The primary feature of C4 photosynthesis is suppression of O2 inhibition through concentration of CO2 around Rubisco. This pathway is estimated to have evolved almost 50 times across 19 angiosperm families (Muhaidat et al. 2007), a remarkable example of evolutionary convergence. In several C4 lineages, there are species with photosynthetic traits that are intermediate between the C3 and C4 states, known as C3-C4 intermediates. In two eudicot genera, Flaveria (Asteraceae) and Alternanthera (Amaranthaceae), there is evidence that these species represented an intermediate state in the evolution of the C4 pathway (McKown et al. 2005; Sanchez-del Pino 2009). The purpose of this thesis is to ascertain the specific benefits to plant carbon balance and resource-use efficiencies of the C3-C4 pathway relative to C3 species, particularly at low CO2 concentrations and high temperatures, factors which are thought to have been important in selecting for C3-C4 traits (Ehleringer et al. 1991). This will provide information on the particular advantages of the C3-C4 pathway in warm, often arid environments and how these advantages may have been important in advancing the initial stages of C4 evolution in eudicots. This thesis addresses the physiological intermediacy of previously uncharacterized C3-C4 species of Heliotropium (Boraginaceae); the water- and nitrogen-use efficiencies of C3-C4 species of Flaveria; and the photosynthetic performance and acclimation of C3, C4 and C3-C4 species of Heliotropium, Flaveria and Alternanthera grown at low and current ambient CO2 levels and high temperature.

Alternative oxidase (AOX) is a non-energy conserving branch of the mitochondrial electron transport chain (ETC) which has been hypothesized to modulate the level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plant mitochondria. The aim of the research presented herein is to provide direct evidence in support of this hypothesis and to explore the implications of this during plant-pathogen interactions in Nicotiana tabacum. We observed leaf levels of ROS and RNS in wild-type (Wt) tobacco and transgenic tobacco with altered AOX levels and we found that plants lacking AOX have increased levels of both NO and mitochondrial O2 - compared Wt plants. Based on the results we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of electrons from the ETC to O2 or nitrite. We characterized multiple responses of tobacco to different pathovars of the bacterial pathogen Pseudomonas syringae. These included a compatible response associated with necrosis (pv tabaci), an incompatible response that included the hypersensitive response (HR) (pv maculicola) and an incompatible response that induced defenses (pv phaseolicola). We show that the HR is accompanied by an early mitochondrial O2 - burst prior to cell death. Also, we found iii that the appearance of HR and the appearance of the mitochondrial O2 - burst are delayed in transgenic plants lacking AOX. A similar delay is seen in transgenic plants treated with the complex III inhibitor antimycin A. In Wt plants, expression of Aox1a is suppressed during the HR response to pv maculicola despite the accumulation of signaling molecules known to induce Aox1a transcription. Also, MnSOD activity declined during the HR. We suggest that the mitochondrial ROS burst controlled by AOX and MnSOD is an important component for the induction and coordination of the HR during plant-pathogen interactions.

The C3 photosynthetic pathway uses light energy to reduce CO2 to carbohydrates and other organic compounds and is a central component of biological metabolism. In C3 photosynthesis, CO2 assimilation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which reacts with both CO2 and O2. While competitive inhibition of CO2 assimilation by oxygen is suppressed at high CO2 concentrations, O2 inhibition is substantial when CO2 concentration is low and O2 concentration is high; this inhibition is amplified by high temperature and aridity (Sage 2004). Atmospheric CO2 concentration dropped below saturating levels 25-30 million years ago (Tipple & Pagani 2007), and the C4 photosynthetic pathway is hypothesized to have first evolved in warm, low latitude environments around this time (Christin et al. 2008a). The primary feature of C4 photosynthesis is suppression of O2 inhibition through concentration of CO2 around Rubisco. This pathway is estimated to have evolved almost 50 times across 19 angiosperm families (Muhaidat et al. 2007), a remarkable example of evolutionary convergence. In several C4 lineages, there are species with photosynthetic traits that are intermediate between the C3 and C4 states, known as C3-C4 intermediates. In two eudicot genera, Flaveria (Asteraceae) and Alternanthera (Amaranthaceae), there is evidence that these species represented an intermediate state in the evolution of the C4 pathway (McKown et al. 2005; Sanchez-del Pino 2009). The purpose of this thesis is to ascertain the specific benefits to plant carbon balance and resource-use efficiencies of the C3-C4 pathway relative to C3 species, particularly at low CO2 concentrations and high temperatures, factors which are thought to have been important in selecting for C3-C4 traits (Ehleringer et al. 1991). This will provide information on the particular advantages of the C3-C4 pathway in warm, often arid environments and how these advantages may have been important in advancing the initial stages of C4 evolution in eudicots. This thesis addresses the physiological intermediacy of previously uncharacterized C3-C4 species of Heliotropium (Boraginaceae); the water- and nitrogen-use efficiencies of C3-C4 species of Flaveria; and the photosynthetic performance and acclimation of C3, C4 and C3-C4 species of Heliotropium, Flaveria and Alternanthera grown at low and current ambient CO2 levels and high temperature.

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.