Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Sunghun Park / Plants are continuously exposed to numerous abiotic stresses, which adversely affect plant growth, development, and yield. Plants have developed different signaling pathways to cope with abiotic stresses, and some of the pathways converge to help plants tolerate simultaneous stresses. Here, we report ectopic expression of an Arabidopsis glutaredoxin AtGRXS17 that confers tolerance to multiple abiotic stresses in tomato plants. In yeast assays, AtGRXS17 co-localized with yeast ScGrx3 in the nucleus and suppressed the sensitivity of yeast grx3grx4 double mutants to oxidative stress and heat shock. In plants, GFP-AtGRXS17 fusion proteins initially localized in the cytoplasm but migrated to the nucleus during heat stress. Ectopic expression of AtGRXS17 in tomato plants minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane systems under heat stress. Furthermore, expression of the heat shock transcription factor (HSF) and heat shock protein (HSP) genes was up-regulated in AtGRXS17-expressing tomato plants during heat stress when compared to wild-type controls. Under cold, drought, and oxidative stress conditions, AtGRXS17-expressing tomato plants also displayed more vigorous growth and less physiological damage than those of the wild-type control plants. Quantitative real-time PCR (qRT-PCR) analysis indicated that expression of AtGRXS17 alters multiple stress defense signaling pathways, including the Abscisic Acid (ABA) and C-Repeat Binding Factors (CBF) pathways. The results revealed a conserved function for a glutaredoxin protein in abiotic stress adaptation, and manipulation of AtGRXS17 may be a useful approach to improve crop stress tolerance and understand plant signaling under abiotic stress conditions. Deregulated expression of an Arabidopsis H[superscript]+/Ca[superscript]2[superscript]+ antiporter (sCAX1) in agricultural crops increases total calcium (Ca[superscript]2[superscript]+) but may result in yield loses due to calcium-deficiency like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca[superscript]2[superscript]+ binding protein located at endoplasmic reticulum) in sCAX1-expressing plants mitigated these adverse effects while maintaining enhanced Ca[superscript]2[superscript]+ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca[superscript]2[superscript]+ transporters and binding protein at different intracellular compartments can alter the content and distribution of calcium within the plant matrix.

Master of Science / Department of Biology / Jyoti Shah / Plant defense responses are constitutively activated in the Arabidopsis thaliana ssi2 mutant plant. In addition, the ssi2 mutant allele confers a dwarf phenotype. The SSI2 gene encodes a stearoyl-ACP-desaturase, which converts stearic acid (18:0) to oleic acid (18:1), suggesting a role for lipids in plant defense. Microarray analysis identified several genes which encode putative acyl hydrolases/lipases that are expressed at elevated levels in the leaves of ssi2, in comparison to the wild type plant. One gene in particular, At5g14180, was expressed at 60-fold greater level in ssi2 than in the wild type plant. To study the involvement of At5g14180 in plant defense and lipid metabolism, two transgenic lines containing T-DNA insertions within the At5g14180 gene were identified. These two T-DNA insertional alleles of the At5g14180 gene attenuate the ssi2-conferred heightened resistance to a virulent strain of Pseudomonas syringae pv. maculicola in the ssi2 At5g14180 double mutant plant. Furthermore, pathogen growth was enhanced in the At5g14180 single mutant plants, as compared to the wild type plant. Profiling of lipid composition in leaf tissue identified changes in the lipid composition between the At5g14180 mutant and wild type plants, suggesting that the At5g14180 encoded protein may impact lipid metabolism in Arabidopsis leaves.

Master of Science / Department of Agronomy / William T. Schapaugh Jr / Crops research moving forward faces many challenges to improve crop performance. In breeding programs, phenotyping has time and economic constraints requiring new phenotyping techniques to be developed to improve selection efficiency and increase germplasm entering the pipeline. The objectives of these studies were to examine the changes in spectral reflectance with soybean breeding from 1923 to 2010, evaluate band regions most significantly contributing to yield estimation, evaluate spectral reflectance data for yield estimation modeling across environments and growth stages and to evaluate the usefulness of spectral data as an optimized phenotyping technique in breeding programs. Twenty maturity group III (MGIII) and twenty maturity group IV (MGIV) soybeans, arranged in a randomized complete block design, were grown in Manhattan, KS in 2011 and 2012. Spectral reflectance data were collected over the growing season in a total of six irrigated and water- stressed environments. Partial least squares and multiple linear regression were used for spectral variable selection and yield estimation model building. Significant differences were found between genotypes for yield and spectral reflectance data, with the visible (VI) having greater differences between genotypes than the near-infrared (NIR). This study found significant correlations with year of release (YOR) in the VI and NIR portions of the spectra, with newer released cultivars tending to have lower reflectance in the VI and high reflectance in the NIR. Spectral reflectance data accounted for a large portion of variability for seed yield between genotypes using the red edge and NIR portions of the spectra. Irrigated environments tended to explain a larger portion of seed yield variability than water-stressed environments. Growth stages most useful for yield estimation was highly dependent upon the environment as well as maturity group. This study found that spectral reflectance data is a good candidate for exploration into optimized phenotyping techniques and with further research and validation datasets, may be a suitable indirect selection technique for breeding programs.

Doctor of Philosophy / Department of Agronomy / Scott A. Staggenborg / Grain sorghum yield has notably increased from the beginning of hybrid production and commercialization in the late 1950s. The yield increases were the result of improved agronomic practices and hybrid advancement. The objectives of my research were: (1) to determine the magnitude of yield change in the hybrid era in irrigated and rain fed sorghum production, (2) to determine the contribution of agronomic and hybrid changes for yield in the hybrid era, (3) to investigate changes in sorghum morphology, physiology, and water use that contributed to yield increases, (4) to investigate changes that accompanied yield increase with hybrid improvement, and (5) to understand sorghum water and nutrient use and variations between hybrids in these regards. Fifty-two years of grain sorghum hybrid performance trial data (1957-2008), were analyzed and greenhouse and field studies were conducted on five selected hybrids to meet our objectives. The greenhouse and field studies were conducted from the summer of 2007 to the fall of 2009 on five selected hybrids, each representing a decade from the past fifty years. Results indicated that there was an increase in hybrid yield of nearly 50 kg ha-1 yr-1 in dryland sites over the 52 yrs (1957-2008) analyzed. Irrigated grain sorghum yields, however, remained unchanged over the same period. Agronomic practices such as planting date, phosphorus fertilizer use, and planting density changed over these years but were not found to contribute to increased dryland sorghum yields. There was no difference found between old and new hybrids tolerance to different densities. Hybrid advancement and increased nitrogen fertilizer application were responsible for changes in dryland yields. Total water use changed with hybrid advancement. New hybrids used the greatest total water and also had greater root-to-total biomass ratio than the old hybrids. Leaf biomass was also greater for the newest hybrid. There was a difference in amount of total nutrients extracted by hybrids, and there were differences among hybrids in allocation of nutrients to different tissues. In general the yield focus of sorghum hybrid development was effective in dryland sorghum production, likely because of intentional or inadvertent selection of hybrids with better drought tolerance. Results indicated that breeding programs created hybrids with improved morphological characteristics that might have resulted in better resource use (water and nutrient) and ultimately increased yield.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Lawrence C. Davis / Synthetic dyes are capable of producing the whole color spectrum on account of their structural diversity but this diversity poses challenges in the degradation of dyeing wastes. Laccases and peroxidases from bacterial or fungal sources and parts of plants in the presence of hydrogen peroxide (H₂O₂) plus a mediator have been exploited in the bioremediation of synthetic dyes. However, intact plants have not found much favor despite their phytoremediation potential. The goal of this research was to further clarify ways by which whole plants bring about decolorization of different types of synthetic dyes. Hydroponically cultivated plants from two dicot families namely Arabidopsis thaliana and sunflowers (Helianthus annuus) were exposed to representative dyes from several classes: monoazo (Methyl Red and Methyl Orange), disazo (Trypan Blue, Evans Blue and Chicago Blue 6B), and arylmethane (Brilliant Blue G, Bromocresol Green, Malachite Green and Phenol Red). Tests were done in presence or absence of externally added H₂O₂, with or without a free radical mediator, 1-hydroxybenzotriazole, using UV-Visible spectrophotometry. The initial rate of decolorization and the overall percentage decolorization was calculated for each dye in the different treatments. Decolorization of the dyes from different classes varied between plant species and depending on the treatment. Except for Methyl Red, all dyes required added H₂O₂ as well as mediator to achieve rapid decolorization. Added H₂O₂ was found to be the limiting factor since it was degraded by plants within a few hours. Both species were able to slowly decolorize dyes upon daily addition of fresh dye even in the absence of added H₂O₂ and mediator, provided that nutrients were supplied to the plants with the dye. A. thaliana was found to be more effective in dye decolorization per gram tissue than sunflower when treated under similar conditions. Analysis of the residual dye solution by ESI/MS did not reveal any potential by-products following the decolorization treatment with plants, suggesting that the plant roots might be trapping the by-products of dye decolorization and preventing their release into the solution. All these findings support the potential application of whole plants for larger scale remediation.

Doctor of Philosophy / Horticulture, Forestry, and Recreation Resources / Sunghun Park / Abiotic stresses, including chilling and drought stresses, are considered to be major limiting factors for growth and yield of agricultural and horticultural crops. One of the inevitable consequences of abiotic stresses is the accumulation of reactive oxygen species (ROS) in plants. ROS can either act as an alarm signal to induce the defense pathway when kept at a low level or cause oxidative damage to various cellular components when increased to a phytotoxic level. Glutaredoxins (GRXs) are members of ROS scavenging system that can maintain the cell redox homeostasis by using the reducing power of glutathione. In this research, we characterized the roles of GRXs in protecting tomato (Solanum lycopersicum) from chilling stresses and rice (Oryza sativa L.) from drought stresses. Our results indicated that ectopic expression of an Arabidopsis gene AtGRXS17 in tomato could enhance the chilling tolerance by increasing antioxidant enzyme activities and reducing H₂O₂ accumulation to ameliorate oxidative damage to cell membranes and photosystems. Furthermore, AtGRXS17-expressing tomato plants had increased accumulation of soluble sugars to protect plant cells from dehydration stress. In rice, silenced expression of a rice glutaredoxin gene OsGRXS17 was used as a reverse-genetic approach to elucidate the roles of OsGRXS17 in drought stress tolerance. Our results showed that silenced expression of OsGRXS17 conferred improved tolerance to drought stress in rice. ABA-mediated stomatal closure is an important protection mechanism that plants adapt to a drought stress conditions, and H2O2 acts as secondary messenger in ABA signaling to induce the stomatal closure. Silenced expression of OsGRXS17 gave rise to H₂O₂ accumulation in the guard cells and promoted ABA-mediated stomatal closure, resulting in reduced water loss, higher relative water content, and consequently enhanced drought tolerance in rice. This research provides a new perspective on the functions of GRXs in chilling and drought stress tolerance of tomato and rice, and an important genetic engineering approach to improve chilling and drought stress tolerance for other crop species.

Doctor of Philosophy / Department of Agronomy / Clenton E. Owensby / Most rangelands are harvested at some point during the year and removal of plant leaf area and biomass alters a host of ecosystem processes including gas exchange. An experiment was conducted in 2005 and 2006 to study the effects of clipping tallgrass prairie at different dates on water vapor and CO2 fluxes. A portable, non-steady-state chamber was designed to measure CO2 and water vapor fluxes from small plots in less than 40 s. A combination of sunlit and shaded readings allowed measurements of net carbon exchange (NCE) and ecosystem respiration (RE); by summing NCE and RE, gross canopy photosynthesis (GCP) was calculated. Throughout the two-year study, the chamber had a minimal effect on microclimate, i.e., average chamber temperature increased 2.9° C, while chamber pressure increased only 0.3 Pa during measurements, and photosynthetically active radiation attenuation was 10%. The immediate effect of all clipping treatments was a loss of leaf area that led to reductions in GCP, NCE, and RE and in most cases decreased water vapor flux. Further patterns of carbon flux were governed by the amount of water stress during canopy development, while water vapor flux rates varied with water availability. Canopies that developed during periods of low water stress quickly increased carbon flux rates following precipitation after a mid-season drought. However, flux rates of canopies, which developed during the mid-season drought, responded considerably slower to subsequent water availability. A separate experiment was conducted from June-October of 2006 to estimate GCP, leaf area index (LAI), and total aboveground biomass with a hyperspectral radiometer. Indices such as the Normalized Difference Vegetation Index and the Simple Ratio were used to estimate LAI and biomass had poor correlations with measured values. However, GCP was significantly correlated to all six indices derived in this study. While GCP measured from June-October was significantly correlated with all indices, removal of the senesced canopy scans recorded during October greatly increased the relationship.

Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Channa B. Rajashekar / Studies were conducted to examine the role of antioxidants in adaptation of lettuce (Lactuca sativa L.) to unprotected environmental conditions and various environmental stresses. Antioxidants, in addition to being a plant defense mechanism, are phytochemicals that provide significant health-promoting and nutritive value in human diet. Various approaches involving mild environmental stresses and cultural and management practices have been used to enhance the quality of this commonly consumed leafy vegetable by improving its phytochemical and health-promoting attributes. Lettuce plants grown in protective environments adapt to unprotective environmental conditions by activating antioxidant genes such as phenylalanine ammonia-lyase (PAL), gamma-tocopherol methyl transferase (gamma-TMT), and L-galactose dehydrogenase (L-GalDH) involved in the biosynthesis of phenolic compounds,alpha-tocopherol and ascorbic acid, respectively. Mild environmental stresses such as heat shock, chilling, water stress and high light also activate these genes leading to the accumulation of secondary metabolites and phytochemicals without any adverse effect on biomass accumulation. The phytochemicals included chlorogenic acid, chicoric acid, caffeic acid, quercetin-3-O-glucoside and luteolin-7-O-glucoside. However, under field conditions, application of drought stress did not produce consistent results with regard to the phytochemical composition of lettuce. Plants grown in open field have higher phenolic content and higher antioxidant capacity than those grown in high tunnel. However, these plants also had less biomass accumulation. Many factors such as plant age, variety, fertilization, transplanting shock affected the phytochemical composition of lettuce. The red leaf variety 'Red Sails' had higher antioxidant capacity than 'Baronet' and younger plants had the highest phytochemical content. With regard to the management practices, there was no significant difference in phytochemical composition between organically and conventionally managed crops. Also, low fertility favored the accumulation of phytochemicals and increased the antioxidant capacity. Thus, the results show that mild environmental stresses along with appropriate cultural and management practices can enhance the quality of lettuce by improving their phytochemical composition.

Master of Science / Department of Agronomy / P. V. Vara Prasad / In many countries producers have been growing varieties of hard white winter (HWW) wheat since decades. The cause of concern is most varieties of HWW wheat are susceptible to pre-harvest sprouting (PHS) which affects grain quality. Environmental conditions like high humidity, precipitation, heavy dew and hormonal activity at physiological maturity stimulate PHS in HWW. To alleviate these conditions research was carried out at KSU. KS01HW163-4, a sprouting tolerance line was crossed with Heyne, a sprout susceptible cultivar. A total of 224 doubled haploid (DH) lines thus produced were phenotyped in the present study through experiments conducted in controlled environments. The objectives of this research were to (i) characterize and phenotype the doubled haploid lines for PHS in controlled environments; (ii) understand the impact of growth environment (high temperature and/or drought) and; (iii) impact of exogenous application of growth hormones on tolerance to pre-harvest sprouting in the parental lines of the doubled haploid population. The phenotypic data collected from this research will be ultimately combined with the genotypic data to identify DNA markers related to PHS tolerance and provide DNA markers for marker assisted selection. Based on my results of the germination percentages from the 224 DH lines, the population was distributed as susceptible, and tolerant to PHS showing a bimodal distribution and X[superscript]2 analysis indicating a complimentary gene action. From the study of the influence of environmental factors on PHS, my results confirmed a definite influence of stress on sprouting. Under optimum temperature (OT), KS01HS163-4 was tolerant to PHS, but at HT and/or drought it became susceptible to PHS. Growth under stressed conditions changed the tolerance levels to PHS. Seed dry-weight, and harvest index were also influenced negatively due to stress. Therefore multi-location tests must be conducted with variable environments to test the stability of a variety to PHS. From the study of the influence of phytohormones on PHS, the results suggest that tolerance to sprouting was seen in seeds from plants sprayed with abscisic acid (ABA) and paclobutrazol (GA-inhibitor) treatments where as those from gibberellic acid (GA) treatment showed susceptibility to sprouting.

Master of Science / Department of Agronomy / Robert M. Aiken / Mary B. Kirkham / Transpiration efficiency (TE) is defined as total biomass produced per unit of water transpired. Improvement of TE means maximizing crop production per unit of water used. The objectives of the study were to examine, at the leaf level and the whole plant level, the variation in TE for sorghum [Sorghum bicolor (L.) Moench] accessions, previously screened for TE and to test physiological mechanisms that may account for differences in TE. Three field studies and two mini-lysimeter studies (one done in pots under greenhouse conditions and one done in pots in the field) were conducted with eight accessions. Instantaneous measurements of assimilation (A), stomatal conductance (gs), and transpiration by gas exchange provided measures of the transpiration efficiency at the leaf level. Growth observations and soil water balance in field plots quantified components of whole-plant TE. Growth and development measurements showed significant difference, explaining the existence of photoperiod sensitivity among the sorghum genotypes. Assimilation (A), stomatal conductance (gs), and maximum quantum efficiency of photosystem II (Fv/Fm) were consistently greater for accession PI533946 (from India) and greater for accession PI295121 (from Australia) in both field and the field-pot studies (p<0.05). Internal carbon dioxide (Ci), an indicator of intrinsic transpiration efficiency, differed among lines under field conditions (p<0.05). Leaf relative water content (RWC), measured in the greenhouse, and did not differ among the eight accessions. No consistent differences in biomass and water use were detected among lines under field conditions. In conclusion, developing reliable selection indices for TE will require a greater understanding of whole-plant physiological processes to utilize the differences in TE observed at the leaf level.