Graduate

Plants can not escape from biotic and abiotic stress factors such as, extreme temperatures, high light intensity, drought, UV radiation, heavy metals, and pathogen attack. Plants have versatile defens systems against such stress conditions. In this study, the role of glutathione S-transferases (GSTs) in cold stress conditions were examined. Glutathione S-transferases are the enzymes that detoxify natural and exogenous toxic compounds by conjugation with glutathione. Glutathione, an endogenous tripeptide, is important as reducing agent, nucleophilic scavenger, and alleviate the chemical toxicity in the plants by the reaction of GSTs. Glutathione conjugates can be transported to the vacuoles or apoplast and are generally much less toxic than the parent compounds. In plants there are four distinct families of the soluble GSTs, namely Phi (F), Type I / Zeta (Z), Type II / Tau (U), Type III / Theta (T), Type IV. By contrast with the mammalian families of GST, relatively little is known about the plant GST families. Up to date, there is not any study on GST isolation and characterization from Turkish red pine, in this respect, this study well play a frontier role the future research dealing with this topic. In this study, some properties of Turkish red pine GST activity towards CDNB (1-chloro-2,4 dinitrobenzene) were examined. The average specific activity of Turkish red pine GST towards CDNB was found as 200&plusmn / 50 (Mean&plusmn / SE, n= 18) nmole/min/mg cytosolic protein. GSTs in cytosol prepared from Turkish red pine needles retained its activity without loss for four weeks at -80&amp / #61616 / C. The rate of conjugation reactions were linear up to 0.8mg of Turkish red pine cytosolic protein and 0.4 mg cytosolic protein was routinely used. The Turkish red pine GST showed its maximum activity at pH 8.0 in 25 mM phosphate buffer and 42 &amp / #730 / C. The measurements were carried out at room temperature (RT) of 25 &amp / #61616 / C. Turkish red pine GST seemed to be saturated at 1 mM CDNB and 1 mM GSH concentrations. The Vmax and Km values of Turkish red pine GST for CDNB was 416nmole/min/mg protein and 0,8 mM, respectively, and for GSH 106.4 nmole/min/mg protein and 0.10 mM, respectively. Turkish red pine cytosol was applied on DEAE-Sepharose fast flow column but almost no purification was achieved with respect GST activity. In order to examine the effects of cold stress on Turkish red pine GST activity, the GST activity was determined in 240 seedlings at &ndash / 3&amp / #61616 / , 0&amp / #61616 / and 13 &amp / #61616 / C environmental temperatures. It was observed that GST activity was the highest at -3&amp / #730 / C and the lowest at 13&amp / #730 / C in both cold resistant and sensitive families with the exception of Yaylaalan and &Ccedil / ameli.

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Indiana University-Purdue University Indianapolis (IUPUI) / Abiotic stresses, such as cold, are serious agricultural problems resulting in substantial crop and revenue losses. Soybean (Glycine max) is an important worldwide crop for food, feed, fuel, and other products. Soybean has long been considered to be cold-intolerant and incapable of cold acclimation. In contrast to these reports, this study demonstrates that cold acclimation improved freezing tolerance in the domestic soybean cultivar ‘Williams 82’ with 50% enhancement of freezing tolerance after 5.2 +\- 0.6 days of cold exposure. Decreases in light dependent photosynthetic function and efficiency accompanied cold treatment. These decreases were due to an increase in photon dissipation likely driven by a decrease in plastoquinone (PQ) pool size limiting electron flow from photosystem II (PSII) to photosystem I (PSI). Cold-induced damage to operational photosynthesis began at 25 minutes of cold exposure and maximal photosynthesis was disrupted after 6 to 7 hours of cold exposure. Cold exposure caused severe photodamage leading to the loss of PSII reaction centers and photosynthetic efficiency. Comparisons of eight cultivars of G. max demonstrated a weak correlation between cold acclimation and northern cultivars versus southern cultivars. In the non-domesticated soybean species Glycine soja, the germination rate after cold imbibition was positively correlated with seedling cold acclimation potential. However, the overall cold acclimation potential in G. soja was equal to that of domestic soybean G. max reducing the enthusiasm for the “wild” soybean as an additional source of genetic diversity for cold tolerance. Despite being relatively cold intolerant, the soybean genome possesses homologs of the major cold responsive CBF/DREB1 transcription factors. These genes are cold-induced in soybean in a similar pattern to that of the cold tolerant model plant species Arabidopsis thaliana. In Arabidopsis, EIN3, a major component of the ethylene signaling pathway, is a negative transcriptional regulator of CBF/DREB1. In contrast to AtEIN3 transcript levels which do not change during cold treatment in Arabidopsis, we observed a cold-dependent 3.6 fold increase in GmEIN3 transcript levels in soybean. We hypothesized that this increase could prevent effective CBF/DREB1 cold regulation in soybean. Analysis of our newly developed cold responsive reporter (AtRD29Aprom::GFP/GUS) soybean transgenic lines demonstrated that inhibition of the ethylene pathway via foliar sprays (AVG, 1-MCP, and silver nitrate) resulted in significant cold-induced GUS activity. Transcripts of GmEIN3A;1 increased in response to ethylene pathway stimulation (ACC and ethephon) and decreased in response to ethylene pathway inhibition in the cold. Additionally, in the cold, inhibition of the ethylene pathway resulted in a significant increase in transcripts of GmDREB1A;1 and GmDREB1A;2 and stimulation of the ethylene pathway led to a decrease in GmDREB1A;1 and GmDREB1B;1 transcripts. To assess the physiological effects of these transcriptional changes; electrolyte leakage, lipid oxidation, free proline content, and photosynthesis were examined. Improvement in electrolyte leakage, a measure of freezing tolerance, was seen only under silver nitrate treatment. Only 1-MCP treatment resulted in significantly decreased lipid oxidation. Transcripts for CBF/DREB1 downstream targets (containing the consensus CRT/DRE motifs) significantly decreased in plants treated with ethylene pathway stimulators in the cold; however, ethylene pathway inhibition generally produced no increase over basal cold levels. To identify if GmEIN3A;1 was capable of binding to GmDREB1 promoters, the negative regulator GmEIN3A;1 and the positive regulator GmICE1A were cloned and expressed in Escherichia coli (E. coli). Preliminary binding results indicated that GmEIN3A;1 can bind to a double stranded section of the GmDREB1A;1 promoter containing putative EIN3 and ICE1 binding sites. GmICE1A is capable of binding to the same section of the GmDREB1A;1 promoter, though only when single stranded. Additional experiments will be required to demonstrate that GmEIN3A;1 and GmICE1A are capable of binding to the GmDREB1A;1 promoter and this work provides the tools to answer these questions. Overall, this work provides evidence that the ethylene pathway transcriptionally inhibits the CBF/DREB1 pathway in soybean through the action of GmEIN3A;1. Yet when GmCBF/DREB1 transcripts are upregulated by ethylene pathway inhibition, no consistent change in downstream targets was observed. These data indicate that the limitation in cold tolerance in soybean is due to a yet unidentified target downstream of CBF/DREB1 transcription.

Living at the northern limits of its geographic range, the Florida manatee is particularly susceptible to cold stress-related mortality during the winter months, with most deaths occurring in the lower two-thirds of the state. Contributing to this cold stress susceptibility is the manatee's limited physiological and behavioral responses available when thermally stressed. While capable of migrating south in response to falling water temperatures, manatees must still find warm water when ambient river temperature drops below 20°C for more than a few days. This is in part due to the species low metabolic rate, limited capacity for thermogenesis, and limited ability to raise its metabolic rate. Prolonged exposure to cold temperatures may result in cold stress syndrome, which involves a number of potentially life-threatening, if not fatal physiological changes. Survival during the winter months is therefore, dependent upon the manatee's ability to balance basic physiological needs, primarily the need to forage and to obtain fresh water with the need to stay warm. When identifying which animals are most susceptible and where, analyses of statewide manatee mortality records from 1996 through 2011 (n = 823) indicated that, size and location matter. Medium to large-sized calves accounted for the majority of documented death from cold stress (46.6%), while subadults and small calves were the least represented size classes (14.3 % and 9.5%, respectively). Adults slightly outnumbered subadults (15.8%). Males outnumbered females in all size classes but gender differences were not statistically significant. With regards to location, two areas of the state, the southwest and central east coasts showed the highest incidents of cold stress-related mortality. Both are regions with no primary, natural warm-water springs and whose principal warm-water refugia are power plant effluents. Brevard County on the central east coast is the area most at risk during cold weather events accounting for more than 25% of all cold stress deaths statewide. Warm-water sites within this region are few and relatively underrepresented in the literature in an area well-studied in terms of manatee abundance and distribution relative to the operational power plant. Results from cold stress data analyses emphasize the importance of identifying and characterizing the physical attributes of both known and suspected secondary warm-sites used by manatees in this region for both long and short term protection of the species, and its critical habitat. Three locations within Brevard County identified as passive thermal basins (PTBs), and classified as secondary warm-water sites, have been documented supporting in excess of 100 manatees on numerous occasions, and during winters of varying severity. Unique in physical appearance, distance to forage, hydrology including thermal profiles, and when it was used by manatees, each site challenged the accepted definitions and criteria of what constitutes an acceptable and appropriate warm-water site. Through analyses of photo-identification records, site fidelity at two of these warm-water sites, the Berkeley Canal and the Desoto Canal, was established for a minimum of 20 highly identifiable animals, 15 of which used adjacent sites within the same year, and 6 that used both sites but during different years. Observations of daily use patterns within the sites supported optimization of thermoregulation through adjustments in both vertical and horizontal movement, the latter of which seemed to follow the path of the sun. Manatees using the sites also made use of bottom sediment presumably to stay warm at all three locations. Temperature data indicated that water temperatures monitored in the sediment at secondary sites were some of the highest in the county. The predictable movements during all but the coldest weather fronts indicated that manatees utilized these sites during the early morning and afternoon hours when ambient river temperatures were coldest, gradually returning to the river to feed as ambient temperatures began to rise later in the afternoon. The availability of PTBs in proximity to primary warm-water sites within the region may provide an important component needed for manatees to successfully balance the need to forage with the need to stay warm by providing a network that allows for more efficient foraging while reducing exposure to sub-critical ambient river temperatures. The challenge of balancing the need to forage and to maintain homeostasis in the face of thermal stress is complex. This complexity was best approached and better understood through use of a manatee energetics model. The model was designed to facilitate simulation of an unlimited number of different case scenarios involving the exposure of virtually created manatees to a variety of winter conditions as might be experienced by real manatees in a natural system. Sixty-four different simulations were run using six virtual manatees of differing ages, gender, physical parameters, and knowledge of warm-water sites. Simulations were conducted using actual winter water temperature data from Brevard secondary sites and the ambient river from both a mild and a severe winter season. Outcomes, measured as changes in physical parameters indicative of body condition (i.e. mass, percent body fat, blubber depth, girths, etc.), showed that all else being equal, calves in the 2 year-old range fared poorly in all scenarios when compared to individuals of larger size. Subadults fared better than larger adults. This outcome illustrates the complex relationship between size, energy requirements and the synergistic effects of body mass, body fat and blubber thickness on SA:V ratio. Model outcomes agree closely with manatee cold stress mortality analyses predicting that medium to large-sized calves are most susceptible to CS, followed by adults, then subadults. Because all models are simplifications of complex systems, the manatee energetics model is not without its flaws and limitations. The current version of the model could not predict the point at which cold stress mortality would occur. However, a cold stress warning system incorporated into the design alerts the user if potential CSS is likely based on changing physical parameters. Another limitation was the inability of the model to account for the behavioral plasticity of individual subjects since virtual manatees respond to water temperatures based on the user defined rules. A number of additional limitations related to gaps in existing manatee data the gaps were identified and defined. Despite these gaps, the model is designed to allow for incorporation of additional interactions, feedback loops and relevant data as it becomes available and as additional physiological interactions and energy requirements are more clearly defined. Sensitivity analyses, a feature of the model that allowed for modifications in a number of physical as well as environmental parameters, provided an otherwise unlikely opportunity to see how incremental changes in input values, specifically the starting values for mass, percent body fat and blubber depth affected the model's outcome. Ultimately the goal of the model was to facilitate a better understanding of complex relationships by challenging our preconceived understanding of the manatee and its environment.

<p> As the effects of climate change become more evident, the development of effective strategies for improving livestock climatic adaptation and the long-term sustainability of animal food production have become key priorities around the world, including in the US. Together with nutrition, infrastructure, and management practices, genetically improving animals is an effective and lasting alternative to simultaneously improve productive efficiency and climatic adaptation of animals. Genetic improvement requires basic understanding of the genomic architecture of the indicator traits of interest and the availability of large-scale datasets. Understanding the role of evolution and selection (both natural and artificial) on shaping animal genomes is of paramount importance for the optimization of breeding programs and conservation of genetic resources. In addition, properly quantifying environmental stress and individual animal responses to thermal stress are still important challenges in breeding programs. Thus, the identification of optimal statistical methods and traits that better capture key biological mechanisms involved in the heat stress response has the potential to enable more accurate selection for thermal tolerant individuals. Therefore, this thesis aimed to investigate complementary topics related to thermal tolerance in livestock species based on genomic information. A total of 946 genotypes from 34 cattle breeds, as well as Datong yak (<em>Bos grunniens</em>) and Bali (<em>Bos javanicus</em>) populations, adapted to divergent climatic conditions, were used to investigate the genetic diversity and unravel genomic regions potentially under selection for thermal tolerance, with a focus on Chinese local cattle breeds and yak. Different signature of selection analyses and a comprehensive description of genetic diversity in 32 worldwide cattle and Datong yak populations was presented. Moderate genetic diversity was observed within each Chinese cattle population. However, these results highlighted the need to adopt strategies to avoid further reduction in the genetic diversity of these populations. Several candidate genes were identified as potentially under selection for thermal tolerance, and important biological pathways, molecular functions, and cellular components were identified, which contribute to our understanding of the genetic background of thermal tolerance in <em>Bos</em> species. Secondly, 8,992 genotyped individuals were used to provide a comprehensive description of genotype-by-environment interaction effects, defining optimal environmental variables based on public weather station data, and critical periods to evaluate heat tolerance for various reproduction, growth, and body composition traits in US Large White pigs. The period of 30 days before the measurement date was suggested to analyze genotype-by-environment interaction for off-test weight, muscle depth, and backfat thickness. While for number of piglets weaned and weaning weight, the suggested period ranged from the last trimester of gestation until weaning. This same population was used to access the genomic predictive ability of heat tolerance based on routinely-measured traits and explore candidate regions involved in the biological mechanisms that underlie heat stress response in pigs. Genotype-by-environment interaction was identified for most of the traits evaluated, and moderate (>0.36 ± 0.05) breeding values prediction accuracy were achieved using genomic information. Lastly, various behavioral, anatomical, and physiological indicators of heat stress were measured in a population of 1,645 multiparous Large White x Landrace lactating sows. This dataset was used to identify the best statistical models and estimate genomic-based genetic parameters for 23 indicators of heat stress, including automatically-measured vaginal temperature, skin surface temperatures, respiration efficiency, respiration rate, panting score, body condition scores, hair density, body size, and ear measurements. All the traits evaluated are heritable, with heritability estimates ranging from 0.04 ± 0.01 to 0.40 ± 0.09. The genetic correlations among these traits ranged from -0.49 (between repeated records of vaginal temperature measured at 0800 hours and caliper body condition score) to 1.0 (between repeated records of vaginal temperature measured at 0800 hours and single record of vaginal temperature measured at 0800 hours; and between repeated records of vaginal temperature measured at 1200 hours and single record of vaginal temperature measured at 1200 hours). These findings indicate that genetic progress for thermotolerance in pigs can be achieved through direct indicators of heat stress in selection schemes. However, special attention is needed due to complex relationship between these traits as evidenced by their genetic correlations. In conclusion, this thesis provides important information to be used when designing breeding strategies for improving thermal tolerance in cattle and pigs, important genomic regions and metabolic pathways that are important for understanding the biological mechanism regulating thermal tolerance, as well as future directions for investigations in the area of livestock climatic adaptation.</p>

<p dir="ltr">Most studies on molecular aspects of cold tolerance in plants have focused on Arabidopsis, a temperate species well adapted to cold environments. However, many vital global vegetable crops are tropical species (e.g., Tomato), and there is a lack of understanding of their cold tolerance mechanisms. Even mild cold stress, such as chilling (<10°C), can lead to adverse effects in tomato, including chlorosis, reduced leaf expansion, and necrosis. These differential consequences of cold stress between temperate and tropical plants are poorly understood. To address this knowledge gap, I compared the cold-responsive genes between Arabidopsis and tomato. Identified conserved early response genes, especially Transcription Factors (TFs), and validated their roles in tomato cold response. By identifying conserved mechanisms, I aimed to enhance tomato cold tolerance through targeted manipulation of regulatory genes. I hypothesized that a TF that consistently responds to cold in both species is an essential regulator of cold tolerance mechanisms, and altering the expression of such TF(s) will improve tomato cold tolerance. In this project, I employed a combination of computational approaches, such as regulatory network inference and molecular techniques like gene editing, to predict and validate the critical TFs that regulate cold response. I identified ~20 TFs that consistently respond to cold in Arabidopsis and tomato. Then, I generated tomato mutant lines using CRISPR/Cas9 and experimentally validated their phenotype using the knockout mutant plants. RNA-Seq was performed using the three cbfs and five additional early cold-responsive TFs mutant lines, and the results are discussed separately in chapters 3 and 4. We concluded that CBFs play a modest role in tomato cold response but a broader role in plant physiology under ambient conditions. Moreover, the regulation of tomato cold response is mediated by a complex network with multiple hubs with overlapping roles. This research enhanced our understanding of plant cold stress response and offered candidate genes/loci for improving the resilience of tropical crops to low temperatures, thereby mitigating yield losses and extending their geographical distribution and growing seasons.</p>

Climate change is expected to increase the frequency of mild winters and warm springs, which can induce premature plant development. This premature development results in a high risk of exposure of young plant tissues to cold stress leading to severe reductions in plant growth and agricultural production. Plants are associated with complex bacterial communities that can activate acclimation processes and positively affect plant performance at low temperatures. Beneficial effects of plant colonization by cold-tolerant bacteria include the modulation of cold-related genes and the reduction in cellular damage under cold stress, but scarce information is available on mechanisms stimulated by bacterial endophytes in tomato plants against cold stress. The aims of this work were i) to analyze the taxonomy and potential functions of plant-associated microbial communities in cold regions, ii) to understand metabolic changes stimulated by cold-tolerant endophytic bacteria in tomato plants exposed to cold stress, and iii) to identify possible genomic traits of cold-tolerant endophytic bacteria responsible for plant growth promotion and cold stress mitigation. The first chapter includes an introduction on cold stress and acclimation processes in plants, and the second chapter defines the aims of the project. In the third chapter, the taxonomic and functional characterization of plant-associated microbial communities of alpine, Arctic, and Antarctic regions was reviewed, highlighting the main environmental factors affecting their taxonomic structure. e. The key findings of this chapter are the functional roles of microbial communities in plant growth and survival in cold environments, and the suggestion of potential biotechnological applications of ubiquitous and endemic cold-tolerant microorganisms. In the fourth chapter, metabolic changes stimulated by cold-tolerant endophytic bacteria in tomato plants exposed to cold stress were studied by metabolomic analyses, and compounds possibly associated with cold stress mitigation were found. 14 Tomato seeds were inoculated with two bacterial endophytes isolated from Antarctic Colobanthus quitensis plants (Ewingella sp. S1.OA.A_B6 and Pseudomonas sp. S2.OTC.A_B10) or with Paraburkholderia phytofirmans PsJN, while mock-inoculated seeds were used as control. The metabolic composition of tomato plants was analyzed immediately after cold stress exposure (4°C for seven days) or after two and four days of recovery at 25°C. Under cold stress, the content of malondialdehyde, phenylalanine, ferulic acid, and p-coumaric acid was lower in bacterium-inoculated compared to mock-inoculated plants, indicating a reduction of lipid peroxidation and the stimulation of phenolic compound metabolism. The content of two phenolic compounds, five putative phenylalanine-derived dipeptides, and three further phenylalanine-derived compounds was higher in bacterium-inoculated compared to mock-inoculated samples under cold stress. Thus, the presented work suggests that psychrotolerant endophytic bacteria can reprogram polyphenol metabolism and stimulate the accumulation of secondary metabolites, like 4-hydroxybenzoic and salicylic acid, which are involved in cold stress mitigation, and phenylalanine-derived dipeptides possibly involved in plant stress responses. In the fifth chapter, functional and genomic traits of Ewingella sp. S1.OA.A_B6 and Pseudomonas sp. S2.OTC.A_B10 were studied. In the framework of the present study, Ewingella sp., Pseudomonas sp., and the bacterial consortium showed plant growth-promoting activity on tomato seedlings at low temperatures. Ammonia was produced by both bacterial isolates and their consortium, while indole-3-acetic acid and proteases were produced by Ewingella sp. and Pseudomonas sp., respectively. Ewingella sp. and Pseudomonas sp. genomes (51.57% and 60.63% guanine-cytosine, 4,148 and 5,983 predicted genes, respectively) encompassed genes related to amino acid metabolism, plant hormone metabolism (auxin, cytokinins, ethylene, and salicylic acid), nitrogen metabolism, lytic activities (amylases, cellulases, and proteases). Traits related to plant growth promotion included genes for iron transport, phosphate metabolism, potassium transport, siderophore metabolism and 15 transport, and zinc transport. Moreover, Ewingella sp. and Pseudomonas sp. encompassed genes related to cold tolerance, such as cold shock and heat shock-related proteins, lipid desaturases, and genes related to polyamine metabolism, proline metabolism, proline and glycine betaine transport, reactive oxygen species detoxification, and trehalose metabolism. Thus, in this chapter, it was discovered that Antarctic cold-tolerant endophytes include multiple genomic and functional traits to survive under cold conditions and some of them can contribute to promote the host plant growth at low temperatures. These findings indicate that plant-associated bacteria of cold regions have a great biotechnological potential to mitigate cold stress in crop plants. In particular, Antarctic bacterial endophytes encompass genomic traits responsible for plant growth promotion and protection against cold stress, and they can mitigate cold stress in tomato plants by a complex reprogramming of plant metabolism. Although further metabolomic and functional studies are required to verify compound annotations and to better clarify the role of phenylalanine-derived compounds and phenylalanine-derived dipeptides in cold stress mitigation, these results provided a better understanding of metabolic changes stimulated by psychrotolerant endophytic bacteria in cold-stressed tomato plants. Thus, the validation of cold stress mitigation activated by psychrotolerant endophytic bacteria under field conditions will pave the way for the further development of endophytic bacterial inoculants as sustainable products to protect crops against cold stress.

<p dir="ltr">Tomato, being a tropical species, is sensitive to temperatures below 10°C, thus limiting its growth to warmer regions and greenhouses. Understanding the cold response pathways in tomato will help improve its climate resiliency through breeding and biotechnology. Reportedly, plant genes undergo alternative splicing (AS) in response to various environmental stresses, however, the scope and dynamics of alternative splicing events in response to cold are unknown in tomato. To fill this knowledge gap, a fine-scale time-series cold (4°C) experiment was performed followed by RNA-sequencing of shoot and root tissues in tomato. Computational analysis revealed that various AS events occur within the first 20 minutes of temperature reduction and later on. Many AS genes were common between shoots and roots, however, the majority of the changes were organ-specific. Circadian rhythm and photosynthesis were the most significant among the various impacted biological processes, highlighting their importance in cold stress response. This study will help us gain insights into cold response pathways of tomato and other commercially important, closely related Solanaceae species.</p>