Isolation of plant growth promoting bacteria from torch lake sediments and their role in phytoremediation of metal contaminated soil

In this study, we isolated eight copper-resistant bacteria from Torch Lake sediment contaminated by copper mine tailings (stamp sand). Sequence analysis of gyrB and rpoD genes revealed that these organisms are closer to various Pseudomonas species. These eight bacterial isolates were also resistant to zinc, cesium, lead, arsenate and mercury. Further characterization showed that all the strains produced plant growth promoting indole-3-acetic acid (IAA), iron chelating siderophore and solubilized mineral phosphate and metals. The effect of bacterial inoculation on plant growth and copper uptake by maize (Zea mays) and sunflower (Helianthus annuus) was investigated using one of the isolates (Pseudomonas sp. TLC 6-6.5-4) with higher IAA production and phosphate and metal soubilization, which resulted in a significant increase in copper accumulation in maize and sunflower, and an increase in the total biomass of maize. Genes involved in copper resistance of Pseudomonas sp. TLC 6-6.5-4 was analyzed by transposon mutational analysis. Two copper sensitive mutants with significant reduction in copper resistance were identified: CSM1, a mutant disrupted in trp A gene (tryptophan synthase alpha subunit); CSM2, a mutant disrupted in clpA gene (ATP-dependent Clp protease). Proteomic and metabolomic analysis were performed to identify biochemical and molecular mechanisms involved in copper resistance using CSM2 due to its lower minimum inhibitory concentration compared with CSM1 and the wild type. The effect of different bacterial inoculation methods on plant growth, copper uptake and soil enzyme activities was investigated. Four different delivery methods were used including soil inoculation (before or after plant emergence), seed coating and root dipping. Soil inoculation before sowing seeds and coating seeds with PGPB led to better growth of maize, higher copper uptake and an increase in soil invertase and dehydrogenase activities. Proteomic and metabolomic analyses were performed to investigate the effect of bacterial inoculation on maize grown in normal soil and stamp sand. Our results revealed that bacterial inoculation led to environment-dependent effects on maize proteome and metabolome.

INTEGRATED MANAGEMENT OF THE INVASIVE SPECIES CIRSIUM ARVENSE, CANADA THISTLE, AND PHRAGMITES AUSTRALIS, COMMON REED: USING ECOLOGICALLY BASED CONTROL MEASURES AT EACH STAGE OF THE INVASION PROCESS

Invasive plant species threaten natural areas by reducing biodiversity and altering ecosystem functions. They also impact agriculture by reducing crop and livestock productivity. Millions of dollars are spent on invasive species control each year, and traditionally, herbicides are used to manage invasive species. Herbicides have human and environmental health risks associated with them; therefore, it is essential that land managers and stakeholders attempt to reduce these risks by utilizing the principles of integrated weed management. Integrated weed management is a practice that incorporates a variety of measures and focuses on the ecology of the invasive plant to manage it. Roadways are high risk areas that have high incidence of invasive species. Roadways act as conduits for invasive species spread and are ideal harborages for population growth; therefore, roadways should be a primary target for invasive species control. There are four stages in the invasion process which an invasive species must overcome: transport, establishment, spread, and impact. The aim of this dissertation was to focus on these four stages and examine the mechanisms underlying the progression from one stage to the next, while also developing integrated weed management strategies. The target species were Phragmites australis, common reed, and Cisrium arvense, Canada thistle. The transport and establishment risks of P. australis can be reduced by removing rhizome fragments from soil when roadside maintenance is performed. The establishment and spread of C. arvense can be reduced by planting particular resistant species, e.g. Heterotheca villosa, especially those that can reduce light transmittance to the soil. Finally, the spread and impact of C. arvense can be mitigated on roadsides through the use of the herbicide aminopyralid. The risks associated with herbicide drift produced by application equipment can be reduced by using the Wet-Blade herbicide application system.

DISSECTION OF STRESS RESPONSE NETWORKS REGULATING MULTIPLE STRESSES IN RICE

Important food crops like rice are constantly exposed to various stresses that can have devastating effect on their survival and productivity. Being sessile, these highly evolved organisms have developed elaborate molecular machineries to sense a mixture of stress signals and elicit a precise response to minimize the damage. However, recent discoveries revealed that the interplay of these stress regulatory and signaling molecules is highly complex and remains largely unknown. In this work, we conducted large scale analysis of differential gene expression using advanced computational methods to dissect regulation of stress response which is at the heart of all molecular changes leading to the observed phenotypic susceptibility. One of the most important stress conditions in terms of loss of productivity is drought. We performed genomic and proteomic analysis of epigenetic and miRNA mechanisms in regulation of drought responsive genes in rice and found subsets of genes with striking properties. Overexpressed genesets included higher number of epigenetic marks, miRNA targets and transcription factors which regulate drought tolerance. On the other hand, underexpressed genesets were poor in above features but were rich in number of metabolic genes with multiple co-expression partners contributing majorly towards drought resistance. Identification and characterization of the patterns exhibited by differentially expressed genes hold key to uncover the synergistic and antagonistic components of the cross talk between stress response mechanisms. We performed meta-analysis on drought and bacterial stresses in rice and Arabidopsis, and identified hundreds of shared genes. We found high level of conservation of gene expression between these stresses. Weighted co-expression network analysis detected two tight clusters of genes made up of master transcription factors and signaling genes showing strikingly opposite expression status. To comprehensively identify the shared stress responsive genes between multiple abiotic and biotic stresses in rice, we performed meta-analyses of microarray studies from seven different abiotic and six biotic stresses separately and found more than thirteen hundred shared stress responsive genes. Various machine learning techniques utilizing these genes classified the stresses into two major classes' namely abiotic and biotic stresses and multiple classes of individual stresses with high accuracy and identified the top genes showing distinct patterns of expression. Functional enrichment and co-expression network analysis revealed the different roles of plant hormones, transcription factors in conserved and non-conserved genesets in regulation of stress response.

INSTANTANEOUS PHOTOSYNTHETIC RESPONSE TO TEMPERATURE OF MATURE FOREST CANOPIES AND EXPERIMENTALLY WARMED SEEDLINGS

Tropical trees have been shown to be more susceptible to warming compared to temperate species, and have shown growth and photosynthetic declines at elevated temperatures as little as 3oC above ambient. However, regional and global vegetation models lack the data needed to accurately represent physiological response to increased temperatures in tropical forests. We compared the instantaneous photosynthetic responses to elevated temperatures of four mature tropical rainforest tree species in Puerto Rico and the temperate broadleaf species sugar maple (Acer saccharum) in Michigan. Contrary to expectations, leaves in the upper canopy of both temperate and tropical forests had temperature optima that are already exceeded by mean daily leaf temperatures. This indicates that tropical and temperate forests are already seeing photosynthesis decline at mid-day temperature. This decline may worsen as air temperatures rise with climate change if trees are unable to acclimate, increasing the likelihood that forests may shift from carbon sinks to sources. A secondary study was conducted on experimentally warmed sugar maple seedlings to determine if photosynthesis had been able to acclimate to +5oC air temperature over four years. Species abundance models had predicted a decline of sugar maple within the Upper Peninsula of Michigan over the next 100 years, due to elevated temperature and altered precipitation. Instantaneous photosynthetic temperature response curves on both control and heated seedlings showed that the differences between treatments were not statistically significant, though there was a 16% increase in temperature optima and a 3% increase in maximum rates of photosynthesis in warmed plots. Though evidence of acclimation was not significant, the seedlings did not fare poorly as the models suggest.

Effects of Ethylene on Secondary Xylem Formation in Arabidopsis thaliana

Ethylene has myriad roles as a plant hormone, ranging from senescence and defending against pathogen attacks to fruit ripening and interactions with other hormones. It has been shown to increase cambial activity in poplar, but the effect on wood formation in Arabidopsis hypocotyl has not previously been studied. The Auxin-Regulated Gene involved in Organ Size (ARGOS), which increases organ size by lengthening the time for cell division, was found to be upregulated by ethylene. We tested the effect of ethylene treatment at 10 and 100 µM ACC on three genotypes of Arabidopsis, Col0 (wild-type), an ARGOS deficient mutant (argos), and ein3-1, an ethylene insensitive mutant. ARGOS expression analysis with qPCR indicated that ACC does induce ARGOS and ARGOS-LIKE (ARL) in the hypocotyl. As seen in poplar, ethylene also decreases stem elongation.Histochemical staining, showed that ethylene changes the way secondary xylem lignifies, causing gaps in lignification around the outer edge of secondary xylem. Our results also implied that ethylene treatment changes the proportion of secondary to total xylem, resulting in less secondary, whereas in poplar, ethylene treatment caused an increase.

The influence of concurrent disturbances on plant community dynamics in northern hemlock-hardwood forests

Throughout the Upper Great Lakes region, alterations to historic disturbance regimes have influenced plant community dynamics in hemlock-hardwood forests. Several important mesic forest species, eastern hemlock (Tsuga canadensis), yellow birch (Betula alleghaniensis), eastern white pine (Pinus strobus), and Canada yew (Taxus canadensis), are in decline due to exploitive logging practices used at the turn of the 20th century and the wave of intense fires that followed. Continued regeneration and recruitment failure is attributed to contemporary forest management practices and overbrowsing by white-tailed deer (Odocoileus virginianus). Therefore, I examined the influence of two concurrent disturbances, overstory removal and herbivory, on plant community dynamics in two hemlock-hardwood forests. I measured the post-disturbance regeneration response (herbaceous and woody species) inside and outside of deer exclosures in 20 artificial canopy gaps (50 – 450 m2) and monitored survival and growth for hundreds of planted seedlings. The results of this research show that interacting disturbances can play a large role in shaping plant community composition and structure in hemlock-hardwood forests. White-tailed deer herbivory homogenized the post-disturbance plant communities across the experimental gradient of gap areas, essentially making species compositions in small gaps “look like” those in large gaps. Deer browsing also influenced probability of survival for planted Canada yew cuttings; all else being equal an individual was nearly seven times more likely to survive if protected from herbivory (P < 0.001). In contrast, the ability of sugar maple (Acer saccharum) to persist under high levels of herbivory and respond rapidly to overstory release appears to be related to the presence of stem layering(i.e., portions of below-ground prostrate stem). Layering occurred in 52% of excavated saplings (n = 100) and was significantly associated with increased post-disturbance height growth. Understory light was also important to planted seedling establishment and height growth. Higher levels of direct under-canopy light negatively impacted survival for shade-tolerant hemlock and Canada yew, while an increase in diffuse light was linked to a higher probability of survival for yellow birch and height growth for hemlock and Canada yew. Increases in white pine height growth were also significantly associated with a decrease in canopy cover.

Effects of management on native and exotic plant communities in Pictured Rocks National Lakeshore in the Upper Peninsula of Michigan

Ecological disturbances may be caused by a range of biotic and abiotic factors. Among these are disturbances that result from human activities such as the introduction of exotic plants and land management activities. This dissertation addresses both of these types of disturbance in ecosystems in the Upper Peninsula of Michigan. Invasive plants are a significant cause of disturbance at Pictured Rocks Natural Lakeshore. Management of invasive plants is dependent on understanding what areas are at risk of being invaded, what the consequences of an invasion are on native plant communities and how effective different tools are for managing the invasive species. A series of risk models are described that predict three stages of invasion (introduction, establishment and spread) for eight invasive plant species at Pictured Rocks National Lakeshore. These models are specific to this location and include species for which models have not previously been produced. The models were tested by collecting point data throughout the park to demonstrate their effectiveness for future detection of invasive plants in the park. Work to describe the impacts and management of invasive plants focused on spotted knapweed in the sensitive Grand Sable Dunes area of Pictured Rocks National Lakeshore. Impacts of spotted knapweed were assessed by comparing vegetation communities in areas with varying amounts of spotted knapweed. This work showed significant increases in species diversity in areas invaded by knapweed, apparently as a result of the presence of a number of non-dune species that have become established in spotted knapweed invaded areas. An experiment was carried out to compare annual spot application of two herbicides, Milestone® and Transline® to target spotted knapweed. This included an assessment of impacts of this type of treatment on non-target species. There was no difference in the effectiveness of the two herbicides, and both significantly reduced the density of spotted knapweed during the course of the study. Areas treated with herbicide developed a higher percent cover of grasses during the study, and suffered limited negative impacts on some sensitive dune species such as beach pea and dune stitchwort, and on some other non-dune species such as hawkweed. The use of these herbicides to reduce the density of spotted knapweed appears to be feasible over large scales.

Regulation of woody plant development by gibberellin catabolic and signaling genes

Gibberellin (GA) is a growth promoting hormone implicated in regulating a diversity of plant processes. This dissertation examines the role of GA metabolic and signaling genes in woody plant growth and development. Transgenic modifications, expression analysis, physiological/biochemical assays, biometric measurements and histological analysis were used to understand the regulatory roles these genes play in the model woody plant, Populus. Our results highlight the importance of GA regulatory genes in woody perennial growth, including: phenology, wood formation, phenotypic plasticity, and growth/survival under field conditions. We characterize two putative Populus orthologs of the SHORT INTERNODES (SHI) gene from Arabidopsis, a negative regulator of GA signaling. RNAi-mediated suppression of Populus SHI-like genes increased several growth-related traits, including extent of xylem proliferation, in a dose-dependent manner. Three Populus genes, sharing sequence homology to the positive regulator of GA signaling gene PHOTOPERIOD-RESPONSIVE 1 (PHOR1) from Solanum, are up-regulated in GA-deficient and insensitive plants suggesting a conserved role in GA signaling. We demonstrate that Populus PHOR1-like genes have overlapping and divergent function(s). Two PHOR1-like genes are highly expressed in roots, predominantly affect root growth (e.g., morphology, starch quantity and gravitropism), and induced by short-days (SD). The other PHOR1-like gene is ubiquitously expressed with a generalized function in root and shoot development. The effects of GA catabolic and signaling genes on important traits (e.g., adaptive and productivity traits) were studied in a multi-year field trial. Transgenics overexpressing GA 2-oxidase (GA2ox) and DELLA genes showed tremendous variation in growth, form, foliage, and phenology (i.e., vegetative and reproductive). Observed gradients in trait modifications were correlated to transgene expression levels, in a manner suggesting a dose-dependent relationship. We explore GA2ox and DELLA genes involvement in mediating growth responses to immediate short-term drought stress, and SD photoperiods, signaling prolonged periods of stress (e.g., winter bud dormancy). GA2ox and DELLA genes show substantial up-regulation in response to drought and SDs. Transgenics overexpressing homologs of these genes subjected to drought and SD photoperiods show hypersensitive growth restraint and increased stress resistances. These results suggest growth cessation (i.e., dormancy) in response to adverse conditions is mediated by GA regulatory genes.

INTERACTIVE CONTROLS OF WATER TABLE POSITION AND PLANT FUNCTIONAL TYPES ON PEAT POREWATER CHARACTER IN NORTHERN BOG ECOSYSTEMS: IMPLICATIONS FOR CARBON CYCLING DYNAMICS

Northern wetlands, and particularly peatlands, have been shown to store around 30% of the world's soil carbon and thus play a significant role in the carbon cycle of our planet. Changes in climate are altering peatland hydrology and vegetation communities. These changes are possibly resulting in declines in the ability of peatlands to sequester carbon because losses through carbon oxidation and mineralization are likely to increase relative to C inputs from net primary production in a warmer, drier climate. However, the consequences of interactive effects of altered hydrology and vegetation on carbon storage are not well understood. This research evaluated the importance of plant species, water table, and their interactive effects on porewater quality in a northern peatland with an average pH of 4.54, ranging from 4.15 to 4.8. We assessed the effects of plant functional group (ericaceous shrubs, sedges, and bryophytes) and water table position on biogeochemical processes. Specifically, we measured dissolved organic carbon (DOC), total dissolved nitrogen (TDN), potential enzyme activity, organic acids, anions and cations, spectral indexes of aromaticity, and phenolic content. Our results indicate that acetate and propionate concentrations in the sedge-dominated communities declined with depth and water table drawdown, relative to the control and ericaceous treatments. DOC increased in the lowered water table treatments in all vegetation community types, and the peat porewater C:N ratio declined in the sedge-dominated treatments when the water table was lowered. The relationship between DOC and ferrous iron showed significant responses to vegetation type; the exclusion of Ericaceae resulted in less ferrous iron per unit DOC compared to mixed species treatments and Ericaceae alone. This observation was corroborated with higher mean oxidation redox potential profiles (integrating 20, 40, and 70 cm) measured in the sedge treatments, compared with the mixed and Ericaceae species treatments over a growing season. Enzymatic activities did not show as strong of a response to treatments as expected; the oxidative enzyme peroxidase and the hydrolytic enzyme phosphatase were the only enzymes to respond to water table, where the potential activity of both enzymes increased with water table drawdown. Overall, there were significant interactive effects between changes in vegetation and water table position on peat porewater composition. These data suggest that vegetation effects on oxidation reduction potentials and peat porewater character can be as important as water table position in northern bog ecosystems.