Doctor of Philosophy / Department of Biology / David C. Hartnett / Grazing and drought are instrumental in the development and maintenance of perennial grasslands. In this research I tested the belowground bud bank contribution to tallgrass prairie resistance and resilience when perturbed by grazing and drought. First, I tested the bud bank role in vegetation response to and recovery from severe drought (Chapter 2). I compared above- and belowground responses of experimentally droughted plots to ambient controls and irrigated plots during two years of severe drought and two years of recovery. I found that although aboveground net primary productivity declined 30-60% during drought, bud bank density and demography were insensitive to drought. These results suggest that grassland resistance and resilience when perturbed by drought may be mediated by stability of belowground bud banks. Second, I investigated vegetation and soil nutrient legacies following release from long-term grazing (Chapter 3). I documented a relatively rapid shift in aboveground vegetation within four years of grazer exclusion, with productivity, stem density, and diversity becoming relatively more similar to ungrazed than grazed prairie. The density and composition of the belowground bud bank and soil seed bank shifted more slowly, remaining more similar to grazed than ungrazed prairie. Responses of soil nutrients to removal of grazers varied, and in some cases was affected by recent fire history. These results demonstrate the contribution of belowground propagules to the maintenance of a diverse plant community both during grazing and after grazers are removed. Finally, I examined short-term vegetation responses to both drought and grazing (Chapter 4). Despite extreme drought and simulated grazing that reduced productivity and increased mortality of individual stems, the dominant C4 grasses maintained a stable bud bank. Aboveground net primary productivity and bud bank density of sedges and forbs, however, were reduced by both drought and grazing. This differential response of species to extreme drought and grazing led to shifts in community composition and species diversity over one growing season. Across drought and grazing treatments, live rhizome biomass was highly correlated with bud bank density and may be a useful, more easily measured index of bud bank density.

Since the dawn of agriculture man has been genetically modifying crop plants to increase yield, quality and utility. In addition to selective breeding and hybridization we can utilize mutant populations and biotechnology to have greater control over crop plant modification than ever before. Increasing the production of plant oils such as soybean oil as a renewable resource for food and fuel is valuable. Successful breeding for higher oil levels in soybean, however, usually results in reduced protein, a second valuable seed component. We show that by manipulating a highly active acyl-CoA: diacylglycerol acyltransferase (DGAT) the hydrocarbon flux to oil in oilseeds can be increased without reducing the protein component. Compared to other plant DGATs, a DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in yeast, insect cells and soybean. Soybean lines expressing VgDGAT1A show a 4% increase in oil content without reductions in seed protein contents or yield per unit land area. Furthermore, we have screened a soybean fast neutrino population derived from M92-220 variety and found three high oil mutants that do not have reduced levels of protein. From the F2 plant populations we quantitatively pooled the high oil and low oil plants and performed comparative genomics hybridization (CGH). From the data it appears that two families have a 0.3 kb aberration in chromosome 14. We are performing further analysis to study this aberration and develop markers for molecular breeding. Mutagenic techniques are also useful for developing other traits such as early flowering varieties and adapting new high oil crops to a new region. Chia (Salvia hispanica) is an ancient crop that has experienced an agricultural resurgence in recent decades due to the high omega 3 fatty acid (ω-3) content of the seeds and good production potential. The area of cultivation has been expanded to Kentucky using mutagenized populations and the composition traits are similar to that of the original regions of cultivation in Central and South America.

<p></p><p></p><p>Abiotic stresses, such as cold, are serious agricultural problems resulting in substantial crop and revenue losses. Soybean (<i>Glycine max</i>) 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.</p> <p>Comparisons of eight cultivars of <i>G. max</i> demonstrated a weak correlation between cold acclimation and northern cultivars versus southern cultivars. In the non-domesticated soybean species <i>Glycine soja</i>, the germination rate after cold imbibition was positively correlated with seedling cold acclimation potential. However, the overall cold acclimation potential in <i>G. soja</i> was equal to that of domestic soybean <i>G. max</i> reducing the enthusiasm for the “wild” soybean as an additional source of genetic diversity for cold tolerance. </p> <p> </p> <p>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 <i>AtEIN3</i> transcript levels which do not change during cold treatment in Arabidopsis, we observed a cold-dependent 3.6 fold increase in <i>GmEIN3 </i>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 (<i>AtRD29Aprom::GFP/GUS</i>) 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 <i>GmEIN3A;1</i> 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 <i>GmDREB1A;1</i> and <i>GmDREB1A;2</i> and stimulation of the ethylene pathway led to a decrease in <i>GmDREB1A;1</i> and <i>GmDREB1B;1</i> 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. </p> <p> </p> <p>To identify if GmEIN3A;1 was capable of binding to <i>GmDREB1</i> 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 <i>GmDREB1A;1</i> promoter, though only when single stranded. Additional experiments will be required to demonstrate that GmEIN3A;1 and GmICE1A are capable of binding to the <i>GmDREB1A;1</i> promoter and this work provides the tools to answer these questions. </p> <p> </p> <p>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 <i>GmCBF/DREB1</i> 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.</p><p></p><p></p>

As espécies de plantas classificadas no gênero Amaranthus são frequentemente encontradas infestando áreas agrícolas brasileiras, contudo existem poucos trabalhos que avaliaram as características biológicas e o controle destas espécies. Assim sendo, este trabalho foi conduzido com o objetivo de analisar a germinação, o crescimento e o desenvolvimento, estimar a área foliar, a competitividade e a susceptibilidade a herbicidas de cinco espécies de plantas daninhas do gênero Amaranthus As espécies de Amaranthus estudadas foram: A. deflexus (caruru-rasteiro), A. hybridus (carururoxo), A. retroflexus (caruru-gigante), A. spinosus (caruru-de-espinho) e A. viridis (caruru-de-mancha). O experimento que avaliou a germinação foi conduzido no Laboratório de Análise de Sementes e os demais em casa-de-vegetação do Departamento de Produção Vegetal da ESALQ/USP, Piracicaba - SP, no período compreendido entre março e dezembro de 2005. Com relação à germinação, constatou-se que variações na disponibilidade de luz e temperatura interferem em todas as espécies de Amaranthus, em que as maiores taxas de germinação foram obtidas em condição de fotoperíodo com alternância de temperatura (8h-luz-30°C / 16h-escuro-20°C). Em condições menos favoráveis, A. viridis e A. hybridus obtiveram maiores taxas de germinação que as demais espécies. Em geral, A. deflexus e A. spinosus foram as espécies de plantas daninhas que apresentaram os menores índices de velocidade de germinação. Sobre o crescimento e desenvolvimento, constatou-se que A. deflexus foi a espécie com ciclo vegetativo mais curto, menor acúmulo de massa seca e área foliar; por outro lado, A. retroflexus e A. hybridus foram aquelas que alcançaram os maiores valores para estas variáveis. A estimativa de área foliar demonstrou que a equação linear passando pela origem (Ar=a.(C.L)) foi adequada para ajustar a relação entre as medidas lineares do limbo e a área foliar real de todas as espécies. Com relação à competição, concluiu-se que a cultura do feijoeiro é melhor competidora que todas as espécies de plantas daninhas do gênero Aramanthus que foram utilizadas neste trabalho, quando cultivadas em igualdade de proporções. A. deflexus e A. viridis foram as espécies com a fenologia menos afetada pela competição com o feijoeiro; a competição intraespecífica foi a mais prejudicial à cultura do feijoeiro, o que sugere que os danos causados pelas plantas daninhas estão mais relacionados com as altas densidades em que estas ocorrem do que com a habilidade competitiva intrínseca das espécies. O controle químico obtido para as espécies de Aramanthus avaliadas neste trabalho demonstrou diferenças de susceptibilidade aos herbicidas aplicados em pósemergência, principalmente ao trifloxysulfuron-sodium e ao chlorimuron-ethyl, em que A. deflexus foi a espécie menos suscetível, seguido por A. spinosus, A. viridis, A. hybridus e A. retroflexus. / Plant species classified in the Aramanthus genus are frequently found infesting brazilian agricultural areas, although there are few researches that evaluated the biological characteristics and the control of these species. Therefore, this work was conducted with the objective of analyzing the germination, the growth and the development, estimating the leaf area, the competitiveness and the susceptibility to herbicides of five weed species of the Aramanthus genus. The species of Aramanthus studied were: A. deflexus, A. hybridus, A. retroflexus, A. spinosus and A viridis. The experiment that evaluated the germination was conducted in the Laboratory of Seed Analyses and the others in the greenhouse of the Crop Science Department of ESALQ/USP, Piracicaba - SP, in the period comprehended between March to December 2005. About the germination, it was established that variation in the availability of light and temperature interferes in all the species of Aramanthus and the highest levels of germination were obtained in condition of photoperiod with alternating temperature (8h-light-30°C / 16h-dark-20°C). In less favorable conditions, A. viridis and A. hybridus obtained higher germination levels than all the other species. In general, A. deflexus and A. spinosus were the weed species that presented the lowest rates of germination speed. About the growth and development, it was observed that A. deflexus was the species with shortest vegetative cycle, the lowest dry mass and leaf area accumulation; however, A. retroflexus and A. hybridus were the species which reached the highest values for these variables. The leaf area estimation showed that the linear equation crossing the origin (Ar=a.(C.L)) was adequate to adjust the correlation between the linear blade dimensions and the real leaf area for all the species. About competition, it was concluded that the crop of common bean is more competitive than all the weed species of the Aramanthus genus that were studied in this work, when grown in equivalent proportion; A. deflexus and A. viridis were the species which phenology was less affected by the competition with common bean; the intraspecific competition was the most damaging to the crop of common bean, what suggests that the damages caused by the weeds are more connected with its high density of infestation than the intrinsic competitive ability of the species. The control obtained for the Aramanthus species evaluated in this research presented differences of susceptibility to post-emergence applied herbicides, mainly to trifloxysulfuron-sodium and chlorimuron-ethyl, which A. deflexus was the least susceptible species, followed by A. spinosus, A. viridis, A. hybridus and A. retroflexus.

Certain chemicals are known to inhibit growth in many plant species. These chemicals possess a common trait, the ability to inhibit stem elongation by suppressing the activity of the subapical meristematic region (11). These growth retardants have been extensively studied during the past several years in an effort to determine the biochemical mechanism responsible for reduction in plant height. The characteristic effect of these chemical retardants on the growth pattern has been described as producing plants with shorter, thicker stems and broader, darker green leaves. Tolbert, however, noted that although plants treated with the plant growth retardant, Cycocel, (2-chloroethyltrimethlammoniumchloride) and some of its related compounds were shorter and exhibited the above characteristics, he also found there was no loss in weight of the treated plants (25). Thus it appears that these chemicals cause growth to be manifested in a manner slightly altered from the normal pattern for that species. In living systems growth can be correlated with an increase in protein content; and since plants must synthesize their own amino acids, the metabolism of nitrogen is a vital factor in determining the rate of growth. Nitrogen is absorbed by the plant in the form of nitrate and must be converted into a usable form (14). The first step of this conversion is accomplished enzymatically by nitrate reductase (2). Although Kahn and Faust (8) have determined the effect of Cycocel on the soluble protein level in barley seedlings, information concerning other aspects of nitrogen metabolism has not been reported in the literature. The purpose of this study was to determine the effects of two growth retardant chemicals, Cycocel and B-Nine (succinic acid 2, 2-dimethylhydrazide), upon the nitrate level, the soluble protein nitrogen content, and the activity of the nitrate reductase enzyme of young barley plants.

Low nutrition availability in the soil can be a major limitation of plant growth. To improve nutrient acquisition, the majority of land plants engage in symbiosis with arbuscular mycorrhizal (AM) fungi. The accommodation of fungal colonisation structures in the roots requires their radical reprogramming. This starts during pre-symbiotic communication, where signals are exchanged between the fungus and plant across the rhizosphere. The receptor D14-LIKE emerged as a vital component of this pre-symbiotic communication when it was found to be absolutely required for symbiosis in rice. However, the broader relevance of the receptor, both in terms of functional conservation across plant species and its relation to other pre-symbiotic plant signalling components, remained unclear. The aim of this thesis was to elucidate these two key points. To address the fragmented picture of fungal signals, plant receptors and signalling pathways, a large scale transcriptomic experiment in rice was conducted to tie D14L together with other distinct pre-symbiotic components. In the absence of D14L-mediated signalling, rice was found to be compromised in the perception of germinated spore exudates, as well as specific chitinaceous signals, meaning that normal transcriptional reprogramming could not be achieved in response to any of these treatments. In addition, the functional conservation of D14L signalling was explored using trans-species complementation experiments. It was found that the Arabidopsis homolog AtKAI2 could complement the developmental phenotype of the d14l rice mutant, but not symbiosis. Likewise, D14La from early diverging Marchantia polymorpha and Marachantia paleacea could rescue developmental phenotypes in d14l rice, but again failed to complement symbiosis. This demonstrated a functional separation between developmental and symbiotic signalling. The data generated during my PhD foster D14L as a central node for multiple inputs to pre-symbiotic reprogramming, and provides new insights into pre-symbiotic communication mechanisms which are required for the successful establishment of symbiosis.

Sciadopitys verticillata was subjected to three propagation treatments designed to inhibit coagulation of its latex-like sap at the cut ends of the stem cuttings. Twenty-four hour soaking in water prior to rooting hormone application significantly enhanced production of adventitious roots. Old wood stem cuttings from shade-grown trees rooted at higher proportions than stem cuttings collected from sun-grown trees. Height, age, and place of origin of the source trees were not important factors in successful rooting. Antibacterial activity against some human pathogens and soil bacterial species was detected in latex application trials but the antibiotic activity was not related to the bacterial Gram reaction. The latex-like sap inhibited none of four plant pathogens tested. A suspension of the water insoluble latex-like sap of S. verticillata had a pH of 5.8. Antibacterial activity of S. verticillata sap was heat stable, which indicates the activity was not protein-based.

Plant secondary metabolites are essential for normal growth and development in plants ultimately affecting crop yield. They play roles ranging from appearance of the plants to defending against pathogen attack and herbivory. They have been used by humans for medicinal and recreational purposes amongst others. Glycosyltransferases catalyze the transfer of sugars from donor substrates to acceptors. Glucosyltransferases are a specific type of glycosyltransferases known to transfer glucose molecules from a glucose donor to a glucose acceptor (aglycone) producing the corresponding glucose secondary metabolite or glycone, in this case glucosides. It was hypothesized that SBIP68, a tobacco putative glucosyltransferase-like protein glucosylated salicylic acid. Salicylic acid is an essential plant defense secondary metabolite. SBIP68 was cloned and heterologously expressed in both prokaryotic and eukaryotic systems. Results from activity screening suggest that SBIP68 is a UDP-glucose flavonoid glucosyltransferase with broad substrate specificity. Further studies are required to fully characterize SBIP68.

We monitored a population of Carolina Hemlocks in northwestern North Carolina for four years to examine the rate and pattern of decline in response to infestation by Adelges tsugae (Hemlock Woolly Adelgid). Our yearly census of hemlock condition and severity of the adelgid infestation included trees of all sizes. We estimated declines in condition as the portions of the leaf canopy that were lost. Initially, infestation occurred throughout the population but was severe in only a small cluster of individuals. Within 1 year, the area of severe infestation increased in size to encompass 48% of the population. In another region of the population, there was a cluster of relatively healthy individuals comprised largely of seedlings. Of the 4 size-classes of trees, sapling-sized individuals experienced the highest rates of decline in condition. Most trees declined to poor health within 3 years of an observation of moderate to severe infestation.

A demographic census and disease assessment was conducted in 2007 and 2015 in three Tennessee populations of the dioecious shrub,;Buckleya distichophylla (Santalaceae). Population sizes were relatively stable over time and plant heights and numbers of stems per clump were similar among populations. Seedlings were present in all populations, where they represented 14–19% of individuals. Two populations had an equal male:female sex ratio, but a third population was male-biased. Nonflowering individuals comprised 33–41% of individuals in a population. The majority of plants in all populations had high vigor. Spatial analyses revealed clusters of seedlings in two populations and a cluster of low-vigor plants in one population. Cronartium appalachianum, a rust fungus dependent upon;Pinus virginiana and B. distichophylla as primary and alternate hosts, respectively, was present in all populations with prevalences on B. distichophylla of 19–29%, but there was no spatial clustering of disease in populations. The tree species nearest to B. distichophyllavaried among populations with Tsuga canadensis predominant in one population, T. caroliniana in another, and P. virginiana in the third. Buckleya distichophylla in proximity to P. virginiana had a higher than expected prevalence of C. appalachianum infection.