Plants species interactions via pollinators are a model system to understand the mechanisms that generate plant diversity in nature. However, most studies have focused on plant-plant interactions via pollinator attraction while ignoring the role of plant-plant interactions via pollen transfer. Heterospecific pollen transfer (henceforth HP) can be common and have negative fitness effects. Negative HP fitness effects may prompt the evolution of adaptive strategies to minimize them. However, the extent of spatial variation in HP load size within and among populations, a tenet for natural selection, remains unexplored. Such knowledge would hence constitute a first step in advancing our understanding of the importance of HP transfer as an evolutionary force promoting plant diversification. For instance, the opportunity for natural selection would only be expected under strong among population variation in HP load size. In this study we aim to answer the following specific questions: Is there variation in the amount and diversity of HP load in Lobelia siphilitica? How is the variation partitioned across different levels of organization (populations, individuals, and flowers among an individual)? Greater among-population variance would suggest that community attributes, such as plant density and diversity are the major drivers of HP load size. Greater among-plant variance would indicate plant traits that affect pollinator foraging behavior may play an important role. Greater variance among flowers within an individual plant, would suggest stochastic events may underlie variation in HP load size and diversity. In order to test these hypotheses, samples of the native perennial Lobelia siphilitica were taken from 10 populations in the Northeast Tennessee region (500 total). The styles were processed in the lab and pollen grains counted separating them into two categories, heterospecific and conspecific pollen. There was variation in the amount and diversity of HP load received. Populations are expected to have the largest variation among them due to different environments (disturbance levels, pollinators, plant communities, etc.) Since populations are expected to have the largest variation in HP received, they are also expected to have the greatest opportunity for natural selection to act. Looking at HP receipt within-species is important for identifying the mechanisms that can generate diversity in plant communities.

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Development of genetic improvement procedures for the bioenergy crop, Miscanthus × giganteus (M×g; triploid), are desired for trait improvement. Agrobacterium tumefaciens has been used in other Miscanthus spp. for genetic improvement, but not in M×g, nor have more than a few genes been introduced at a given time. Transformation procedures for M×g were developed; studies included use of A.tumefaciens (strain LBA4404) and A. rhizogenes (strain A4) for introduction of seven genes isolated (by other researchers) from Sorghum bicolor involved in sorgoleone biosynthesis (an allelopathic compound), and selectable marker gene neomycin phosphotransferase II. Procedural development included generation of embryogenic calli from immature inflorescences, attempts to minimize generation of phenolics by these tissues, transformation procedures for both Agrobacterium spp., selection of transgenic tissues, and tissue screenings via polymerase chain reaction to identify putative transgenic tissues/plants. Although four M×g putative transgenic plants were generated, none were proven to be transgenic.

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In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 20C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, their metabolism and functional implications in plants however, remain incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. FAAH orthologs, putative PpFAAHs (Physcomitrella patens FAAH) were identified based on the sequence blast of ratFAAH, and named as PpFAAH1 to PpFAAH10. Based on the highest mRNA expression of the PpFAAH homologs upon NAE treatment, PpFAAH1 was selected for further in vitro characterization, which shares 31% sequence identity with ratFAAH. PpFAAH1 was heterologously expressed in E. coli and purified for characterization. Highest amidohydrolysis activity of PpFAAH1 was observed in vitro at pH 8.0 and temperature 37°C. Methoxy arachidonyl fluorophosphonate (MAFP), an inhibitor showed highest inhibition with 10mM concentration, however, one of the principal classes of FAAH inhibitor O-aryl carbamates (URB597) exhibited only 22% inhibition with the same concentration. Both in vivo and in vitro studies showed that unsaturated NAE substrate (NAE 20:4) is hydrolyzed faster than the saturated NAE (NAE16:0); more than 50- and 10-fold higher in vitro and in vivo assays, respectively. Amidohydrolase activity in vivo was mostly associated with microsomes compared with cytoplasmic fractions. Additionally, microsomal fraction of mature gametophytes showed higher amidohydrolase activity than of the protonemal or early gametophyte stages; however, PpFAAH expression was not significantly different between the developmental stages. Further functional characterization of NAE metabolic pathway is ongoing by generation of PpFAAH knock out (KO) and overexpressor (OE) to understand the biological implications of FAAH in growth and development of early land plants.

Co-flowering plants within communities typically share pollinators which can lead to the deposition of pollen grains from varying species onto stigmas; or heterospecific pollen transfer (HPT). It has been shown that HPT can have varying effects on plant reproductive success. These effects can range from an 80% reduction on seed production to no effect. However, to date, there are no studies that have evaluated the causes underlying variability of these effects. We investigated this variability through a greenhouse experiment. Hand-pollinations were conducted with a mix of conspecific and heterospecific pollen and a control (conspecific pollen only). We used six pollen recipient and four species as donors for the heterospecific pollen (5 treatments total) We conducted hand-pollinations on a minimum of 10 plants per species (584 total pollinations). After pollination seed set data was recorded for each treatment and the amount of conspecific and heterospecific pollen on stigmas was recorded. We evaluated if the effects of HPT can be explained by the recipient species, the donor species or the interaction between donor and recipient. The results show high variability in the magnitude of HPT(2-93) but this variability is not a result of donor characteristics or even recipient characteristics but a result of the interactions between donors and recipients (df=20, α=.05, p=0.015). We will further investigate if the interaction can be explained by stigma/pollen size ratio, degree of co-flowering, or phylogenetic distance between donor and recipient species. This study will contribute to advance our understanding of how co-flowering plant communities interact amongst themselves after pollination.

Weedy rice is a persistent weed of cultivated rice (Oryza sativa) fields worldwide, which competes with the crop and drastically reduces yields. Within the US, two main populations of genetically differentiated weedy rice exist, the straw-hulled (SH) group and the black-hulled awned (BHA) group. Current research suggests that both groups are derived from Asian cultivated rice. However, the weeds differ from the cultivated groups in various morphological traits. My research focus is on the genetic basis of two such traits: seed shattering ability and differences in flowering time. The persistence of weedy rice has been partly attributed to its ability to shatter (disperse) seed prior to crop harvesting. I have investigated the shattering phenotype in a collection of US weedy rice accessions and find that all US weedy rice groups shatter seeds easily. Additionally, I characterized the morphology of the abscission layer at the site where seed release occurs and find that weeds begin to degrade their abscission layers at least five days prior to wild plants. I also assessed allelic identity and diversity at the major shattering locus, sh4, in weedy rice and find that all cultivated and weedy rice share similar haplotypes at sh4. These haplotypes contain a single derived mutation associated with decreased seed shattering during domestication. The combination of a shared cultivar sh4 allele and a highly shattering phenotype suggests that US weedy rice have re-acquired the shattering trait after divergence from their crop progenitors through alternative genetic mechanisms. Additionally, my investigation into flowering time in weedy rice shows that weed populations differ in their flowering times. I also assessed allelic identity and diversity at two genes involved in the transition to flowering, Hd1 and Hd3a, and again found haplotype sharing between weeds and cultivars with Hd1 only accounting for some of the flowering time differences between weeds. In order to locate genomic regions containing additional candidate genes I conducted a QTL mapping study on two F2 populations derived from crosses of weedy rice with cultivated rice. My results show sharing of QTL for flowering time between populations, yet lack of sharing of QTL for shattering.

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