Research interests of the faculty affiliated with the Plant Biology Program at the University of Kentucky 

Expression and Activity of Sorbitol Dehydrogenase in Apple

Sorbitol dehydrogenase (SDH, EC 1.1.1.14) has been identified as the primary enzyme that metabolizes sorbitol, the major phloem-transported carbohydrate in apple. Thus SDH may play a critical role in defining apple fruit set, development, and postharvest quality. We are currently examining SDH expression in fruit flesh versus seed, developing techniques to determine at what developmental times and in which tissues the isoforms might be expressed, and assessing how sorbitol availability may influence SDH expression and activity during fruit set.

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Pawpaw Fruit Ripening and Postharvest Storage

The pawpaw [ Asimina triloba (L.) Dunal] is a promising tree fruit crop for Kentucky and the United States . Pawpaw grows wild in the mesic hardwood forests of 26 states in the eastern United States and is the largest edible fruit native to North America. This project is a collaboration with Dr. Kirk Pomper at Kentucky State University, who directs a comprehenisve program towards developing pawpaw as a new commercial tree fruit crop. The goals of our collaborative project are to fully characterize pawpaw fruit ripening, and to develop recommendations for maximizing postharvest storage life while maintaining high fruit quality. For further information on pawpaw, visit the Pawpaw Information Website

Plant Volatile Compounds and Microbial Development on Strawberry

Strawberry fruit produce a diverse group of wound volatile compounds, including aldehydes, alcohols, and esters, derived from the lipoxygenase-hydroperoxide lyase pathway. The volatile ( E)-2-hexenal, a 6-carbon aldehyde, is a major volatile product produced in response to bruising. We have determined that this volatile can influence the development of the major fungal mold on strawberry fruit, Botrytis cinerea L. or gray mold. Our current goals are to determine how bruising alters biosynthesis of the volatile compound and how the volatile interacts with the fungus.

The physiological basis for herbicide selectivity, herbicide mode of action (including absorption, translocation, and mechanism of action), and environmental effects on herbicide activity. Present research areas include: 1) the effect of herbicide protectants on enzyme activities, protein synthesis and herbicide metabolism; 2) environmental effects on herbicide efficacy and crop injury; 3) genetic variation in herbicide response and 4) interactions between herbicides when applied as herbicide mixtures.

Understanding and modification of plant quality. Much of the research involves alkaloid metabolism in tobacco and tall fescue. We are looking at the enzymes, pathways and genetic regulation of nicotine synthesis in attempts to understand limiting parameters and techniques of modification of alkaloid amount and composition that accumulates in the leaf. Some alkaloids in tall fescue are the result of plant metabolism but others are formed only when an endophytic fungus is present. The presence of the endophyte also is associated with insect and large animal toxicity. One project is aimed at understanding host-fungus interactions and the chemicals involved in toxicity.

My laboratory is dedicated to understanding the mechanisms that plants use to make the dizzying array of terpene/isoprenoid compounds. For many years, and like many laboratories, we focused our attention on how plants regulate the biosynthesis of antimicrobial terpene-based phytoalexins. Our interests have expanded from there. Our work utilizes a wide range of experimental strategies including genetic engineering, structure-function comparisons of genes and proteins, and cross-comparisons between many different plants and other organisms.

Our lab focusses on two main areas:

1. Characterization of genes and the proteins they encode involved in cell wall synthesis. our interest lies in cell shape and morphogenesis as well as the question of how extracellular cues are translated through the cell wall to respond to abiotic or biotic interaction.

2. Examination of how do plants respond to their regional and local environment? we are actively involved in both basic and applied research based on the concept of the farm of the future. our goal is assess value of building soil and plant biodiversity, increasing plant productivity at the systems level, water quality, nutrient use efficiency, carbon, nitrogen and renewable energy. a 60 acre research farm nearby to UK Campus and collaborate with both industry partners and other research faculty.

Seeds comprise 70% of the human diet world-wide and make up the bulk of the feed used to produce livestock that constitute a considerable proportion of the other 30% of our diet. On a practical level seed longevity and germination are of extreme importance to the establishment of nearly all plants and are the foundations of modern agriculture. On a fundamental level, seed germination is the stage of the life cycle when the plant undergoes a rapid transition from being most impervious (the seed), to being most susceptible (the seedling) to environmental stress. The orchestration of the switch from seed development to seed maturity and then to the germinative mode each involves a radical alteration of gene transcriptional activity. These alterations in transcriptional activity results in profound physiological changes that permit most seeds to survive dehydration to 5% moisture content, extend longevity in this dry state for considerable periods and finally undergo a fascinating alteration upon imbibition commencing with an intense metabolic activity and culminating in the completion of seed germination and the establishment of the next generation of plants. Using mutant screens followed by physiological and molecular investigations into the pathways thus uncovered, I examine how the seed thus fulfills its function as a propagule.

Production and physiology of woody perennials. The research relates to the physiological, biochemical and molecular aspects of growth and development in woody perennials. Specific projects include: understanding the control of in vitro morphogenesis related to juvenility in woody plants; the involvement of ethylene in growth and development of woody perennials including embryogenesis, dormancy and seed germination.

Our research program focuses on the general area of plant biochemistry and genetics and the application of biotechnology to crop improvement with particular emphasis on food, lipid and oil quality and new uses of agricultural commodities. This research involves the identification, isolation, cloning and manipulation by plant genetic engineering of agriculturally important genes. The major research thrust is the understanding and manipulation of fatty acid metabolism and triglyceride synthesis.


We are improving triglycerides of oilseeds with emphasis on soybeans for enhanced edible and industrial quality. For improved edible quality we are changing the ratios of the mix of vegetable oil fatty acids reducing both the saturated and polyunsaturated fatty acid percentages with a corresponding increase in monounsaturated fatty acids. This will result in a healthier and more stable product and eliminate trans-fatty acid formation. For industrial uses we are tailoring the triglycerides towards high triunsaturated fatty acid level which would make vegetable oils much more valuable in several industrial products such as "drying oils" as well as a superior source of w-3 fatty acids.


We are also are working toward developing oilseed oils high in epoxy fatty acids which will greatly increase their value for a number of industrial products. Epoxy fatty acids are examples of "oxylipins", or oxygenated products of fatty acids. Another major thrust of this research program is the detailed understanding of oxylipin formation in plant tissues. Most plant tissues form a range of oxylipins. Some oxylipins are very important in the flavor and aroma and therefore general quality of plant derived foods. Some are also important in plant pest defense and defense signaling systems.  Others we are working with can be useful new antibiotics and for prevention of food-borne illnesses.

Mechanism and significance of post-translational modifications in the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase as investigated using biochemical and molecular approaches. Specific projects are currently targeted towards understanding the functional significance of trimethyllysine-14 formation in the LS, and determination of the molecular and enzymological characteristics of LS N-methyltransferase and its interaction with the des(methyl) forms of rubisco.

A) Messenger RNA 3' end formation and post-transcriptional events in plants. Current emphasis is on the delineation of protein interaction networks involving plant polyadenylation factor subunits, and of the different RNA-binding activities of these proteins. B) Expression of foreign genes in plants. Several collaborative projects involving the expression of foreign genes in plants for particular purposes are in progress. These projects seek to use foreign genes as tools for analyzing biochemical and physiological phenomena in plants.

My lab is interested in identifying components of regulatory networks operating during plant embryogenesis. As a starting point, we are isolating genes that are regulated by AGL15 (for AGAMOUS-like 15). AGL15 is a member of the MADS-domain family of regulatory factors that is preferentially expressed during embryo development. We will use a combination of biochemical, molecular, genetic and structural techniques to identify genes regulated by AGL15 and to understand how the products encoded by these genes operate during seed development.

Molecular biology and evolution of plant symbionts. Endophytic fungi provide natural biological control to forage, turf, and wild grasses against nematodes, insects, and disease, but can also be toxic to livestock and mammalian wildlife. These fungi are seed transmissible, thus maternally inherited, and are ecologically important for persistence of several grass hosts. Together with the related Epichloe species, causative agents of grass choke disease, they provide a model for the evolution of mutualism, which we investigate with DNA sequence comparisons. We also genetically engineer fungal endophytes for use in forage grass cultivars. Transformation systems have been developed and genes involved in the synthesis of toxic ergot alkaloids have been identified. Such genes are targeted for disruption to reduce or eliminate endophyte toxicity to livestock and wildlife.

My lab studies the functions of the ubiquitin (Ub)/26S proteasome proteolytic pathway in the developmental and stress response pathways of Arabidopsis thaliana. The Ub/26S proteasome pathway is essential for cellular housekeeping as well as regulation 1. Its housekeeping and stress-defense functions involve the proteolysis of misfolded proteins, products of mistranslation and stress-induced damage that are highly toxic for the cell and need to be detected and removed rapidly. The regulatory functions of the Ub/26S proteasome pathway are based on the conditional degradation of activator and repressor proteins of various signal transduction systems. In response to external or internal stimuli, many regulatory proteins undergo posttranslational modifications that either prevent or trigger their attachment to Ub, leading to their stabilization or accelerated degradation by the proteasome.


Problems that are studied in my lab are:
1. Developmental and environmental control of 26S proteasome abundance: The 26S proteasome is a multisubunit protease and the coordinated expression levels of the gene set that encodes for its subunits ultimately determines the capacity of the Ub/26S pathway to degrade both misfolded and regulatory proteins. Our aim is to identify the cellular components that control the developmental and tissue specific variations in the expression levels of proteasome subunits.
2. 26S proteasomal control of hormone regulation: We have identified proteasome subunits that are needed for the responses to the hormones cytokinin and abscisic acid 2,3. Our aim is to understand the molecular basis of these proteasome-dependent steps in hormone regulation, by identifying the target proteins involved, as well as the mechanisms that control their ubiquitination and delivery to the proteasome for degradation.

Population and evolutionary biology in forest trees. Current work includes: (i) the molecular basis and population genetic structure of chloroplast and mitochondrial DNA polymorphisms; (ii) tests of cytonuclear disequilibrium (as well as the geographical structure of this disequilibrium) among the three major eucaryotic genomes (chloroplast, mitochondrial, and nuclear); (iii) spatial patterns of organellar and nuclear genetic diversity within populations; and (iv) molecular phylogeny. Taxa of interest include North American, European, and Asian species of pine (Pinus), hemlock (Tsuga), and oak (Quercus) genera.

Research on Pollutant metal accumulation and tissue partitioning in plants.
We are studying mechanisms that control Cd accumulation and tissue partitioning in plants. About 70% intake of the pollutant metal Cd in humans is derived from vegetable foods in the diet. Restricting accumulation of Cd in roots could significantly reduce Cd burden in humans. We are investigating Cd transport into root cell vacuoles, a primary site for transient and accumulated metal in this tissue. Cd accumulation and tissue partitioning are being studied in tobacco plants overexpressing Arabidopsis CAX genes that encode Cd/H antiporters, which are primarily responsible for vacuolar Cd accumulation under low Cd exposure conditions as occur in agricultural and natural environments. Studies include analysis of Cd transport in isolated tonoplast vesicles, Cd accumulation and root/shoot partitioning in solution cultured seedlings, and field studies conducted in a real-world fashion.


Research on the Biochemistry and Metabolic Engineering of Plant Trichomes.
Glandular, secreting trichomes are surface structures that occur on many plants (Annals of Botany, 93: 3-11, 2004). Principally, they produce and secrete to aerial surfaces compounds that provide for insect and pathogen resistance. We are studying metabolic biochemistry responsible for production of diterpenes and sugar esters of tobacco glandular trichomes. Gene knockdown and overexpression have been used to alter trichome exudates to enhance aphid resistance (demonstrated in the laboratory and field), and create novel trichome exudate chemistries. We have discovered a novel family of surface secreted proteins called phylloplanins that inhibit disease caused by the fungus-like oomycete, Peronospora tabacina.

The Yuan laboratory is interested in studying the mechanisms of and engineering new functions for transcription factors (TFs) and metabolic enzymes such as cytochrome P450. Transcription factors are sequence-specific DNA binding proteins that interact with the promoter regions of target genes and modulate the rate of initiation of transcription. Plant pathways controlling biosynthesis of many bioactive secondary metabolites are regulated by one or more TFs.  Understanding how TFs recognize specific DNA sequences and the ability to utilize the knowledge to create so called “designer TFs” will greatly facilitate many aspects of bioengineering.  The desired protein functions are being generated by novel protein engineering approaches, including laboratory directed evolution, mutagenesis, and combinatorial protein synthesis.  The P450 enzymes catalyze reactions for synthesis of many high value secondary metabolites in plants and are involved in drug metabolism in mammals.  The P450 superfamily is one of the largest protein families, making it an ideal target for exploiting the gene sequence space by laboratory directed evolution.

 

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