Performing Department
Plant Biology
Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) of sustainable agriculture and biotechnology.Our research program is focused on understanding mechanisms that regulate plant cell division and cell growth by analyzing the cytoskeletal element of microtubules in model and crop plants. We employ approaches of biochemistry, cell biology, genetics, genomics, and molecular biology to meet our goals. Because plant growth and reproduction are brought about by cell division and cell expansion that both are dependent on microtubules, results garnered from our investigations will shed light on designing strategies aimed at improvement of crop plants in the field. Three aspects connect our research program with crop production, breeding, and management. 1) Manipulations of cell division event can be applied for developing strategies aimed at increasing the biomass of crops. 2) Knowledge of cell division regulation during pollen formation can be applied to the production of male sterile mutants or haploid offspring for breeding purposes. 3) Understanding how basic mechanisms underlying microtubule-dependent plant growth will bring insights into the development of crop plants that are resistant to microtubule-targeted herbicides. In summary, our research program is aimed at advancing basic plant biology knowledge that serves for crop improvement in the agricultural field.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We aim to discover cytoskeleton-based mechanisms that regulate plant growth and reproduction. We employ both the model organism Arabidopsis thaliana and the crop rice (Oryza sativa) and focus on the cytoskeletal network of microtubules that govern cell division and cell expansion in response to internal and external cues. Microtubules also function as a critical factor for plants cells to respond to biotic and abiotic stresses. In addition, the dynamic network of microtubules serves as one of the primary targets of herbicides like oryzalin and pendimethalin. Therefore, our investigations of microtubule behaviors will not only advance the fundamental knowledge of plant growth and development but also can be translated into practices aimed at crop improvement and protection. Our research program covers the following two major aspects.MICROTUBULES AND PLANT CELL DIVISION. Plant growth and development are brought about by cell division that is followed by expansion and differentiation. There are splendid forms of cell division that allow simple cell propagation as well as the generation of cells acquiring distinct fates. During cell division, microtubules are organized into dynamic arrays that ensure the segregation of chromosomes and the partition of the cytoplasm. Microtubule reorganization is controlled by a spectrum of proteins that are expressed in spatiotemporally regulated manners. Our first specific objective is to discover microtubule-associated proteins that specifically function during plant cell division so that we can gain insights into mechanisms that regulate cell division progression. Results garnered from such investigations will serve as a foundation for practices like increases of biomass in crop production. The second objective is to elucidate principles that govern specialized cell division events in a plant's life. We have been focusing on what is called pollen mitosis I that leads to the production of two sperm cells during reproduction. Unlike a typical somatic cell division, this mitosis takes place after four identical microspores are produced by male meiosis. During pollen mitosis I, the microtubule spindle is assembled towards the cell cortex so that an asymmetric cell division takes place to produce the generative cell which is the mother of two sperm cells and the large vegetative cell which forms the pollen tube to deliver the sperm after pollination. The elucidation of the molecular mechanism underlying this division not only advances our knowledge of plant sexual reproduction but also has practical implications. For example, the conversion of the asymmetric pollen mitosis I into a symmetric division is desired for the production of haploid embryos from the microspore. Seed companies devote tremendous efforts to making double haploids that are derived from haploids. Besides, breeding can significantly benefit from the production of male sterile plants that are the result of unsuccessful pollen mitosis I. The third objective is to investigate microtubule-based cytokinesis in plants which is mechanistically different from that in most organisms of other kingdoms. Our investigations are centered at discovering different motors that act on microtubules to regulate their organization and function to transport materials for cell plate assembly. We investigate both general mechanisms of plant cytokinesis as well as cellularization of the endosperm, a specialized cytokinesis event, in plants, especially rice.MICROTUBULE DYNAMICS IN PLANT GROWTH. Because of the essential contribution of microtubules to plant growth, we aim to study functions of proteins that allow microtubules to undergo reorganization when cells respond to internal and external growth cues as well as various stresses. Plants employ the microtubule network to build the cell wall in order for their tissues and organs to achieve the desired forms and strengths. There are increasing numbers of examples that reveal connections between microtubules and trait improvement in crops. For example, duplication of a gene encoding a microtubule-associated protein leads to the formation of long grains in rice. Specifically, we aim to discover novel proteins that regulate both plant cell expansion and cell responses to challenges. By generating mutant plants that have either gained or lost functions of microtubule properties, we test them under physiological challenges like salt or drought stresses in order to assay how their responses may change accordingly. Besides, we also routinely test their sensitivities to common microtubule-targeted herbicides like oryzalin and pendimethalin in order to discover mutations that confer hypersensitivity or resistant to these herbicides. One of the practical goals is to introduce novel mutations into corresponding conserved genes in crops like rice and wheat so that weeds may be effectively eliminated in the crop fields upon herbicide application.
Project Methods
We primarily work on both the model plant Arabidopsis thaliana and the crop rice in our laboratory because they are genetically tractable and have rich resources established in the past few decades. Methods and tools developed by colleagues and us in these plants allow us to advance our knowledge that applies to all plants most rapidly. Our methods are summarized as follows:1. GENOMICS-BASED GENE DISCOVERY. Genome-wide gene expression analyses in Arabidopsis, rice, and maize by the plant biology community provide us with lists of genes that are up-regulated during cell division and endosperm development. By analyzing their amino acid sequences of proteins encoded by these genes, ones that are predicted to have microtubule-related functions will be selected for further experiments.2. MUTATIONAL ANALYSIS BY MOLECULAR GENETICS. Mutations at the targeted genetic loci in Arabidopsis and rice will be isolated in community-generated mutant pools or by genome editing tools. The isolated mutants will provide insights into the functions of the genes of interest after their phenotypes are analyzed macroscopically and microscopically.3. DNA-MEDIATED TRANSFORMATION OF PLANTS. Recombinant DNA techniques will be applied to assemble plasmid constructs that will be used in agrobacteria-assisted transformation experiments. Plant transformants will be identified based on selection markers. The transformation experiment will allow us to determine the linkage between a phenotype and a mutation that is based on genetic suppression of mutant phenotypes. It will also generate experimental materials for observations of protein behaviors after they are tagged by genetically coded fluorescent proteins.4. TRANSIENT EXPRESSION IN A HETEROLOGOUS HOST. Tobacco leaves are used in our transient expression experiments after agrobacteria carrying expression vectors are infiltrated into tobacco leaves. Transient expression experiments permit rapid assays of protein expression when compared to stable transformation.5. MICROSCOPIC OBSERVATION OF PROTEINS. We will apply state-of-the-art imaging techniques to observe the subcellular localization and dynamics of the proteins under investigation. Plant cells will be observed under fluorescent and confocal microscopes. Their subcellular localization and especially dynamic behavior during cell division and cell expansion will provide clues regarding their functions in vivo.6. PROTEIN PRODUCTION IN BACTERIA. We use bacteria as a convenient host for protein production. Because bacteria can be easily handled and grown into large quantities, proteins of interest can be expressed there so that desired amounts can be obtained for further studies.7. PROTEIN BIOCHEMISTRY-BASED INVESTIGATIONS. Proteins expressed in bacterial or plant cells will be purified by various protein separation methods. These proteins will be subject to functional studies like their interaction with microtubules. We also aim to investigate protein-protein interaction when target proteins are isolated. Furthermore, we will apply proteomics tools to investigate how functions of targeted proteins may be regulated by posttranslational modifications.Taken together, we take multifaceted approaches to gain insights into functions of novel proteins that regulate plant cell division and ultimately plant growth and reproduction.