Peter K. Hepler - US grants

In this proposal the PI will address physiolgical and structural questions concerning the contribution of calcium (Ca) to three specific plant systems: 1) cytokinin-induced bud formation in Funaria, 2) phytochrome-stimulated spore germination in Onoclea, and 3) germination and pollen tube growth in Tradescantia and Nicotiana. In the physiolgical studies he will determine if the intracellular free Ca changes in response to a stimulus. The kinetics of Ca changes will be examined micro- spectrophotometrically in target cells of Funaria that have been microinjected with the absorbant Ca-indicator dye arsenazo-III. Particular attention will also be given to the role of the phosphatidyl inositol (PI) cycle in mediating development. Pharmacological agents that enhance or inhibit portions of the PI cycle will be tested for their effect on development. Structural studies will focus on cytoskeletal and membrane components. The ability of fluxes in Ca to modify these components will be analyzed at the light microscope level using fluorescent stains and antibodies, and at the electron microscope level using material that has been prepared by a rapid freeze fixation method, in which cytoplasmic structure is preserved with exceptional fidelity. Calcium ions act as intracellular messengers in many organisms. They are thought to couple the stimulus to the response and thus to mediate or trigger many important developmental events. The role of calcium ions as intracellular messengers has not been as well studied in plants. These studies will contribute to an understanding of the role of Ca in stimulus/response coupling in plants.

It is widely assumed that Ca is a regulator of mitosis. Changes in the intracellular free ?Ca! could modulate a variety of processes that control the formation and/or function of the mitotic apparatus. Recent studies show that free ?Ca! changes during mitosis; however, there are reported differences concerning the timing, kinetics, and location of these changes. The problem will first be clarified by comparing the timing and kinetics of Ca transients in a single cell type (Tradescantia stamen hair) using two different Ca reporting techniques: differential absorbance measurements from arsenazo-III-loaded cells, and light emission from aequorin-injected cells. Second, the timing and kinetics of Ca transients will be compared in cell types of different origin, including Tradescantia stamen hair, Haemanthus endosperm, newt lung epithelium, and cultured kangaroo rat (PtK1) cells, to see if there are biological variations. Third, the role of G-binding proteins and components of the phosphatidylinositol cycle in regulating the metaphase/anaphase transition will be examined. Fourth, the roles of Ca and calmodulin in regulating the rate of chromosome movement will be studied, by microinjecting Ca, calmodulin, GTP-gamma-S, inositol triphosphate, or Ca chelators directly into the living cells. Fifth and finally, the possible occurrence of ultrastructural changes in the mitotic apparatus during anaphase that might be related to chromosome movement will be addressed, using rapid freezing and freeze-substitution techniques to elucidate the structure and periodicity of microtubule connections to other microtubules or to the membrane and to see if changes can be related to chromosome movement. Cell division is a fundamental aspect of life on earth. In eukaryotic cells, the orderly distribution of genetic material in the form of mitotic chromosomes to daughter cells at each division event is absolutely necessary for normal maintenance of individual organisms and populations at all stages of life. The mechanism whereby this orderly distribution occurs is a complex phenomenon involving structural and regulatory aspects. In the proposed studies, Dr. Hepler will extend his previous work on mitosis, as well as definitively address a current technical controversy in the field.

9304953 Hepler Pollen tube elongation delivers the male gametes to the egg apparatus and is thus essential for sexual reproduction in plants. Within this highly polarized cell, vesicles are produced throughout and transported by cytoplasmic streaming to the apical region. By a process that is not well understood, these vesicles fuse specifically at the tip, providing new wall material that supports unidirectional elongation. Recent work has shown that a growing pollen tube of Llium has a sharply defined gradient in free calcium focused at the extreme apex of the tube. The further observation that dissipation of this calcium gradient leads to cessation of growth, and that reinitiation of growth is accompanied by its reformation provide evidence that the gradient is essential for tip growth. The purpose of this research is to analyze the relationship between the calcium gradient and tip extension in detail. Using indicator dyes that have been microinjected into pollen tubes the spatial profile of the calcium gradient will be examined in living cells and directly correlated with the rate of tube elongation. Cells will then be experimentally treated with a variety of agents including calcium buffers, channel agonists, antagonists, osmotic modulators, and growth inhibitors to allow a careful exploration of the relationship between the position and magnitude of the calcium gradient with the modulated growth behavior, and with the underlying cell structure and patterns of cytoplasmic streaming. The position and activity of calmodulin will also be studied in living cells using confocal laser scanning microscopy and fluorescence anisotropy. Finally, the relationship between the calcium gradient and the actin cytoskeleton will be examined in living pollen tubes using confocal microscopic analysis of cells that have been microinjected with fluorescent actin monomer. By correlating the calcium gradient with these several factors, a new and greater understanding of the mechanism of pollen tube elongation should result. %%% In higher plants, the male reproductive cells (gametes) are carried in pollen grains. For fertilization to take place, the pollen must deliver the male gametes to the egg apparatus within a flower by producing a tube, along which the male gametes move toward the egg. The pollen tube is the fastest growing of all plant cells. As it grows, it becomes several thousand times longer than it is wide. Because of its importance to plant reproduction and its dramatic growth characteristics, the mechanisms regulating pollen tube are important topics of research. The goal of this project is to understand the role of calcium ions in pollen tube growth. Sophisticated biophysical and structural techniques will be used to study the gradient of internal calcium along a growing pollen tube. The results should determine whether the calcium gradient plays a role in the organization and movement of the sub-cellular structures involved in pollen tube growth. ***

Award Abstract - 9970191


This award will support the acquisition of a suite of plant growth and environmental chambers for a plant growth facility. The Plant Growth Chamber Facility at the University of Massachusetts, Amherst, MA will be used by faculty in the Plant Biology Graduate Program for the growth and reproduction of plants under conditions of controlled light, temperature, humidity, and day length. Specific projects will examine clonal growth and resource capture in Fragaria (strawberries), molecular and biochemical analysis of plant reproduction in Nicotiana (tobacco), the role of calcium and the cytoskeleton during division and growth in Tradescantia (spiderwort) and Lilium (lily), the developmental regulation of auxin biosynthesis in Arabidopsis, virus replication and symptom reproduction in Brassica (turnip) and Arabidopsis, and the structural analysis of paramutation at the R locus of Zea (corn). The growth chambers will be located in the head house in close association with the Biology Department Greenhouses. Day to day operation and maintenance will be undertaken by experienced staff, which includes expertise in both plant growth and in heating and refrigeration. The facility will be governed by a core of participating faculty.

Pollen tube growth is the process by which flowering plants deliver the male gamete to the egg apparatus for fertilization, a necessary prerequisite to seed production. Characterized by rapid rates and a high degree of polarity, the elongation process is of great interest, both because of its economic implications and its relevance to fundamental aspects of plant cell growth and development. This project focuses on the role of calcium (Ca2+), protons (H+), and the actin cytoskeleton, and builds upon the recent demonstration that pollen tube growth rate oscillates, and that there are underlying oscillations in both ions and cytoskeletal activity. Using pollen tubes of both lily and tobacco, an attempt will be made to correlate at high temporal resolution the oscillations in intracellular Ca2+ and H+ with those in the growth rate. In addition, an exploration will be made on how these oscillations are changed when cell wall properties are modified by various agents, e.g., Ca2+, H+, boron, pectin methyl esterase, or Yariv phenyl glycoside. By determining if and how these agents modify the oscillatory behavior, and its relationship to both the intracellular gradients of Ca2+ and H+ as well as the extracellular fluxes of these ions, it seems possible that a temporal hierarchy can be developed for the activity of factors that control growth. Techniques involved here include ratiometric imaging for intracellular Ca2+ and H+, and ion selective vibration probe analysis to determine the magnitude and direction of extracellular fluxes. In studies on the cytoskeleton, attention will be given to the structure and dynamic distribution of F-actin in the clear zone, where rapid turnover and formation of these elements is presumed to occur. It will be particularly interesting to correlate these turnover processes with the oscillatory pattern of growth, again with the view of determining which events precede the rapid growth phases and which events follow. For these studies the newly developed GFP-talin fusion will be used to fluorescently label F-actin in living pollen tubes.

When taken together it is anticipated that these studies will provide insight into the physiological and molecular factors that control growth and development of the pollen tube. To the extent that other tip growing cells, e.g., root hairs, fungal hyphae, fern and moss protonemata, use similar mechanisms, the results from pollen tubes could be pertinent, and help us gain a general understanding of basic mechanisms involved in cell growth.

Research Activity: Pollen tube growth delivers the sperm cells to the egg apparatus in higher plants, and is essential for sexual reproduction and food production. Because the growth rate oscillates, as do the underlying physiological processes, it is possible, using cross-correlation analysis, to determine if a process precedes or follows the change in growth rate. The main objective of this project is to focus on anticipatory events, because they are thought to represent primary regulators of growth.

Three apical events that anticipate increases in growth rate will receive attention as follows: increase in cell wall thickness, increase in pH in the alkaline band, and the oscillatory inward movement of endoplasmic reticulum (ER). As a working model it is suggested that secretion anticipates growth and accounts for the cell wall thickening, that changes in alkalinity modulate the structure of the actin cytoskeleton, and that changes in actin dictate the movement of ER and secretory vesicles.

Using different microscopic methods, including ratiometric ion imaging, fluorescence confocal imaging, and electron microscopy (EM), secretion, pH, and actin structure will be examined. Secretion dynamics will be probed using a pollen specific pectin methylesterase, linked to GFP. Oscillations in pH will be determined in cells injected with BCECF-dextran. Experimental modification of pH and/or the activity of the H+-ATPase will characterize the relationship of the alkaline band to growth. The structure of actin will be examined at the EM level. Finally, the activity of the actin cytoskeleton will be probed by examining the motion of ER.

Broader Impact: This project emphasizes the inclusion of underrepresented minorities as primary investigators in the research/discovery process. Dr. S. McKenna, who is a faculty member at Long Island University (LIU), where there are many minority students, is a Senior Collaborator. His involvement is two fold: firstly, as a researcher, he shares in the discovery process, and greatly improves the upward trajectory of his career. Secondly, as a mentor, he will help identify qualified minority students and engage them in research.

Intellectual merit. This project focuses on the control of pollen tube growth in flowering plants. Pollen tube growth, which is confined to the extreme apex of the tube and is fast, occupies a crucial position in plant development because it delivers the sperm cells to the egg apparatus for fertilization. The process thus is essential for sexual reproduction and also for food production because the development of the fruits, nuts, seeds and grains that we eat requires a prior fertilization event. Successful tube growth and sperm delivery can only occur if expansion at the tip, driven by high turgor pressure, is continuously balanced by deposition of new wall materials. An important observation has been the discovery that the growth rate and the underlying physiological processes oscillate, making it possible to determine which processes lead and which follow the increase in growth rate. The ultimate goal is to elucidate the mechanisms that regulate cell wall expansion. The processes that seem particularly important include secretion of new wall material, mitochondrial production of energy, actin organization, calcium influx, and pH changes. Although it is possible that a single process acts as the sole pacemaker, it seems more likely that the oscillatory behavior is an emergent property dependent on the interaction of many different processes. As a consequence a multifaceted approach will be employed to determine how parallel events interact. Firstly, the above five processes will be probed during growth reorientation. The goal is to determine which process begins first, and where in the cell it becomes evident. To perturb pollen tube growth endogenous chemotropic factors, imposed ion gradients, and elevated osmoticum will be used. A structural analysis of cytoplasmic components and cell shape will be included. Secondly, energy metabolism, specifically the oxidation/reduction of nicotinamide adenine dinucleotide phosphate (NAD(P)H), will be examined, and its effect on growth assessed. Companion studies with inhibitors and with an extracellular oxygen-selective electrode will further probe the characteristics of energy metabolism. Thirdly, the role of calcium in mitochondrial energy metabolism will be examined. Because some NAD(P)H dehydrogenases are calcium sensitive, the inter-relationship between mitochondrial calcium, cytosolic calcium, and NAD(P)H will be explored. Intellectual merit accrues from the ability of the research to elucidate structures and physiological processes that contribute to the control of pollen tube growth. The research takes advantage of the accessibility of pollen tube growth in vitro, the apical location of the growth events, and the oscillatory nature of the growth rate and underlying physiological processes. It further exploits the extensive toolkit of genetic and fluorescent probes, high resolution imaging and microelectrode techniques to decipher cause and effect relationships between interacting events. Although the pollen tube is specialized for the delivery of sperm cells, it shares mechanisms with other tip-growing cells, notably root hairs, and also very likely with diffusely growing plant cells. Information gained from this study will therefore have wide reaching relevance.

Broader impacts. The broader impacts of the research activity derive in large part from its significant educational components. The Principal Investigator has had extensive experience guiding students. In addition, he holds a close connection to a consortium of 10 universities in the northeast US involved in the recruitment and retention of under-represented minorities into graduate education. The co-Principal Investigator has worked extensively in promoting science education with inner city school students, undergraduate and graduate students, and middle school teachers. He currently directs several undergraduates in honors research. Finally, the investigators have involved a pool of research motivated undergraduates at nearby Hampshire College, including under-represented minorities in their Baldwin Scholars Program. Through these different avenues, interested undergraduate students from all ethnicities will be encouraged to participate in the research/discovery process.