During plant reproduction, signals from female tissues (pistils) guide the sperm-carrying pollen tube to the egg cell to achieve fertilization and initiate seed development. Essential for production of many crops, including all grains and fruits, this signaling process is still poorly understood. This project will develop next-generation microdevices that are necessary for identifying pollen tube guidance signals in the model plant Arabidopsis thaliana. Existing pollen tube guidance bioassays are not sensitive enough to detect the signals that are likely produced in minute quantities by pistils. Consequently, despite extensive research, the pistil signals that mediate this essential process remain elusive. Microdevices have been used in numerous biological applications, can realize 3-D structures ranging from 1-1000 micrometers and can amplify hard to detect biological signals. Thus, microsystem technology represents a radically different approach to replicate the complex configuration of the pistil micro-environment and faithfully recapitulate pistil signal micro-gradients. In an interdisciplinary collaboration between engineers and biologists, this project will develop the first ever microsystem-based assay to identify pollen tube guidance signals. Microdevices will be designed and fabricated to resemble the in-planta micro-environment of A. thaliana fertilization and subsequently used to define chemotropic signals from pistils. Pollen tube guidance leads to successful fertilization and seed formation in plants, and identifying the guidance signals may aid in increasing seed yield and quality of crops essential for human nutrition. Consequently, the project could have a major global impact since seeds of crop plants supply nearly 80% of world's staple food. Furthermore, this project may spur development of automated microsystems for investigating plant cell-cell signaling at an unprecedented nano-scale resolution. This project will promote synergistic interactions between biologists and engineers; undergraduate students will carry out some of the experiments, and post graduate researchers will gain interdisciplinary scientific training.
Scientific accomplishments of this study: During plant reproduction, signals from female tissues (pistils) guide the sperm-carrying pollen tube to the egg cell to achieve fertilization and initiate seed development. Existing pollen tube guidance bioassays are performed in an isotropically diffusive environment (for example, in petri dishes) instead of anisotropically diffusive conditions required to characterize guidance signal gradients. Lack of a sensitive pollen tube guidance bioassay has therefore compounded the difficulties of identifying and characterizing the guidance signals that are likely produced in minute quantities by the ovules. The goal of this project is to develop a novel microsystem-based assay that mimics the in vivo micro-environment of ovule fertilization by pollen tubes in the model research plant Arabidopsis thaliana. We have designed and fabricated PDMS microdevices (Image 1) and developed a novel microsystem-based assay to assess and quantify pollen tube behavior in response to pistil tissues (Image 2). In these microdevices, the pollen tube growth rate, length and ovule targeting frequencies were similar to those obtained using a previously published semi in vivo plate assay. As a direct measure of the microdevice utility in monitoring pollen tube guidance, we demonstrated that in these microdevices, pollen tubes preferentially enter chambers with unfertilized ovules, suggesting that the pollen tubes sense the concentration gradient and respond to the chemoattractants secreted by unfertilized ovules (Image 3). Furthermore, in this microsystem-based assay, preferential turning of pollen tubes toward attractants secreted by unfertilized ovules was achieved consistently (Image 3). We further validated this assay by demonstrating that in this assay pollen tubes showed dramatically low preference to mutant ovules deficient in pollen tube attraction (Image 3). During the course of the study, we identified a novel mutant that is affected in pollen tube attraction to ovules, devised a novel map-based cloning strategy to identify the mutation, and performed a detailed study to understand the function of this gene (FEMale gametophyte gene 137 – FEM137), the first RNA Polymerase III subunit (which is critical for translation of proteins in a cell) to be functionally characterized, especially during reproduction, in Arabidopsis. Broader impact of this study: In vivo pollen tube guidance to excised Arabidopsis ovules has clearly been recapitulated in fabricated microdevices and, thus, micro-environments for studying Arabidopsis pollen tube growth and guidance can be recapitulated in vitro. Moreover, these results con?rm that microdevices are an excellent tool, far superior to a plate assay, for generating microgradients of guidance cues. This microsystem-based assay removes a signi?cant barrier to obtaining a comprehensive understanding of pollen tube–ovule interactions and will facilitate key experiments such as measurement of attractant micro-gradients secreted by ovules, as well as detection of repulsion signals, which are impossible to conduct in vivo and dif?cult to control using in vitro plate assays. Additionally, the assay is robust and ?exible enough that it can be adapted to characterize, analyze and quantify subtle behavioral changes of thin pollen tubes in species other than Arabidopsis. The novel map-based cloning strategy developed and demonstrated in this study can be immediately adapted and used in all plants. Thus, this work will lead to easy map-based cloning of gametophytic and zygotic lethal genes in Arabidopsis, which has long remained difficult to be cloned, as mutant plants are not viable. Thus this study has overcome this major hurdle and may facilitate identification of genes that are important for plant reproduction, a major source of human staple food. This project had a high impact on human resources. A total of 7 researchers in engineering and plant science disciplines (2 post doctroal fellows in plant sciences, 2 graduate students – one each in engineering and plant sciences, and 3 undergraduate students – 1 each in engineering and plant sciences) gained invaluable interdisciplinary experience in not only scientific research, including experimental procedures, data analysis, and scientific data presentation but also communicating their research to others outside of their disciplines in one on one meetings, lab meetings, scientific conferences, and community gatherings. Experience from this project will help the personnel involved in this project while pursuing higher studies and continuing to work in STEM fields. During the duration of this award many outreach activities in multiple venues by setting up activities on microscopes, videos, and displays and demonstrating the importance of pollen in our lives, ranging from seed formation to forensic palynology. The activities conducted during the three-year period reached more than 1000 people ranging from elementary school children to science enthusiasts in Tucson, Arizona.
