Previous research by the Principal Investigator (PI) has derived, developed, and established quantitative biophysical equations that describe expansive growth of the large cylindrical single-celled sporangiophores of Phycomyces blakesleeanus and other cells with walls, i.e. algal, fungal, and plant cells. The objective of the current research project is to obtain insight into relationships between the biophysical variables and associated biological processes that control expansive growth and growth behavior by using mutant sporangiophores that exhibit abnormal growth behavior. The research will focus on strains of stiff and hypertropic mutant sporangiophores which exhibit weak and enhanced tropic responses and growth responses, respectively. Biophysical experiments will be conducted to determine the magnitude and behavior of relevant biophysical variables for stiff and hypertropic mutant sporangiophores. The obtained results will be compared to those of wild type sporangiophores. Research conducted for the prior NSF Grant (MCB-0640542) proposes a fungal wall chemistry hypothesis which can explain the observed behavior of relevant biophysical variables during anoxia, during light and avoidance growth responses, and for different stages of sporangiophore development during steady growth. In the current research project, the fungal wall chemistry hypothesis is complemented with new theoretical findings that provide quantitative relationships between relevant biophysical variables and biological processes that are integral to expansive growth. Importantly, the fungal wall chemistry hypothesis together with these newly proposed quantitative relationships can reconcile the previously established quantitative biophysical equations for expansive growth with other previously proposed biological models for growth of algal, fungal, and plant cells. The fungal wall chemistry hypothesis and the newly proposed quantitative relationships will be used to analyze and interpret the experimental results obtained from stiff and hypertropic mutant sporangiophores and wild type sporangiophores. In the near future, the results of the proposed research and the sequencing of the genome can provide new insight into the functions of genes and their products in expansive growth and regulation of expansive growth of fungal cells. As the sporangiophores exhibit growth responses to sensory stimuli, the results can provide new insight into the functions of genes and their products in sensory transduction pathways.
Broader Impacts
The results of the proposed research will be integrated into two new graduate bioengineering courses that the PI introduced, developed, and teaches: Cellular Bioengineering I and II. Participating graduate students will be encouraged to teach one of the undergraduate laboratory courses so that the PI may mentor them through the course for the first semester and develop their confidence and teaching skills so that they can teach the second semester more independently. Graduate and undergraduate students, which include members of underrepresented groups, will participate in all aspects of the research project. The PI and participating students will conduct workshops for the Society of Hispanic Professional Engineers student chapter's annual outreach conference for high school and middle school students in the Denver metropolitan area.
During the past three decades, the PI (Dr. J.K.E. Ortega, Professor in the Mechanical Engineering Department, University of Colorado Denver) and his students have conducted theoretical and experimental research, supported by prior NSF Grants, that focus on deriving, validating, developing, and establishing biophysical equations that describe and predict expansive growth behavior of algal, fungal, and plant cells, i.e. cells with walls. A major result of their research is the establishment of Augmented Growth Equations that describe and predict growth rate behavior of cells with walls. Many predictions made by the Augmented Growth Equations for algal, fungal, and plant cells have been validated with experimental research, including the results of in vivo stress relaxation experiments and in vivo creep experiments. Most of the experiments employ the pressure probe that measures and controls the magnitude and behavior of the turgor pressure within individual cells. Overall, the results of the theoretical and experimental research contributes to a better understanding of how algal, fungal, and plant cells grow and how their growth rate is regulated. It is envisioned that in the future this research will lead to the development of mathematical models that can assist in the improvement of crop production rates and crop production in adverse climates and conditions, and facilitate genetic tailoring of algae, fungi, and plants for specific purposes. The major objective of the current research project is to obtain insight into relationships between the biophysical variables within the Augmented Growth Equations and associated biological processes that control expansive growth and growth behavior by using mutant sporangiophores that exhibit abnormal growth behavior. Because expansive growth occurs within a limited region of the cell wall of the sporangiophores, the growth zone, the Augmented Growth Equation describing the cell wall mechanical behavior was theoretically re-derived to include a "growth zone". In vivo creep pressure probe experiments were conducted on strains of stiff and hypertropic mutant sporangiophores, which exhibit weak and enhanced tropic and growth responses, to measure the inclusive biophysical variables. The obtained results are compared to those of wild type sporangiophores. It is found that the wall extensibility biophysical variable within the modified Augmented Growth Equation was considerably smaller for the stiff mutants compared to wild type. The wall extensibility biophysical variable reflects the viscous nature of the wall and the length of the growth zone, both of which are smaller in stiff mutant sporangiophores. These findings can explain why stiff mutants exhibit weak tropic and growth responses, because the viscous nature and growth zone length are considerably smaller compare to respective values obtained from wild type sporangiophores. Interestingly, the results reveal that the turgor pressure was larger and the biophysical variable, critical turgor pressure, was smaller for stiff mutants, thus explaining why the elongation rate of the stiff mutant sporangiophores were nearly the same as those of wild type sporangiophores. Unfortunately, good experimental results could not be obtained using hypertropic mutant sporangiophores which are small in diameter (making it difficult to measure the turgor pressure) and sporadic in growth rate. Another theoretical objective is to extend the application of the Augmented Growth Equations to plant cells in tissue. Cell walls are part of the apoplasm pathway that transports water, solutes, and nutrients to cells within plant tissue. Pressures within the apoplasm, PA (in cell walls and xylem), are often times different from atmospheric pressure during expansive growth of plant cells in tissue. The Augmented Growth Equations were re-derived to include PA and to evaluate the turgor pressure, water uptake, and expansive growth of plant cells in tissue when PA is lower and higher than atmospheric pressure. The modified Augmented Growth Equations were validated with some experimental results and make new predictions that require new experiments to verify them. A review of mathematical models for cells with walls was conducted. The review highlights the strengths and weaknesses of different models and makes recommendations for future theoretical and experimental research. Broader Impacts Three undergraduate mechanical engineering students (one underrepresented minority woman) were involved in all of the experimental research and in some of the theoretical research. All three students obtained their B.S. in Mechanical Engineering and coauthored a peer-reviewed journal article with the PI. Two of the students (one underrepresented minority woman) continued their education and obtained their M.S. in Mechanical Engineering, coauthored a book chapter, and are now Ph.D. students in Mechanical Engineering. These two students also participated (for three years) as judges for middle and high school students' science and engineering projects submitted to the Colorado Math and Engineering Association (CMEA) outreach programs. In addition to disseminating the results of the theoretical and experimental research with three peer-reviewed journal articles and two book chapters, the PI disseminated the results to both undergraduate and graduate mechanical engineering students in the graduate level "Cellular Bioengineering" course that he teaches every year.
