Connecting Research and Teaching Introductory Cell and Molecular Biology Using an Arabidopsis Mutant Screen

Abstract

A complex research project was translated into a Course-based Undergraduate Research Experience (CURE), which was implemented in sections of an introductory Cell and Molecular Biology laboratory course. The research laboratory generated an engineered plant line producing a growth-inhibiting, lipid-derived plant hormone and mutagenized this line. Students in the CURE cultured the mutagenized plant population and selected and characterized suppressor mutants. They learned to observe phenotypes related to the biosynthesis and perception of the plant hormone and explored the genetic and biochemical basis of these phenotypes. As the students studied the relevant genetic, molecular and biochemical concepts during this CURE, they were able to translate this knowledge into practice and develop scientific arguments. This CURE was a successful collaboration between the teaching lab and the research lab. It benefited both parties as the students had a real-life, deep learning experience in scientific methodology, while the research lab gathered data and materials for further studies.

Introduction

Course-based Undergraduate Research Experiences (CUREs) are known for their positive effects in helping students with conceptual understanding, scientific thinking, scientific communication, raising interest in research, and career decisions [1-6]. In 2009, the National Science Foundation along with the American Association for the Advancement of Science issued the call to action for improving undergraduate biology education [7], in which “integrated core concepts and competencies throughout the curriculum” and a “focus on student-centered learning” were emphasized. Responding to this vision and mission change, institutions have developed and implemented more CURE courses [8-11], and assessment instruments and learning outcomes for CUREs have been evaluated [2, 6, 12-14]. In addition, large scale partnerships, like SEA-PHAGES and the Genomics Education Partnership, have been established [12, 15]. These efforts illustrate the value instructors, researchers, and institutions see in CUREs as a method of providing students opportunities to practice critical thinking, collaboration, and communication, the transferrable skills needed to excel in future courses and careers.

A CURE curriculum has certain characteristics, a few of which include students making scientific discoveries, generating novel data for potential publications, team collaboration, and communication [6, 13]. In light of these characteristics, here we report a CURE collaboration between two units at Michigan State University: The Biological Sciences Program as the teaching unit offering an entry-level Cell and Molecular Biology Laboratory course, and the research laboratory headed by Christoph Benning. A crucial component of the research project, in which the knowledge and techniques are aligned with the core concepts of the laboratory course, is conducted by the students in class, meeting the CURE laboratory course mission for undergraduates to attain a deeper understanding and application of fundamental biological concepts and to gain a broader view of the underlying scientific methodology.

Specifically, this research project was built upon the functional description of a plastid lipase gene, PLIP3, by the Benning lab in 2018 [16]. The lipase PLIP3 degrades chloroplast lipids and its over-production in Arabidopsis, a small model plant extensively used in basic plant research, releases fatty acids that are subsequently converted to the bioactive compound JA-Ile (isoleucine), an amino acid conjugate of jasmonic acid (JA) [16]. Overabundance of JA activates plant defense mechanisms against herbivores, which strongly inhibit plant growth, alter leaf morphology, and lead to increased accumulation of the pigment anthocyanin [16, 17]. To identify new genes encoding enzymes or transporters that are required for the biosynthesis of this hormone, or receptors and signaling components mediating the hormone-initiated responses (Figure 1), the research lab developed a suppressor mutant screen in the PLIP3-overexpressing (PLIP3-OX) plants that can be readily transferred to a course laboratory. With this CURE, undergraduates were provided with hands-on experience in plant cultivation, genetics, molecular biology, and biochemistry allowing them to learn fundamental principles of modern biological science such as connecting genotypes with phenotypes, as they participated directly in real-life scientific research project. Therefore, beyond the educational aspects, the materials and data generated by the students from the CURE allowed for further studies by the research lab and interacting in this CURE project, undergraduate students and researchers together produced novel scientific insights into lipid-derived plant hormone biosynthesis and perception.

Figure 1.

Background hypothesis. The plastid lipase PLIP3 initiates JA biosynthesis. The JA signal transduction pathway in Arabidopsis is shown as well. PLIP3 protein is imported into the chloroplast where it associates with the thylakoid membrane. It cleaves an 18:3 fatty acid from the polar lipid MGDG. The free 18:3 is converted into OPDA which is then transported out of the chloroplast and into the peroxisome where JA is synthesized. JA-Ile, the active form of JA, triggers the signal transduction process, to which the plant responds showing the respective phenotypes. Abbreviations: 18:3: fatty acid with 18 carbons and three double bonds. PLIP3: Plastid Lipase 3; PI: Protein importer; MGDG: monogalactosyldiacylglycerol; PUFA: polyunsaturated fatty acids; OPDA: 12-oxo-phytodienoic acid; JA: jasmonic acid; JA-Ile: JA-Isoleucine; T: transporter; TF: transcription factor.

Method

This CURE implements components of an ongoing science laboratory research project in a Cell and Molecular Biology laboratory class taught to undergraduate students. Both are summarized below.

The Science Laboratory Project

The plastid lipase PLIP3 is encoded in the nucleus, translated from its RNA in the cytosol, and imported into the chloroplast by the chloroplast protein import machinery. It is associated with the thylakoid membranes, where it functions as a lipase and releases a polyunsaturated 18:3 fatty acid (18 carbons : 3 double bonds) from the thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) [16]. The free 18:3 fatty acid is further converted into 12-oxo-phytodienoic acid (OPDA), which is then transported out of the chloroplast and processed in the cytosol and peroxisome to synthesize JA. JA is conjugated with isoleucine to produce its active form, JA-Isoleucine (JA-Ile), which is bound by a receptor complex in the nucleus, initiating a signal transduction process that leads to plant responses such as reduced growth (Figure 1) [17, 18]. During the biosynthesis of the active hormone, multiple enzymes and transporters (T) are necessary. Some, but not all, transporters have been reported [19, 20]. The research goal is to gain a more complete understanding of factors involved in the biosynthesis of JA by identifying additional transporters at the chloroplast envelope membranes or the peroxisome and enzymes or auxiliary proteins involved in the process. In addition, the approach has the promise to identify novel components of the signal transduction process, possibly linking the action of this hormone to that of other plant hormones. The approach is based on a suppressor mutant screen in the engineered PLIP3 overexpression mutant (PLIP3-OX) and a detailed analysis of these suppressor mutants that can occur in factors along the pathway between signal generation, perception and transduction into plant responses. Overexpression of the PLIP3 cDNA leads to increased levels of JA and reduced growth, shorter leaf petioles, and darker plant color due to accumulation of anthocyanin pigments [16]. Suppressor mutations eliminating JA overproduction, perception or signal transduction are expected to appear more similar to the wild-type plants.

The Cell and Molecular Biology Teaching Laboratory

This is an introductory course required for all life science major undergraduates at Michigan State University. Students need to take college algebra and general chemistry 1 and the cell and molecular biology lecture before or at the same time as this cell and molecular biology course. Each semester, about 700 students enrolling in this laboratory course are divided into sections with a maximum enrollment of 28 students per section. When enrolling, students in the CURE sections lack prior knowledge that they selected a CURE section. During the semester, the students are divided into teams of 3 or 4 members. Each week students meet for a 3 h laboratory period and a 50 min recitation the following day. The learning objectives and assessments of this CURE course align with the non-CURE sections of the course.

Curriculum Design:

This CURE course is designed in a semi-flipped style. The students need to study the underlying knowledge of the techniques and the lab protocols and finish the pre-lab before the class. A pre-lab quiz is usually held at the beginning of the lab period to ensure the students’ preparation for the class. In-lab lectures are held after the quiz to further explain experimental procedures and how the techniques can be applied to the research questions. Recitation time is used to reflect on the experiment, further introduce research project related background, discuss the research project, and other course related activities (Supplemental Table 1).

The CURE mainly covers three major modules: Knowledge and skill-building (Week 1-5), a genotype-phenotype module (Week 6-8), and a lipid analysis module (Week 9-10). While each of these modules serves its own purpose, the genotype-phenotype module plays a central role in this CURE. This module ties the knowledge of cell and molecular biology taught in the accompanying lecture course, such as the flow of genetic information, DNA replication, mutations and mutants, genotype and phenotypes, membranes and transport, signal transduction and cell communication, into this research project and further expands the concepts, such as forward genetics, reverse genetics, primary mutation, and secondary (suppressor) mutation, which are fundamental concepts that help students understand the genetic background of the provided plants. A diagram about this module is shown in Figure 2. The background knowledge regarding the research project has been interwoven into the course materials throughout the semester (weekly topics and assessments are listed in Supplementary Table 1), to help the students connect the concepts and gain an overall understanding of the research project.

Figure 2.

Genotype-Phenotype relationship in different genetic backgrounds of Arabidopsis plants. There are three different genotypes included in this figure: the original unmutated “Wild-type DNA” and two type of mutations, “Engineered DNA” and “Randomly mutagenized DNA”. The “engineered DNA” is the genetic background of PLIP3-OX mutant which is generated by introducing the PLIP3 gene into wild type Arabidopsis by the reverse genetics method, and the “Randomly mutagenized DNA” is the genetic background for suppressor mutant which mutation is induced by mutagen through forward genetics approach. The very top of the figure shows the genotypes of the experimental plants used in this research, while the phenotypes row at the bottom of this figure illustrates the phenotypes of different plants. The middle section of this figure lays out how the DNA changes affect gene expression and further causes the metabolic and downstream signaling changes to the JA biosynthesis and plant response pathways. Beyond the concept of connecting phenotypes and genotypes, this figure also includes different genetics methods in generating engineered DNA or randomly mutated DNA by Reverse Genetics and Forward Genetics methods. Abbreviations as in the legend to Figure 1.

Course Implementation

Implementing the CURE, the students started their semester by sowing Arabidopsis seeds and observing seedling development. While the plants developed, the students learned the necessary lab techniques and the background science needed for the project. Students were guided in developing a proposal and design experiments for phenotyping and genotyping to initiate the research process. When the plants were about 4-5 weeks old, the students started the genotype-phenotype module of the course, during which they selected plants with phenotypes indicative of suppressor mutants (Figure 2) and designed experiments to genotype them to confirm that the suppressor mutants were in the correct genetic background containing the over-expressing PLIP3 transgene. Furthermore, the students started on a lipid module designed to capture a biochemical feature of the transgenic plants, because overproduction of PLIP3 affects thylakoid membrane lipid composition in subtle ways [16]. Towards this end, they extracted pigment and lipids from the mutant leaves and ran thin-layer chromatography to identify different polar lipid components in the plants. A classroom-wide discussion about future experiments and research interests was also included at the end of the semester. A flow chart of the curriculum can be found in Supplemental Figure 1.

One of the challenges of the CURE project was that there was more knowledge to be introduced while the students had limited time. To succeed, the students not only had to learn the lab techniques and their underlying science, but also had to have a good understanding of the research project and the underlying concepts. To help with these learning goals, lectures covering these concepts were given during labs and in recitations throughout the semester, in coordination with the practical lab work.

Course Assessment

This CURE used multiple layers of assessments to measure students’ learning. Formative assessments included pre-lab notebooks, post-lab notebooks, short-quizzes and worksheet evaluations. Summative assessments included two or three individual, one-page reports in which students developed a scientific argument, three team presentations with subsequent reflections, a final team poster, and a cumulative quiz as the final evaluation. Lab techniques were evaluated by mid-term and in final lab practicals. The one-page reports were scaffolded using the claim, evidence, reasoning format for scientific argumentation [21, 22]. The Survey of Undergraduate Research Experience (SURE) was administered at the end of each semester to collect students’ self-perception after taking the CURE [1]. The SURE survey is a 1-to-5 Likert scale instrument with 1 no gain or very small gain as the lowest and 5 very large gain as the highest scale. The data collected during our CURE was compared with the national CURE database containing data ranging from 2011 to 2018 [23], and the bar graph was shown in Figure 3. A t-test was performed based on the suggestions for data analysis of Likert scale data [24]. The research and informed consent protocols used to collect student data were approved by the institutional review board.

Figure 3.

Students’ learning outcomes based on the SURE Likert Scale data. “BS171” represents data from this CURE, “National” are data from all students in the complete SURE database including data from 2011-2018. The p values from t-test analysis are labelled for those factors showing difference, and confidential intervals (0.95) are also labeled on the factors evaluated in this research.

Results

Research Hypothesis Formulation

The biosynthesis of the lipid-derived plant hormone JA and its perception and signal transduction as shown in Figure 1, involves many steps mediated by enzymes and regulatory proteins encoded by numerous genes. In PLIP3-OX engineered plants, PLIP3 was overexpressed constitutively under the cauliflower mosaic virus 35S promoter, while normally its expression is induced by factors such as another plant hormone, abscisic acid (ABA). The resulting high PLIP3 activity leads to an increased release of 18:3 fatty acids, which then serve as the starting substrate for the JA biosynthesis pathway to produce increased amounts of JA-Ile, the active form of JA. The high amount of this hormonal signal is sensed by the plant and triggers a plant defense response, directing resources from plant growth to defense and ultimately causing plants to be small, dark and having altered leaf shape as shown in Figure 2. The Benning lab is interested in identifying transporters and new accessory factors involved in the production of JA and its precursors. In addition, it is expected that new factors connecting different hormone regulatory networks involved in biotic (JA) and abiotic (ABA) stress responses can be discovered using this suppressor screen. For this purpose, the PLIP3-OX plants overexpressing the PLIP3 cDNA were chemically mutagenized by the Benning lab to generate random point mutations in different genes. The working hypothesis is that these secondary mutations disrupting any gene encoding proteins involved in the biosynthesis, perception or signal transduction of JA, should lead to a suppression of the growth phenotype. Hence, these secondary mutations are called suppressor mutations and the screen a suppressor screen. The logic of this screen and expected outcomes are presented in Figure 2. There are also some caveats to be considered that could lead to the discovery of false positive mutants or mutations with no visible suppressor phenotypes if redundant steps of the process were disrupted. If the mutagenesis would disrupt the transgene, the phenotype of the PLIP3-OX plants would reverse, which can be tested by targeted sequencing of the transgene in a putative suppressor line. It is also possible that the mutant seed stock was contaminated with wild-type seeds leading to normal looking plants similar to the expected suppressor mutant plants. To tell wild type apart from genuine suppressor mutants, all putative mutant lines were tested for the presence of the original PLIP3-OX transgene. The ultimate goal was to identify the suppressor mutation in the disrupted gene, which will be done by sequencing a pooled mapping population [25]. This step goes beyond the current CURE project, but the underlying bioinformatics analysis could be conducted by students and integrated into future iterations of this CURE project.

Module Example: The Connection Between Concepts and Their Applications in This CURE

One of the modules in this CURE was focused on the distinction between genotype and phenotype, and the students were expected to confirm the genotypes with regard to the PLIP3 locus of the controls (the wild-type plant and PLIP3-OX engineered plant), as well as the putative suppressor mutants they had identified. To help students better connect underlying concepts and techniques, and to translate this knowledge into practice in identifying true putative mutants, the major concepts were introduced during the class as listed in Figure 2 to allow students to make sound claims as the result of their analysis. In this module, the underlying concepts of DNA-RNA-protein and accompanying replication, transcription and translation as well as the concepts of hormone signal generation and transduction were scaffolded into the process of information flow. For example, when the students learned about DNA extraction and practiced the technique, the three types of plants were included in their analysis. The students visually examined the plants and recorded the phenotypic differences, followed by a discussion of the reasons for this difference, and how the different PLIP3 genotypes in the wild-type and in particular the PLIP3-OX overexpression line and the suppressor mutants can affect the phenotypes. During the subsequent PCR analysis class, the students learned how to use the Arabidopsis database (www.arabidopsis.org) to access and export different types of PLIP3 nucleotide sequences, then to locate the primers on these sequences and further predict the length of the PCR product. By doing so, the students had a visual experience and gained a better understanding of the different genotypes in these plants by looking at the two different DNA forms of PLIP3, the native genomic sequence and the cDNA introduced by engineering. Concepts such as exons, introns, transcription, and reverse transcription were all integrated in the conversation, as shown in Supplemental Figure 2. Formative assessments, such as pre-labs and post-labs for the bench techniques, worksheets, and short quizzes were applied to assist the students in their understanding of these techniques and concepts, while the students were building their knowledge during the research time. The summative assessments related to this module included the mutant selection team presentation, mutant genotyping team presentation, and two individual scientific arguments using reasoning and evidence from the DNA extraction and PCR-agarose gel electrophoresis results to support claims about the genotype of the plants being analyzed. These assignments not only provided the students with opportunities to translate their knowledge into practice, e.g. by analyzing and evaluating their data, drawing conclusions from the data and finally presenting their findings to the class or developing their scientific argument during their individual assignments. They also provided opportunities for the students to practice their scientific writing and communication skills.

Learning Outcomes

During the Spring and Fall semesters of 2019, 97 students from 31 majors took this course and 91 students submitted the SURE III survey. Among these students, 52.7% were second-year college students, 24.2% first-year students, and third and fourth-year students made up 16.5% and 6.6%, respectively. We learned that 74.7% of the students did not have research experience prior to taking this CURE, and 25.3% had research experience during one or multiple academic semesters.

Based on the SURE III survey results, students from this CURE reported similar or greater perceived gains compared to students participating in other research experiences (Figure 3). Among the twenty factors that were evaluated, four factors had t-test p-values <0.001 with students in this CURE reporting greater perceived gains in all four of these factors (Figure 3). Notably, three of these factors, skills in the interpretation of results (p=0.0002, Cohen’s d = 0.36), understanding that scientific assertions require supporting evidence (p=.0001, Cohen’s d = 0.39), and skills in scientific writing (p<<0.0001, Cohen’s d = 0.44) are all learning outcomes supported by the claim evidence reasoning scientific argument framework. Since students in this CURE completed several highly scaffolded reports that required developing a scientific argument using the claim, evidence, reasoning framework, something not commonly incorporated into CUREs or other undergraduate research experiences, it is possible the highly scaffolded reports provided students a simple but useful framework that allowed them to connect evidence they generated in the lab to claims in their report and build ability and confidence in these areas. Because the data in the national SURE database was collected from students that participated in a variety of research experiences that differed in the length, structure, and the types of assessments used, and because data from this CURE is limited to a few iterations of this course, and because the effect size is small, this connection needs to be further corroborated as more date become available in the future.

The Successful Collaboration Between the Undergraduate Lab and Scientific Research

In this CURE, the students learned the techniques and the underlying science of cell and molecular biology and biochemistry, including DNA extraction, PCR, agarose gel electrophoresis, pigment extraction, lipid extraction, thin layer chromatography, and math in biology. Students also practiced literature search, proposal development, experimental design, report writing, team presentations, and poster writing and presentations. Related concepts beyond a typical cell and molecular biology laboratory course were also introduced to help students understand the research project, inspire their thinking, and expose them to plant based biological research often not available to students at early stages of their education. We have implemented this CURE for four semesters at Michigan State University and about 3300 plants from 48 different mutagenized pools have been planted and screened. About 30 of these plants were brought back to the research lab for further analysis, which will provide knowledge for new modules of this CURE and make it sustainable and evolvable.

Discussion and Future Modification

This CURE demonstrates how scientific research and undergraduate education can be integrated in a way that benefits both the research laboratory and the students. CUREs by their very nature are depending on the respective research environment at a given institution and specific research topics will vary depending on the participating research groups. However, the underlying concepts are transferable, and the process outlined here in developing this CURE can be repeated for other projects. It is also possible for others to participate in this project as seed stocks for the lines used here together with the course materials will be made available to course instructors upon request and the latter deposited at CUREnet (https://serc.carleton.edu/curenet/index.html).

Among the three major modules in this CURE, the techniques and knowledge-building module and the genotyping module were more correlated to each other and the students were able to reach a clear answer about whether the potential suppressor mutants were in the correct genetic background. The purpose of the lipid module in the curriculum was to characterize the mutants at the small molecule level and was more independent of the other two modules and leaned more towards biochemistry knowledge and techniques. As the research moves forward in the research laboratory, we will place more focus on the knowledge and techniques leading to the identification of the mutant DNA sequence which will be based on the sequencing of a bulked mapping population and the bioinformatics analysis and interpretation of the sequencing data. In fact, the bioinformatics analysis leading to the identification of the respective gene candidates can be recapitulated by the students in a future CURE module. Similarly, as candidate genes become available, a module focused on the protein encoded by the wild-type gene disrupted in the suppressor mutant will be included in the future covering all aspects of the information flow from genotype to phenotype. We have not yet factored in the influence of the environment on the phenotype and with appropriate mutants, an additional module could be developed to introduce this additional concept.

The student survey provided us with new insights into how to further evaluate the students’ learning. Additional assessment instruments, such as the Laboratory Course Assessment Survey [26], could be applied for a deeper evaluation of the students’ learning outcomes.

Supplementary Material

1

Supplemental Figure 1. Course design flow chart

Supplemental Figure 2. Different forms of PLIP3 that exist in the plants in this CURE.

Supplemental Table 1: Course syllabus and assessments.