Integrating disciplines in a plant chemistry laboratory module to enhance interdisciplinary and scientific thinking in undergraduate students
To meet the complex global challenges workers in STEM fields face, it is critical that today's students develop integrative technical skills and cognitive competencies. As a uniquely interdisciplinary field, medicinal plant chemistry provides an exceptionally rich environment for such training. Here, we describe a hands-on medicinal plant chemistry laboratory module (Phytochemical Laboratory Activities for iNtegrative Thinking and Enhanced Competencies; PLANTEC) for undergraduates that targets the development of core competencies in (i) critical thinking and analysis of text and data, (ii) interdisciplinary and systems thinking, (iii) oral and written communication of science, and (iv) teamwork and collaboration. Each student determines the natural product profile of a plant species using thin-layer-chromatography and gas chromatography-mass spectrometry. Students work in pairs and small groups to analyze their data and interpret their findings in chemical, biochemical, and biological contexts. PLANTEC is scalable and so can be offered in laboratory sections or in lecture courses. We implemented this module in an undergraduate biology lecture course over six fifty-minute lessons in the fall semesters of both 2018 and 2019. We also implemented modifications of PLANTEC to tailor learning objectives and thus emphasize different disciplines during data interpretation (e.g., plant chemistry, ecology, evolution). Students consistently responded that PLANTEC increased not only their confidence in analyzing, interpreting, discussing, and writing about new kinds of data and complex ideas, but also their interest in medicinal plant chemistry. Interdisciplinary laboratory modules of this type will be particularly useful in developing an innovative and versatile STEM workforce of the future.
Introduction<!>Module Design<!>Introduction, background research, and research objectives<!>Data collection and analysis<!>Interpretation and communication of results<!>Results and Discussion<!>Critical thinking and analysis of text and data<!>Interdisciplinary and systems thinking<!>Oral and written communication of science<!>Teamwork and collaboration<!>Setting and Assessment<!>References
<p>To meet the complex challenges facing the world today, it is critical that educators provide highest quality training to tomorrow's STEM work force. To accomplish this, national scientific organizations (e.g. AAAS, AMC) recommend that undergraduate educators use scientific teaching approaches in which students develop core cognitive and interpersonal competencies required for future innovation and decision making 1-3 . These competencies include, (i) critical thinking and analysis of text and data, (ii) interdisciplinary and systems thinking, (iii) oral and written communication of science, and (iv) teamwork and collaboration (Fig. 1A). It is also recommended that students develop these competencies in the context of the scientific process, making scientific laboratory-based activities ideal training venues. However, while many courses in STEM fields include laboratory sections, instructors often wish to include authentic scientific experiences in lecture courses lacking a specific laboratory section. Medicinal plant chemistry is a field of study at the intersection of chemistry, biochemistry, ecology, evolution, physiology, and human medicine. It is uniquely interdisciplinary and requires researchers to exercise core cognitive and interpersonal competencies to make novel inferences. Accordingly, our aim was to leverage the interdisciplinary nature of medicinal plant chemistry in developing a scalable laboratory module that can be incorporated in undergraduate laboratory or lecture courses and that can be tailored to emphasize different disciplines, according to the focus of the course. The module reported here (Phytochemical Laboratory Activities for iNtegrative Thinking and Enhanced Competencies; PLANTEC) uses the scientific teaching framework 4 to develop learning goals that achieve the above core competencies. PLANTEC thus provides a versatile laboratory activity offering students scientific experiences that promote the development of skills that the next generation of scientists must master to tackle emerging global challenges in health and environment-related fields.</p><!><p>Here we describe PLANTEC, a medicinal plant chemistry laboratory module in which students analyze the natural products present in plant species and develop their technical and cognitive core competencies. Prior to administering PLANTEC, we identified a unique plant species or tissue for each student that would participate by screening plant extracts using gas chromatography-mass spectrometry (GC-MS). Since students work in pairs in PLANTEC, we sought to identify pairs of closely related plant species or pairs of tissues from a single plant that yielded chromatograms with a manageable number of identifiable peaks. We anticipate that within a two-day period a skilled teaching assistant or technician would be capable of performing this screening in preparation for 20 students. Although GC-MS instruments are nearly ubiquitous in research universities, instructors may not always have cost-effective access to them. We therefore provide several alternative ways to implement PLANTEC when the GC-MS data cannot be generated in the course itself (see Supporting Information), including through the use of GC-MS data from online databases (e.g. datadryad.org/stash) or from one or more of the recommended publications 5 .</p><p>The workflow of activities in PLANTEC is centered around the scientific process (Fig. 1B). The module thus includes several phases: (i) introduction and background research, (ii) the development of research objectives, (iii) collecting observational data, (iv) analyzing and interpreting data in pairs and small groups, (v) communicating research in a student-focused scientific symposium, and a mini-scientific manuscript. Here we describe each phase of PLANTEC. A student handout (see Supporting Information) serves as a detailed guide to the module and includes grading rubrics, as well as descriptions of assignments and example frameworks for data analysis. We also provide a document for the instructor (see Supporting Information) that provides additional details on administering PLANTEC in the classroom.</p><!><p>At the beginning of the module, we use the module workflow diagram as a visual aid to introduce students to the specific learning goals and how they are achieved through each activity (Fig. 1C), as well as how those activities contribute to development of core competencies. We explain that (i) each student will apply the scientific method to the study of a plant species' natural products to meet a research objective of their design, (ii) the methods used will be thin layer chromatography (TLC) and GC-MS, and (iii) the students will communicate their results in a presentation and mini manuscript. Each student then finds a partner, and each pair chooses one of the pairs of plant species that we had pre-selected. As partners, students perform background research on their species and the methods they will use, guided by a set of thought questions (see Supporting Information). Students also read and summarize a scientific article that serves as a model for what they will do in PLANTEC, namely, in which TLC and GC-MS are used to study medicinal plant chemicals (for article suggestions, see references 6-9 and Supporting Information). Students are guided in this assignment by a set of key questions to answer in their article summaries. As a class, we discuss the scientific article, emphasizing the objective(s) of the study and how the methods were used to meet those aims. With this example fresh in mind, the students confer with their partners and develop a 'partner research objective' based on their plant species. We guide students toward feasible research objectives (i.e. that can be answered using the methods in PLANTEC), then ask several groups to share their research objectives aloud with the class. Depending on how PLANTEC is deployed in the classroom, the instructor can also aggregate students into small groups that will eventually pool their results to create larger datasets documenting chemical profiles from a set of multiple species pairs. In these small groups, students develop a 'small group research objective'. This objective should be unanswerable using a single pairs' data alone and instead require the larger data set (for examples, see Supporting Information). Having both a 'partner research objective' and a 'small group research objective' provides an opportunity for students to gain experience distinguishing between primary and secondary research objectives in the context of a single study, as well as the close correspondence between research objectives and the data that can address them.</p><!><p>We next meet in a teaching laboratory and each student is given tissue from their plant species that they then grind or muddle in organic solvent using a glass rod (for detailed protocols see Supporting Information). The released metabolites are partitioned between the solvent and an aqueous phase, and the extract is obtained as the top layer in the extraction tube (Fig. 1D). One aliquot of the extract is transferred to a gas chromatography vial using a Pasteur pipette (Fig. 1E), and another is loaded onto a TLC plate that is then developed (Fig. 1D). Students visualize TLC bands with a UV active stain. We collect the students' GC vials and, before the next class period, analyze them with a GC-MS system using a method designed to detect a wide range of compounds (for details and alternatives, see Supporting Information; we used a previously reported GC-MS method 10 ). We then export each student's raw data as a .cdf file (standard export format for both Shimadzu and Agilent systems) and send each student their raw data file as an email attachment. We then guide students through data analysis and interpretation, starting with their own data, then progressing to their partner dataset, and finally to their small group's dataset. Students record TLC band data (R f values, band size, and band color) in a table provided in their handout. Using R Shiny 11,12 , we have built an application (part of the R package 'phylochemistry' github.com/LucasBusta/phylochemistry) that students then use to analyze their GC-MS data. Students load their .cdf file and can extract mass spectra from any peak, perform spectral matching using the MassBank of North America (>18,000 spectra; mona.fiehnlab.ucdavis.edu), integrate peaks to estimate relative abundances of different compounds, adjust baseline parameters, and more (Fig. 1G). Although the R Shiny app is straightforward to use and requires no prior knowledge of R, some alternatives are to provide GC-MS results as print outs or to analyze GC-MS data on the instrument's native software, class size permitting. In analyzing their GC-MS data, students determine total peak area in their chromatogram, the percent of that area that can be identified, the area under each peak, and the identity of as many peaks as possible. They tabulate this information in their handouts and calculate the relative abundance of each identifiable chemical in their extract. Finally, students meet in their small groups and create their small group datasets by filling in the small group data table in their handout. Ultimately, they transfer their data to an excel spreadsheet in which to visualize their partner dataset (grouped bar chart, Fig. 1H) and their group dataset (annotated phylogenetic tree, template provided in Supporting Information, Fig. 1I). The spreadsheet can also be used to conduct quantitative and statistical analyses to support research objectives, for example, t-tests, ANOVA, normalization against an internal standard, or phylogenetic comparative analyses, though specifics of downstream analyses will depend on the disciplinary focus of the module.</p><!><p>Finally, the students use their partner and small group data to address their research objectives, interpret their findings from various perspectives, and determine if any new questions now arise. We guide students in identifying as many aspects of the data as possible that could be incorporated into a holistic interpretation. Since PLANTEC is based on scientific teaching, it is critical to allow students time and liberty to be creative about data analysis and interpretation, while being guided by the instructor to make scientifically correct inferences. We allow several class meetings for data analysis and interpretation so that students become familiar with their data and how to use it to address their research objectives. Students often find this process challenging: they are accustomed to undergraduate lab activities with pre-determined outcomes, of which the students are often aware. It can be difficult for students to understand that, depending on the particular emphasis of the research objective, there could be multiple "correct answers", and that the "most correct answer" is determined by their own scientific rationale and justification. PLANTEC challenges students to be creative in approaching their data and to discover unknown outcomes and interpretations on their own. After students overcome the initial intellectual hurdle of understanding they (not the instructor or the lab activity) are in control of discovering what messages the data convey, we consistently observed that students are enthusiastic and eager to embrace the challenge of exploring their data in new ways. The science communication component of PLANTEC provides an opportunity for each pair of students to showcase their interpretations as they prepare a scientific abstract, give a presentation, and write a miniature scientific manuscript. Guidelines in the handout describe how the activities in the module align with the segments of their abstracts, presentations, and manuscripts, thereby providing a framework within which students can initiate their writing. Grading rubrics for each assignment are also included in the handout. While running PLANTEC in 2018 and 2019, we used several sessions for in-class writing.</p><!><p>Our primary objective was to develop a laboratory module that leverages the interdisciplinary nature of medicinal plant chemistry to help students develop core competencies. Here, we discuss learning goals associated with the module's activities and how they address these competencies (illustrated in Fig. 1).</p><!><p>One of the first learning goals in the module is for students to conduct background research on their plant species and be able to describe several contexts in which natural products produced by their plant might function. Answering should require students to draw upon other material taught in the course implementing PLANTEC (i.e. lessons leading up to the module) when formulating their answers. In 'Plants in Human Medicine', the medicinal plant chemistry course in which we ran PLANTEC, students often describe how a plant's chemicals might function in plant defense or a system of traditional medicine. In PLANTEC, students also analyze an assigned scientific article describing a study that is similar to the one they carry out. They should be able to identify key sections of the article -the objective, methods used, as well as the main results and their key implications. When analyzing their partner data, students should be able to calculate and explain how the R f values and retention times of the compounds on their TLC plates in their GC-MS chromatograms relate to chemical structures and properties. Building on this, students should be able to connect the concepts of polarity and boiling point such that they can make reasonable predictions about correspondence between bands on their TLC plate and peaks in their GC-MS chromatograms (Fig. 2, upper portion of "Chemistry" semicircle). In analyzing their partner data, students should be able to think critically about the connections that exist between their background research and their findings (their species' natural products). For example, a student pair found that the carrot root epidermis, but not root cortex (root core), contains known antifungal compounds (Fig. 2, upper portion of "Biology" semicircle). With guidance, they should also be able to connect this difference to their knowledge of the root's environment, identify that only the epidermis is exposed to the soil, and a perhaps there is evolutionary pressure on the epidermis to protect the root from pathogenic soil fungi. Students should be able to subsequently connect this concept with information from their background research -that wild carrot plants are biennials with tap roots that need to survive for two seasons if the plant is to reproduce, further underscoring the importance of protecting the root from fungal pathogens. We find that reminding students about their background information and other course material can help them think more critically about their data by enabling them to identify connections between their chemical measurements and the potential biological functions of those chemicals.</p><!><p>Medicinal plant chemistry is a uniquely integrative field requiring multifaceted thinking about biological systems. It provides an excellent framework for learning goals involving interdisciplinary thinking and integration of information into a systems context. Working with plant chemical data in PLANTEC, students should be able to interpret their small group dataset in a way that connects concepts in chemistry, biochemistry, and/or biology (including ecology, evolution, or physiology). Consider the example of a small group identifying that one group of species had terpenoid chemicals on its leaf surfaces, while a very closely related group of species bore fatty acid derivatives on its leaf surfaces (Fig. 2, central annotated phylogeny). The group should be able to describe, for example, potential differences the biochemical systems that might underlie this difference: that the relative activities of fatty acid and triterpenoid biochemical pathways in the epidermal cells of these two clades may have diverged (Fig. 2, "Biochemistry" semicircle). With instructor guidance, they should also be able to assess this data in an evolutionary context, for example, by quantitatively determining whether more closely related species have more similar chemistries by regressing chemical similarity against the phylogenetic distance between each pair of species (Fig. 2, "Biology" semicircle). Students could also highlight their findings in a chemical context, comparing fragmentation patterns of compounds from different chemical classes (Fig. 2, "Chemistry" semicircle). Though the amount of instructor guidance needed for students to appreciate the multifaceted nature of their datasets is initially high, interpreting a single dataset in the light of multiple disciplines is an excellent means by which to develop student competencies in interdisciplinary thinking.</p><!><p>It is imperative that scientists develop finely honed communication skills so they may both share information with one another and engage in public discussions about the real-life problems that science endeavors to solve. Accordingly, PLANTEC includes several learning goals related to oral and written science communication. The first two relate to the scientific article prereading. Students should be able to concisely and accurately describe the key features of the article in a written summary, and then describe and evaluate these key features in a classroom discussion. When communicating about partner and group datasets, each student should be able to compose a scientific abstract that accurately describes the key elements in their own study. As partners, they should be able to deliver a short, conference-style oral presentation that communicates to the class their findings and interpretations in a way that sparks audience questions because it creates a "story" with a clear, logical flow and scientifically valid, well-justified conclusions. To help create a scholarly atmosphere, we run the class presentations as a student-focused Medicinal Plant Chemistry Symposium featuring abstract booklets and invite other department members to attend. Using feedback from the instructors and the questions posed during the symposium to enhance their interpretations, each student should be able to prepare a miniature scientific manuscript that describes their study and elaborates their findings and conclusions in clear and concise language.</p><!><p>As scientific pursuits become more interdisciplinary, it is increasingly necessary for STEM field members to work in collaborative, team settings to achieve project goals. Several PLANTEC learning goals address this core competency. First, students work in pairs to identify and list potential research objectives, then deliberate and identify which of the objectives are feasible given the measurement tools at their disposal. Working in small groups, students should be able to describe to one another their ideas for how an expanded dataset may enable new objectives, then assess these objectives and choose one that is unanswerable with a single pair's data alone but can be met using the small group dataset. When interpreting data, partners should compare the chemical profiles from their two species, explain to each other how the profiles differ or are similar, and discuss possible ways in which the comparison could be interpreted in different contexts (e.g. chemical, biochemical, biological, etc.). Each pair should then be able to explain to their small group how they interpreted their partner dataset. Working as small groups, students should discuss if and how their interpretations could be expanded with the information in the small group dataset. They should then be able construct arguments for the expanded interpretations and ideally arrive at an agreement about which reasonable interpretation is the most impactful. Finally, when presenting as partners in the student-focused Medicinal Plant Chemistry Symposium, students should identify discrete segments in the presentation in which to take turns narrating such that they spend a roughly equal amount of time taking and create no awkward transitions during the presentation.</p><!><p>We administered this module over six 50-minute class sessions during the fall semesters of each 2018 and 2019 in 'BIOS 368: Plants in Human Medicine', a lecture course on medicinal plant chemistry with average annual enrollment of 20 undergraduate students at the University of Nebraska -Lincoln. By surveying students that participated, we learned that (i) the learning goals associated with each section of the module were clear to the students, (ii) PLANTEC increased student confidence in analyzing, interpreting, discussing, and writing about new kinds of data and complex ideas, and (iii) the module increased student interest in medicinal plant chemistry (Fig. 3). Based on its success in the classroom, PLANTEC will help educators meet our needs for an innovative and versatile STEM workforce of the future and may also serve as a model for the development of similar scalable, versatile laboratory modules that can be implemented in a range of classroom settings. Hazards PLANTEC involves only a few hazards, including organic solvents, applying gentle pressure to glass rods, and, potentially, bioactive plant natural products (allergens, etc.). Students wear safety glasses, long pants, and close-toed shoes, work in fume hoods, and are warned about hazards before the extraction and TLC separations. Acknowledgement This work was supported in part by a Postdoctoral Research Fellowship in Biology (NSF PRFB IOS-1812037) to LB. We wish to thank Edgar B. Cahoon for use of his GC-MS system and Samantha Link for access to greenhouse plants. We would also like to thank the students of the Fall 2018 and Fall 2019 'Plants in Human Medicine' course at the University of Nebraska -Lincoln for their suggestions on how to improve this module.</p><!><p>(1) Vision and Change in Undergraduate Biology Education: A Call to Action. American Association for the Advancement of Science. 2011.</p><p>(2) Science for All Americans. American Association for the Advancement of Science. 1989.</p><p>(3) Transforming Undergraduate Education in Science, Mathematics, Engineering, and Technology. National Research Council. 1999. (4) Couch, B. A.; Brown, T. L.; Schelpat, T. J.; Graham, M. J.; Knight, J. K. Scientific Teaching: Defining a Taxonomy of Observable Practices. Cbe-life Sci Educ 2015, 14 (1), ar9. https://doi.org/10.1187/cbe.14-01-0002 .</p><p>(  The annotated phylogenetic tree in the center of the wheel indicates the distribution of six chemical compounds across eight species using colored circles. The color and position of each circle indicates the species in which that particular chemical was found, and circles are grouped according to compound class (terpenoids or fatty acyl compounds). It is an example of a small group dataset that was generated during one implementation of this module. This data can be interpreted in many different lights, broadly falling into categories of chemistry, biochemistry, and biology, indicated by the colored, truncated rings. For example, a student could discuss correlations between phylogenetic relatedness and chemical relatedness (biology perspective; blue truncated ring), relationships between polarity and boiling point and how TLC bands may be related to particular GC-MS peaks (chemical perspective; purple truncated ring), and/or how the distribution of chemical across the phylogeny may be underpinned by divergent biochemical or metabolic processes in the different plant lineages (biochemical perspective; yellow truncated ring).</p>