Progress 01/15/23 to 01/14/24
Outputs
Target Audience:During this reporting period, our foundational research efforts again targeted plant biologists and biophysicists whose interests focus on the protection of staple crops and the development of bio-inspired materials. Additionally, our target audiences included student trainees, including underrepresented groups from City University of New York's City College and Borough of Manhattan Community College as well as a more general group at a local private college. We reached these audiences through invited lectures on Research in Plant Biophysics and Research in Molecular Biophysics. Changes/Problems:Growth of tomato plants from the CRISPR lines and consistent AFM-based nanomechanical measurements required time consuming optimization. What opportunities for training and professional development has the project provided?Postdoctoral Research Associate Ekram Wassel joined the lab, working with PI Ruth Stark and AFM Facility Manager Tai-de Li to apply her expertise in the polymer chemistry of bioactive surfaces to the wild type and genetically modified tomato cuticles produced by our collaborators. She obtained new training in ssNMR technology from NMR Facility Managers Hsin Wang and Eric Keeler at CCNY and the New York Structural Biology Center, respectively. A summer Research Experiences for Undergraduates trainee (Ms. Eleejah Rosa) and a new Physics Ph.D. student (Mr. Austin Laviano) were guided by Dr. Wassel in the operation and analysis of AFM and ssNMR methods for biophysical studies of tomato cuticles; both students also developed skills in oral, poster, and written communication of their scientific findings. Dr. Wassel and Mr. Laviano, along with other Stark Group members and a Visiting Scientist from Spain, participated in a series of tutorial workshops on ssNMR methods, applications to macromolecular structure of native plants, and biological inspiration for engineered plastic materials. They improved their oral communication skills through short presentations followed by questions and discussion. How have the results been disseminated to communities of interest?The PI gave three invited talks on research including this project to undergraduate researchers: (1) a keynote at Sarah Lawrence College's Undergraduate Research Symposium; (2) a lecture- demonstration-discussion session for visiting interns in an NSF-sponsored Undergraduate Science Education curriculum development program; and (3) a lecture for trainees in the NIH-supported CCNY- Borough of Manhattan Community College Bridges to the Baccalaureate program. She also introduced the project to new Ph.D. students in Biochemistry, Chemistry, and Physics who were undertaking researcher rotations to choose mentors and laboratory projects. In each instance, our oral presentations exposed undergraduate and Ph.D. level students to the investigative power of molecular biophysics approaches for investigations of how tomato crop protection can be compromised or enhanced. A manuscript in review after revision is listed elsewhere in this report. What do you plan to do during the next reporting period to accomplish the goals?We anticipate greater involvement of Mr. Laviano as he finishes his classwork and develops his technical expertise further. We plan to add Ms. Melanie Garcia as an undergraduate trainee to our lab. We hope to submit manuscripts for publication with each of our collaborators during the next granting period.
Impacts
What was accomplished under these goals?
Food staples including fruits, vegetables, and grains can suffer losses of 50% or more during growth, post-harvest storage, and processing due to abiotic threats (desiccation; thermal, radiative, or mechanical stress) and biotic infection (bacteria, fungi, oomycetes). Using the tomato as a model organism and focusing on its protective outer skin (cutin polymer with waxes), we are seeking to mitigate these challenges through an understanding of the molecular, thermodynamic, and biomechanical factors that underlie crop protection. Our findings will impact plant biologists and biophysicists who are working to improve the agricultural yield of food crops and to design bio-inspired sustainable materials. During this reporting period, we have conducted a range of biological and biophysical experiments that advance our overall project goals and specific objectives: Our partners in the Nawrath lab extended their production of CRISPR lines in which both genes involved in transport of cutin intermediates across the plasma membrane (SlABCG36 and SlABCG42) were mutated to modify tomato fruit cuticles of the Sweet 100 variety, a robust cherry tomato that is now used increasingly for gene editing and genomic studies. They have analyzed the amounts and molecular compositions of both cutin monomer and wax constituents in these tomato cuticles to look for differences with respect to wild type and MicroTom varieties. Estimates of glycerol content and ester crosslinks that could alter the protective capability of these fruit cuticles are in progress. To examine how the genetic modifications altered the surface topography, stiffness, resistance to deformation, and adhesion of the tomato cuticles, we conducted an extensive series of measurements using Atomic Force Microscopy (AFM). During this granting period, our experiments focused on comparisons of MicroTom double mutants and wild type reference materials, also exploring the variation of these cuticular properties with field conditions such as temperature and humidity typical of field conditions. Despite uncertainties attributable to the heterogeneous topography of the surfaces as well as variations for biological and technical replicates, several intriguing trends were evident. For fruits that were isolated 50 days past anthesis (DPA), the double mutant exhibited twice as strong adhesion to the surface and stiffness that was elevated by 20-100%. All three surface properties showed significant variations with temperature at this developmental stage; the double mutant lines followed steeply rising sigmoidal curves with inflection points between 45 and 55 , a temperature range that corresponds to a previously reported phase transition in wild type tomatoes. These findings suggest that the mutation produces cuticular surfaces that are stiffer and better able to adhere to substances or organisms, especially at high temperatures. Preliminary nanomechanical measurements have also been conducted for the 20 DPA stage, at lower temperatures where additional phase transitions have been reported, and as a function of humidity. Although the soluble cuticular materials obtained from chemical depolymerization reveal differences in amount and monomer proportions for the double mutant, solid-state 13C NMR spectra of the intact cuticles show the same relative proportions of each carbon functional group (e.g.,(CH2)n vs CHO vs. C=C) in comparisons of wild type and double mutant lines, for both 20 and 50 DPA developmental stages. These results indicate that subtle differences in the cutin building blocks can nevertheless favor different patterns of molecular organization that in turn alter the magnitude and temperature sensitivity of their functionally important surface mechanical and adhesion parameters. To test the impact of these mutations on cuticular heat transfer and verify the possible phase transitions deduced from AFM, we piloted differential scanning calorimetry measurements in collaboration with CCNY's Prof. George John. Previously, our partners in the Rose lab produced a CRISPR line in the M82 tomato that had a striking fruit phenotype in which the skin had rough brown 'scars' that displayed a phenolic spectroscopic signature and chemical recalcitrance typical of suberin polymers found in the skin of potato tubers. Thus, these findings suggest that metabolic disruption from the CDEF mutation could provide an independent pathway to form a suberin-like protective layer in tomato fruits. The Rose group is further characterizing the CDEF1 gene modifications that are thought to impact the polymerization steps required to form the cutin polyester. We received a gift of transgenic fruits from Dr. Hagai Cohen (Agricultural Research Organization, Volcani Institute, Israel), which were designed to enhance cutin and cutinase production, respectively, hypothetically altering resistance to the necrotrophic fungus Botrytis cinerea. A mutant line that up-regulated a cutinase gene exhibited brown patches that were reminiscent of the Rose group's CDEF1 mutant; those cuticles displayed distinctive molecular signatures in their solid-state 13C NMR spectra and altered nanomechanical characteristics. Along with the Nawrath and Rose materials, these tomato fruits comprise a genetically diverse set of study candidates that are designed to alter the biosynthetic steps, amount, macromolecular architecture, and pathogen resistance of their protective cuticles.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
K. Dastmalchi, V.C. Phan, S. Chatterjee, B. Yu, M. Figueras, O. Serra, and R.E. Stark, A Comprehensive Approach to Phytochemical Analysis of Macromolecular Composites that Protect Tubers: case studies in suberized potato periderm tissues, Phytochemistry Reviews, lightly revised manuscript under review.
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:During this reporting period, our foundational research efforts have targeted plant biologists and biophysicists whose interests focus on the protection of staple crops and the development of bio-inspired materials. Additionally, our target audiences have included student trainees at our minority-serving institution and from other colleges who are underepresented in STEM disciplines due to race, ethnicity, first-generation college attendance, and/or economic disadvantage. We reached these audiences through scientific partnerships, invited talks at both regional and international conferences, a research internship, and a Introduction to Plant Biophysics Research workshop for college students. Changes/Problems:Postdoctoral recruiting was made more challenging than expected by the COVID pandemic, but our research plan will be maintained with the hire of a polymer physical chemist who has a track record with biomaterials. Optimization and analysis of CRISPR/Cas9 procedures was more time consuming than anticipated. What opportunities for training and professional development has the project provided?Postdoctoral Research Associate Christine Chrissian worked with PI Ruth Stark to obtain new training in plant biophysics, applying her prior expertise in biological solid-state NMR to evaluate genetically induced changes in macromoleular composition of tomato cuticles and developing new exposure to atomic force microscopy though our collaboration with the group of Prof. Xi Chen. Biophysics Ph.D. student Austin Laviano undertook a summer internship to explore his interests in our tomato cuticle project. These individuals, along with other Stark Group members, participated in a 5-week solid-state NMR short course that included both spectroscopic fundamentals and biophysical applications. The PI attended the 5th international conference on Plant Apoplastic Diffusion Barriers (PADiBa) in Dundee, Scotland, where she held a planning meeting with the other team members and bolstered her knowledge of plant molecular and cellular biology. How have the results been disseminated to communities of interest?The PI gave several invited talks on research results related to this project: seminars in Chemistry Departments at Rhode Island and Kenyon Colleges, for CCNY's Bridge to the Baccalaureate Program, at the Bio-inspired and Green (BIG) Science & Technology Symposium hosted by CUNY, and at the PADiBa meeting described in the professional development section. In each instance, our oral presentations have exposed undergraduate and Ph.D. level students, in disciplines spanning biology, biochemistry, and physics, to the investigative power of molecular biophysics appraoches for investigations of how tomato crop protection can be compromised or enhanced. What do you plan to do during the next reporting period to accomplish the goals?We anticipate bringing a new Postdoctoral Research Associate on board in January, 2023 and affiliating Mr. Laviano with our lab as a Ph.D. student starting in June, 2023, augmenting our investigative team and allowing us to supervise the training of several undergraduate researchers on this project. We plan to submit a manuscript for publication on the phenotypic and structural changes achieved by modification of CDEF1 gene expression in tomato cuticles during the next reporting period.
Impacts
What was accomplished under these goals?
Food staples including fruits, vegetables, and grains can suffer losses of 50% or more during growth, post-harvest storage, and processing due to abiotic threats (desiccation; thermal, radiative, or mechanical stress) and biotic infection (bacteria, fungi, oomycetes). Using the tomato as a model organism and focusing on its protective outer skin (cutin polymer with waxes), we are seeking to mitigate these challenges through an understanding of the molecular, thermodynamic, and biomechanical factors that underlie crop protection. Our findings will impact plant biologists and biophysicists who are working to improve the agricultural yield of food crops and to design bio-inspired sustainable materials. During this reporting period, the Stark-Nawrath-Rose team has conducted a range of biological and biophysical experiments that advance our overall project goals and specific objectives: We employed RNA interference and CRISPR/Cas9 technologies to produce tomato fruits with two types of gene modifications. For the SlABCG36 or SlABCG42 genes involved in transport of cuticular precursors to their sites of polymerization, we used RNAi to down-regulate the expression of each homolog. Their double mutant, with properties described below, was obtained by both RNAi and CRISPR methods. For the CDEF1 gene that was hypothesized to compromise formation of the cutin biopolymer, we produced several mutants by CRISPR methods. To probe the impact of these mutations on transport of water though the fruit cuticle, Dr. Aurore Gerault, who produced the SlABCG36-RNAi and SlABCG42-RNAi mutants under the direction of Nawrath, received training to conduct water permeability measurements in the Rose lab. To test the impact of these mutations on cuticular heat transfer, we piloted differential scanning calorimetry measurements in the laboratory of CCNY's Prof. George John. Our plan to test how the mutations could alter the surface stiffness and mechanical strength of the tomato cuticles was initiated with CCNY's Prof. Xi Chen using Atomic Force Microscopy to assess surface nanomechanical properties. We conducted gas chromatography - mass spectrometry (GC-MS) on alkaline hydrolysis products from tomato cuticles along with light microscopy and solid-state 13C NMR (ssNMR) of the intact materials in order to evaluate the effects of the gene mutations on the molecular and macromolecular structure of the tomato fruit cuticles at two developmental stages. Whereas down-regulation of the SlABCG36 or SlABCG42 transporter genes left the cuticle thickness and molecular composition nearly unchanged, the double mutant displayed changes in the total amount, surface area, and proportions of chemical constituents; these differences were validated by both GC-MS analysis of the monomeric products and quantitatively reliable ssNMR of the intact biopolyester. Moreover, these molecular changes altered the cell shape and resulting organization of the cuticle embedded within the tomato fruit cell wall. In the case of the CDEF1 mutants, we discovered a remarkable fruit phenotype in which the skin had rough brown 'scars' that displayed a phenolic spectroscopic signature and chemical recalcitrance typical of suberin polymers found in the skin of potato tubers. Given the hypothesis of common initial metabolic steps in the formation of cutin and suberin protective plant polymers in tomatoes and potatoes that are both classified in the Solanum genus, these findings suggest that metabolic disruption from the CDEF mutation could provide an independent pathway to form a suberin-like protective layer in tomato fruits.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Norma A. Alcantar, Scott Banta, Gregg T. Beckham, Anthony Cak, Xi Chen, Taraka Dale, Christopher DelRe, Leila Deravi, Jonathan S. Dordick, Brian M. Giebel, Dianne Greenfield, Peter M. Groffman, Mand� Holford, George John, Neel S. Joshi, Nick A. Kotov, Jin Kim Montclare, Bradley S. Moore, Julia Ortony, Andrew Reinmann, Jiye Son, Ruth E. Stark, Rein V. Ulijn, Charles J. V�r�smarty, and Corey J. Wilson, Matter of Opinion: Bioinspired Green Science and Technology Symposium in NYC, invited submission, Matter, 5(7), 1980-84 (2022).
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