Source: UNIVERSITY OF GEORGIA submitted to NRP
ASHLEY SNOUFFER: INVESTIGATING THE CELLULAR AND BIOCHEMICAL MECHANISMS OF OFPS AND TRMS IN TOMATO FRUIT SHAPE REGULATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1019218
Grant No.
2019-67012-29596
Cumulative Award Amt.
$154,954.00
Proposal No.
2018-08033
Multistate No.
(N/A)
Project Start Date
May 1, 2019
Project End Date
Apr 30, 2022
Grant Year
2019
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
(N/A)
Non Technical Summary
Highly distinguishable characteristics between plant species are often the result of differences in organ shapes and sizes.Leaves, flowers, and fruits can take on a wide variety of shapes in the plant kingdom, however, the biological processes responsible for generating the diversity of shape during plant growth and development remain elusive. Differences in organ shape can also occur within a species, such as the diverse fruit shapes of cultivated tomato,Solanum lycopersicum. Different shapes and sizes of tomatoes have been selected for during breeding to give tomatoes broad culinary uses throughout the world and to facilitate more efficient harvesting and packaging of the fruits. For example, large round tomatoes are excellent for slices on sandwiches while small round tomatoes are better for a salad. Elongated tomatoes can be easily packaged together in a can and are often used for sauces while flat tomatoes can be easily stacked for transport.Research has determined that many elongated tomato varieties are the result of breeders selecting for mutations in two Ovate Family Protein (OFP) genes, OVATE and OFP20. Furthermore, the ovary, which will develop into the tomato fruit after pollination, is also elongated by mutations in these two genes. This result indicates that these genes controlling elongated fruit shape are functioning in the growing ovary during flower development.This project aims to understand the molecular mechanisms that are regulated by these two OFP genes and to determine how loss of these genes results in elongated ovaries and fruits. This project will investigate OFP protein interactions, subcellular localization, and the orientation of cell divisions during tomato flower development to determine potential molecular mechanisms OFPs may regulate to control fruit shape.In additional to their role in tomato fruit shape, OFP genes have been implicated in shape regulation of multiple economically important vegetable and crop species including potato, melon, and rice. Tomato has excellent genetic and molecular resources making it an ideal system to study how fruit shape is regulated, and since OFP genes are present in nearly all land plants, determining the molecular mechanisms of OFPs in tomato may broaden our knowledge of how all plant organ shapes are regulated. Furthering our understanding of how plant organ shape is regulated will allow for breeders and producers to modulate harvestable organ shapes for a desired purpose, and can lead to improvements in harvest efficiency, packaging, and crop yield.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20114601050100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1050 - Developmental biology;
Goals / Objectives
GoalsThe overall goal of this project is to gain a better understanding of how organ shape is regulated in plants. I will investigate the role of Ovate Family Proteins (OFPs) and TON1 Recruitment Motif (TRM) proteins in ovary and fruit shape regulation to elucidate the cellular mechanisms by which they control organ shape in tomato. Modern tomato fruits have very diverse shapes that have different functions for consumers from slices to sauces. Furthering our understanding of the genes and cellular mechanisms that control different shapes in tomato fruits will give breeders and producers the ability to more finely regulate their desired tomato fruit shape. Furthermore, this research will broaden our understanding of how organ shapes are regulated in plants and may be used in other crop species to regulate other edible organ shapes and sizes.Objectives1.Determine the interactome of OFPs and TRMs in tomato flower development to better understand the protein modules that control fruit shape during development.2.Investigate the interactions between OFPs, TRMs and TON1 with microtubules to understand if they control cellular shape or division through microtubules.3.Investigate the orientation and timing of cell divisions in early tomato flower development and the sub-cellular localization of OVATE, OFP20, and TRM5. This will allow for a better understanding of these proteins are regulated and interact during development. Furthermore, it may help to elucidate the cellular function of these proteins in controlling ovary and tomato fruit shape.
Project Methods
MethodsAim 1. Protein interactions will be investigated through Western blot, Immunoprecipitation and, mass spectrometry. Results will be interpreted to determine the timing of OFP and TRM interactionsin vivoand the identify potentially new interacting partners of these proteins in the developing tomato flower.Aim 2. Protein interactions will be further investigated through Agrobacterium infiltration of tagged proteins intoN. benthamiana.Confocal microscopy will be used to evaluate the subcellular localization of these proteins and their relationship to microtubules.Aim 3. The cellular mechanisms by which OFPs and TRMs will be investigated by EdU labeling and confocal imaging to assess the role of cell division orientation and frequency in the altered tomato ovary shape.Evaluation PlanMilestone 1: Gain proficiency in proteomics and tomato transformation and apply my molecular biology knowledge and imaging skills to plants. I will use these skills to determine if OVATE interacts with TRM5in vivo, the cellular localization of OVATE, OFP20, and TRM5 proteins, and the impact on cell division orientation in the developing fruit.Milestone 2: Data presentation - I will present my findings at internal lab meetings and at conferences such as the annual ASPB and the Solanaceae conferences.Milestone 3: Science communication and public outreach - I will communicate my research to the scientific community through publication at the conclusion of this project. I will also present my data to the general public through Athens Science Café where I will emphasize the importance of understanding the functions of genes in controlling fruit shape in crops.

Progress 05/01/19 to 04/30/22

Outputs
Target Audience: Target audience: Plant developmental biologists and plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Professional development Three undergraduate reserachers were trained on this project Workshops Workshop covering using R in biology was taken Plant Center Retreat Professional Development Workshops Seminars IPBGG monthly seminars and journal club The University of Georgia Department of Plant Biology weekly seminars The University of Georgia Department of Genetics weekly seminars The University of Georgia Department Cell Biology weekly seminars How have the results been disseminated to communities of interest? This work has been presented at meetings as posters, within lab meetings, and institutional talks were give. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective:Determine the interactome of OFPs and TRMs in tomato flower development to better understand the protein modules that control fruit shape during development. Investigation of the interactome of OFPs and TRMs was carried out through RNAseq and WGCNA analysis offloral and developmental stages. OFPs and TRMs have dynamic expression patterns and the ability to interact biochemically may not represent biologically relevant interactions. Therefore, we assessed the expression pattern correlations of OFPs with TRMs known to impact fruit shape. While subsets of OFPs and TRMs were correlated in their expression during developmental stages only SlTRM19 and SlTRM26a were present within the same WGCNA cluster. GO terms correlated with the WGCNA clusters for each OFP and TRM assessed may also give indications on the biological functions. The SlTRM5 cluster was enriched for microtubule-based movement and processes, cell division and the regulation of cell cycle as well as DNA replication. The SlTRM19 and SlTRM26a cluster also have GO terms associated with the cytoskeleton and cell division. Interestingly, the OVATE cluster was more associated with GO terms which included gene regulation and regulation of biosynthesis processes, some of which are shared with SlTRM25. The cluster includingSlOFP20 had GO terms enriched for growth, cell wall organization, and carbohydrate metabolism which were shared with SlTRM19/SlTRM26a and SlTRM17/20a clusters. While peak expression for some SlTRMs occurs during fruit development the highest correlations with OFPs was observed during floral development. These results directed the assessment of interactions of OFPs and TRMs to ensure more biologically relevant assessments. Objective:Investigate the interactions between OFPs, TRMs and TON1 with microtubules to understand if they control cellular shape or division through microtubules. Co-expression of fluorescently tagged OVATE, OFP20, TRM3/4, and TRM5 with TON1b was done in N. benthamiana. Single expression of OVATE and OFP20 was cytosolic and cytosolic and nuclear respectively. Both TRM3/4 and TRM5 signally expressed are microtubule localized. TON1b signally expressed in cytosolic with some cytosolic strands. Co- expression of TRM3/4 or TRM5 with TON1b results in relocalization of TON1b to the microtubules. Co-expression of OVATE or OFP20 with TON1b all remain cytosolic and more localization of OVATE and OFP20 to the cytosolic strands with TON1b. Assessment of the relocalization of TON1b as a result of the OVATE and TRM5 interaction did not impact the microtubule structure found within the cells as measured by a microtubule marker. Additionally, Other OFPs were not found to co-localize to cytosolic strands as OFP20 does with TON1b so no further evidence that this subcellular localization is relevant was found. Analysis of mcherry tubulin mutants did not present any clear differences in microtubule structure within cells of the meristem The interaction between additional microtubule TRMs known to regulate organ shape have also be examined. TRM19 or TRM26a co-expressed with OVATE relocalizes these TRMs from the microtubules to the cytosol. However co-expression of TRM19 and TRM26a with OFP20 results in microtubule localization of OFP20. These results recapitulate those seen for OVATE, OFP20 and TRM5 suggesting this may be the conserved mechanism for these OFPs in regulation of subcellular localization. The impact of this relocalization may go on to regulate the cell cycle progression, although the molecular mechanism by which this occurs remains elusive. Objective: Investigate the orientation and timing of cell divisions in early tomato flower development and the sub-cellular localization of OVATE, OFP20, and TRM5. This will allow for a better understanding of these proteins are regulated and interact during development. Furthermore, it may help to elucidate the cellular function of these proteins in controlling ovary and tomato fruit shape. These results revealed that while the orientation of cell divisions in the anticlinal and periclinal directions is relatively equal early in development, it is biased to more periclinal divisions as the organ elongates. However, there was no significant difference in the number of periclinal vs anticlinal cell divisions in the WT and ovate mutants. There was a significant decrease in the frequency of cell divisions between the WT and ovate mutants at early floral stages from the meristem to 7 days post initiation (dpi) and an even further decrease in cell division frequencies in the ovate/sov1 (ofp20) double mutant at these stages. The decrease in cell division frequencies was partial rescued by the trm5 mutant in the ovate/sov1/trm5 triple mutant. Antibody labeling for OFP20 was successful and was found in the cytosol and nucleus of mid-staged floral buds. Staging of protein expression in younger and older buds is in progress. TRM5 antibody labeling was also detected but further fixation methods to preserve microtubule architecture are necessary and in progress. Cell size and shape are not altered early in floral development in the ovate and ovate/sov1 mutants but cells are significantly smaller and flatter in the trm5 mutant starting around 5dpi. There are also smaller cells and flatter cells in the triple mutant. In the mature ovary the cell shapes and sizes are larger and more elongated in the ovate and ovate/sov1 mutants but remain smaller in the trm5 and ovate/sov1/trm5 mutant. The lack of significant difference in cell division orientations indicates that changes in the orientation of cell divisions is not likely the primary mechanism resulting in the change in organ shape. However, the decrease in the cell divisions at these stages could result in an organ with a reduced width suggesting this could be the initial event that results in a change in organ patterning and the pear-shaped proximal end of the tomato fruit. While there are no alterations of cell shape or size early in floral development for ovate/sov1 the larger and more elongated cells in the proximal end of the mature ovary may contribute to the final pear-shaped fruit. The smaller and flatter cells in the early trm5 mutant ovary, however, may be causative for the flatter fruit shape and the pattern persists in the ovate/sov1/trm5 triple mutant, suggesting trm5 may have an independent role in regulating cell shape and this may contribute to the partial rescue of the elongated shape observed in the ovate/sov1/trm5 triple mutant. Together these results suggest that specific subsets of TRMs interact with OFPs to regulate organ shape during floral development in tomato with distinct co-expression patterns. This regulation occurs through modulation of cell division frequencies likey through sequestration at specific subcellular compartments. This regulation does not impactthe mechanism that regulates the orientation of cell divisions during organ outgrowth but alteration to the numer of cells dividing at specific times cna result in changes toovary shape and ultimately tomato fruit shape.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ashley Snouffer, Carmen Kraus, Esther van der Knaap (2020) The shape of things to come: ovate family proteins regulate plant organ shape. Current Opinion in Plant Biology 53:98-105 DOI: 10.1016/j.pbi.2019.10.005
  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Biyao Zhang, Qiang Li, Neda Keyhaninejad, Nathan Taitano, Manoj Sapkota, Ashley Snouffer, and Esther van der Knaap (2022) A combinatorial TRM-OFP module is required to fine-tune tomato fruit shape New Phytologist
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Qiang Li, Qian Feng, Ashley Snouffer, Biyao Zhang, Gustavo Ruben Rodriguez, and Esther van der Knaap (2022) Increasing Fruit Weight by Editing a Cis-Regulatory Element in Tomato KLUH Promoter Using CRISPR/Cas9 Frontiers in Plant Science 13:879642 DOI: 10.3389/fpls.2022.879642
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Edgar Sierra-Orozco, Reza Shekasteband, Eudald Illa-Berenguer, Ashley Snouffer, Esther van der Knaap, Tong Geon Lee and Samuel F. Hutton (2021) Identification and characterization of GLOBE, a major gene controlling fruit shape and impacting fruit size and marketability in tomato Horticulture Research 8: 138 DOI: 10.1038/s41438-021-00574-3
  • Type: Journal Articles Status: Other Year Published: 2022 Citation: OFP-TRM module regulation of cell division in early floral development regulates organ shape in tomato. Ashley Snouffer, Carmen Kraus, Yanbing Wang, and Esther van der Knaap (2022)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: The role of OFPs and TRMs in patterning tomato fruit shape and identification of additional fruit shape modifiers. Snouffer, A, Zhang, B., Kraus, C., van der Knaap, E. Plant Center Retreat poster, Young Harris, GA. December 15-16, 2021(2nd place poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Investigation of the cellular function and comparative expression analysis of OFPs, TRMs, and SUNs in floral organ shape. Ashley Snouffer, Plant Functional Genomics Talk, Athens GA. November 10th, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Cell Division Patterns in Early Floral Development Shape the Tomato Ovary and Fruit. Snouffer, A. , Zhang, B. , Kraus, C., van der Knaap, E. Institute of Plant Breeding Genetics and Genomics Retreat. Virtual Talk and poster. May 27-28, 2021 (1st place Postdoc talk)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Early Floral Development Regulates Shape of the Tomato Ovary and Fruit. Snouffer, A., Zhang, B. , Kraus, C., Taitano N. , van der Knaap, E. UGA Developmental Biology Retreat poster. Virtual May10th, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Regulation of cell division and cell shape by tomato OFPs and TRMs to control early ovary shape. Snouffer, A., Zhang, B. , Kraus, C., Taitano N. , van der Knaap, E. Sol International Online Meeting Nov. 9th-11th 2020 Talk
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Developmental mechanisms regulated by tomato OFPs and TRMs to control early ovary shape. Snouffer, A., Zhang, B. , Kraus, C., Taitano N. , van der Knaap, E. Sainsbury Laboratory Symposium '20 Pluripotency in Plant Development Sept. 22nd-25th 2020


Progress 05/01/21 to 04/30/22

Outputs
Target Audience: Target audience: Plant developmental biologists and plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Professional development Three undergraduate reserachers were trained on this project Workshops Workshop covering using R in biology was taken Plant Center Retreat Professional Development Workshops Seminars IPBGG monthly seminars and journal club The University of Georgia Department of Plant Biology weekly seminars The University of Georgia Department of Genetics weekly seminars The University of Georgia Department Cell Biology weekly seminars How have the results been disseminated to communities of interest?This work has been presented at meetings as posters, within lab meetings, and institutional talks were give. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? ? Completion of Aim 1:Investigation of the interactome of OFPs and TRMs was carried out through RNAseq and WGCNA analysis offloral and developmental stages. OFPs and TRMs have dynamic expression patterns and the ability to interact biochemically may not represent biologically relevant interactions. Therefore, we assessed the expression pattern correlations of OFPs with TRMs known to impact fruit shape. While subsets of OFPs and TRMs were correlated in their expression during developmental stages only SlTRM19 and SlTRM26a were present within the same WGCNA cluster. GO terms correlated with the WGCNA clusters for each OFP and TRM assessed may also give indications on the biological functions. The SlTRM5 cluster was enriched for microtubule-based movement and processes, cell division and the regulation of cell cycle as well as DNA replication. The SlTRM19 and SlTRM26a cluster also have GO terms associated with the cytoskeleton and cell division. Interestingly, the OVATE cluster was more associated with GO terms which included gene regulation and regulation of biosynthesis processes, some of which are shared with SlTRM25. The cluster includingSlOFP20 had GO terms enriched for growth, cell wall organization, and carbohydrate metabolism which were shared with SlTRM19/SlTRM26a and SlTRM17/20a clusters. While peak expression for some SlTRMs occurs during fruit development the highest correlations with OFPs was observed during floral development. These results directed the assessment of interactions of OFPs and TRMs to ensure more biologically relevant assessments. Completion of Aim 2:Assessment of the relocalization of TON1b as a result of the OVATE and TRM5 interaction did not impact the microtubule structure found within the cells as measured by a microtubule marker. Additionally, Other OFPs were not found to co-localize to cytosolic strands as OFP20 does with TON1b so no further evidence that this subcellular localization is relevant was found. Analysis of mcherry tubulin mutants did not present any clear differences in microtubule structure within cells of the meristem. Completion of Aim 3: Further replication supports that the cell division frequency is reduced early in floral development in ovate/sov1 mutants and this is likely the causative effect which results in the initial alteration to organ shape. At anthesis there are alterations to cell size and shape which may also impact the final organ shape. The addition of the trm5 mutation partially rescues the reduction in cell divisions frequencies. Additionally, thetrm5mutant also has significantly smaller cells early in floral development which may also contribute to the rescue of organ shape but may represent a functionally independent role for TRM5. Analysis of analternative mutantwhich elongate fruit shape, SUN, did not present with the same reduction in cell division frequencies early in development, suggesting this mechanism is specific to the OFP-TRM module for organ shape regulation. Together these results suggest that rather than specific cellular and microtubule architecture regulation, ovate/sov1 regulate fruit shape through regulation of cell division progression in early in floral development.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Biyao Zhang, Qiang Li, Neda Keyhaninejad, Nathan Taitano, Manoj Sapkota, Ashley Snouffer, and Esther van der Knaap (2022) A combinatorial TRM-OFP module is required to fine-tune tomato fruit shape New Phytologist
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Qiang Li, Qian Feng, Ashley Snouffer, Biyao Zhang, Gustavo Ruben Rodriguez, and Esther van der Knaap (2022) Increasing Fruit Weight by Editing a Cis-Regulatory Element in Tomato KLUH Promoter Using CRISPR/Cas9 Frontiers in Plant Science 13:879642 DOI: 10.3389/fpls.2022.879642
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Edgar Sierra-Orozco, Reza Shekasteband, Eudald Illa-Berenguer, Ashley Snouffer, Esther van der Knaap, Tong Geon Lee and Samuel F. Hutton (2021) Identification and characterization of GLOBE, a major gene controlling fruit shape and impacting fruit size and marketability in tomato Horticulture Research 8: 138 DOI: 10.1038/s41438-021-00574-3
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: The role of OFPs and TRMs in patterning tomato fruit shape and identification of additional fruit shape modifiers. Snouffer, A, Zhang, B., Kraus, C., van der Knaap, E. Plant Center Retreat poster, Young Harris, GA. December 15-16, 2021(2nd place poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Investigation of the cellular function and comparative expression analysis of OFPs, TRMs, and SUNs in floral organ shape. Ashley Snouffer, Plant Functional Genomics Talk, Athens GA. November 10th, 2021
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Cell Division Patterns in Early Floral Development Shape the Tomato Ovary and Fruit. Snouffer, A. , Zhang, B. , Kraus, C., van der Knaap, E. Institute of Plant Breeding Genetics and Genomics Retreat. Virtual Talk and poster. May 27-28, 2021 (1st place Postdoc talk)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Early Floral Development Regulates Shape of the Tomato Ovary and Fruit. Snouffer, A., Zhang, B. , Kraus, C., Taitano N. , van der Knaap, E. UGA Developmental Biology Retreat poster. Virtual May10th, 2021


Progress 05/01/20 to 04/30/21

Outputs
Target Audience:Target audience: Plant developmental biologists and plant breeders. Efforts: Presentations at conferences and publishing papers. This work was presented as a poster and a selected talk at the virtual The XVII International Conference on the Plant Family of Solanaceae (SOL2020) meeting, September 20-24th 2020 Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Professional development Workshops Plant Center Retreat Professional Development Workshops Seminars IPBGG monthly seminars and journal club The University of Georgia Department of Plant Biology weekly seminars The University of Georgia Department of Genetics weekly seminars The University of Georgia Department Cell Biology weekly seminars How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Objective: Determine the interactome of OFPs and TRMs in tomato flower development to better understand the protein modules that control fruit shape during development. 70 plants will be grown in smaller pots to induce early flowering. The first floral bud will be isolated from 60-70 plants to increase the tissue collected to isolate protein. The GFP-Trap magnetic beads will be used to enrich the OVATE-GFP protein Expression co expression of OVATE-GFP in N. benthamiana with other TRMs to test in vivo interaction. Analysis of co-expressed genes from RNAseq data available to generate potential candidates for interaction. Objective: Investigate the interactions between OFPs, TRMs and TON1 with microtubules to understand if they control cellular shape or division through microtubules. Further replication to support current results Co-expression of different OFPs and TRMs and TON1 in cell culture to assess subcellular localization during cell division along with microtubules Objective: Investigate the orientation and timing of cell divisions in early tomato flower development and the sub-cellular localization of OVATE, OFP20, and TRM5. This will allow for a better understanding of these proteins are regulated and interact during development. Furthermore, it may help to elucidate the cellular function of these proteins in controlling ovary and tomato fruit shape. Further replication of current results Analysis of EdU labeling in additional shape regulating genes and the genetic interactions with OVATE, OFP20 and TRM5 3D modeling of cell shape and size during early floral development Antibody labeling to analyze sub-ceullular localization in sections of early floral buds

Impacts
What was accomplished under these goals? Objective: Investigate the interactions between OFPs, TRMs and TON1 with microtubules to understand if they control cellular shape or division through microtubules. Co-expression of fluorescently tagged OVATE, OFP20, TRM3/4, and TRM5 with TON1b was done inN. benthamiana. Single expression of OVATE and OFP20 was cytosolic and cytosolic and nuclear respectively. Both TRM3/4 and TRM5 signally expressed are microtubule localized. TON1b signally expressed in cytosolic with some cytosolic strands. Co-expression of TRM3/4 or TRM5 with TON1b results in relocalization of TON1b to the microtubules. Co-expression of OVATE or OFP20 with TON1b all remain cytosolic and more localization of OVATE and OFP20 to the cytosolic strands with TON1b.Co-expression of OVATE and TRM3/4 results in relocalization of TRM3/4 from the microtubules to the cytosol and is co-expressed with OVATE. Co-expression of OVATE and TRM5 also results in relocalization of TRM5 from the microtubules to the cytosol with OVATE. Co-expression of OVATE, TRM3/4 or TRM5, and TON1b results in relocalization of both TRMs and TON1b to the cytosol. Co-expression of OFP20 with TRM3/4 results in relocalization of OFP20 to the microtubules to with TRM3/4. Co-expression of OFP20 with TRM5 results in relocaiztion of OFP20 to the microtubules with TRM5.Co-expression of OFP20 with TRM3/4 and TON1b results in relocalization of all three to the microtubules These results indicate that there is an interaction between TRM3/4 or TRM5 and TON1b and these TRMs recruit TON1b to the microtubules. OVATE and OFP20 seem to have different impacts on these interactions where OVATE pulls the complex between the TRMs and TON1b off the microtubules while OFP20 relocalizes with the TRM-TON1b complex to the microtubules. While both modes of interaction may impact TRM-TON1b microtubule functions similarly it also may indicate alternative modes of regulation resulting in alterations to microtubule architecture and cellular function. Objective: Investigate the orientation and timing of cell divisions in early tomato flower development and the sub-cellular localization of OVATE, OFP20, and TRM5. This will allow for a better understanding of these proteins are regulated and interact during development. Furthermore, it may help to elucidate the cellular function of these proteins in controlling ovary and tomato fruit shape. Experiments were early floral buds were cultured with EdU to label cells in S phase were conducted. The protocol was optimized and buds were cultured for 10 hours under light in MS media. Floral buds were then fixed and paraffin sectioned and slides were labeled for EdU and Hoechst to detect nuclei. Antibodies were generated against OVATE, OFP20, and TRM5 to detect subcellular localization in vivo. Floral buds were fixed and sectioned to label tissue with each antibody. Additionally cell size and area were measured in both developing ovaries and ovaries at anthesis. Confocal microscopy was used to generate z-stack images of floral buds labeled with either EdU or antibodies. To investigate the cell division frequency the proportion of EdU labeled cells to the total number of cells per section of a bud was calculated for each mutant (ovate,sov1,ovate/sov1,trm5,ovate/sov1/trm5). To investigate the cell division orientation the direction of cell division was scored indirectly by the position of labeled daughter cells in proximity to one another. Antibody labeling was tested sections of different staged floral buds to determine subcellular localization. Confocal z-stack images of cleared floral buds were used to measure cell shape and size during development and confocal images of PI stained ovaries were used to measure cell shape and size at anthesis. These results revealed that while the orientation of cell divisions in the anticlinal and periclinal directions is relatively equal early in development, it is biased to more periclinal divisions as the organ elongates. However, there was no significant difference in the number of periclinal vs anticlinal cell divisions in the WT andovatemutants. There was a significant decrease in the frequency of cell divisions between the WT and ovate mutants at early floral stages from the meristem to 7 days post initiation (dpi) and an even further decrease in cell division frequencies in theovate/sov1(ofp20) double mutant at these stages. The decrease in cell division frequencies was partial rescued by thetrm5mutant in theovate/sov1/trm5triple mutant. Antibody labeling for OFP20 was successful and was found in the cytosol and nucleus of mid-staged floral buds. Staging of protein expression in younger and older buds is in progress. TRM5 antibody labeling was also detected but further fixation methods to preserve microtubule architecture are necessary and in progress. Cell size and shape are not altered early in floral development in theovateandovate/sov1mutants but cells are significantly smaller and flatter in thetrm5mutant starting around 5dpi. There are also smaller cells and flatter cells in the triple mutant. In the mature ovary the cell shapes and sizes are larger and more elongated in the ovate and ovate/sov1 mutants but remain smaller in the trm5 and ovate/sov1/trm5 mutant. The lack of significant difference in cell division orientations indicates that changes in the orientation of cell divisions is not likely the primary mechanism resulting in the change in organ shape. However, the decrease in the cell divisions at these stages could result in an organ with a reduced width suggesting this could be the initial event that results in a change in organ patterning and the pear-shaped proximal end of the tomato fruit. While there are no alterations of cell shape or size early in floral development for ovate/sov1 the larger and more elongated cells in the proximal end of the mature ovary may contribute to the final pear-shaped fruit. The smaller and flatter cells in the early trm5 mutant ovary, however, may be causative for the flatter fruit shape and the pattern persists in the ovate/sov1/trm5 triple mutant, suggesting trm5 may have an independent role in regulating cell shape and this may contribute to the partial rescue of the elongated shape observed in the ovate/sov1/trm5 triple mutant. Preliminary antibody labeling with OFP20 suggests that it is also expressed both in the cytosol and nucleus in vivo similar to the expression pattern in N. benthamiana cells. Further analysis with fixation methods to preserve microtubule architecture will be used to determine if there is variation in subcellular localization of OFP20 from the cytosol to microtubules in vivo as well suggesting a biological function for this localization. Additionally mutants with mCherry tagged tubulin have been generated and live microtubule dynamics will be measured in vivo.

Publications


    Progress 05/01/19 to 04/30/20

    Outputs
    Target Audience:Target audience: Plant developmental biologists and plant breeders. Efforts: Presentations at conferences and publishing papers. This work was presented as a poster at FASEB Mechanisms in Plant development Conference July28-August2, 2019 Olean, NY and at the University of Georgia Plant Center Retreat October 24-25, 2019 Helen, GA. This work was presented as a talk at the Developmental Biology Retreat May 13 -14 2019 Athens, GA and the Plant functional Genomics Meeting April 8th, 2020 Athens, GA. The basis of this work was also used in a review written for Current Opinion in Plant Biology on which PI Snouffer is the first Author (DOI: 10.1016/j.pbi.2019.10.005). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Professional development Workshops Plant Center Retreat Professional Development Workshops Seminars IPBGG monthly seminars and journal club The University of Georgia Department of Plant Biology weekly seminars The University of Georgia Department of Genetics weekly seminars The University of Georgia Department Cell Biology weekly seminars How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Objective: Determine the interactome of OFPs and TRMs in tomato flower development to better understand the protein modules that control fruit shape during development. 70 plants will be grown in smaller pots to induce early flowering. The first floral bud will be isolated from 60-70 plants to increase the tissue collected to isolate protein. The GFP-Trap magnetic beads will be used to enrich the OVATE-GFP protein Expression co expression of OVATE-GFP in N. benthamiana with other TRMs to test in vivo interaction. Analysis of co-expressed genes from RNAseq data available to generate potential candidates for interaction. Objective: Investigate the interactions between OFPs, TRMs and TON1 with microtubules to understand if they control cellular shape or division through microtubules. Further replication to support current results Co-expression of different OFPs and TRMs and TON1 in cell culture to assess subcellular localization during cell division along with microtubules Objective: Investigate the orientation and timing of cell divisions in early tomato flower development and the sub-cellular localization of OVATE, OFP20, and TRM5. This will allow for a better understanding of these proteins are regulated and interact during development. Furthermore, it may help to elucidate the cellular function of these proteins in controlling ovary and tomato fruit shape. Further replication of current results Analysis of EdU labeling in additional shape regulating genes and the genetic interactions with OVATE, OFP20 and TRM5 3D modeling of cell shape and size during early floral development Antibody labeling to analyze sub-ceullular localization in sections of early floral buds

    Impacts
    What was accomplished under these goals? Objective: Determine the interactome of OFPs and TRMs in tomato flower development to better understand the protein modules that control fruit shape during development. OVATE-GFP plants grown and protein isolated using GFP-Trap magnetic beads to enrich OVATE-GFP protein. Western blots to detect OVATE-GFP generated and tested to see if TRM-5 was pulled down along with the GFP beads. OVATE-GFP protein was likely too lowly expressed to detect on Western blot from IP. However, OVATE antibody was developed and works on Western blot Experiment being repeated with higher tissue input and repeated with transient expression inN. benthamianato confirm technical efficiency with higher protein concentration. Objective: Investigate the interactions between OFPs, TRMs and TON1 with microtubules to understand if they control cellular shape or division through microtubules. Co-expression of fluorescently tagged OVATE, OFP20, TRM3/4, and TRM5 with TON1b was done inN. benthamiana. Single expression of OVATE and OFP20 was cytosolic and cytosolic and nuclear respectively. Both TRM3/4 and TRM5 signally expressed are microtubule localized. TON1b signally expressed in cytosolic with some cytosolic strands. Co-expression of TRM3/4 or TRM5 with TON1b results in relocalization of TON1b to the microtubules. Co-expression of OVATE or OFP20 with TON1b all remain cytosolic and more localization of OVATE and OFP20 to the cytosolic strands with TON1b.Co-expression of OVATE and TRM3/4 results in relocalization of TRM3/4 from the microtubules to the cytosol and is co-expressed with OVATE. Co-expression of OVATE and TRM5 also results in relocalization of TRM5 from the microtubules to the cytosol with OVATE. Co-expression of OVATE, TRM3/4 or TRM5, and TON1b results in relocalization of both TRMs and TON1b to the cytosol. Co-expression of OFP20 with TRM3/4 results in relocalization of OFP20 to the microtubules to with TRM3/4. Co-expression of OFP20 with TRM5 results in relocaiztion of OFP20 to the microtubules with TRM5.Co-expression of OFP20 with TRM3/4 and TON1b results in relocalization of all three to the microtubules These results indicate that there is an interaction between TRM3/4 or TRM5 and TON1b and these TRMs recruit TON1b to the microtubules. OVATE and OFP20 seem to have different impacts on these interactions where OVATE pulls the complex between the TRMs and TON1b off the microtubules while OFP20 relocalizes with the TRM-TON1b complex to the microtubules. While both modes of interaction may impact TRM-TON1b microtubule functions similarly it also may indicate alternative modes of regulation resulting in alterations to microtubule architecture and cellular function. Objective: Investigate the orientation and timing of cell divisions in early tomato flower development and the sub-cellular localization of OVATE, OFP20, and TRM5. This will allow for a better understanding of these proteins are regulated and interact during development. Furthermore, it may help to elucidate the cellular function of these proteins in controlling ovary and tomato fruit shape. Experiments were early floral buds were cultured with EdU to label cells in S phase were conducted. The protocol was optimized and buds were cultured for 10 hours under light in MS media. Floral buds were then fixed and paraffin sectioned and slides were labeled for EdU and Hoechst to detect nuclei. Antibodies were generated against OVATE, OFP20, and TRM5 to detect subcellular localization in vivo. Floral buds were fixed and sectioned to label tissue with each antibody. Additionally cell size and area were measured in both developing ovaries and ovaries at anthesis. Confocal microscopy was used to generate z-stack images of floral buds labeled with either EdU or antibodies. To investigate the cell division frequency the proportion of EdU labeled cells to the total number of cells per section of a bud was calculated for each mutant (ovate,sov1,ovate/sov1,trm5,ovate/sov1/trm5). To investigate the cell division orientation the direction of cell division was scored indirectly by the position of labeled daughter cells in proximity to one another. Antibody labeling was tested sections of different staged floral buds to determine subcellular localization. Confocal z-stack images of cleared floral buds were used to measure cell shape and size during development and confocal images of PI stained ovaries were used to measure cell shape and size at anthesis. These results revealed that while the orientation of cell divisions in the anticlinal and periclinal directions is relatively equal early in development, it is biased to more periclinal divisions as the organ elongates. However, there was no significant difference in the number of periclinal vs anticlinal cell divisions in the WT andovatemutants. There was a significant decrease in the frequency of cell divisions between the WT and ovate mutants at early floral stages from the meristem to 7 days post initiation (dpi) and an even further decrease in cell division frequencies in theovate/sov1(ofp20) double mutant at these stages. The decrease in cell division frequencies was partial rescued by thetrm5mutant in theovate/sov1/trm5triple mutant.Antibody labeling for OFP20 was successful and was found in the cytosol and nucleus of mid-staged floral buds. Staging of protein expression in younger and older buds is in progress. TRM5 antibody labeling was also detected but further fixation methods to preserve microtubule architecture are necessary and in progress.Cell size and shape are not altered early in floral development in theovateandovate/sov1mutants but cells are significantly smaller and flatter in thetrm5mutant starting around 5dpi. There are also smaller cells and flatter cells in the triple mutant. In the mature ovary the cell shapes and sizes are larger and more elongated in the ovate and ovate/sov1 mutants but remain smaller in the trm5 and ovate/sov1/trm5 mutant. The lack of significant difference in cell division orientations indicates that changes in the orientation of cell divisions is not likely the primary mechanism resulting in the change in organ shape. However, the decrease in the cell divisions at these stages could result in an organ with a reduced width suggesting this could be the initial event that results in a change in organ patterning and the pear-shaped proximal end of the tomato fruit. While there are no alterations of cell shape or size early in floral development for ovate/sov1 the larger and more elongated cells in the proximal end of the mature ovary may contribute to the final pear-shaped fruit. The smaller and flatter cells in the early trm5 mutant ovary, however, may be causative for the flatter fruit shape and the pattern persists in the ovate/sov1/trm5 triple mutant, suggesting trm5 may have an independent role in regulating cell shape and this may contribute to the partial rescue of the elongated shape observed in the ovate/sov1/trm5 triple mutant. Preliminary antibody labeling with OFP20 suggests that it is also expressed both in the cytosol and nucleus in vivo similar to the expression pattern in N. benthamiana cells. Further analysis with fixation methods to preserve microtubule architecture will be used to determine if there is variation in subcellular localization of OFP20 from the cytosol to microtubules in vivo as well suggesting a biological function for this localization.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ashley Snouffer, Carmen Kraus, Esther van der Knaap (2020) The shape of things to come: ovate family proteins regulate plant organ shape. Current Opinion in Plant Biology 53:98-105 DOI: 10.1016/j.pbi.2019.10.005