Source: TENNESSEE STATE UNIVERSITY submitted to NRP
PROFILING CELL SPECIFIC TRANSCRIPTOMES AND PROTEOMES TO UNDERSTAND POLLEN DEVELOPMENT UNDER HEAT STRESS IN TOMATO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1020855
Grant No.
2020-65114-30766
Cumulative Award Amt.
$200,000.00
Proposal No.
2018-06370
Multistate No.
(N/A)
Project Start Date
Apr 15, 2020
Project End Date
Apr 14, 2023
Grant Year
2020
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Agriculture and Environmental
Non Technical Summary
Tomato is a major crop worldwide.For tomato to properly set fruits it requires 21-22°C at night and 24-25°C during the day. Tomato pollen production and viability is highly vulnerable to higher temperature (32/26?C day and night). Hot summers with temperature reaching above 32°C can disrupt production of viable pollens and fruit set, resulting in yield loss, as plants deal with the heat stress (HS). Pollens are developed through the microsporogenesis and micro-gametogenesis stages. The sensitivity of pollen to HS varies over the course of development. The most heat sensitive period is from the meiotic process of the pollen mother cells (PMC), at the young microspore stage (uninucleate stage of microspore) to pollen mitosis.Pollen development and maturation is also affected by the tapetum, disturbance in tapetum development and degeneration has been known to cause pollen injury. Studies have shown that pollen metabolites, transcriptomes and proteomes are all affected by HSand pollens at different development stages would express a cell-type specific proteome. However, the lack of research capability to access these individual cells has been hindering the investigation and understanding of cell-specific functions within complex tissues. In this project, we will utilize the laser capture microdissection (LCM) method to isolate homogeneous tissue samples of cells of interest that affect pollen development and viability. These tissues will be analysed for transcriptomeand proteome expression using next generation sequencing and quantitative proteomics analysis. We expect these analysis will identify genes/proteins that can be targeted to improve developmental stage-specific pollen heat tolerance.
Animal Health Component
50%
Research Effort Categories
Basic
30%
Applied
50%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20314601060100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1060 - Biology (whole systems);
Goals / Objectives
The major researchgoal isto identify a cohort of candidate genes/proteins fromseveral heat-sensitive and heat-toleranttomato vatieties inclduing 'Micro-Tom' and 'Maxifort'. The four objectives are: 1). Profiling the heat-induced transcriptomes and proteomes of thefollowing tissues: a) pollen mother cells (PMC) at meiosis, tetrads, and microspore stages; b) tapetal cells from the same tissue section; and c) viable and non-viable pollen separately; 2). Identifying pollen heat stress genes/proteins through systemic analysis of the transcriptome/proteome datasets, and 3). Developing methods for validation of gene (transcript and protein) expression of heat tolerance; 4). Using these results to prepare a research proposaltargeting to explore strategies to improve pollen heat stress, and reproductive traits in tomato.
Project Methods
Experimental MethodsObjectives A. Profiling heat-induced transcriptomes and proteomes #A1 Heat treatmentThree tomato varieties will be studied in this project: the rootstock F1 'Maxifort', miniature tomato 'Micro-Tom' and 'Black Vernissage'. The heat treatment will be set as: 26/22oC (non-HS-control), 32/22oC (Mid-HS), and 36/22oC (High-HS) at a 12/12 hr (day/night) cycle. For each treatment conditions, three replicates each comprising of 10-20 plants will be grown to produce enough flower/flower buds/pollen for all the analysis. Wewill collect PMC at meiosis, tetrad, free microspore (prior to pollen release), and the tapetum on the same cross-sections of flower buds. In the first step, the correlation between anther length and pollen developmental stages will be determined under heat-treated and non-heat-treated conditions. Then flower buds of corresponding sizes will be collected, and embedded in the Optimum Cutting Temperature (OCT) solution (Fisher Scientific). The tissue blocks will be stored at -80oC or immediately sectioned into 20µm thickness using a cryostat at -20oC. To separate live from dead pollen grains, newly opened flowers will be picked at 8-9 am, pollen will be squashed in a 100 mM PBS (pH 7.6) solution and collected by centrifugation for 5 min at 3000g. Pollen grain will be re-suspended in FDA (10 μg/ml in PBS) and immediately transferred onto PEN membrane slides. Viable pollen will be picked under thefluorescencemode in LCM. Dead pollen stained with PI will be picked into separate batches.Objective B: Identification of heat-induced cell-specific transcriptomes and proteomes# B1. Transcriptome analysisTotal RNA will be isolated using an RNeasy Micro kit (Qiagen), and in vitro transcription will be using a TargetAmp two-round aRNA amplification kit (Epicentre). Strand-specific libraries will be constructed using Illumina TruSeq RNA Library Prep Kit, and sequenced using the paired-end 75bp on the Illumina HiSeq 3000 at Vanderbilt University. Three replicates will be included for each treatment condition/cell type. After trimming and removing adapter sequences using FastX toolkit, resulting high-quality cleaned reads will be used for assembly and expression analysis using the Cufflinks RNA-Seq workflow (reads aligned against the ITAG 3.20 version of tomato genome annotation using TopHat followed by assembly and expression analysis using Cufflinks and cuffcompare packages). Differentially Expressed Genes (DEGs) will be selected based on the reads per million mapped reads (RPM) annotated to each gene.Quantitative PCR will be used in validation assay of a few selected genes.# B2. LCM- TMT quantitative proteomics analysisThe LCM-tissues are washed into a PCT buffer and transferred to a micro-tube. Protein extraction will be done on a Barocycler (Pressure Biosciences), by running for 60 cycles at 25 °C under 45 kPsi pressure. Supernatants containing proteins will be loaded onto S-TRAP column for desalting followed by on-column trypsin digestion. For quantitative proteomics analysis, we will use ten isotopically coded, isobaric tags (TMT Reagent-10-Plex Kit; Thermo Fisher, MA), where pairs of treatment conditions are considered independently. Since each of the mass tags is chemically identical but distinguishable by their characteristic fragment ions in MS2and MS3(due to the isotope coding) up to 10 different samples can be multiplexed in a single analytical sample. Tryptic peptides from these samples will be fractionated using a 2-dimensional separation strategy involving high pH reverse phase followed by low pH reverse phase chromatography. The mass spectral analysis will be carried out using our recently purchased Orbitrap Eclipse Trybrid mass spectrometer (to be installed and commissioned August 2019).This instrument is capable of carrying out synchronous precursor selection (SPS) and MS3quantification. Furthermore, it incorporates a Real-Time Search (RTS) algorithm that identifies MS2fragment ion spectra as they are acquired. Used in the RTS-MS3mode the Eclipse only triggers quantitative MS3analysis on confidently identified peptides, saving much of the time previously spent in the analysis of non-productive ions. A recent study incorporating this new strategy shows that the RTS-MS3approach provides dramatic performance improvements for experiments involving multiplexed quantitative analysis, reducing abundance ratio distortion while simultaneously maintaining proteome coverage. Proteome Discoverer 2.3 will be used to identify and quantify proteins from these experiments. The MS2spectra will be used to interrogate the tomato protein database (ITAG3.20-proteins) in real-time. The criteria used to define differentially expressed proteins (DEPs) will be based on ratio of abundance (treated/control) andp< 0.05 value. If none of the distributions tested is found to be sufficiently normal we will use appropriate nonparametric approaches such as the Mann-Whitney or Wilcoxon Rank Sum to test the null hypothesis.#B3,Integrated analysis of transcriptomes and proteomes, and development of associated datafilesFor each cell type, the identified transcriptome and proteome datasets will be processed(using SQLite) to list the unigene accessions that are identified using the two analysis platforms, and those in only one of them, as well as quantitative changes from heat-treated to non-treated conditions. Two separate files will be generated: one for all the unigene accessions, and one for the heat induced significantly changed genes and proteins (DEGs and DEPs). Eventually, we will generate a datafile which will allow users to investigate gene/protein expression across pollen developmental stages in association with tomato varieties. Objective C. Developing validation and screening methods of HS biomarkersDuplicates of the LCM sections will be prepared on glass slides and used for gene-expression validation experiments. We will select a list of DEGs that are directly relevant to pollen development, and the priority will be given to those associated with the heat tolerance properties. Thein-situmRNA hybridization will be carried out using probes designed against the gene-unique regions of the mRNA sequences. For protein validation, we will use antibodies that are readily available for tomato proteins to develop a standard protocol. We have antibodies against HSPs, SODs, peroxidases stored in the lab. These proteins will be the priority for the first batch of analysis.Objective D. Preparation of a full research NIFA proposal The project team will prioritize research objectives to ensure relevant data to be generated in 2020 and a competitive proposal to be submitted to NIFA in 2021.

Progress 04/15/22 to 04/14/23

Outputs
Target Audience:We have shared the research results with scientific communities through making several presentations at conferences and seminars. We have maintained a close connection withlocal tomato growers; our students wouldexplain thescience and technology that are developed through the project, and the potential impacts on tomato breeding; at the same time these growers helped us to identify new research goals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five graduate students majorin Plant Biotechnology at Tennessee State University received trainingin plant physiology, tomato greenhouse production managment, singel cell typle proteomics and machine learning, at the collaborating USDA-ARS lab and Tennessee State University. These students have participated in two-three conferences/per student and learnt how to make presentations and preparing manuscripts. Four MS students graduated with relevant skills. One undergraduate student worked on the project to learn proteomics data analysis and bioinformatics. How have the results been disseminated to communities of interest?Participating students have madeconference presentations. The research materials were integrated in TSU Graduate andundergraduate courses "Principle and Methods of Propagation of Horticultural Plants" to help students understand the reproduction process of plants. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have developed a new method for counting pollen number as well as differetiatingviable/non-viable pollenuisng machine learning.Two tomato varieties'Black Vernissage' and 'Micro-Tom' were evaluated for heat tolerance in pollen productivity and pollen viability under heat stress. Data confirmed that 'Black Vernissage' is more tolerant to heat stress, especially at the mid-heat (30-32oC) temperature level; the pollen productivity of 'Black Vernissage'wasless affacted by heat stress, compared to 'Micro-Tom'.The number of pollen produced per flower is largely determined by the meiotic activity of pollen mother cells. Thenwe focused on the meiotic pollen mother cellsto identify genes/proteins affacting pollen productionunder high temperature.Using laser capture microdissection (LCM), single cell-type tissues were collected forpollen mother cells at the meiotic stage, and the microspore stage. Quantitative proteomics analysis were conducted to identifycell type-specific differentially abundant proteins induced by heat stress in the two tomato varieties, one graduate student completed thesis using these experimental results. Manuscripts to publishthese data/results are in preparation.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dibya Thapa Magar*, Jun Guo, Kajol Pradhan, Pyria Thapa, Hui Li. Suping Zhou Collection of germinating tomato pollen using laser capture micro-dissection for identification of heat-induced proteomes. 2023 TSU symposium
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Priya Thapa*, Shourav Bin Rabbani*, Kajol Pradhan, Jun Guo, Dibya Thapa Magar, Suping Zhou, Manar Samad, Hui Li, Sonali Roy, "DEVELOPING NEW POLLEN GRAIN COUNT METHOD USING MACHINE LEARNING TO EVALUATE HEAT TOLERANCE OF TOMATOES" (Oral Presentation), 45th Annual University-Wide Research Symposium, Tennessee State University, Nashville, TN, March 28- March 30, 2023


Progress 04/15/20 to 04/14/23

Outputs
Target Audience:The target audience includes thescientific communities, research and extenstion faculty, graduate and undergraduate students,local high school studentsand Ag teachers, andowners and workers on local farms growing tomato and other vegetabkecrop. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five graduate students major in Plant Biotechnology at Tennessee State University received training in plant physiology, tomato greenhouse production managment, singel cell typle proteomics and machine learning, at the collaborating USDAARS lab and Tennessee State University. These students have participated in two-three conferences/per student and learnt how to make presentations and preparing manuscripts. Four MS students graduated with relevant skills. One undergraduate student worked on the project to learn proteomics data analysis and bioinformatics. How have the results been disseminated to communities of interest?Participating students have made conference presentations. The research materials were integrated in TSU Graduate and undergraduate courses "Principle and Methods of Propagation of Horticultural Plants" to help students understand the reproduction process of plants. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have developed a new method for counting pollen number as well as differetiating viable/non-viable pollen uisng machine learning. Two tomato varieties 'Black Vernissage' and 'Micro-Tom' were evaluated for heat tolerance in pollen productivity and pollen viability under heat stress. Data confirmed that 'Black Vernissage' is more tolerant to heat stress, especially at the mid-heat (30-32oC) temperature level; the pollen productivity of 'Black Vernissage' was less affacted by heat stress, compared to 'Micro-Tom'. The number of pollen produced per flower is largely determined by the meiotic activity of pollen mother cells. Then we focused on the meiotic pollen mother cells to identify genes/proteins affacting pollen production under high temperature. Using laser capture microdissection (LCM), single cell-type tissues were collected for pollen mother cells at the meiotic stage, and the microspore stage. Quantitative proteomics analysis were conducted to identify cell type-specific differentially abundant proteins induced by heat stress in the two tomato varieties, one graduate student completed thesis using these experimental results. Manuscripts to publish these data/results are in preparation.

Publications


    Progress 04/15/21 to 04/14/22

    Outputs
    Target Audience:1. Scientific communities to share the research results; 2. Local middle to high schools to utilize the project to attract students to the Ag program; 3. Tomato growers to introduce the new development in science and technology and alsoseek their inputs/advices on the major issues the research should focuson. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training to two post-docs and three graduate stduents on heat treatments, laser capture microdissection (LCM) technology, single cell proteomics, bioinformatics, data analysis, preparation of manuscripts and research proposals. TSU Faculty from Computer Science have joined the project to develop reliable/easierapproaches for pollen counts using articifical intelligence. Post-docs and students have exposed to the emerging technology. How have the results been disseminated to communities of interest?We have published one journal article, and graduate students have made two conference presentations. Project materials/results were included in lecturesfor undergraduate course" Principle and Methods of Propagation of Horticultural Plants" to help students understand the reproduction process of plants and the impacts from envrionmental conditions. What do you plan to do during the next reporting period to accomplish the goals?To complete the analysis of heat-treated pollen samples.

    Impacts
    What was accomplished under these goals? Completed proteomics analysis for meioticpollen mother cells from tomato 'Maxifort'; Completed collection of single cell type samples forheat-treated pollen cells at different developmental stages using laser capture microdisscetion (LCM); Have developed plan for the analysis of these samples in summer, 2022in the USDA/ARS collabrator's lab; Completed develpong the technical procedures for analysis of pollen viability integrating imaging and machine learning (ML); Started preparation of a NIFA proposal to continue the resarch projectsto be submitted in 2022/2023.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Pyria Thapa, : Suping Zhou, Hui Li. SINGLE CELL PROTEOMICS ANALYSIS OF TOMATO POLLEN MICROSPORE UNDER HEAT STRESS. 44TH ANNUAL TENNESSEE STATE UNIVERSITY-WIDE RESEARCH SYMPOSIUM. April 2-3, 2022.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Pyria Thapa, Proteomics Analysis of Heat-Induced Proteins in Tomato Male Gametophyte Cells. ARD Research Symposium 2022. Atlanta, GA April 2  5, 2022
    • Type: Journal Articles Status: Published Year Published: 2022 Citation: Hui Li, Yaoguo Qin, Xingbo Wu, Joshua O'Hair, Jesse Potts, Suping Zhou, Yong Yang, Tara Fish, Theodore W. Thannhauser. Identification of heat-induced proteomes in meitoic pollen mother cells of tomato 'Maxifort' using single-cell-type tandem mass tag (TMT) proteomics. Vegetable Research (2022)2: 2.


    Progress 04/15/20 to 04/14/21

    Outputs
    Target Audience:The project activities related to heat stress on pollen development and seed/fruit productionwere included in undergraduate and graduate courses at Tennessee State University. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students andtwo graduate students have been trained to conduct pollen viability/pollen germinationassay experiments on this project. One undergraduate student conducted a training using laser capture microdissection to collect pollen samples in the USDA/ARS collaborator's lab. 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?1. To complete heat treatment experiments for tomato 'Black Vernisage' and 'Microt-Tom'; 2. To collect tissue samples using laser capture microdissection; 3. To complete transcriptome and proteomics analysis of tissue samples; 4. To publish the data in journal articles (1-2 papers) and conference presntations.

    Impacts
    What was accomplished under these goals? 1.The size offlower buds containing pollen at different developmental stages has been determined for aheat sensitive varietyMicro-Tomand heat tolerant'Black Vernisage' and 'Maxifort'. InSpring-Summer, 2020, flower buds were collected and frozen tissue blocks prepared for'Maxifort', but plants for the other two varieties were all killed by a tornado which damaged the greenhouses.A new treatment experiment was set-up for tomato'Micro-Tom' and'Black Vernisage' in Spring, 2021. Flower buds are harvested to prepare frozen tissue blocks as they grow to the sizes required for isolating anther tissues for further analysis. 2.One proteomics analysis for mother pollen cells from'Maxifort' was completed. The manuscript is under internal review. 3.Genes encoding heat-responsibe proteins identfied in 'Maxifort' were selected to develop CRISPR/Cas9-induced mutants for functional genomics analysis and producing new traits for heat tolerance.

    Publications