Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The target audience for communicating this work is other researchers working on temperature stress and its effects on reproductive and vegetative development in both tomatoes and other model and crop species. The results from this work will also be of interest to breeders and farmers as we work to engineer and breed high temperature stress resistant lines of tomato and other crop plants. The PI and CoPI will attend the International Conference on Sexual Plant Reproduction during this next project year to share results from this work. Changes/Problems:We experienced delays in receiving requested transgenic tomatolines and await T2 plants that can be used for experiments. What opportunities for training and professional development has the project provided?This project supported one graduate student at Wake Forest University this year, Anthony Postiglione, and five undergraduate students: Eric Wang, Stacy Hahn, Colleen Roark, Iain Newman, and Ashley Lockwood. These students received training on all aspects of working with tomato pollen. These WFU students optimized and applied methods for visualization of flavonols and ROS in pollen using specific dyes, quantification of pollen tube elongation and rupture with elevated temperature, and quantification of flavonol metabolites using liquid chromatography-mass spectroscopy. They learned experimental skills and how to communicate their work in oral and poster presentations. At Brown University, this project has supported Sorel Ouonkap Yimga (PhD student), Rasha Althiab Almasaud (postdoctoral researcher), Octavia Rowe (undergraduate student), and Camilo Ramirez (undergraduate student) and Atticus Henry (undergraduate student). Sorel has analyzed changes in the tomato pollen tube transcriptome in response to temperatures stress (RNA-seq) and has worked to develop an automated live imaging analysis workflow important for analysis of pollen performance under heat stress. Octavia has developed methods to chronically heat stress plants that have allowed her to collect pollen samples for analysis of flavanol content. Camilo worked to identify heat-stress-induced promoters that are active in pollen tubes. Rasha has begun to identify candidate genes for reverse genetic analysis and has worked with Atticus to develop transgenic tomato lines to generate novel mutants. Rasha's goal is to define signaling events that lead directly from perception of high temperature to changes in ROS levels and consequent changes in pollen tube cell wall integrity. How have the results been disseminated to communities of interest?During this year, two team members gave oral presentations and one presented at poster at the 26th International Conference on Plant Reproduction. Gloria Muday presented seminars at the North American Phytochemical Society meeting and the University of Missouri Interdisciplinary Plant Groups on Plant Hormones and the Intersection of Stress and Development Conference. We have not yet shared results from this new project in a publication but are in the final stages of submission of this work. What do you plan to do during the next reporting period to accomplish the goals?An important goal for this year is to submit several publications on temperature effects on pollen germination using four tomato genotypes with reduced and elevated flavonol levels and another group of genotypes with natural variation in thermotolerance that may also be tied to levels of reactive oxygen species. A goal of this project is to engineer tomatoes to be more thermotolerant by engineering them to overproduce flavonols. During our first year of the project, we designed a strategy to engineer plants to produce elevated levels of flavonols in pollen only during heat stress. We are using this more complicated approach (to go beyond our constitutively overproducing lines) as flavonols at super-optimal concentrations may impair pollen performance if they are at sufficiently high levels to reduce ROS below the minimum needed for pollen germination and fertilization. An important goal for this year is to complete the transformation of plants with these inducible constructs and test their function in controlling pollen response to elevated temperature. The Johnson lab will contribute to this effort by evaluating promoters that are active in pollen and that respond robustly and reproducibly to high temperature stress. An additional post-doctoral research was hired this year, M. Foteh Ali, who has working todefine the source of ROS during high temperature stress using inhibitors of specific enzymes that generate ROS. Once we define the pertinent source of ROS, we will then begin engineering constructs to inhibit ROS synthesis using the lessons learned from our construction of transgenics designed to control flavonol synthesis. Rasha Althiab Almasaud (postdoctoral fellow, Johnson lab) has significant experience with production and analysis of CRISPR-mediated mutants of tomato that affect pollen tube function. Rasha will use our RNA-seq data set to help identify potential ROS producing enzymes and to test hypotheses about their function using reverse genetic approaches.
Impacts
What was accomplished under these goals?
The Muday lab has been examining the ability of flavonols to enhance pollen germination under optimal and elevated temperature stress conditions. We are examining germination and pollen tube growth of wildtype, the are mutant containing a mutation in the F3H gene resulting in impaired flavonol synthesis, the are mutant genetically complemented with a wildtype copy of the mutated gene, and a wildtype that is overexpressing a wildtype copy of the gene. The pollen was germinated under both optimal (28°C) and heat stress (34°C) conditions. The impaired germination rate of are, especially under higher temperature, was rescued by genetic complementation with a F3H transgene, reaching near wild-type levels. Most importantly, under heat stress conditions, the overexpression lines are less sensitive to the heat stress, consistent with flavonols offering a protective effect. We have quantified levels of the flavonols in pollen using liquid chromatography-mass spectroscopy and shown that thermotolerance of these lines is proportional to levels of flavonols. We have also chemically complemented pollen germination medium with flavonols to assess the effects of specific flavonols on pollen germination and pollen tube growth. The addition of the flavonols quercetin, kaempferol, and myricetin reversed the high temperature effect on germination in both are and VF36 pollen at 34°C. At 28°C, the flavonol concentrations used had no effect on the wildtype pollen. These results suggest that all three flavonols are able to protect pollen from high temperature stress, without impairing pollen germination at ideal temperatures. A graduate student in the Muday laboratory, Anthony Postiglione, has quantified intracellular and extracellular ROS changes in pollen during germination and tube elongation at optimal and elevated temperatures. He has shown that ROS increases with elevated temperature in germinating pollen grains, looking at this response in elongating pollen tubes. Using the tomato genotypes with altered flavonol levels described above, it is clear that the levels of ROS are inversely proportional to the levels of flavonol antioxidants across multiple developmental stages. The increases in ROS in response to elevated temperature is greatly accentuated in mutants with impaired flavonol synthesis, while lines containing wildtype flavonol levels or engineered to overproduce flavonols display a reduced ROS increase. This work is a chapter in Anthony's PhD thesis that is in the final stages of preparation for submission as a publication. We have also published two review articles exploring the role of flavonols in development and the role of ROS in development during this year. Ongoing experiments are determining the roles of levels of flavonols on pollen tube growth through the pistil. Eric Wang has shown that are pistils have reduced flavonols and elevated ROS. He has developed methods to quantify the number of pollen tubes that grow through a pistil and is using this to quantify pollen tube number in are and its parental line, and sees evidence of impaired pollen tube growth of are in vivo, as well as in the in vitro assays described above. He is also asking whether wildtype pistils complement the growth of are pollen tubes, by providing flavonols to modulate ROS levels in vivo. Sorel Ounkap Yimga (PhD student in the Johnson lab) has developed a live imaging system with semi-automated image analysis to determine how pollen tube growth responds to high temperatures stress. Sorel has found that pollen tube cell wall integrity, an aspect of pollen performance known to be modulated by ROS, is the key feature that determines pollen performance at high temperature. Importantly, Sorel has also identified varieties of tomato that have been selected for enhanced pollen tube cell wall integrity at high temperature. Collaborating with members of the Muday lab, Sorel tested the hypothesis that cultivars of tomato with enhanced pollen tube integrity have distinct pollen tube ROS profiles. Interestingly, pollen tube ROS levels increased in all cultivars tested. This leads to the interesting idea that some cultivars have a larger range of ROS levels that promote successful pollen tube growth. To begin to understand that molecular basis for the ability to maintain pollen tube growth across a wider range of ROS levels, Sorel and postdoctoral fellow, Rasha Althiab Almasaud have determined how the transcriptome and proteomes change in response to heat stress in thermotolerant and thermosensitive cultivars. Intriguingly, we find evidence that thermotolerant cultivars have higher basal levels of expression of flavanol biosynthesis and enzymatic scavengers of ROS. These results lead us to propose that thermotolerant varieties are adapted to growth at higher temperature by modulation of the response to ROS. Reverse genetic analysis of the signaling pathway that integrates ROS and pollen tube integrity is underway to test this idea in thermosensitive and thermotolerant cultivars.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Althiab-Almasaud R, Teyssier E, Chervin C, Johnson MA, Mollet JC. (2023) Pollen viability, longevity, and function in angiosperms: key drivers and prospects for improvement. Plant Reprod. 2023 Nov 5. doi: 10.1007/s00497-023-00484-5. PMID: 37926761
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Daryanavard, Hana, Postiglione, Anthony E, M�hlemann, JK, Muday, GK (2023) Flavonols modulate plant development, signaling, and stress responses. Current Opinion in Plant Biology; doi.org/10.1016/j.pbi.2023.102350
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Joelle K. Muhlemann and Gloria K. Muday, Flavonols take the heat out of heat stress. 26th International Conference on Sexual Plant Reproduction, Prague, Czech Republic, June 2022
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Sorel Ouonkap Yimga and Mark A. Johnson. Enhanced pollen tube integrity was selected during breeding of tomatoes that set fruit at elevated temperatures. 26th International Conference on Sexual Plant Reproduction, Prague, Czech Republic, June 2022
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Gloria Muday: Flavonol antioxidants modulate reactive oxygen species signaling to control development and modulate pollen stress responses. Keynote speaker: Phytochemical Society of North America Meeting July 2022, Blacksburg, Va
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Gloria Muday: Flavonol antioxidants modulate reactive oxygen species signaling to control development and modulate pollen stress response. Keynote speaker: Redox Regulation of Plant Stress and Development Conference, Columbia, MO, May 2023
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:The target audience for communicating this work is other researchers working on temperature stress and its effects on reproductive and vegetative development in both tomatoes and other model and crop species. The results from this work will also be of interest to breeders and farmers as we work to engineer and breed high temperature stress resistant lines of tomato and other crop plants. The PI and CoPI will attend the International Conference on Sexual Plant Reproduction during this next project year to share results from this work. Changes/Problems:Progress on this project has been limited by delays in recruiting senior personnel. A post-doc hired in the Muday laboratory had a family emergency and had to resign her position. A post-doc hired by the Johnson laboratory (Rasha Althiab Almasaud) was delayed for 6 months due to visa delays but has now begun her work. What opportunities for training and professional development has the project provided?This project has supported two graduate students at Wake Forest University, Allison DeLange and Anthony Postiglione. They both received training on all aspects of working with tomato pollen. Both students enrolled in a microscopy course and have applied the skills from that class to imaging pollen germination, pollen tube elongation, and pollen viability using both stereo microscopy and confocal microscopy. Both have also optimized and applied methods for visualization of flavonols and ROS in pollen using specific dyes and quantification of flavonol metabolites using liquid chromatography-mass spectroscopy. At Brown University, this project has supported Sorel Ouonkap Yimga (PhD student), Rasha Althiab Almasaud (postdoctoral researcher), Octavia Rowe (undergraduate student), and Camilo Ramirez (undergraduate student). Sorel has analyzed changes in the tomato pollen tube transcriptome in response to temperatures stress (RNA-seq) and has worked to develop an automated live imaging analysis workflow important for analysis of pollen performance under heat stress. Octavia has developed methods to chronically heat stress plants that have allowed her to collect pollen samples for analysis of flavanol content. Camilo is working to identify heat-stress-induced promoters that are active in pollen tubes. Rasha has begun to identify candidate genes for reverse genetic analysis. Rasha's goal is to define signaling events that lead directly from perception of high temperature to changes in ROS levels and consequent changes in pollen tube cell wall integrity. How have the results been disseminated to communities of interest?During this year, Allison DeLange presented work on this project at the Plant Biology conference. Gloria Muday presented seminars at Brown University and the University of Arizona describing this work. Allison DeLange presented a public seminar on her results as part of her MS thesis defense. We have not yet shared results from this new project in a publication but are in the process of editing Allison's thesis for publication. Mark Johnson gave seminars at the University of Maryland, Vaparaiso University, and Bayer Crop Sciences symposium. Sorel Ounkap Yimga presented his work at the annual ASPB Plant Biology meeting and in Brown University seminars (The MCB Graduate Program Retreat and MCB Graduate Program Data Club). What do you plan to do during the next reporting period to accomplish the goals?An important goal for this year is to submit a publication on temperature effects on pollen germination using four genotypes with reduced and elevated flavonol levels and treatment with exogenous flavonols. Second, the levels of ROS will be quantified in these genotypes and treatment conditions to ask if the protective effect of flavonols is mediated by maintaining ROS homeostasis in developing, germinating, and elongating tubes of pollen. Additionally, flavonol levels will be quantified in pollen germinated under optimal temperature or heat stress and in pollen produced in plants with more long-term heat stress to ask if plants increase flavonol synthesis as a protection from high temperature stress. Parallel experiments will also be performed in roots under control and heat stress, as well as during recovery from heat stress. A goal of this project is to engineer tomatoes to be more thermotolerant by engineering them to overproduce flavonols. During our first year of the project, we designed a strategy to engineer plants to produce elevated levels of flavonols in pollen only during heat stress. We are using this more complicated approach (to go beyond our constitutively overproducing lines) as flavonols at super-optimal concentrations may impair pollen performance if they are at sufficiently high levels to reduce ROS below the minimum needed for pollen germination and fertilization. An important goal for this year is to complete the transformation of plants with these inducible constructs and test their function in controlling pollen response to elevated temperature. The Johnson lab will contribute to this effort by evaluating promoters that are active in pollen and that respond robustly and reproducibly to high temperature stress. We are currently searching for a post-doctoral researcher who will carry out many of the planned experiments in Aim 1, to define the source of ROS during high temperature stress using inhibitors of specific enzymes that generate ROS. Once we define the pertinent source of ROS, we will then begin engineering constructs to inhibit ROS synthesis using the lessons learned from our construction of transgenics designed to control flavonol synthesis. Rasha Althiab Almasaud (postdoctoral fellow, Johnson lab) recently joined the team (April 2022). Rasha has significant experience with production and analysis of CRISPR-mediated mutants of tomato that affect pollen tube function. Rasha will use our RNA-seq data set to help identify potential ROS producing enzymes and to test hypotheses about their function using reverse genetic approaches.
Impacts
What was accomplished under these goals?
The Muday lab has been examining the ability of flavonols to enhance pollen germination under optimal and elevated temperature stress conditions. We are examining germination and pollen tube growth of wildtype, the are mutant containing a mutation in the F3H gene resulting in impaired flavonol synthesis, the are mutant genetically complemented with a wildtype copy of the mutated gene, and a wildtype that is overexpressing a wildtype copy of the gene. The pollen was germinated under both optimal (28°C) and heat stress (34°C) conditions. The impaired germination rate of are, especially under higher temperature, was largely rescued by genetic complementation with a F3H transgene, reaching near wild-type levels. Most importantly, under heat stress conditions, the overexpression lines are less sensitive to the heat stress, consistent with flavonols offering a protective effect. Pollen from each of the lines has been collected to quantify levels of the flavonols quercetin, kaempferol, and myricetin using liquid chromatography-mass spectroscopy. This will determine if one or all of these molecules are correlated with pollen thermotolerance in the different genetic lines. We have also chemically complemented pollen germination medium with flavonols to assess the effects of specific flavonols on pollen germination and pollen tube growth. The addition of the flavonols quercetin, kaempferol, and myricetin reversed the high temperature effect on germination in both are and VF36 pollen at 34°C. At 28°C, the flavonol concentrations used had no effect on the wildtype pollen. These results suggest that all three flavonols are able to protect pollen from high temperature stress, without impairing pollen germination at ideal temperatures. Additionally, we have been testing the possibility that high temperature stress induces flavonol synthesis in pollen to protect them from elevated ROS induced by high temperature. The Johnson lab collected pollen from chronically heat stressed plants (multiple days of treatment at elevated temperatures) to ask whether heat increases flavonol levels in pollen relative to plants grown at optimal temperatures. Allison DeLange, a graduate student in the Muday laboratory, quantified flavonols by LC-MS and found that heat stress significantly increased levels of one flavonol, quercetin. To understand whether these changes are also part of short-term temperature stress, we queried an RNA-seq data set generated by the Johnson lab using tomato pollen tubes growing in vitro. Control pollen were grown at 28ºC for 6 hours and heat stress pollen were 28ºC for 3 hours and 37ºC for 3 hours. Analysis of this data set did not reveal significant changes in abundance of transcripts encoding flavonol biosynthesis enzymes or ROS generating enzymes. Our subsequent experiments examining the time course of effects of heat stress on pollen germination and pollen tube growth reveals that heat stress responses are more profound earlier in these processes. Therefore, the Muday lab prepared pollen tube samples which were exposed to heat stress (34ºC) for 90 min after an initial germination at 28ºC for 30 min and then compared to pollen tubes grown at 28ºC for the entirety of the time course. Analysis of this sample preparation also found no evidence that transcripts encoding flavonol biosynthetic enzymes are induced during a shorter heat stress treatment. A second graduate student in the Muday laboratory, Anthony Postiglione, has begun optimizing staining conditions to measure intracellular and extracellular ROS changes in pollen during germination and tube elongation at optimal and elevated temperatures. He has expanded analyses began by Allison DeLange to show that ROS increases with elevated temperature in germinating pollen grains, looking at this response in elongating pollen tubes. Using the tomato genotypes with altered flavonol levels described above, it is clear that the levels of ROS are inversely proportional to the levels of flavonol antioxidants across multiple developmental stages. The increases in ROS in response to elevated temperature is greatly accentuated in mutants with impaired flavonol synthesis, while lines containing wildtype flavonol levels or engineered to overproduce flavonols display a reduced ROS increase. Allison DeLange has completed experiments asking whether flavonols protect roots of young seedlings from temperature stress. She designed a protocol for heat stress treatments that impair root development. Young seedlings were grown at optimal temperatures of 25°C for 72 hours and then split into three groups: control, heat stress, and recovery. The heat stress and recovery groups were subjected to high temperature stress of 37°C while the control remained at the optimal temperature. After 24 hours, the recovery group were moved back to the optimal temperature for 48 hours, while the heat stress group was exposed to high temperature stress for the full 72 hours. The numbers of lateral root primordia and emerged lateral roots were quantified for each genotype in the presence and absence of heat stress and recovery across this time course. For both are and wildtype seedlings, heat stress reduced the number of lateral roots. After a period of recovery, the wildtype seedlings recovered to approximately 60% of the lateral root number of its control. The are mutant, however, only recovers to 25% of its control, which forms fewer lateral roots than wildtype. The over expression lines do not have a significantly different response from that of VF36. The complementation lines are currently being assessed under these conditions. Analysis of flavonol metabolites in seedlings via liquid chromatography mass spectrometry have shown that flavonols increase in VF36 during the recovery phase, but not are. These results are consistent with flavonols mediating heat stress recovery and we are working on a publication on these results. Sorel Ounkap Yimga (PhD student in the Johnson lab) has developed a live imaging system with semi-automated image analysis to determine how pollen tube growth responds to high temperatures stress. Sorel has found that pollen tube cell wall integrity, an aspect of pollen performance known to be modulated by ROS, is the key feature that determines pollen performance at high temperature. Importantly, Sorel has also identified varieties of tomato that have been selected for enhanced pollen tube cell wall integrity at high temperature. These data will set the stage for coming work to define how ROS levels are modulated in distinct varieties of tomato to achieve enhanced pollen tube integrity under heat stress. Camilo Ramirez (undergraduate in the Johnson lab) has used our pollen tube RNA-seq data set to identify promoters that are active in pollen tubes and that respond to high temperature stress. The next goal of this project is to prepare reporter constructs to test whether these promoters will be useful in approaches to engineer temperature-responsive expression of flavanol biosynthesis genes. The Johnson lab has also used our pollen tube heat stress response RNA-seq dataset to identify the most abundant pollen-tube expressed ROS generating enzymes, with a focus on Respiratory Burst Oxidase Homologs. Interestingly, this RBOH transcript is significantly elevated in a thermotolerant variety. These data set the stage for planned reverse-genetics experiments (postdoctoral fellow Rasha Althiab Almasaud) to test whether this gene is an important contributor to pollen tube ROS homeostasis and cell wall integrity.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sorel Ouonkap Yimga, SO; Draycott, A; Blakley, IC; Loraine, A; Johnson, MA
Defining natural variation contributing to reproductive success under temperature stress. ASPB 2021 Worldwide Summit, July 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
DeLange, A, Muhlemann, JK, and Muday, GK (2021) Flavonols protect tomato pollen and roots from heat stress induced reactive oxygen species synthesis. ASPB 2021 Worldwide Summit, July 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
M�hlemann, JK, Younts, TLB, Lopez, B.S., DeLange, A, Johnson,MA, Muday, GK (2021) Flavonols take the heat out of heat stress to protect pollen from elevated ROS. VIB plant science for climate emergency, June 2021VIB plant science for climate emergency
|
Progress 06/01/20 to 05/31/21
Outputs
Target Audience:The target audience for communicating this work is other researchers working on temperature stress and its effects on reproductive and vegetative development in both tomatoes and other model and crop species. The results from this work will also be of interest to breeders and farmers as we work to engineer and breed high temperature stress resistant lines of tomato and other crop plants. Changes/Problems:This grant was funded in spring 2020 and the funding was followed closely by a COVID19 shut down of research laboratories. Both Wake Forest and Brown University laboratories were closed between mid March and mid June 2020. The timing of this shut down had significant impacts on our ability to do pollen experiments with greenhouse grown tomatoes at WFU, as we could not do any experiments with our spring tomato crop and our greenhouses are not suitable for tomato growth during the summer. We were able to begin pollen experiments in the fall of 2020, but a poorly timed greenhouse malfunction truncated this experimental season. We developed a new related question during summer of 2020 in which we asked about the effect of temperature stress on root development in young seedlings. These experiments used the same set of mutants and transgenics with reduced or elevated flavonols that we have been using in our pollen studies, revealing a role of these specialized metabolites in both reproductive and vegetative development. Additionally, our progress during this year was limited by COVID19 impact on team members who had to work around childcare and school closures, which impacted their available time in the laboratory for the majority of this year. What opportunities for training and professional development has the project provided?This project has supported a graduate student at Wake Forest University, Allison DeLange, who received training on all aspects of working with tomato pollen. She took a microscopy course and has now applied the skills from that class to imaging pollen germination, pollen tube elongation, and pollen viability using both stereo microscopy and confocal microscopy. She has also optimized and applied methods for visualization of flavonols and ROS in pollen using specific dyes and quantification of flavonol metabolites using liquid chromatography-mass spectroscopy. At Brown University, this project has supported Sorel Ouonkap Yimga (PhD student) and Octavia Rowe (undergraduate student). Sorel has analyzed changes in the tomato pollen tube transcriptome in response to temperatures stress (RNA-seq) and has worked to develop an automated live imaging analysis work flow important for analysis of pollen performance under heat stress. Octavia has developed methods to chronically heat stress plants that have allowed her to collect pollen samples for analysis of flavanol content. How have the results been disseminated to communities of interest?Results from this project were shared in oral presentations at the Plant Biology conference (which was remote during summer 2020) presented by Joelle Muhlemann and Mark Johnson. They were also shared in poster presentation by undergraduate Suprene Mohamdzein at this conference. We have not yet shared results from this new project in a publication. What do you plan to do during the next reporting period to accomplish the goals?An important goal for this year is to expand the pollen experiments with these four genotypes with reduced and elevated genotypes to determine if flavonol levels are protective in pollen viability and pollen tube germination. Similarly, treatment with exogenous flavonols will be performed in all of these assays. Second, the levels of ROS will be quantified in these genotypes and treatment conditions to ask if the protective effect of flavonols is mediated by maintaining ROS homeostasis in developing, germinating, and elongating tubes of pollen. Additionally flavonol levels will be quantified in pollen germinated under optimal temperature or heat stress and in pollen produced in plants with more long term heat stress to ask if plants increase flavonol synthesis as a protection from high temperature stress. Parallel experiments will also be performed in roots under control and heat stress, as well as during recovery from heat stress. A goal of this project is to engineer tomatoes to be more thermotolerant by engineering them to overproduce flavonols. During our first year of the project we designed a strategy to engineer plants to produce elevated levels of flavonols in pollen only during heat stress. We are using this more complicated approach (to go beyond our constitutively overproducing lines) as flavonols at super-optimal concentrations may impair pollen performance if they are at sufficiently high levels to reduce ROS below the minimum needed for pollen germination and fertilization. An important goal for this year is to complete the transformation of plants with these inducible constructs and test their function in controlling pollen response to elevated temperature. We made limited progress on Aim 1 of this grant during year 1. We are currently searching for a post-doctoral researcher who will carry out many of the planned experiments in Aim 1, to define the source of ROS during high temperature stress using inhibitors of specific enzymes that generate ROS. Once we define the pertinent source of ROS, we will then begin engineering constructs to inhibit ROS synthesis using the lessons learned from our construction of transgenics designed to control flavonol synthesis.
Impacts
What was accomplished under these goals?
The Muday lab has been examining the ability of flavonols to enhance pollen germination under optimal and elevated temperature stress conditions. The germination of pollen tubes of wildtype, the are mutant containing a mutation that impairs flavonol synthesis, the are mutant genetically complemented with a wildtype copy of the mutated gene, and a wildtype that is overexpressing a wildtype copy of the gene. The pollen was germinated under both optimal (28°C) and heat stress (34°C) conditions. The impaired germination rate of are, especially under higher temperature, was largely rescued by complementation, reaching near wild-type levels. Most importantly, under heat stress conditions, the overexpression lines are less sensitive to the heat stress, consistent with flavonols offering a protective effect. Pollen from each of the lines has been collected to quantify levels of the flavonols quercetin, kaempferol, and myricetin using liquid chromatography-mass spectroscopy. This will determine if one or all of these molecules are correlated with pollen thermotolerance in the different genetic lines. We have also chemically complemented the germination medium with flavonols to assess the effects of specific flavonols on pollen germination and pollen tube growth. The addition of the flavonols quercetin, kaempferol, and myricetin reversed the high temperature effect on germination in both are and VF36 pollen at 34°C. At 28°C, the flavonol concentrations used had no effect on the wildtype pollen. These results suggest that all three flavonols are able to protect pollen from high temperature stress, without impairing pollen germination at elevated levels. Additionally, we have been testing the possibility that high temperature stress induces flavonol synthesis in pollen to protect them from elevated ROS induced by high temperature. The Johnson lab collected pollen from chronically heat stressed plants (and controls) to ask whether heat increases flavonol levels in pollen. The analysis of the flavonol levels is currently in progress in the Muday lab. Second, the Johnson lab generated an RNA-seq data set using tomato pollen tubes growing in vitro. Control pollen were grown at 28ºC for 6 hours and heat stress pollen were 28ºC for 3 hours and 37ºC for 3 hours. Analysis of this data set did not reveal significant changes in abundance of transcripts encoding flavonol biosynthesis enzymes or ROS generating enzymes. Our subsequent experiments examining the time course of effects of heat stress on pollen germination and pollen tube growth reveals that heat stress responses are more profound earlier in these processes. Therefore the Muday lab has prepared pollen tube samples which were exposed to heat stress for a shorter time point and we await the results of the RNA Seq analysis, which is in the hands of the sequencing facility. This analysis includes a wild-type parental line, the are mutant and overexpression lines of the enzyme that is deficient in the are mutant. Allison DeLange, a graduate student in the Muday laboratory, also began new experiments to ask if flavonols protect roots of young seedlings from temperature stress. First, we designed a protocol for heat stress treatments that impair root development. Young seedlings were grown at optimal temperatures of 25°C and split into three groups: control, heat stress, and recovery. The heat stress and recovery groups were subjected to high temperature stress of 37°C while the control remained at the optimal temperature. After 24 hours, the recovery group were moved back to the optimal temperature for 48 hours. The numbers of lateral root primordia and emerged lateral roots were quantified for each genotype in the presence and absence of heat stress and recovery across this time course. For both are and wildtype seedlings, heat stress reduced the number of lateral roots. After a period of recovery, the wildtype seedlings recovered to approximately 60% of the lateral root number of its control. The are mutant, however, only recovers to 25% of its control, which forms fewer lateral roots than wild-type. The over expression lines do not have a significantly different response from that of VF36. The complementation lines are currently being assessed under these conditions. Analysis of flavonol metabolites in seedlings via liquid chromatography-mass spectrometry have shown that flavonols increase in VF36 during the recover phase, but not are. These results are consistent with flavonols mediating heat stress recovery.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Flavonols take the heat out of heat stress to protect pollen from elevated ROS
Jo�lle K. M�hlemann, Trenton L.B. Younts, Allison DeLange, Gloria K. Muday. Plant Biology Worldwide Summit (virtual) July 2020
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Understanding the molecular basis of thermotolerant reproduction in tomato.
Sorel Ouonkap Yimga, Robert W. Reid2, Benjamin Styler, Nathaniel Ponvert, James Pease, Jo�lle K. Muhlemann, Gloria K. Muday, Ann Loraine, Ravishankar Palanivelu, Mark A. Johnson. Plant Biology Worldwide Summit (virtual) July 2020
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Testing the hypothesis that flavonols modulate high temperature induced reactive oxygen species. Suprene Mohamedzein, Sheena Gayomba, and Gloria Muday. Plant Biology Worldwide Summit (virtual) July 2020
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