Progress 03/01/24 to 02/28/25
Outputs
Target Audience:The target audience for communicating this work is other researchers working on temperature stress and reproduction. This work will be of interests to researcher who study pollen tube development in both tomatoes and other model and crop species. I will also share results with plant biologists and cell biologists studying the cytoskeleton. This work was shared in oral and poster presentations at 2 conferences. The PI will attend the Plant Biology 2025 conference during this next project year and has been invited to give an oral presentation on this work. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided significant opportunities for me to improve in key professional areas. Specifically, I have improved my project management skills, developed enhanced adaptability in research planning by successfully implementing alternative strategies, and refined my mentorship abilities through guiding and supporting undergraduate student. One undergraduate student Noor Ahmidouch has worked in this project. She has received training on all aspects of working with tomato pollen. She optimized and applied methods to inhibit F-actin via drug treatment and gained skills on microscopy, image quantification to measure pollen tube elongation. She learned experimental skills and how to communicate her work in oral presentations. How have the results been disseminated to communities of interest?During this year, I will share my research findings, I communicated with scientific audiences by presenting my work at two conferences. I gave one oral presentation at 35th Annual North Carolina Plant Molecular Biology Retreat conference and prepared a poster for presentation at the 27th International Congress on Sexual Plant Reproduction conference. During this period, I also shared my work in two publications. What do you plan to do during the next reporting period to accomplish the goals?A goal of this project is to utilize transgenic lines expressing F-actin markers. Due to the negative effect of proLAT52::LifeAct-Venus or proLAT52::LifeAct-mRuby3 on pollen gemination and pollen tube growth I generated new plasmid constructs with UBQ10 promoter (proUBQ10::LifeAct-Venus or proUBQ10::LifeAct-mRuby3). Evidence suggests that that the expression level of UBQ10 is lower compared to LAT52. UBQ10 gene encode the highly conserved 76-amino acid protein ubiquitin that targets protein for degradation and usually has mild relative expression in most of the plant tissues (https://bar.utoronto.ca). To understand how temperature affects the dynamics of F-actin and pollen tube growth, I will monitor pollen tube growth at optimal (28°C) and elevated temperatures (34°C). I will collect pollen grains from reporter lines and pollen will be subjected to either optimal or elevated temperatures. Our confocal microscope is equipped with a tightly controlled heat chamber providing a consistent temperature environment. This setup will offer us the unique capability to conduct time-lapse live imaging under temperature-controlled conditions. To understand temperature affects in the dynamics of F-actin I will cross F-actin marker lines with temperature sensitive are mutant, with a defect in the F3H gene, and a transgenic line that overproduces F3H. In our 1st transformation round we realized that plant transformation is very difficult into are due to its poor seed germination rate. Therefore, we will transform the parental line, VF36, with the F-actin reporter and cross this reporter into the are mutant. Furthermore, I will monitor pollen tube F-actin dynamics at optimal (28°C) and elevated temperatures (34°C). I will also cross the Hyper7 reporter line with the F-actin reporter line (proUBQ10::Lifeact-mRuby3) to observe ROS and F-actin dynamics simultaneously. HyPer7 is fused with the mutated cpYFP (yellow fluorescent gene), and F-actin marker line will be with mRuby3 (red fluorescent gene). This double reporter line will allow us to visualize ROS and F-actin dynamics within a same experiment. ROS levels and F-actin dynamics will be observed under optimal (28°C) and elevated temperatures (34°C). To further investigate the relationship between F-actin and ROS, I will monitor the dynamics of F-actin under high temperature in the presence of VAS2870, a highly specific inhibitor of the enzyme respiratory burst oxidase/NADPH oxidase, which controls the increase in ROS in response to elevated temperature.
Impacts
What was accomplished under these goals?
ROS homeostasis and signaling are essential for the pollen tube growth and the tube's penetration into ovary for successful fertilization. At the beginning of this project, I helped contribute an understanding of how high temperature stress impact tomato pollen performance. In the tomato are mutant with reduced flavonol production, we detected increases ROS levels which had detrimental effects on pollen development and germination, and pollen tube elongation. Furthermore, we showed that high temperature treatment increased the level of ROS in VF36 with greater increases in the are mutant, which also had increased prevalence of pollen tube impairment phenotypes. To test if the elevated ROS in response to temperature stress was mediated by NADPH oxidase (NOX)/respiratory burst oxidase homolog (RBOH) enzymes, I treated pollen with VAS2870, a highly specific inhibitor of RBOH enzymes. I found that at elevated temperature ROS level was reduced in both VF36 and the are mutant after VAS2870 treatment, suggesting a potential increase in RBOH enzyme activity within the pollen tube in response to heat stress. This work was published last year in an article in the Plant Cell (Postiglione et al., 2024). I also examined whether tomato genotypes with pollen tube thermotolerance had smaller increases in ROS levels following high temperature treatment. For this experiment, I used the tomato varieties Heinz, Malinka, Nagcarlag, and Tamaulipas, with Heinz being thermosensitive and Tamaulipas being thermotolerant. I allowed pollen tubes to grow for 1 h under optimal conditions (28°C) and high temperature (37°C) for 1 h and measured ROS levels in the pollen tube after temperature treatment. High temperature significantly increased ROS levels in thermosensitive cultivars, but with significantly smaller increases in Tamaulipas, the most thermotolerant cultivar This work was published in Current Biology (Ouonkap et al., 2024). In both articles described above, total ROS levels in pollen tube was detected by application of a cell permeable marker, the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), which is commonly used chemical to visualize ROS. However, this probe has some limitations as it may only visualize the gradual buildup of ROS over time, lacking the ability to offer dynamic or real time changes. However, total H2O2 levels in pollen tube can be detected by the genetically encoded hydrogen peroxide sensor, HyPer7. I have successfully transformed this construct into tomato and obtained 8 independent transformant lines. I have also tested the efficiency of this reporter line and detected H2O2 in the tomato pollen tube. To observe the changes of localized ROS in real time I am performing live cell imaging under a range of temperatures condition. Utilizing this line, I aim to monitor spatial and temporal ROS dynamic changes during pollen tube growth and tip rupture under control and elevated temperatures in the absence and presence of drugs that perturb the actin cytoskeleton. These experiments are the foundation upon which I will overlay changes in actin networks during this fellowship. To investigate F-actin dynamics in the tomato pollen tube I generated tomato transgenic reporter lines expressing a protein fusion in which fluorescent proteins are linked to the "Lifeact" peptide, which binds to F-actin. I used the LAT52 promoter, whose transcriptional activity is specific to the pollen tube to drive these reporters (proLAT52::Lifeact-Venus and proLAT52::Lifeact-mRuby3). I utilized the University of Nebraska-Lincoln Plant Transformation Core Research Facility to generate transgenic lines and obtained 12-15 individual transformants for each construct. I screened all transformants to identify stable lines with high signal and wild-type pollen tube dynamics. I identified multiple positive transformants with strong F-actin signal in the pollen grain. Unfortunately, I also found that the pollen grains transformed with the transgene are unable to germinate and to develop a pollen tube. The cytoplasmic dynamics in the pollen are completely inhibited in the transgenics, which is likely due to overproduction of Lifeact peptide. Lifeact, is a peptide consisting of 17 amino acids, which binds to F-actin in eukaryotic cells and tissues, which can be visualized when this peptide fused to fluorescent proteins. We hypothesize that transcriptional activity of proLAT52 is producing sufficiently large amounts of Lifeact peptide, which is impairing the process of cytoplasmic streaming, which requires actin dynamics. Lat52 is a tomato gene and in Arabidopsis proLAT52::Lifeact-GFP was transformed to visualize F-actin and it did not affect pollen germination, so we hypothesize that the promoter may be active in tomato. Currently I am generating new reporter lines replacing LAT52 promoter with Ubiqutin10 (UBQ10). The UBQ10 gene encodes the highly conserved 76-amino acid protein ubiquitin that targets protein for degradation and usually expressed in all tissues but at a relatively low level in most of the plant tissues. To understand how F-actin controls pollen tube growth, I have been mentoring Noor Ahmidouch, a Wake Forest University undergraduate student, who treated the pollen tube with the actin depolymerizing drug Latrunculin B (LatB). She used wild type VF36 pollen while we were waiting for the F-actin reporter lines. She treated growing pollen tubes with LatB at concentrations ranging from 10-50 nM for a duration of 30 minutes. She found that LatB concentration as low as 10 nM were sufficient to reduce pollen tube elongation and doses over 25 nM completely blocked pollen tube elongation. She also found dose dependent effects of LatB on pollen tube elongation.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Ouonkap SVY, Palaniappan M, Pryze K, Jong E, Foteh Ali M, Styler B, Althiab Almasaud R, Harkey AF, Reid RW, Loraine AE, et al (2024) Enhanced pollen tube performance at high temperature contributes to thermotolerant fruit and seed production in tomato. Curr Biol 34: 5319-5333.e5
- Type:
Peer Reviewed Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Postiglione AE, Delange AM, Ali MF, Wang EY, Houben M, Hahn SL, Khoury MG, Roark CM, Davis M, Reid RW, et al (2024) Flavonols improve tomato pollen thermotolerance during germination and tube elongation by maintaining reactive oxygen species homeostasis. Plant Cell 36: 45114534
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