DEVELOPMENTAL RESPONSES TO INCREASED TEMPERATURES DURING PLANT GAMETOGENESI

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1030206
• Grant No.: 2023-67013-39412
• Cumulative Award Amt.: $649,636.00
• Proposal No.: 2022-10970
• Project Start Date: May 1, 2023
• Project End Date: Apr 30, 2027
• Grant Year: 2023
• Program Code: [A1152]- Physiology of Agricultural Plants

Project Director
Begcy Padilla, K.

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
(N/A)

Non Technical Summary
Historically, pollen development studies under heat stress are performed across several developmental stages and occasionally through the entire pollen formation. However, high temperature spikes have become more extreme and with increasing frequency, and in many cases with greater intensity. These temperature spikes impact single developmental stages and in rare occasions two or more of them. Therefore, it is necessary to elucidate the most critical stages of plant reproduction to understand the underlying molecular stress responses, and to use the knowledge generated with the aim of developing heat stress tolerant crops. How developmentally male gametophyte is perturbed by heat stress has remained largely unexplored. The goal of the proposed work is to fill a significant gap in our understanding on how heat stress perturbs developmentally each stage of pollen formation and ultimately affects productivity in maize. Our preliminary results indicate a negative effect of heat stress during the microsporogenesis-to-microgametogenesis transition. Whether each pollen developmental stage responds similarly to heat stress episodes remain to be determined. We hypothesize that heat stress does not induce the same responses when only individual developmental stages are exposed to heat stress. The proposed work aims to identify the genetic and epigenetic factors that need to be tightly regulated for normal pollen development but are perturbed when plants experience heat stress. Elucidating the molecular basis of pollen development under stress will help frame the model needed for improving the resilience of pollen viability and hence yield when plants experience heat stress during this highly sensitive developmental window.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1510	1020	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1510 - Corn;

Field Of Science
1020 - Physiology;

Keywords

heat stress

epigenetics

maize

pollen development

yield

Goals / Objectives
How developmentally male gametophyte is perturbed by heat stress has remained largely unexplored. The long-term goal of our research is to elucidate the molecular events that are responsible for the reduced yield during pre-fertilization events under heat stress. Recently, we identified two critical pathways affected by heat stress during tetrad stage of pollen development resulting in reduced pollen germination and yield. Both pathways are required for normal pollen development and/or pollen viability. Genes of both pathways are potential candidates for stress resilience. However, not all pollen developmental stages respond in the same way when impacted by heat stress. Because male gametophyte development events are largely conserved among cereals, an opportunity exists to translate this knowledge to other economically important crops. The goal of the proposed work is to fill a significant gap in our understanding on how heat stress perturbs developmentally each stage of pollen formation and ultimately affects productivity in maize. Therefore, it is necessary to elucidate the most critical stages of plant development and reproduction, to understand the underlying molecular stress responses, and to use the knowledge generated with the aim of developing heat stress tolerant crops.The proposed work aims to identify the genetic factors that need to be tightly regulated during normal pollen development but are perturbed when plants experience heat stress. What role do epigenetic factors play in the regulation of these heat-sensitive genes? Elucidating the molecular basis of pollen development under stress will help frame the model needed for improving the resilience of pollen viability and hence yield when plants experience heat stress during this highly sensitive developmental window.Objective 1: Elucidate how heat stress affects pollen development gene networks using transcriptome analysis. 1.1. Transcriptome analysis to identify heat stress responsive pollen gene networks in maize. 1.2. Integration of time series analysis and co-expression-based predictions to identify pollen gene networks that are sensitive to heat stress during pollen microsporogenesis and microgametogenesis. Objective 2: Integrate transcriptional regulation with genome-wide bisulfite sequencing and histone methylation (H3K27me3) analysis of each pollen developmental stage to identify critical genes and networks that determine stress adaptability. 2.1. Perform whole genome bisulfite analysis in each pollen developmental stage in maize under optimal and heat stress conditions to correlate with the gene expression dataset. 2.2. Perform whole genome analysis of H3K27me3 modifications in developing pollen grains under optimal and stressful conditions and integrate the data with transcriptome and bisulfite dataset. Objective 3: Functionally characterize a set of candidate genes that are misregulated during heat stress and contribute to stress resilience. 3.1. Determine the role of a set of candidate maize genes in controlling pollen development and fertilization using CRISPR/Cas9 and overexpression lines

Project Methods
Heat stress in maize: Seeds of the maize inbred line B73 will be germinated in an incubator and then transferred to pots (10 cm diameter, 10 seedlings per pot) containing a substrate and soil mixture (1:1, v/v). Maize B73 seedlings will be transferred to 10-L pots to the greenhouse under controlled conditions of 14 h of light at 25 ± 2°C and 10 h of darkness at 21 ± 2°C and a constant air humidity of 60-65%. Supplemented light of 16,000 LUX will be provided to adjust day length duration. From our experience, we have observed that two or three days of heat stress can effectively induce physiological, molecular, and biochemical changes. Gas exchange parameters such as photosynthesis, conductance and transpiration rate will be measured with a Portable Photosynthesis System-LiCor 6400. To apply heat stress treatments, plants at each pollen developmental stage will be identified using the Leaf Collar Method, and then transfer to walk- in growth chambers. For heat stress, growth chamber day/night temperature conditions will be set at 40/30°C and 60% humidity at 25,000 lux for 48 or 72 h. Correspondingly, non-stressed plants will be maintained under a 28/25°C day/night temperature regime and 60% humidity at 25,000 lux.RNA-Seq analysis of developing pollen responsive genes in maize. To identify genes that are expressed specifically at high level during pollen development and are heat stress responsive, we will use an RNA-Seq approach to gain a complete picture of the pollen development transcriptome. The following RNA-Seq methodology will be applied throughout for all RNA-Seq experiments in this project. Total RNA will be isolated using TRIzol method, purified and DNA contamination removed using with Turbo DNase (Ambion). We have successfully used the extraction protocol for developing pollen for high quality RNA. In previous experiments, we have identified several marker genes that are expressed in each pollen developmental stage. We have used some of these genes in our preliminary results to confirm our experimental set-up.Cluster analysis of RNA-Seq data: After genes with time-varying expression profiles have been selected with the aforementioned method, we will identify specific genes in each pollen developmental stage using a multilevel mixture modeling approach similar to the one we previously used in rice. In particular, log-transformed and standardized read counts for expressed genes at each pollen developmental stage will be used to form a time course profile for the entire pollen developmental stages, and the profiles will then be assigned to different clusters representing distinct expression patterns over each single developmental stage. This method provides a convenient mechanism to parameterize the cluster means for easy identification of biologically interpretable profiles. During this analysis, we will focus on genes that are highly active during the transition from pollen meiosis to pollen mitosis. These genes are likely to be associated with proper pollen development and are under epigenetic regulation. Importantly, we expect to identify divergent transcript patterns and novel genes including genes that are unique to maize.Methylome of pollen development under control and heat stress conditions. Non-destructive method to determine pollen development stages in maize. Using the Leaf Collar Method in the maize inbreed line B73, we have determined the duration of each stage from pollen mother cells before meiosis to mature tricellular pollen. Our method allows to precisely isolate large quantities of pollen for further studies. We will use it to isolate homogeneous cell populations at individual stages throughout pollen development.For bisulfite sequencing analysis, homogeneous cell populations of each individual pollen developmental stage will be filtered through a 30-micron mesh into a tube containing 100 μL of glass beads, and vortexed for 3 minutes in order to break the pollen cell wall. The resulted crude fraction will be filtered through Miracloth to remove larger debris, and concentrated by centrifugation (800 g, 5 min). Genomic DNA will be isolated from and fragmented in 10 mM Tris- HCl, pH 8.0. Fragments will be end repaired, A-tailed and ligated to methylated Illumina adaptors. Ligated fragments will be bisulfite treated using the EZ DNA Methylation-Gold Kit, and PCR enriched with Expand High-Fidelity Polymerase (Roche). Amplified fragments of 340- 360 bp will be size selected by gel extraction and sequenced on an Illumina Novaseq6000 platform as paired end 100 nt reads.ChIP-Seq analysis for H3K27me3 marks in each pollen developmental stage under optimal and heat stressed conditions. For chromatin immunoprecipitation, we will harvest large quantities of each pollen developmental stage using the Leaf Collar Method. from several maize plants grown under control conditions for ChIP-Seq assay. Parallel samples will be collected from plants that are under heat stress. Because greenhouse growing conditions vary to some extent throughout the year, we will plan to collect additional pollen materials for future analysis within a five-week window. The ChIP protocol for pollen grains is well-established in our laboratory. Briefly, the collected material will be crosslinked, followed by sonication to fractionate the chromatin. ChIP assay will be performed following the protocol we recently published using the polyclonal antibody for H3K27me3 and magnetic protein A beads. Before we initiate ChIP-Seq experiments, we will first perform some initial validation with ChIP- qPCR assays. We will select ~15 genes from the transcriptome analysis that are differentially regulated in response to heat stress during each stage of pollen development for ChIP using H3K27me3 antibody.Characterization of CRISPR/Cas9 knock-out and overexpression lines for pollen related phenotypes. We will use the CRISPR/Cas9 system to disrupt gene function. CRISPR/Cas9 uses a single guide RNA (sgRNA) coupled with the Cas9 nuclease to create double strand breaks in target genes and hence generate mutations via non-homologous end joining. The CRISPR/Cas9 system has already been shown to work in maize protoplasts and transgenic plants, using a U6 promoter to express a nuclear localized Cas9 nuclease protein codon optimized for maize and a sgRNA expressed from pTaU3. We will generate transgenic maize plants carrying pU6::Cas9-NLS and pTaU3::sgRNA to use in generating sgRNA-guided CRISPR knockouts. The sgRNAs will be designed to target each candidate gene. Due to the time constraints for CRISPR, we can grow three seasons per year in Florida to accelerate the generation of maize and following downstream genetic analysis. For overexpression, transformation constructs will be made in the pCAMBIA binary vectors, which contain the maize Ubiquitin1 promoter driving the expression of the heat stress pollen sensitive candidate genes and the nopaline synthase terminator. The constructs will include a myc epitope tag to facilitate monitoring of expression levels and downstream analyses for future experiments. For each candidate pollen gene, over expression (OE) lines will be generated (~10 T0 lines). Transgenic stable overexpression lines will be characterized for pollen related traits such as pollen viability, sperm cell integrity, pollen tube formation and pollen abundance to identify gene function in pollen development.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:Our primary target audience are breeders, growers and stakeholders related to the production of corn. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A Ph.D. student is currently under training for this project. training involvesidentify research questions, generate hypothesis, define specific objectives, develop research approaches, think about alternative strategies, and construct appropriate timeline and budget. How have the results been disseminated to communities of interest?We published a total of 2 peer reviewed articles as well as 2 presentations were given at various conferences throughout the year. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 - we will explore the impact of heat stress at the tricellular stage of pollen development. We hypothesize that the response to this developmental stage is different to the one at the bicellular stage. Objective 2 - we will isolate tricellular pollen grains and conduct DNA methylation analysis. Objective 3 - we aim to generate T1 edited plants to conduct functional analyses.

Impacts
What was accomplished under these goals? Objective 1 - Elucidate how heat stress affects pollen development gene networks using transcriptome analysis. -We made significant progress on elucidating the effect on heat stress on pollen development. Particularly at the bicellular stage of pollen development. Heat stress at the bicellular stage impaired sperm cell development and transport into pollen tubes. We used marker lines and analyzed the transcriptomes of isolated sperm cells. We monitored the sperm cell journey using an α-tubulin-YFP marker line and found that within 1?h after pollen germination and growth in vitro, about 80% non-stressed sperm cells were visible inside pollen tubes. In contrast, only about 20% sperm cells were visible inside pollen tubes formed by heat stress pollen. Heat stress at the bicellular stage activated spindle assembly check point and meta- to anaphase transition genes in sperm cells. Our data point toward misregulation of genes associated with PM II. During the transition from metaphase to anaphase, where chromosomes aligned at the equator of the cell and migrate to the poles, a developmental process ensures that all chromosomes are aligned at the same level. This process is controlled by the SAC signaling complex, which regulates proper partitioning of chromosomes to daughter cells during mitosis. SAC signaling is a mechanism that it is only active when chromosomes are not properly attached to the kinetochores and thus delays progression of the cell cycle until all kinetochores are correctly assembled. We found upregulation of genes encoding nuclear division cycle 80, minichromosome instability 1, and mitotic arrest deficient 2 in sperm cells generated during heat stress. These proteins are part of the SAC signaling pathway. This resulted in a strong reduction of seed set. Objective 2 - identification of critical genes and networks that determine stress adaptability- At the transcriptional level, our gene network analysis yielded 8 main hubs affected by heat stress in sperm cells. Notably, these hubs are formed by genes related to histones, DNA synthesis, RNA processing, translation, posttranslational modification, cell cycle, signal transduction and cell homeostasis. Notably, the misregulation of histone genes also confirmed previous observations that levels of centromeric histone CENH3 are reduced after heat stress. Objective 3: Functionally characterize a set of candidate genes that are misregulated during heat stress and contribute to stress resilience. We identified a set of candidates genes for functional analyses. We are in the process of cloning and generation of edited plants. What was accomplished under these goals? Objective 1 - Elucidate how heat stress affects pollen development gene networks using transcriptome analysis. -We made significant progress on elucidating the effect on heat stress on pollen development. Particularly at the bicellular stage of pollen development. Heat stress at the bicellular stage impaired sperm cell development and transport into pollen tubes. We used marker lines and analyzed the transcriptomes of isolated sperm cells. We monitored the sperm cell journey using an α-tubulin-YFP marker line and found that within 1?h after pollen germination and growth in vitro, about 80% non-stressed sperm cells were visible inside pollen tubes. In contrast, only about 20% sperm cells were visible inside pollen tubes formed by heat stress pollen. Heat stress at the bicellular stage activated spindle assembly check point and meta- to anaphase transition genes in sperm cells. Our data point toward misregulation of genes associated with PM II. During the transition from metaphase to anaphase, where chromosomes aligned at the equator of the cell and migrate to the poles, a developmental process ensures that all chromosomes are aligned at the same level. This process is controlled by the SAC signaling complex, which regulates proper partitioning of chromosomes to daughter cells during mitosis. SAC signaling is a mechanism that it is only active when chromosomes are not properly attached to the kinetochores and thus delays progression of the cell cycle until all kinetochores are correctly assembled. We found upregulation of genes encoding nuclear division cycle 80, minichromosome instability 1, and mitotic arrest deficient 2 in sperm cells generated during heat stress. These proteins are part of the SAC signaling pathway. This resulted in a strong reduction of seed set. Objective 2 - identification of critical genes and networks that determine stress adaptability- At the transcriptional level, our gene network analysis yielded 8 main hubs affected by heat stress in sperm cells. Notably, these hubs are formed by genes related to histones, DNA synthesis, RNA processing, translation, posttranslational modification, cell cycle, signal transduction and cell homeostasis. Notably, the misregulation of histone genes also confirmed previous observations that levels of centromeric histone CENH3 are reduced after heat stress. Objective 3: Functionally characterize a set of candidate genes that are misregulated during heat stress and contribute to stress resilience. We identified a set of candidates genes for functional analyses. We are in the process of cloning and generation of edited plants.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Li X, Bruckmann A, Dresselhaus T, Begcy K (2024). Heat stress at the bicellular stage inhibits sperm cell development and their transport into pollen tubes. Plant Physiology. https://doi.org/10.1093/plphys/kiae087
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Begcy K, Mendes MA, De Storme N (2024). Plant reproduction under environmental stress. Front. Plant Sci. 15:1369070. https://doi.org/10.3389/fpls.2024.1369070
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Li X, Bruckmann A, Dresselhaus T, Begcy K (2024). Heat stress at the bicellular stage inhibits sperm cell development and their transport into pollen tubes. 66th maize genetic meeting. Raleigh, North Carolina. USA.