EARLY REPRODUCTIVE DEVELOPMENT IN PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1014128
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824

Performing Department
Plant Science

Non Technical Summary
Seeds are the primary source of calories for humans and livestock, and thus one of the most critical aspects of agricultural productivity is early reproductive development in plants, which includes gametophyte development, fertilization, endosperm development and provisioning, and embryogenesis. Even small changes in early reproductive development can have profound impacts on seed yield and quality with cascading repercussions through the entire food chain.This proposal addresses several aspects of early reproductive development in a variety of plant systems, including important crops and their close relatives. As the human population increases and climate change threatens the stability of our agricultural systems, we must understand the molecular underpinnings of reproductive development if we are to increase crop yield and quality while minimizing environmental impact.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2499	1080	100%

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

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1080 - Genetics;

Keywords

pollen tube

thermotolerance

transcription factor network

opaque2

rna-directed dna methylation

small rna

genomic imprinting

brassicaceae

brassica rapa

genome balance

Goals / Objectives
Temperature sensitivity during fertilization. Pollen tube-mediated double fertilization is the feature that defines angiosperms including our critical crop plants. The pollen grain germinates on a stigma, producing a cellular extension called a pollen tube, which navigates the ovary to deliver a pair of sperm to the female gametes inside an ovule. In recent years, we have learned a great deal about this process using a variety of model systems. Now is the time to apply this basic knowledge to a critical agricultural question: How will reproductive signaling systems function in crop plants when temperatures are abnormally high during the critical window of vulnerability when pollination occurs?Understand the gene regulatory networks during early endosperm development in maize. Early reproductive development requires exquisite control of gene expression to coordinate development and provisioning between maternal and filial tissues. Development of endosperm is of particular agricultural importance because the endosperm acts as a sink for the developing embryo or the germinating seedling (Lopes and Larkins, 1993). The endosperm of cereals in particular occupies a large portion of the mature grain, expresses a high level of storage products such as carbohydrates and storage proteins, and is an important source of food, feed, and industrial feedstock (FAO, 2012; Sabelli and Larkins, 2009).RNA-directed DNA methylation and successful seed development. Establishment and maintenance of DNA methylation is critical to proper development of the embryo and endosperm during sexual reproduction. For example, differential DNA methylation on matrigenic and patrigenic genomes is critical for correct expression of endosperm growth regulators (Gehring, 2013). DNA methylation can be initiated by 24 nt short interfering (si)RNAs in a process called RNA-directed DNA methylation (RdDM). These siRNAs are most abundant in gametophytes and developing seeds (Mosher et al., 2009; Rodrigues et al., 2013), and mutants defective in RdDM have defective seed production in multiple species (Gouil & Baulcombe, 2016; Hollick, 2010). We will use RdDM mutants in Brassica rapa, which have a specific defect in seed production, to characterize the genetic, developmental, and molecular requirements underlying the requirement for RdDM during seed production.Develop a comparative genetic approach for seed development. Mutant analysis is a powerful approach to understand the molecular events underlying early reproductive development. Similarly, an evolutionary perspective has been useful in understanding changes in developmental trajectory ("evo-devo" studies). Here we propose to combine these ideas by producing cognate mutations in multiple members of the Brassicaceae family to test hypotheses explaining the requirement for RdDM during seed production.

Project Methods
Objective 1. Define the transcriptional response of the pollen tube and pistil to heat stress in thermotolerant and thermosensitive varieties of tomato.Use SNP-informed RNA-seq to simultaneously define the pollen tube and pistil responses to heat stress.We will use single-nucleotide polymorphisms (SNPs) in RNA-seq reads obtained from cross-pollinations to unambiguously define pollen tube and pistil gene expression changes occurring in response to high temperatures. We will use thermosensitive M82 as the pistil parent in cross-pollinations with thermosensitive Heinz and with thermotolerant Malintka. We will incubate pollinated pistils at optimal or heat-stress temperatures. Styles will be collected from unpollinated and pollinated pistils at 3 and 8 hours after pollination and application of heat stress (along with unstressed controls). Differential gene expression analysis will identify transcriptional changes and gene networks that influence pollen tube thermotolerance.We will use Malintka as the pistil parent in a second set of experiments like those described above. Comparing these experiments will define pollen- and pistil-expressed genes that respond to heat stress in a pistil adapted to heat stress. These data (48 libraries each for two pistil parents: 2 temperatures, 2 timepoints, 4 pollinations, and 3 replicates) will provide a rich opportunity to define stress signaling interaction networks in a single cell (the pollen tube) as it interacts with multiple pistil cell types. The data will also be valuable to plant breeders working toward new, high-yielding cultivated varieties adapted to high temperature.Objective 2: Reveal transcriptional gene networks underlying endosperm development in maizeIn order to further understand the complexity of the O2-regulated processes, we will explore the nature of TF genes downstream of O2. We will carry out ade novocis-motif discovery (coupled with searches for any knowncis-motif/TF pairings) for the targets of TFs among DEGs that are likely indirect targets of O2 (unbound by O2). We will then test directly for interactions between the individual O2-regulated TFs and their putative targets usingin vitroDNA-protein binding assays, yeast one-hybrid assays (Y1H),in plantatransactivation assays, or a combination thereof.To understand the dynamics of O2 gene network expression during early endosperm development, we will analyze the temporal patterns of gene expression in a representative subset of the O2-regulated genes. Our preliminary data based on mRNA levels indicates that some O2-regulated genes may in fact be activated before O2 itself has been activated. We will explore this further through a real-time RT-PCR (qRT-PCR) analysis of the expression of a selected set of O2-regulated genes during multiple stages of endosperm development.Objective 3: Understand the role of small RNA-directed DNA methylation during seed development in BrassicaceaeTo determine if the defect is maternal, paternal, or zygotic we will cross homozygous RdDM mutants to wild type and measure seed production. Our initial experiments suggest a parental defect and we will further investigate parental effects through reciprocal wild type x heterozygous mutant crosses. To further test for a zygotic defect we will genotype >50 seeds arising from self-crossed heterozygotes. Finally, to determine if the observed mutant phenotype worsens with additional generations, we will inbreed the RdDM mutants and look for enhanced or new phenotypes.To determine how RdDM impacts reproductive programs, we will sequence transcriptomes from critical developmental timepoints. To determine whether the transcripts altered in the RdDM mutants are direct or indirect targets of p4-siRNAs and RdDM, we will also sequence siRNA transcriptomes and methylomes and correlate differential small RNA accumulation, differential methylation, and differential transcription. To determine if transcripts are misregulated specifically during reproduction, we will also sequence RNA and sRNA transcriptomes and methylomes of vegetative tissue. Libraries will be sequenced with the Illumina platform and processed in the iPlant Discovery Environment (DE). After processing, EPIC-CoGE (https://www.plant-epigenome.org/) will be used to identify differentially methylated regions and differentially expressed transcripts.Objective 4: Identify the life-history factors that determine whether RdDM is required during early reproductive development.To test the impact of RdDM in inbreeding vs. outbreeding species we will generatenrpd1andnrpe1mutants inC. rubellaandC. grandiflora, sister species that differ in reproductive strategy (Slotte et al., 2013). More specifically, we will use the CRISPR/Cas9 genome editing system with two guide RNAs and the 'nickase' mutants of Cas9 (Shen et al., 2014). Guide RNAs will be expressed behind the ArabidopsisUBQ10promoter, which we have successfully used for expression in other Brassicaceae species. Mutations in target genes will be assessed by PCR. Following identification, mutant lines will be bred to homozygosity, and seed set and seed weight will be measured to determine the extent to which RdDM affects reproductive success.To test the hypothesis that RdDM influences reproductive success in species whose genomes are the result of relatively recent duplications and thus composed of subgenomes, we will generatenrpd1andnrpe1mutants inCamelina sativa. BothB. rapaandC. sativaare mesopolyploids, however they arose through independent whole genome triplication events (Kagale, Koh, et al., 2014a; Kagale, Robinson, et al., 2014b), and are separated from each other by ~42 million years (MY) of evolution.We will simultaneously knockout all threeNRPD1andNRPE1paralogs by searching for conserved regions that will be targeted by the same CRISPR/Cas9 guide RNA (Y. Wang et al., 2014). Simultaneously we will mine the TILLING population publicly available forC. sativato identify mutantnrpd1andnrpe1alleles. Taken concurrently, these approaches will allow us to more readily generate the desired triple mutants through a combination of transformation and cross-breeding. As above, mutant lines will be bred to homozygosity, and seed set and seed weight will be measured to determine the extent to which RdDM affects reproductive success.How can we distinguish the mode of action of RdDM in maintaining genome balance, imprinting, or both processes? We expect to find that reproduction is impacted innrpd1andnrpe1mutants fromC. grandiflora,C. sativa, or both species. Using the list of genes misregulated inB. rapaplants lacking RdDM (Objective 3) we will query whether the orthologous gene(s) is misregulated inC. grandifloraand/orC. sativa. We will choose the top 20 candidates with GO annotations indicating a role in reproduction from Objective 3 and test by RT-PCR whether their expression is similarly altered innrpd1andnrpe1lines ofC. grandifloraandC. sativa.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience of this work is the scientific community, which is reached through peer-reviewed publications, university seminars, and presentations at scientific conferences.Results have been disseminated to communities of interest through eight peer-reviewed scientific publications, four oral presentations, and poster presentations at national and international conferences. Details of publications can be found in the Products section. Changes/Problems:The coronavirus pandemic has caused significant disruption to research programs across the country. We have mitigated this as much as possible by focusing on bioinformatic analysis that could be completed remotely, however our progress has inevitably slowed. Mentorship of undergraduate researchers has been especially difficult "at a distance". We continue to seek safe and effective ways to conduct research that respond to the ever-changing landscape of the pandemic. What opportunities for training and professional development has the project provided?Before the pandemic restricted such events, students in the project team were able to attend scientific conferences to develop their presentation skills and build their professional networks. • In August 2019, the team attended the AZ RNA Salon annual meeting, where graduate students Tania Chakraborty, Cecilia Chow, Jeff Grover, and Kelly Dew-Bud presented posters. • Kelly Dew-Bud also presented her research at the Plant and Animal Genomes (PAG) Conference in January 2020. In addition to a poster, she gave an oral presentation in the Small RNA workshop at this meeting. • Also in January 2020, undergraduate researchers in the team presented posters at the University of Arizona Undergraduate Biology Research Program (UBRP) annual meeting. • Finally, Maya Bose created a video description of her UBRP research project in summer 2020 (available at https://www.youtube.com/watch?v=MVFeofaltJs&feature=youtu.be). We are proud to report that two graduate students in this project successfully defended their dissertations this year. Jeffrey Grover has graduated and taken a position with Seven Bridges Genomics in Boston, while Kelly Dew-Bud also graduated and took a position with Integral Molecular. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through eight peer-reviewed scientific publications, four oral presentations, and poster presentations at national and international conferences. Publications are listed in the Products section. Invited seminars: Oct, 2019 Department of Plant Biology, Michigan State University, East Lansing, MI, USA. Evolutionary and Functional Analysis of a Multifunctional Plant Cell Signaling Complex. Nov, 2019 Plant Biology Graduate Seminar, University of California; Davis, CA, USA. RNA-directed DNA Methylation during seed development. Oral conference presentations: Jan, 2020 Plant and Animal Genomes Conference XXVIII (Comparative Genomics workshop); San Diego, CA, USA. Using orthologous mutations to understand the role of RNA-directed DNA Methylation during seed development. Jan, 2020. Plant and Animal Genomes Conference XXVIII; San Diego, CA, USA. Genomic Analysis of Thermotolerance During Tomato Pollination Jan, 2020 Plant and Animal Genomes Conference XXVIII; San Diego, CA, USA. Elucidating the influence of RdDM during reproduction through comparative small-RNAseq in two Capsella species. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we are continuing our analysis of siren siRNAs, with an emphasis on the functional role of these molecules during seed development. We are also completing analysis of RdDM mutants in Camelina sativa, Capsella grandiflora, and Capsella rubella. Finally, we will define the transcriptional response of the pollen tube and pistil to heat stress in thermotolerant and thermosensitive varieties of tomato.

Impacts
What was accomplished under these goals? Goal 1: Temperature sensitivity during fertilization Development of each seed within a fruit requires successful sperm delivery by a pollen tube and there is a strong relationship between seed number and fruit weight in tomato and other fruit crops. To test how decreased pollen tube growth affects tomato fruit production, the Palanivelu group applied acute heat stress (37°C during 12 hours of pollen tube growth) following manual self-pollination of Heinz or Malintka pistils. Unstressed controls (maintained at 24°C for 12 hours) and stressed plants were returned to optimal greenhouse conditions until fruit set and maturation. Heinz fruit dry weight (14.57g, n=3) decreased 51% relative to unstressed controls (28.4g, n=4). Importantly, in thermotolerant Malintka, fruit weight (29.47g, n=6) decreased only 16% relative to unstressed controls (35.62g, n=5). These results show that even a short exposure to heat stress during the critical period of pollen tube growth in the pistil reduces fruit yield of thermosensitive varieties far more than thermotolerant varieties. Goal 3: RNA-directed DNA methylation and successful seed development We recently published our analysis of "siren" siRNA loci during seed development (Grover JW, et al, PNAS 2020). The key conclusions from this work are: (1) Siren loci account for 90% of the siRNA accumulation in ovules, vastly overwhelming all other sources of small RNAs. (2) Siren siRNAs resemble canonical Pol IV-dependent siRNAs with respect to their biogenesis, and we confirmed that their accumulation is associated with abundant de novo methylation. (3) Siren loci can be detected in diverse angiosperms, however there is little to no conservation of sequence or position. Similarly, there is little conservation of siren loci, even when comparing the three homeologous positions in B. rapa. (4) Siren siRNAs are expressed most highly in the maternal integuments, both before and after fertilization, explaining the observation of maternally-biased siRNA populations in whole seeds. (5) Siren siRNAs are expressed orders of magnitude lower in endosperm and embryo, however they are still dominate the transcriptomes in these fertilized tissues. In embryo, siren siRNAs are expressed from both matrigenic and patrigenic alleles, but in endosperm siren siRNA accumulation is entirely maternal. Several groups have reported that mature embryos (in dry seeds) are hypermethylated in the CHH context, and RdDM has been proposed as the mechanism. We sought to better understand the role of RdDM in embryo methylation, including testing the prevailing hypothesis that embryo methylation might be reinforced by siRNAs from non-embryogenic tissues. We have made several discoveries: (1) Like other dicot species, B. rapa mature embryos are hypermethylated in the CHH context. This hypermethylation occurs across the genome, in both euchromatin and heterochromatin. (2) Embryo hypermethylation is eliminated in the rdr2 mutant, indicating that it occurs via RdDM. However, siRNA accumulation at hypermethylated regions is not substantially higher than in tissues which do not exhibit hypermethylation. (3) There is no correlation between endosperm demethylation and embryo hypermethylation, providing no support for the model of endosperm demethylation triggering siRNA production that causes embryo methylation. (4) rdr2 mutant embryos from RDR2/rdr2 heterozygous mothers have no change in hypermethylation, indicating that siRNA from the maternal sporophyte (including the abundant siren siRNAs) do not influence embryo methylation. Goal 4: Develop a comparative genetic approach for seed development We generated knock-out mutant lines for two RdDM components (rdr2 and nrpe1) in three different species of Brassicaceae - Camelina sativa, Capsella grandiflora, and Capsella rubella. We collected seed set and seed weight data for two mutant lines of each RdDM component in all three species and found that C. grandiflora plants showed significantly reduced seed set; we are currently analyzing seed weight data. Interestingly, C. grandiflora is an obligate outcrosser while the two other species can inbreed. Thus, our data, along with data collected by the Mosher lab suggests that RdDM is required for proper seed development in species that are obligate or historical outcrossers. In addition, in collaboration with the Mosher lab, we sequenced small-RNAs from the ovules and leaves of rdr2 and wildtype lines for the inbreeder/outcrosser species pair C. rubella/C. grandiflora. To improve the mapping rate of siRNAs recovered from our sequencing, we used long-read sequencing to generate a less fragmented genome for C. grandiflora. Our comparative mapping results showed that the majority of RDR2 dependent siRNAs in ovules were produced from ~200 loci in both C. grandiflora and C. rubella, similar to observation of abundant siren siRNAs observed in other species.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Chakraborty T, Kendall T, Grover JW, and Mosher RA. Embryo CHH hypermethylation is mediated by RdDM and is autonomously directed in Brassica rapa BioRxiv, 2020. https://www.biorxiv.org/content/10.1101/2020.08.26.268573v1
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Grover JW, Burgess D, Kendall T, Baten A, Pokhrel S, King GJ, Meyers BC, Freeling M, and Mosher RA. Abundant expression of maternal siRNAs is a conserved feature of seed development. Proceedings of the National Academy of Sciences, USA, 117(26): 15305-15315, 2020. https://doi.org/10.1073/pnas.2001332117
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Chow HT, Chakraborty T, and Mosher RA. RNA-directed DNA Methylation and sexual reproduction: expanding beyond the seed. Current Opinion in Plant Biology, 54: 11-17, 2020. https://doi.org/10.1016/j.pbi.2019.11.006
• Type: Theses/Dissertations Status: Other Year Published: 2020 Citation: Grover JW. Investigating The Role Of RNA-directed DNA Methylation in Plant Reproduction and Development of Computational Resources Enabling Epigenomics Research. PhD Dissertation, May 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Desnoyer N, Palanivelu R. (2020). Bridging the GAPs in plant reproduction: a comparison of plant and animal GPI-anchored proteins. Plant Reprod. https://doi.org/10.1007/s00497-020-00395-9.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Desnoyer N, Howard G, Jong E, Palanivelu R. (2020). AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase subunit, is critical for pollen tube growth in Arabidopsis. BMC Plant Biol. 20 (1):380. https://doi.org/10.1186/s12870-020-02587-x.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Noble JA, Palanivelu R. (2020). Workflow to Characterize Mutants with Reproductive Defects. Methods Mol Biol. 2160:109-128. DOI: 10.1007/978-1-0716-0672-8_8.
• Type: Theses/Dissertations Status: Other Year Published: 2020 Citation: Dew-Bud, K. Evolution and Genetic Dissection of Two RNA-Dependent Processes in Brassicaceae. PhD Dissertation, August 2020.
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Dew-Budd K, David B, Beilstein MA (2019) Agrobacterium-mediated floral-dip transformation of the obligate outcrosser Capsella grandiflora DOI: 10.1101/757328
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Jennifer A. Noble, Ming-Che James Liu, Thomas A. DeFalco, Martin Stegmann, Kara McNamara, Brooke Sullivan, Khanhlinh K. Dinh, Nicholas Khuu, Sarah Hancock, Shin-Han Shiu, Cyril Zipfel, Mark A. Beilstein, Alice Y. Cheung, Ravishankar Palanivelu. Conserved Molecular Function and Regulatory Subfunctionalization of the LORELEI Gene Family in Brassicaceae. bioRxiv 2020.04.27.062893; doi: https://doi.org/10.1101/2020.04.27.062893

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience of this work is the scientific ommunity, which is reached through peer-reviewed publications, university seminars, and presentations at scientific conferences. During the reporting period, the target audience was reached through five peer-reviewed publications andfive conference presentation (listed in the "Products" section). In addition, the work was disseminated at four seminar visits: Oct, 2018 School of Integrated Plant Sciences, Cornell University; Ithaca, NY, USA. Evolution of RNA Polymerases in plants: understanding duplication of multi-subunit complexes. Jun, 2019 Indian Institute of Sciences, Bengaluru, India. Changes in gene expression underlie the diversification of the LORELEI gene family in Brassicaceae Aug, 2019 Department of Genetics, University of Georgia; Athens, GA, USA. Evolution of RNA Polymerases in plants: understanding duplication of multi-subunit complexes. Sep, 2019 School of Plant Sciences, University of Arizona; Tucson, AZ, USA. RNA-directed DNA methylation: evolution and roles in reproduction. Changes/Problems:Dr. Ramin Yadegari has asked to be removed from the project, and therefore Goal 2 (Understand the gene regulatory networks during early endosperm development in maize) has not been included in this report, and will not be included in future reports. What opportunities for training and professional development has the project provided?Members of the project team attended the Plant and Animal Genome conference in San Diego, CA during January 2019. Graduate students Jeff Grover and Kelly Dew-Budd presented posters of their most recent findings to a broad audience of researchers at this meeting, and also at the Arizona RNA Salon, a gathering of RNA researchers from across the state. In addition, Ms. Dew-Budd attended informational meetings on long-read pore-based sequencing platforms and is developing skills in data analysis of both short read small RNAseq data and long read genomic data. Graduate students Nick Desnoyer and Jennifer Noble submitted their Masters and PhD thesis, respectively, and graduated from the School of Plant Sciences, University of Arizona in Summer 2019. Nick is a PhD student in the University of Zurich and Dr. Noble is a Scientific Services Scientist at HTG Molecular Diagnostics, Tucson. Undergraduate researchers Brandon David and Jack Stearns presented posters at the University of Arizona Undergraduate Biology Research Program annual meeting held on campus in January 2019. Both students worked closely with Ms. Dew-Budd, providing her an opportunity to improve her management and mentoring skills. Finally, through our interactions with Dr. Michael Frank, an educator at Empire High School in Vail, Arizona, undergraduate and graduate students in the Beilstein lab were provided the opportunity to mentor high school students in a collaborative research project to screen CRISPR generated mutants. How have the results been disseminated to communities of interest? Results have been disseminated to communities of interest through five peer-reviewed scientific publications and five oral presentations at national and international conferences. Details of these can be found in the Products section. In addition, the work was discussed during four university seminar visits (detailed in the Target Audience section). This work also enabled five classroom visits that included hands-on activities about flowers and seeds to Kindergarten and 2nd -grade students at Annie W. Kellond Elementary School in Tucson, AZ and Walter Douglas Elementary School in Tucson. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Temperature sensitivity during fertilization Development of each seed within a fruit requires successful sperm delivery by a pollen tube and there is a strong relationship between seed number and fruit weight in tomato and other fruit crops. We will test how decreased pollen tube growth affects tomato fruit production, and whether even a short exposure to heat stress during the critical period of pollen tube growth in the pistil reduces fruit yield of thermosensitive varieties far more than thermotolerant varieties. Goal 3: RNA-directed DNA methylation and successful seed development During the next reporting period, we will finish our analysis of "siren" loci, including investigating whether maternal siren production impacts siRNA production, DNA methylation, genomic imprinting, or genome dominance in filial tissues.We are also extending this analysis beyond B. rapa to see whether our conclusions will be generalizable in plants. Goal 4: Develop a comparative genetic approach for seed development Now that breeding is completed for nrpe1 and rdr2 alleles in Capsella species, we will finish analyzing seed set and seed weight data, as well as extend our sRNA sequencing to include these mutants, providing a first-pass analysis of the inbreeder/outbreeder effect.

Impacts
What was accomplished under these goals? Goal 1: Temperature sensitivity during fertilization We examined the effect of heat stress on pollen tube growth in pistils in vivo. In self-crosses, when an acute heat stress of 37°C was applied only during the pollen tube growth phase (12 hours), there was a significant (p<0.0001) reduction in pollen tube length at 37°C compared to 25°C in thermosensitive varieties. Importantly, at 37°C, pollen tubes in thermotolerant varieties grew significantly (p<0.004) longer than the pollen tubes from thermosensitive varieties, revealing the genetic control of enhanced pollen tube growth in thermotolerant varieties under heat stress. We performed reciprocal crosses between thermotolerant and thermosensitive varieties, subjected the crossed pistils to acute heat stress (37°C for 12 hours), and measured pollen tube length. A three-way ANOVA showed that all three factors (pollen genotype, pistil genotype, and temperature) significantly (p<0.05) influenced pollen tube growth. When Heinz was the female parent, pollen from all varieties performed poorly. However, when Heinz was used as a pollen donor, Heinz pollen grew significantly better in Malintka and Tamaulipas pistils, suggesting these pistils with Heinz pollen can be used to identify pistil factors conferring thermotolerance. Strikingly, thermotolerant Malintka pollen out-performed M82 pollen in the M82 pistil under stress. These analyses identified a useful combination (M82 female x Malintka male) to identify genes underlying pollen tube thermotolerance. These data clearly indicated that pollen and pistil jointly contribute to thermotolerant reproduction and that tomato genetic diversity can be exploited to define thermotolerance mechanisms. Goal 3: RNA-directed DNA methylation and successful seed development We have discovered that the B. rapa ovule transcriptome is dominated by about 200 loci that account for 90% of all sRNAs in the sample. These loci, which we call siren loci, resemble canonical RNA-directed DNA Methylation (RdDM) loci, but with some subtle differences. Siren loci are less repetitive than canonical RdDM loci and have more uniquely-mapping siRNAs. Like canonical RdDM loci, sirens overlap annotated TEs more frequently than expected by chance, however they do not show depletion for genic sequences as is observed for canonical RdDM loci. Like most 24-nt siRNAs, siren siRNAs begin with a 5' A and are NRPD1- and RDR2-dependent but NRPE1-independent, indicating that these siRNAs feed into the canonical RdDM mechanism. Indeed, CHH methylation is very high at siren loci only in tissues where siren siRNAs accumulate. Siren siRNAs continue to be expressed following fertilization, and are found at high levels in the developing seed coat. Siren siRNAs are also detected in the developing endosperm and embryo at lower levels, although at this point it is not clear whether these siRNAs are produced in filial tissues, transmitted to filial tissues, or contaminate filial tissues following dissection. The presence of abundant siRNAs from the seed coat is intriguing as the seed coat is an ephemeral tissue that does not genetically contribute to the embryo. This raises the possibility that sirens produced in the seed coat are intended for activity in the filial tissues and loss of these sirens might explain seed abortion from RdDM mutant mothers. A similar process occurs with piRNAs in Drosophila - deposition of piRNAs from maternal sporophytic tissue triggers production of additional piRNAs and suppression of transposons in the developing fly embryo. Goal 4: Develop a comparative genetic approach for seed development We have identified probable loss-of-function mutations in both RDR2 and NRPE1 in the inbreeder/outbreeder pair Capsella rubella and Capsella grandiflora. Loss of RdDM in C. rubella might have a minor impact on seed production. In contrast, C. grandiflora mutants have high rates of seed abortion and lower seed weight, supporting a model where RdDM is required for seed development specifically in outbreeding species. Similar to the seed development defect in B. rapa, seed abortion in C. grandiflora mutants is controlled by maternal genotype rather than zygotic genotype. In addition, we have generated homozygous rdr2 mutants in Camelina sativa and are breeding homozygous lines of nrpe1. Our preliminary results in C. grandiflora prompted us to look more closely at embryo and endosperm development in C. grandiflora. To accomplish this goal, we began a collaboration with Dr. Pablo Jenik, Professor at Franklin and Marshall College in Pennsylvania. Dr. Jenik visited the lab and trained graduate student Kelly Dew-Budd on sample tissue preparation and imaging. Our preliminary results indicate that embryos undergo normal development but may abort due to improper endosperm development. Currently we are working to characterize endosperm development in C. grandiflora to better understand the impact of rdr2 and nrpe1 mutations.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kirkbride RC, Lu J, Zhang C, Mosher RA, Baulcombe DC, and Chen ZJ. Maternal small RNAs mediate spatial and temporal regulation of gene expression and seed development in Arabidopsis. Proceedings of the National Academy of Sciences, USA, 116: 2761-2766, 2019. https://www.pnas.org/content/116/7/2761
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Trujillo JT, Seetharum AS, Hufford MB, Beilstein MA, and Mosher RA. Evidence for a sixth DNA-dependent RNA polymerase in cereal crops. Molecular Biology and Evolution, 35(10): 2454-2462, 2018. https://doi.org/10.1093/molbev/msy146
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Johnson M.A., Harper J.F., Palanivelu, R. (2019). A Fruitful Journey: Pollen Tube Navigation from Germination to Fertilization. Annu Rev Plant Biol. 70:80937
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Desnoyer, N. GAPs in Plant Reproduction: Uncovering the Role of Glycosylphosphatidylinositol-Anchoring of Proteins in Arabidopsis Gametophyte Function. MS Thesis. May 2019.
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Noble, JN. Changes in gene expression underlie the diversification of the LORELEI gene family in Brassicaceae. PhD Dissertation, Aug 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Jan, 2019 Plant and Animal Genomes Conference XXVII (Brassica workshop); San Diego, CA, USA. Using Brassica rapa to understand epigenetic dynamics in the seed.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Aug, 2019 3rd Annual Arizona RNA Salon Symposium; Tucson, AZ. Evolution of RNA Polymerases in Plants
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Aug, 2019 Chair & speaker, Mini Symposium  Reproductive Biology, American Society of Plant Biologists, San Jose, CA. LORELEI and its most closely related paralog, LLG1, show evidence of regulatory subfunctionalization in Brassicaceae.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Sep, 2019 22nd Annual PGRP Awardee Meeting; Alexandria, VA. Using parallel mutations to understand the role of RNA-directed DNA Methylation during seed development
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Jun, 2019 Algal model systems on the rise: Understanding and exploiting the algae to land plant transition (SEB satellite meeting); Seville, Spain. Origin of plant-specific polymerases in Charophycean algae.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience of this work is the scientific community, which is reached through peer-reviewed publications and presentations at scientific conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project is providing training in genomics, epigenetics, and genome editing for graduate students, as well as training for a postdoctoral fellow in analysis of genome-wide ChIP-Seq data. In addition, graduate students Kelly Dew-Budd (Beilstein Lab) and Jen Noble (Palanivelu Lab) presented posters at the 2018 American Society of Plant Biologists meeting in Montreal, Canada, graduate student Nick Desoyner (Palanivelu Lab) presented his research at the 2018 Plant Reproduction meeting in Gifu, Japan, and graduate student Joshua Trujillo (Mosher Lab) gave an oral presentation at the 2018 Gordon Conference on Plant Molecular Biology. This project also involves the training of undergraduate researchers. The Mosher, Beilstein, and Palanivelu labs each trained two undergraduates in the past year. Two of these students presented posters in January 2018 for the Undergraduate Research Program symposium at the University of Arizona, and two will submit Honors Theses this academic year. Finally, this project enabled professional development of Michael Frank, a local high school teacher working in the Beilstein lab. How have the results been disseminated to communities of interest?Results have been dissemintaed to communities of interest through peer-reviewed scientific publications (4), oral presentations (7), and poster presentations at national andinternational conferences (4). Details of these can be found in the Products section. Results are also disseminated through the website www.grainendosperm.org. What do you plan to do during the next reporting period to accomplish the goals?Goal 1:Temperature sensitivity during fertilization To address the effect of heat on fertilization, we will first identify a heat stress regime by evaluating the pollen tube growth in pistil assay with two thermosensitive (M82, Heinz) and two thermotolerant (Nagcarlang, Malintka) varieties. Using this temperature regime and assay, our next goal is to define the transcriptional response of the pollen tube and pistil to heat stress in thermotolerant and thermosensitive varieties of tomato. Goal 2:Understand the gene regulatory networks during early endosperm development in maize Our major goals include (1) analysis of function of 5-7 transcription factor proteins that likely function downstream of O2 using the reverse-genetic resources of maize, (2) temporal analysis of gene networks regulated by O2 during endosperm development, and (3) analysis of the cis-regulatory-sequence targets of O2 to understand the nature of its protein partners in regulation of downstream genes. Goal 3:RNA-directed DNA methylation and successful seed development We have generated a variety of NGS libraries to better understand how RdDM influences seed development, including additional siRNA and mRNA libraries as well as whole genome bisulfite libraries. In the coming year we will use these libraries to understand how siRNAs might enable communication between the distinct genomes in a developing seed - the diploid maternal genome in the integuments, the 2:1 matrigenic:patrigenic genome in the endosperm, and the 1:1 matrigenic:patrigenic genome in the embryo. Goal 4:Develop a comparative genetic approach for seed development In the coming year we plan to build on our considerable progress and finish identification of CRISPR mutant alleles in each of the threeRDR2andNRPE1homoeologs inCamelina. These alleles will then be used to generate triple mutants. We will continue to analyze seed set and seed weight in CRISPR mutants as we develop the appropriate knock-out plant lines. We will also explore the possibility of extending our siRNA analysis to include theCapsella rubellaandCapsella grandifloragenomes.

Impacts
What was accomplished under these goals? Goal 1:Temperature sensitivity during fertilization To study pollen tube-synergid interactions, which are key to understand the process of double fertilization in plants, we developed tools to study the localization of membrane-bound proteins and the components of the endomembrane system in synergid cells. We demonstrated the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells of Arabidopsis thaliana. These markers were used in co-localization experiments to investigate the subcellular distribution of pollen tube reception LORELEI, a GPI-anchored surface protein, and NORTIA, a MILDEW RESISTANCE LOCUS O protein. These secretory markers will be very useful tools for us when we analyze the fertilization defects that occur in plants experiencing heat stress. Goal 2:Understand the gene regulatory networks during early endosperm development in maize We continued our efforts in understanding the regulatory mechanisms that control accumulation of storage proteins and starch during mid to late stages of endosperm development in maize (Zea mays). Development of the cereal endosperm involves cell differentiation processes that enable nutrient uptake from the maternal plant, accumulation of storage products and their utilization during germination. We set out to use the previously studies Opaque-2 (O2) bZIP transcription-factor protein, a major regulator of storage protein gene expression in maize, as an opening to dissect further these regulatory programs. We mapped in vivo binding sites of O2 in B73 endosperm, and compared the results with genes differentially expressed in B73 and B73o2. We identified 186 putative direct O2 targets and 1,677 indirect targets, encoding a broad set of gene functionalities. Examination of the temporal expression patterns of O2 targets revealed at least two distinct modes of O2-mediated gene activation. Two O2-activated genes, bZIP17 and NAKED ENDOSPERM2 (NKD2), encode transcription factors, which can in turn co-activate other O2-network genes with O2. NKD2 (with its paralog NKD1) was previously shown to be involved in regulation of aleurone development. Collectively, our results have provided insights into the complexity of the O2-regulated network and its role in regulation of endosperm cell differentiation and function. We plan to further characterize the functions of some of the key downstream target genes of O2 during the next reporting period. Goal 3:RNA-directed DNA methylation and successful seed development To better understand the role of RdDM during seed development, we are characterizing siRNA production in both developmental time and space. Initial assessment indicates that 24 nt siRNAs are abundant in unfertilized ovules and decrease in relative abundance during the early stages of seed development (10 dpf, globular embryos). Relative abundance of 24 nt siRNAs then increases as seed development progresses. 24 nt siRNAs accumulate to their lowest relative level in endosperm. This is surprising as prevailing models of RdDM in the seed include robust production of 24 nt siRNAs in endosperm. However, this observation is consistent with the temporal dynamics we observed - as endosperm proliferates following fertilization, overall 24 nt siRNA accumulation decreases (because endosperm cells now represent a larger fraction of the seed); and as embryo growth increases during later seed development 24 nt siRNA accumulation is restored. We are currently including additional timepoints and further datasets to better understand the dynamics of siRNA production in the seed. Goal 4:Develop a comparative genetic approach for seed development We have identified probable loss-of-function mutations in bothRDR2andNRPE1in the inbreeder/outbreeder pairCapsella rubellaandCapsella grandiflora. As in Arabidopsis, multiple independent mutations of each gene inC. rubellahad no obvious defect in seed production. Conversely, for the obligate outbreederC. grandiflora, homozygous mutations inNRPE1andRDR2have higher rates of seed abortion and potentially lower seed weight. We are currently breeding theC. grandiflorapopulation to generate sufficient homozygous mutants for repeated and careful measurements of seed development in these mutants, but these preliminary observations support a model where RdDM is required for seed development in outbreeding species. We are also testing whether RdDM is required for seed developing in mesopolyploids by generating NRPE1 and RDR2 mutations inCamelina sativa. We have identified multiple frameshift mutations for each of the threeC. sativaNRPE1homeologs, and we are searching for mutations in each of the threeRDR2homeologs. None of theNRPE1single or double mutants show overt seed development defects, and we are currently generating homozygous triple mutants.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Grover JW, Kendall T, Baten A, Burgess D, Freeling M, King GJ, and Mosher RA. Maternal components of RNA-directed DNA methylation are required for seed development in Brassica rapa. The Plant Journal, 94: 575-582, 2018.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhao L, Cai H, Su Z, Wang L, Huang X, Zhang M, Chen P, Dai X, Zhao H, Palanivelu R, and Chen X. KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis. Proceedings of the National Academy of Sciences. 115(3):E526-35, 2018.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Jones DS, Liu X, Willoughby AC, Smith BE, Palanivelu R, and Kessler SA. Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana. The Plant Journal. 94(1):192-202, 2018.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhan J, Li G, Ryu C-H, Ma C, Zhang S, Lloyd A, Hunter BG, Larkins BA, Drews GN, Wang X, and Yadegari R  Opaque-2 regulation of endosperm development. Plant Cell, 30(10):2425-46, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Mosher RA. Evolution of RNA Polymerases in plants: understanding duplication of multi-subunit complexes. Earlham Institute, Norwich Biosciences Institute; Norwich, UK. (Oral presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Mosher RA. RNA-directed DNA methylation: mother knows best! Linnean Centre Seminar, Swedish University of Agricultural Sciences; Uppsala, Sweden. (Oral presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Palanivelu R. Closing the deal: Arabidopsis LORELEI is required for pollen tube reception by the female gametophyte. Plant Biology Seminar Series, Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA. (Oral presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Mosher RA. RNA-directed DNA methylation: evolution and roles in reproduction. Institut de Recherche pour le D�veloppement (IRD); Montpellier, France. (Oral presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Palanivelu, R. Closing the deal: Arabidopsis LORELEI is required for pollen tube reception by the female gametophyte. Department Seminar Series, Department of Molecular, Cellular, & Developmental Biology, University of Michigan, Ann Arbor, MI. (Oral presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Dew-Budd K, Mosher RA & Beilstein, MA. Identifying the genomic factors that influence the impact of RNA-directed DNA methylation during seed development. American Society of Plant Biologists Annual Meeting (ASPB 2018). Montreal, Canada. (Poster presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Noble J, Beilstein M, Cheung A, & Palanivelu, R. LORELEI, and its most closely related paralog LLG1, have similar function but divergent expression patterns. Plant Biology 2018. Montreal, Canada. (Poster presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Yadegari, Y. Gene regulatory networks in maize endosperm development. The 25th International Congress on Sexual Plant Reproduction, Gifu, Japan (Oral presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Yadegari, Y. Gene regulatory networks in maize endosperm development. Annual Meeting of the Multistate Research Project: Environmental and Genetic Determinants of Seed Quality and Performance (W3168), Corvallis, OR. (Oral presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Desnoyer N, Liu X, & Palanivelu R. Mutation in a glycosylphosphatidylinositol (GPI) transamidase complex subunit disrupts male gametophyte function in Arabidopsis. The 25th International Congress on Sexual Plant Reproduction 2018. Gifu, Japan. (Poster presentation)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Cheung A, Feijo J, Johnson M, & Palanivelu R. Research Coordination Network on Integrative Pollen Biology: Report and perspectives on fostering a community of pollen biology researchers. The 25th International Congress on Sexual Plant Reproduction. Gifu, Japan. (Poster presentation)