GENETICS OF PLANT REPRODUCTION AND THE GENOMIC EFFECTS OF PLANT MICROBIOMES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0192081
• Project Start Date: Oct 1, 2011
• Project End Date: Sep 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Interdepartmental

Non Technical Summary
This project will investigate two types of effectors, one internal and the other external, that have the potential to have a significant impact on agricultural yields. The internal effector is the embryo sac, a critical unit of plant reproduction within the female organs of the flower, which governs the formation of the seed. The genetic mechanism by which rice plants specify their female gametes (egg cell and central cell) inside the embryo sac will be deciphered using rice plants. The female gametes give rise to the two major components of the seed, the embryo and the endosperm. Genes that control embryo sac development can be used to facilitate production of hybrid seed, and for improving seed size and seed production, which is of agricultural importance to rice and other seed crops. An external effector that can impact yields is the soil microbiome, i.e. the community of microbes in association with the roots of field grown plants, which provides benefits to crops through processes such as conversion of ammonia to nitrate, and protection against pathogens. The project will determine the effects of the soil microbiome on gene expression in rice, to provide an understanding of how the plant metagenome (the composite genomes of the associated microbiome) interacts with the plant genome. Such knowledge can be exploited to select for genes/traits that favor the growth of beneficial bacteria and suppress the growth of harmful bacteria. Utilizing the natural alliances between plants and microbes can lead to an entirely new direction in plant breeding for the future of agriculture.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1530	1080	75%
206	2499	1050	25%

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

Subject Of Investigation
1530 - Rice; 2499 - Plant research, general;

Field Of Science
1080 - Genetics; 1050 - Developmental biology;

Keywords

embryo sac

seed development

gametophytes

auxin

microbiome

metagenome

rice

transcriptome

Goals / Objectives
Knowledge of the mechanisms that control embryo sac development will enable identification of genes for better seed production. We have previously shown the plant hormone auxin is an important determinant of cell-fate specification in the embryo sac. We will now decipher the mechanism by which auxin acts to specify cell fates such as the egg cell. The studies will be performed in Arabidopsis and rice, and will utilize the manipulation of auxin signaling and auxin biosynthetic genes in transgenic plants. Further, we use isolated individual cells from rice embryo sacs to determine the expression of downstream target genes of auxin, such as the auxin response factors, that mediate the specification of cell fates. In order to better understand the effect of the microbiome on plant performance, we will examine the plant responses to the microbiome by studying changes in the transcriptome of hydroponically grown rice plants exposed to microbial extracts from the soil. The microbial populations that are in close association with roots of field grown rice will be first characterized by metagenomic sequencing to identify the species of bacteria that are present in the extracts that will be used as inputs. The changes in the rice transcriptome generated by exposure to these microbes will measure their effects on global gene expression, and identify specific functional classes of genes that would reflect changes in nutrient availability, establishment of plant immunity, or promotion of growth. We will attempt to correlate the changes in expression of specific gene families with the presence of defined species of microbes, with the long term goal of defining and understanding the complex interactions of plants with their microbiomes. This understanding could be translated into new breeding approaches that take advantage of these interactions to improve yields.

Project Methods
To study the basis of cell fate determination in the embryo sac, individual cells (egg cells, synergid cells, central cells) will be dissected from rice embryo sacs by Scott Russell (U. Oklahoma), the RNA extracted in the Sundaresan lab, and tested by quantitative PCR for the expression of auxin response factors. The experiments will reveal whether individual cell-types have a distinct signature of auxin response factors, or whether the same subset of factors is expressed at different levels in the different cell-types. The fine-structure dynamics of auxin signaling during embryo sac development will be studied by confocal microscsopy of fluorescent auxin reporters in transgenic rice. The manipulation of the auxin gradients will be performed using Arabidopsis, where mutants in all the auxin biosynthetic pathway genes are readily available, as well as the ease of transformation to introduce transgenes to alter rates of auxin biosynthesis at specific spatial and temporal locations. The rice plants for the microbiome studies will be grown in sterile hydroponic conditions. As rice is a semi-aquatic plant, the effect of hydroponics on normal growth is minimal compared to most other crop plants. The rice we will use is a japonica rice cultivar M201, that is widely grown in California. The microbiome used for inoculating the rice plants will be obtained from the roots of M201 plants grown in rice fields in Davis. Plant tissues, from both root and leaf, will be harvested after establishment of the inoculated bacteria in the hydroponically grown rice plants, with samples from multiple time points. RNA extracted from these tissues will be sequenced with a high throughput Illumina sequencer at the UCD Genome Center, using a procedure called RNA-seq. The transcript profiles of uninoculated controls and inoculated plants at different time points will be analyzed bioinformatically to determine genes with significantly altered expression in response to the microbes, and formulate clusters of co-ordinately regulated genes that might represent specific metabolic or signaling pathways activated as components of the plant's responses. The collection of microbiome samples will be at UCD, and the hydroponic rice plants will be grown by collaborator Harsh Bais at Delaware, who will perform the inoculations and then send the plant tissues to Davis, for the RNA-seq profiling and analysis. The bacterial species that are present in the inoculum, and are established in the hydroponic system, will be identified by 16S rRNA metagenomic profiling. The DNA from the soil microbiome will be extracted, amplified by PCR using primers specific for bacterial 16S RNA, and sequenced using 454 pyrosequencing at the Genome Center in the lab of Jonathan Eisen, who is a collaborator on this part of the project. Bioinformatics and statistical analysis will be performed to correlate the gene expression responses of the plants with the presence or absence of species or phylogenetic groups of bacteria, and used to construct models of possible interaction networks between plants and the microbiome.

Progress 10/01/11 to 09/30/16

Outputs
Target Audience:Plant geneticists and breeders in academic and government research institutions, seed companies, biotechnology companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training for 3 postdoctoral fellows, 5 graduate students, and 12 undergraduate students. How have the results been disseminated to communities of interest?1. Research publications 2. Scientific Conferences 3. Industry-Academy collaborative conferences 4. Seminars at academic institutions 5. Seminars at companies What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Seed formation requires the fertilization of two female gametes, the egg cell and the central cell by sperm cells in the pollen. The fertilized egg cell and central cell gametes give rise to an embryo and an endosperm respectively, the constituents of a seed. Despite a century of genetic studies in plants, the genes required to specify female gametes or to transition from an egg cell to an embryo, are unknown or poorly characterized. This research project will identify and characterize the genes and underlying mechanisms underlying these biological processes. Because seed production requires the formation of gametes and zygotes, the findings could be used to improve the quality and quantity of seeds in crop plants. The process of double fertilization results in a seed containing a diploid embryo and a triploid nourishing tissue, the endosperm. Despite their importance for seed formation, the molecular mechanisms by which the two female gametes with very distinct characteristics are specified are yet to be characterized, leaving a major gap in our understanding of flowering plant reproduction. We have found that the CYTOKININ INDEPENDENT 1 (CKI1) gene, encoding a histidine kinase activator of the cytokinin signaling pathway plays a key role in female gamete specification (Yuan et al., 2016). In Arabidopsis mutants deficient for this gene, the central cell and proximal cells of the embryo sac exhibit egg cell characteristics. Conversely, mis-expression of this gene results in conversion of egg cells and other distal cells to central cells. These additional central cells are functional, and can be fertilized, resulting in the formation of seeds with two endosperms and no embryo. Expression of the CKI1 gene is female-gametophyte specific, and the CKI1 protein is localized to the proximal domain of the female gametophyte from the four nuclear stage of development onwards. Together, the observations provide a model by which female gamete specification is regulated through the distribution of CKI1 protein within the syncitial female gametophyte. 2. The fertilized egg cell forms a zygote, which is a critical stage in the plant life cycle. In the zygote, the genome is reprogrammed for the initiation of a new diploid generation that arises from this one cell. In animals, the zygotic genome is initially transcriptionally inactive, and early embryogenesis depends exclusively upon maternal transcripts. Previous studies have shown that in plant embryogenesis, the large-scale activation of zygotic genes occurs earlier than in animals, although both the timing and the relative contributions of the maternal and paternal genomes to zygotic genome activation have been controversial (Baroux and Grossniklaus, 2015). However, a detailed characterization of the reprogramming and activation of the zygotic genome at the earliest stages has not been performed in a flowering plant due to the difficulties of inaccessibility, time-staging, and isolation of single-cell zygotes. These difficulties have been overcome in rice by our collaborator, Dr. Scott Russell at the University of Oklahoma. This project will utilize the crop plant rice to characterize genome-wide changes associated with epigenetic mechanisms involved in the transition from differentiated gametes to totipotent zygote. We have previously successfully characterized the transcriptomes of the male and female gametes, the egg cell and the sperm cells of rice, and shown that they are markedly different from each other and from vegetative cells (Anderson et al. 2015). Using these protocols, we have now obtained data showing that the zygotic transition, as defined by large scale transcriptomic changes (e.g., the differential expression of thousands of genes) is underway before the first cell division of the plant embryo, much earlier than in animals. Our preliminary data also show that in the zygote, there is generally delayed activation of the paternal alleles, but some genes, including key promoters of embryogenesis, appear to be exclusively paternally expressed beginning at very early stages. Future experiments will characterize the contribution of paternal and maternal genomes to the zygote. To understand the role of epigenetic processes in zygotic genome activation the small RNA transcriptomes and methylomes of gametes and zygotes will also be characterized. We have also been able to identify transcription factors that appear to play a key role in the zygotic transition. Ectopic expression of one such transcription factor in the egg cell appears to result in the formation of seeds that give rise to haploid plants. This is an exciting result with applications to efficient plant breeding. 3. Plants grow in close association with large communities of microbes called microbiomes, that influence nutrient composition and uptake of nutrients in the soil, as well as disease susceptibility. Major advances in understanding the health effects of human microbiomes have resulted in the utilization of beneficial microbes in medicine. However, relatively little is known about plant microbiomes and their interactions and effects on their plant hosts. We have completed an extensive characterization of the root microbiomes of soil grown rice plants (are modulated by several environmental factors, including location, soil type, and cultivation practice, suggesting that changes to microbiomes constitute part of the plant adaptation to the environment (Edwards et al. 2015). This study also illustrated for the first time, the dynamics of root microbiome assembly, and the role played by the rhizoplane in the process (commentary by van der Heijden and Schlaeppi, 2015). In new research, we have been characterizing the microbiomes that are acquired under drought conditions, and developing systems to test functional consequences of different microbiomes on plant performance. In collaboration with Greg Phillips at Arkansas State University, we have also initiated a project to investigate the association of archaea with cultivated rice. This association is a significant contribution to planetary methane emissions (an estimated ~15% of anthropogenic methane emissions is due to rice paddies), and potentially to climate change due to the potency of methane as a greenhouse gas (25X that of CO2). Computational analysis of our microbial sequence data rice fields revealed the presence of association networks correlated with functions in methane cycling (Edwards et al. 2015). Understanding the interactions of the host plant with its microbiome, and subsequent effects on crop performance and yields, provides possibilities for exploiting plant-microbe associations for future crop improvement.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Leshem Y., Johnson C.A., Wuest S.E., Song X., Ngo Q.A., Grossniklaus U., Sundaresan V. 2012. Molecular Characterization of the glauce Mutant: a Central Cell-Specific Function Is Required for Double Fertilization in Arabidopsis. The Plant Cell, 24: 3264-3277.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Ngo QA, Baroux C, Guth�rl D, Mozerov P, Collinge MA, Sundaresan V, Grossniklaus U., 2012. The Armadillo Repeat Gene ZAK IXIK Promotes Arabidopsis Early Embryo and Endosperm Development through a Distinctive Gametophytic Maternal Effect. The Plant Cell, 24: 4026-4043.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yang S-Y, Gronlund M, Jakobsen I, Grotemeyer MS, Rentsch D, Miyao A, Hirochika H, Kumar CS, Sundaresan V, Salamin N, Catausan S, Mattes N, Heuer S, Paszkowski U. 2013. Nonredundant Regulation of Rice Arbuscular Mycorrhizal Symbiosis by Two Members of the PHOSPHATE TRANSPORTER1 Gene Family. The Plant Cell, 24: 4236-4251.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Tsai H., Missirian V., Ngo K., Tran R.K., Chan S., Sundaresan V., Comai L., 2013. Production of a high efficiency TILLING population through polyploidization. Plant Physiology 161: 1604-1614.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Leshem Y., Johnson C., Sundaresan V., 2013. Pollen tube entry into the synergid cell of Arabidopsis is observed at a site distinct from the filiform apparatus. Plant Reproduction, 26: 93-99.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Anderson, S., Johnson, C., Jones, D., Conrad, L., Gou, X., Russell, S., Sundaresan, V., 2013. Transcriptomes of isolated rice gametes characterized by deep sequencing: Evidence for distinct sex-dependent chromatin and epigenetic states before fertilization. The Plant Journal, 76: 729-741.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Martin, M., Fiol, D., Sundaresan, V., Zabaleta, Pagnussat, G., 2013. oiwa, a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase, Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis. The Plant Cell, 25: 1573-1591.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Spence, C., Alff, E., Johnson, C., Ramos, C., Donofrio, N., Sundaresan,V., Bais, H., 2014. Natural rice rhizospheric microbes suppress rice blast infections. BMC Plant Biology, 14:130.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Conrad, L.J., Khanday, I., Johnson, C., Guiderdoni, E., An, G., Vijayraghavan, U., Sundaresan, V., 2014. The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice. The Plant Journal 80:883-94.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Ravi, M., Marimuthu, M.P., Tan, E.H., Maheshwari, S., Henry, I.M., Marin-Rodriguez, B., Urtecho, G., Tan, J., Thornhill, K., Zhu, F., Panoli, A., Sundaresan, V., Britt, A.B., Comai, L., Chan, S.W., 2014. A haploid genetics toolbox for Arabidopsis thaliana. Nature Commun. 5:5334.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Song, X., Yuan, L., Sundaresan, V., 2014. Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis. Plant Reprod. 27:197-203.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yuan, L., Sundaresan, V., 2015. Spore formation in plants: SPOROCYTELESS and more. Cell Research 25:7-8.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Edwards, J., Johnson, C., Santos-Medellin, C., Lurie, E., Podishetty, N.K., Bhatnagar, S., Eisen, J.A., Sundaresan, V., 2015. Structure, variation and assembly of the root-associated microbiomes of rice. Proceedings of the National Academy of Sciences, doi: 10.1073/pnas.1414592112.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Panoli, A., Martin, M.V., Alandete-Saez, M., Simon, M., Neff, C., Swarup, R., Bellido, A., Yuan, L., Pagnussat, G.C., Sundaresan, V., 2015. Auxin import and local auxin biosynthesis are required for mitotic divisions, cell expansion and cell specification during female gametophyte development in Arabidopsis thaliana. PLoS One, DOI: 10.1371/journal.pone.0126164
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yuan, L., Liu, Z., Song, X., Johnson, C., Yu, X., Sundaresan, V., 2016. The CKI1 histidine kinase specifies the female gametic precursor of the endosperm. Developmental Cell 37:34-46.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Plant scientists, plant breeders and agronomists working in universities and industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training for 4 graduate students, 3 postdoctoral researchers, and 3 undergraduate students at UC Davis. We also trained 3 African-American undergraduate interns and one faculty from the University of Arkansas- Pine Bluff, an HBCU, during the summer of 2015. How have the results been disseminated to communities of interest?Primarily through journal publications. The PI also has given public lectures at conferences, including the UIC (University and Industry Consortium) which involved several major international Agriculture-related companies. What do you plan to do during the next reporting period to accomplish the goals?1. Discoveries on cytokinin signaling in the embryo sac will be pursued to understand the pathways underlying the effects on gamete development. 2. The role of maternal and paternal contributions to embryonic development will be further characterized.. 3. The established germ-free rice system will be utilized to elucidate host-microbiome interactions.

Impacts
What was accomplished under these goals? Change in Knowledge 1. In flowering plant reproduction, seed formation is a critical step. Seeds consist of an embryo and nutritive endosperm, arising from double fertilization of the two female gametes, the egg cell and central cell of the embryo sac, by the two sperm cells of the pollen. We have shown that the formation of female gametes is regulated by hormonoal signaling. Manipulation of auxin signaling or cytokinin signaling in the embryo sac can be used to increase the number of egg cells or central cells. We have discovered a key gene involved in cytokinin signaling that can be used to change egg cells into central cells, resulting in seeds without embryos but containing multiple endosperms. 2. To determine the factors that govern early embryo growth and development, we have investigated zygote transcriptomes in rice to. We find that while the earliest stages depend upon maternal transcripts deposited in the egg cell, the zygote expresses its own genome by the time of the first embryonic division. Using zygotes in hybrid seeds, we have further shown that expression from the zygotic genome is asymmetric, with greater contribution from the maternal alleles as compared to paternal alleles. However, a few genes show greater expression from paternal alleles, including critical transcription factors that promote embryonic development. This is the first comprehensive study undertaken of zygotic genome activation in any plant, and has potentially important applications to seed production. 3. Communities of microbes, called microbiomes, provide beneficial effects to crop plants. Over the past few years, we have analyzed the structural and compositional distribution of the microbial populations that are associated with roots of field grown rice by high throughput sequencing to identify the species of bacteria. We previously showed that different sub-populations of microbes inhabit the soil around the roots, the root surface and the internal tissues of the root. We have now shown that microbial communities enter plant roots through a three step process: (1) Attraction to the root vicinity by root exudates. (2) Attachment to the root surface by a sub-set of microbiota. (3) Selective entry into the internal root tissues by a smaller sub-set of microbiota. Our studies are now focused on identifying specific effects of the microbiomes by establishing a germ-free system for rice plants. We are also collaborating with IRRI (International Rice Research Institute, Philippines) to identify potentially beneficial microbial interactions with rice plants that can be incorporated into future breeding programs.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Edwards, J., Johnson, C., Santos-Medellin, C., Lurie, E., Podishetty, N.K., Bhatnagar, S., Eisen, J.A., Sundaresan, V., 2015. Structure, variation and assembly of the root-associated microbiomes of rice. Proceedings of the National Academy of Sciences, doi: 10.1073/pnas.1414592112
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Panoli, A., Martin, M.V., Alandete-Saez, M., Simon, M., Neff, C., Swarup, R., Bellido, A., Yuan, L., Pagnussat, G.C., Sundaresan, V., 2015. Auxin import and local auxin biosynthesis are required for mitotic divisions, cell expansion and cell specification during female gametophyte development in Arabidopsis thaliana. PLoS One, DOI: 10.1371/journal.pone.0126164
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yuan, L., Sundaresan, V., 2015. Spore formation in plants: SPOROCYTELESS and more. Cell Research 25:7-8.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Plant scientists, plant breeders and agronomists working in universities and industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training for 4 graduate students, 3 postdoctoral researchers, and 2 undergraduate students at UC Davis. How have the results been disseminated to communities of interest? Primarily through journal publications. The PI also provided information on rice microbiomes to the New York Times, for an article published September 15th, 2014. The project was also used for outreach to Langston University, an HBCU in Oklahoma, and a two-week participation in the UC Davis project by by 3 students and 1 faculty from Langston U. during 2014. Additionally, a collaboration with the USDA-ARS Rice Experiment Station in Stuttgart, AR has been initiated in 2014. What do you plan to do during the next reporting period to accomplish the goals? 1. Additional manipulation of hormonal signaling pathways in the female gametophyte will be carried out. 2. Transcriptome analysis of hybrid rice zygotes will be performed. 3. More detailed characterization of the microbiome will be undertaken, especially with respect to responses of plants to acquired microbiomes, and characterization of methanogenic archaea in the rhizosphere.

Impacts
What was accomplished under these goals? Change in Knowledge 1. Seed production is dependent on the formation of the embryo and endosperm, which are the result of double fertilization of the female gametes, the egg cell and central cell of the embryo sac, by the two sperm cells of the pollen. Using the model plant Arabidopsis, we have manipulated hormonal signaling pathways within the embryo sac to demonstrate the production of additional or fewer gametic cells, as well as switches in cell fates, e.g. from central cell to egg cell, as well as egg cell to central cell. We have also investigated the other cell types of the embryo sac in Arabidopsis, and found that the antipodal cells which were assumed to have degenerated at maturity actually persist beyond fertilization. Symplastic connections with the ovule suggest that they may act as a channel for maternal supply of nutrients to the young developing seed. 2. A fundamental question with practical implications is whether the earliest stages of seed development depend upon maternal RNA or whether the zygote begins to produce its own transcripts. A comprehensive transcriptome analysis of isolated time-staged zygotes from rice show that the zygotic transcriptome is activated hours after fertilization, suggesting that the latter model might be correct. Similar experiments on hybrid seeds are now in progress, to understand the contributions of the parental genomes to the transition from a fertilized egg to an embryo. 3. Further progress has been made on the characterization of the microbiome of rice. Cultivated plants live in close association with communities of microbes, called microbiomes, which can provide beneficial effects to plants. We have analyzed the structural and compositional distribution of the microbial populations that are associated with roots of field grown rice by high throughput sequencing to identify the species of bacteria. We found that different sub-populations of microbes inhabit the different compartments of the rhizosphere, the endophytes, the rhizoplane, the distal rhizosphere and bulk soil. We have now shown that germ-free plants can acquire a microbiome from soil rapidly, and that a stable microbiome can be established within two weeks.There appears to be progressive enrichment for particular bacterial species correlated with proximity to the root surfaces, supporting a concept of active recruitment of beneficial bacteria by plants, probably through molecules present in root exudates. Other factors that significantly affect the root microbiome are soil type, genotype, and cultivation method (organic vs. conventional). We are further characterizing the microbial fractions to identify potentially beneficial interactions with the plants that can be incorporated into future breeding programs.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Spence, C., Alff, E., Johnson, C., Ramos, C., Donofrio, N., Sundaresan,V., Bais, H., 2014. Natural rice rhizospheric microbes suppress rice blast infections. BMC Plant Biology, 14:130.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Ravi, M., Marimuthu, M.P., Tan, E.H., Maheshwari, S., Henry, I.M., Marin-Rodriguez, B., Urtecho, G., Tan, J., Thornhill, K., Zhu, F., Panoli, A., Sundaresan, V., Britt, A.B., Comai, L., Chan, S.W., 2014. A haploid genetics toolbox for Arabidopsis thaliana. Nature Communications 5:5334.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Conrad, L.J., Khanday, I., Johnson, C., Guiderdoni, E., An, G., Vijayraghavan, U., Sundaresan, V., 2014. The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice. The Plant Journal 80:883-94.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Song, X., Yuan, L., Sundaresan, V., 2014. Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis. Plant Reprod. 27:197-203.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Researchers in plant sciences and agronomy working in universities and industries Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training for 4 graduate students, 3 postdoctoral fellows, and two undergraduate students at UC Davis. How have the results been disseminated to communities of interest? Primarily through journal publications. The project was also used for outreach, in a visit by the PI to Langston University, an HBCU in Oklahoma, and a two-week participation in the UC Davis project by by 3 students and 1 faculty from Langston U. during 2013. What do you plan to do during the next reporting period to accomplish the goals? 1. Additional manipulation of hormonal signaling pathways in the female gametophyte will be carried out. 2. Transcriptome analysis of isolated rice zygotes will be performed. 3. More detailed characterization of the microbiome will be undertaken, especially with respect to methanogenic archaea in the rhizosphere.

Impacts
What was accomplished under these goals? Change in Knowledge 1. Seed production is dependent on the formation of the embryo and endosperm, which are the result of double fertilization of the female gametes, the egg cell and central cell of the embryo sac, by the two sperm cells of the pollen. By modifying hormonal signaling within the embryo sac we have been able to demonstrate the production of additional or fewer gametic cells, as well as switches in cell fates, e.g. from central cell to egg cell, as well as egg cell to central cell. We have also investigated the process of fertilization, and found that the pollen tube enters the female gametophyte through a different site than previously believed i.e. the side of the synergid cell, rather than the tip, which in turn leads to revision of existing models of fertilization. 2. Using rice, a comprehensive transcriptome analysis of isolated living egg cells, and of the sperm and vegetative cell of the pollen grain has been performed, to identify over 36000 expressed genes by deep sequencing. This represents the first comprehensive transcriptome analysis of the male and female gametes of a flowering plant. We showed that the sperm cell and the egg cell have very divergent transcriptomes, as well distinct epigenetic states based on expression of genes for chromatin and RNA-mediated gene silencing. Studies with isolated zygotes are now in progress, to understand the nature of the transition from a fertilized egg to an embryo. 3. Progress has been made on the characterization of the microbiome of rice. Cultivated plants live in close association with communities of microbes, called microbiomes, which can provide beneficial effects to plants. We have analyzed the structural and compositional distribution of the microbial populations that are associated with roots of field grown rice by high throughput sequencing to identify the species of bacteria. We find that different sub-populations of microbes inhabit the different compartments of the rhizosphere, the endophytes, the rhizoplane, the distal rhizosphere and bulk soil. There appears to be progressive enrichment for particular bacterial species correlated with proximity to the root surfaces, supporting a concept of active recruitment of beneficial bacteria by plants, probably through molecules present in root exudates. We have investigated the effects of soil type, genotype, and cultivation method (organic vs. conventional), and determined the extent to which each of these factors affects the microbiome. We are further characterizing the microbial fractions to identify potentially beneficial interactions with the plants that can be incorporated into future breeding programs.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: 57. Tsai H., Missirian V., Ngo K., Tran R.K., Chan S., Sundaresan V., Comai L., 2013. Production of a high efficiency TILLING population through polyploidization. Plant Physiology 161: 1604-1614.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Leshem Y., Johnson C., Sundaresan V., 2013. Pollen tube entry into the synergid cell of Arabidopsis is observed at a site distinct from the filiform apparatus. Plant Reproduction, 26: 93-99
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Anderson, S., Johnson, C., Jones, D., Conrad, L., Gou, X., Russell, S., Sundaresan, V., 2013. Transcriptomes of isolated rice gametes characterized by deep sequencing: Evidence for distinct sex-dependent chromatin and epigenetic states before fertilization. The Plant Journal, 76: 729-741.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Martin, M., Fiol, D., Sundaresan, V., Zabaleta, Pagnussat, G., 2013. oiwa, a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase, Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis. The Plant Cell, 25: 1573-1591

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Seed production is dependent on the formation of the embryo and endosperm, which are the result of double fertilization of the female gametes, the egg cell and central cell of the embryo sac, by the two sperm cells of the pollen. Our research on the development of these two gametes has shown that they are specified by the hormones auxin and cytokinin. The two hormones appear to be synthesized at opposite ends of the embryo sac, and form opposing concentration gradients. By modifying hormonal signaling within the embryo sac we have been able to demonstrate the production of additional or fewer gametic cells, as well as switches in cell fates, e.g. from central cell to egg cell. We have also investigated the signals required for double fertilization, and identified genes required for fertilization of the central cell. In a second study, we have begun to investigate the changes that occur when the egg cell after fertilization by the sperm cell, changes into a totipotent zygote. Transcriptome analysis of the egg cell and the sperm cell, compared with that of the early zygote, has revealed important genes in this process. These studies are in progress, using later stage zygotes to help us understand the nature of this transition from a fertilized egg to an embryo. A third study is the characterization of the microbiome of rice. Cultivated plants live in close association with communities of microbes, called microbiomes, which can provide beneficial effects to plants. We have begun to analyze the structural and compositional distribution of the microbial populations that are associated with roots of field grown rice by high throughput sequencing to identify the species of bacteria. We find that different sub-populations of microbes inhabit the different compartments of the rhizosphere, the endophytes, the rhizoplane, the distal rhizosphere and bulk soil. There appears to be progressive enrichment for particular bacterial species correlated with proximity to the root surfaces, supporting a concept of active recruitment of beneficial bacteria by plants, probably through molecules present in root exudates. Moreover, we have now obtained evidence for cultivar-specific differences, suggesting that the genotype of the plants plays an important role in the microbiome. PARTICIPANTS: The project helped provide training for 3 graduate students, 4 postdoctoral researchers, and 6 undergraduate students during 2012. There were partnerships with U. of Delaware (land-grant), Langston University (land-grant and HBCU), Delaware Technical Community College, and U. of Oklahoma. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Identification of genes and processes that control the development of gametes and the zygote will enable improvements in seed production. In particular, manipulation processes that control of fertilization can be used for more efficient production of hybrid seeds or for asexual propagation of hybrids. Elucidating the structure and composition of the rice microbiome can be translated into new breeding approaches that take advantage of the microbial interactions to improve crop yields. Our findings show enrichment for particular species of bacteria in different compartments of the rhizosphere which are likely to confer beneficial effects, as well as dependence on the genotype of the cultivar, which can be exploited for this purpose.

Publications

• Leshem Y., Johnson C.A., Wuest S.E., Song X., Ngo Q.A., Grossniklaus U., Sundaresan V. 2012. Molecular Characterization of the glauce Mutant: a Central Cell-Specific Function Is Required for Double Fertilization in Arabidopsis. The Plant Cell, 24: 3264-3277.
• Ngo QA, Baroux C, Guthorl D, Mozerov P, Collinge MA, Sundaresan V, Grossniklaus U. 2012. The Armadillo Repeat Gene ZAK IXIK Promotes Arabidopsis Early Embryo and Endosperm Development through a Distinctive Gametophytic Maternal Effect. 2012, The Plant Cell, e-published doi:10.1105/tpc.112.102384.
• Yang S-Y, Gronlund M, Jakobsen I, Grotemeyer MS, Rentsch D, Miyao A, Hirochika H, Kumar CS, Sundaresan V, Salamin N, Catausan S, Mattes N, Heuer S, Paszkowski U. Nonredundant Regulation of Rice Arbuscular Mycorrhizal Symbiosis by Two Members of the PHOSPHATE TRANSPORTER1 Gene Family. 2012, The Plant Cell, e-published doi:10.1105/tpc.112.104901.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The embryo sac is the key reproductive unit of flowering plants. in the embryo sac. The embryo sac makes the two female gametes, egg cell and central cell, which give rise to the embryo and endosperm of the seed. We have previously shown the plant hormone auxin is an important determinant of gamete specification We have now discovered that a second phytohormone cytokinin is also present in the embryo sac, and that its action is opposite to that of auxin. The two hormones appear to be synthesized at opposite ends of the embryo sac, and form opposing gradients that result in cell specification within the embryo sac. In a second set of studies, we have begun to investigate the changes that occur when the egg cell after fertilization by the sperm cell, changes into a totipotent zygote. By examining transcripts of certain key genes in rice zygotes isolated at different times after fertilization, we have found that the transition to a zygote is accompanied by specific loss of a subset of maternal transcripts, and the expression of a set of zygotic transcripts. Cultivated plants live in close association with communities of microbes, called the microbiome, which can promote plant growth. We have begun to characterize the microbial populations that are associated with roots of field grown rice by metagenomic sequencing to identify the species of bacteria. We find that the distribution of bacterial species present as biofilms on the root surface are very different from those in bulk soil. There appears to be progressive enrichment for particular bacterial species correlated with proximity to the root surfaces. These findings support a concept of active recruitment of beneficial bacteria by plants, probably through molecules present in root exudates. PARTICIPANTS: The project was used to train 1 graduate student, 3 postdoctoral researchers, and 3 undergraduate students during 2011. One of the undergraduate researchers has now progressed to graduate school, while a second is working for a seed biotechnology company. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Knowledge of the mechanisms that control embryo sac development will enable identification of genes for better seed production. We have been able to generate additional egg cells in the same embryo sac through manipulation of hormone concentrations, which might be used in tissue culture to generate haploid plants for breeding. A better understanding of the microbiome of cultivated crop plants is important to decipher the effect of the microbiome on plant performance. Our findings show enrichment for particular species of bacteria in the rhizosphere which are likely to confer beneficial effects. This knowledge can be translated into new breeding approaches that take advantage of the microbial interactions to improve crop yields.

Publications

• Gutjahr C, Radovanovic D, Geoffroy J, Zhang Q, Siegler H, Chiapello M, Casieri L, An K, An G, Guiderdoni E, Kumar CS, Sundaresan V, Harrison MJ, Paszkowski U. (2012) The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice. The Plant Journal 69:906-20.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We have used the model plant Arabidopsis, to perform detailed characterization of selected mutants in which the male gametophyte (pollen) or the female gametophyte mutant (embryo sac) is defective. The mutant seeds have been sent to the Arabidopsis Research Stock Center at Ohio State University for public distribution. The gene sequences and the mutant phenotypes are available through a publicly accessible website at http://www-plb.ucdavis.edu/Labs/sundar/GameDev.htm In a second collaborative project with Dr. at UC Davis, and Dr. Harsh Bais at the University of Delaware, we have been developing methods for elucidating the effects of the soil microbiome of field-grown rice on the transcriptome. These studies will lead to understanding of how microbes in the soil affect gene expression and agricultural yields in rice. The preliminary transcriptomics data are being posted on our website at http://www-plb.ucdavis.edu/labs/sundar/soil.htm PARTICIPANTS: The project was used to train 1 graduate student, 4 postdoctoral researchers, and 4 undergraduate students during 2010. One of the undergraduate researchers has moved on to graduate school. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The following are long-term benefits for agriculture which derive from the project. Genes that control gametophyte development can potentially be used to generate male sterility, which facilitates production of hybrid seeds. Additionally, they might be used to engineer apomixis (reproduction without fertilization) in crop plants, enabling the fixation of hybrid genotypes. This would enable the maintenance of hybrid vigor for many crop plants, without the need for expensive hybrid seed production. Seed formation is critically dependent upon the formation of the egg cell which will give rise to the embryo, as well as the growth of the endosperm after fertilization. We have identified molecular mechanisms that control specification of the egg cell, and can now manipulate the embryo sac to generate additional egg cells. We have also discovered genetic loci that are required by the embryo sac for double fertilization and endosperm formation. Identification of gametophyte genes controlling embryo and endosperm is necessary for improving seed size and yield, and are therefore seed production, which is of agricultural importance. In our recent work on field-grown rice, we have been able to identify the major bacterial constituents of the microbiome associated with the roots of California japonica rice. We are now examining their effects on the rice transcriptome using a controlled hydroponic system. Rice is the only plant other than Arabidopsis for which the genome has been sequenced, and is the best model for the agronomically important cereal crops as well as an important crop in its own right in several states including California and Arkansas. The project to determine the effects of the soil microbiome on gene expression in rice provides an understanding of how the plant metagenome (the composite genomes of the associated microbiome) interacts with the plant genome. Such knowledge can be exploited to select for genes/traits that favor the growth of beneficial bacteria and suppress the growth of harmful bacteria, providing an entirely new direction in plant breeding beyond the current approaches.

Publications

• Sundaresan V., Alandete-Saez, M. 2010. Pattern formation in miniature: The female gametophyte of flowering plants. Development 137:179-189.
• Ma H., Sundaresan V. 2010. Development of flowering plant gametophytes. Curr. Top. Dev. Biol. 91:379-412.
• Ravi M., Kwong P.N., Menorca R.M., Valencia J.T., Ramahi J.S., Stewart J.L., Tran R.K., Sundaresan V., Comai L., Chan S.W. 2010. The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana. Genetics 186:461-71.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: In collaboration with Dr. Sheila McCormick of USDA (PGEC Albany), we have continued to further characterize genes required for the gametophytic phase of plant reproduction. We have used the model plant Arabidopsis, to perform detailed characterization of selected mutants in which the male gametophyte (pollen) or the female gametophyte mutant (embryo sac) is defective. The mutant seeds have been sent to the Arabidopsis Research Stock Center at Ohio State University for public distribution. The gene sequences and the mutant phenotypes are available through a publicly accessible website at http://www-plb.ucdavis.edu/Labs/sundar/GameDev.htm In a second collaborative project with Dr. Vicki Vance and Dr. Lew Bowman of the University of South Carolina we have completed an analysis of the small RNA transcriptomes of rice and maize genomes during plant development. Small RNAs are regulatory RNAs that control gene expression after transcription, as well as the silencing of chromatin. We have now greater than 2,000,000 unique small RNAs in these two crop plants, from different tissues , which are mapped to these genomes and displayed in a browser format on the Cereal Small RNA Database (CSRDB), publicly accessible at http://csrdb.ucdavis.edu This constitutes a major resource for small RNAs in these crop plants. Bioinformatically predicted target genes of the small RNAs are also available from this website. Additionally, the small RNAs expressed at different developmental stages and tissues, and their variation in abundance, are displayed for unrestricted access to the plant research community. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The following are long-term benefits for agriculture which derive from the project. Genes that control gametophyte development can potentially be used to generate male sterility, which facilitates production of hybrid seeds. Additionally, they might be used to engineer apomixis (reproduction without fertilization) in crop plants, enabling the fixation of hybrid genotypes. This would enable the maintenance of hybrid vigor for many crop plants, without the need for expensive hybrid seed production. Seed formation is critically dependent upon the formation of the egg cell which will give rise to the embryo, as well as the growth of the endosperm after fertilization. We have identified molecular mechanisms that control specification of the egg cell, and can now manipulate the embryo sac to generate additional egg cells. We have also discovered genetic loci that are required by the embryo sac for double fertilization and endosperm formation. Identification of gametophyte genes controlling embryo and endosperm is necessary for improving seed size and yield, and are therefore seed production, which is of agricultural importance. In our work on rice and maize, we have been able to identify the large set of regulatory small RNAs which can be used to manipulate gene expression. Rice is the only plant other than Arabidopsis for which the genome has been sequenced, and is the best model for the agronomically important cereal crops as well as an important crop in its own right in several states including California and Arkansas. Maize is the most important field crop in the USA. The project to determine small RNAs in the rice and maize genomes is providing a more detailed understanding of these genomes and how they respond to adverse conditions. Such knowledge can be applied for breeding commercial varieties of rice and maize with improved traits.

Publications

• Krishnan A, Guiderdoni E, An G, Hsing YI, Han CD, Lee MC, Yu SM, Upadhyaya N, Ramachandran S, Zhang Q, Sundaresan V, Hirochika H, Leung H, Pereira A. 2009. Mutant resources in rice for functional genomics of the grasses. Plant Physiology 149:165-70.
• Boavida LC, Shuai B, Yu HJ, Pagnussat GC, Sundaresan V, McCormick S. 2009. A Collection of Ds Insertional Mutants Associated With Defects in Male Gametophyte Development and Function in Arabidopsis thaliana. Genetics 181:1369-85.
• Pagnussat G., Alandete-Saez, M., Bowman, J., Sundaresan, V. 2009. Auxin dependent patterning and gamete specification in the Arabidopsis female gametophyte. Science 324: 1684-1689.
• Johnson C., Kasprzewska A., Tennessen K., Fernandes J., Nan G., Walbot V., Sundaresan V.*, Vance V.*, Bowman L.* 2009. Clusters and Superclusters of Phased Small RNAs in the Developing Inflorescence of Rice. Genome Research 19:1429-1440 (* Joint corresponding author).
• Li N., Yuan L., Liu N., Shi D., Li X., Tang Z., Liu J., Sundaresan V., Yang W.-C. 2009. SLOW WALKER2, a NOC1/Mak21 Homologue, Is Essential for Coordinated Cell Cycle Progression during Female Gametophyte Development in Arabidopsis. Plant Physiology 151:1486-1497.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: In collaboration with Dr. Sheila McCormick of USDA (PGEC Albany), to characterize genes required for the gametophytic phase of plant reproduction. We have used the model plant Arabidopsis, to complete an initial characterization of 80 mutants in which the male gametophyte (embryo sac) is defective, which together with the previously characterized 130 female mutants, represents the largest such collection in the world. The mutant seeds have been sent to the Arabidopsis Research Stock Center at Ohio State University for public distribution. The gene sequences and the mutant phenotypes are available through a publicly accessible website at http://www-plb.ucdavis.edu/Labs/sundar/GameDev.htm In a second collaborative project with Dr. Vicki Vance and Dr. Lew Bowman of the University of South Carolina we have been analyzing the small RNA transcriptomes of rice and maize genomes. Small RNAs are regulatory RNAs that control gene expression after transcription, as well as the silencing of chromatin. We have now molecularly identified greater than1,000,000 unique small RNAs in these two crop plants, which are displayed in the Cereal Small RNA Database (CSRDB), publicly accessible at http://csrdb.ucdavis.edu This constitutes a major resource for small RNAs in these crop plants. Additionally, we have been able to identify smRNAs in rice that are specifically expressed during the transition to flowering. The genomic organization of these small RNAs indicates that a major reprogramming of the small RNA transcriptome might be occurring during the entry of the plants into reproductive growth. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
There are long term benefits for agriculture which derive from the project. Genes that control gametophyte development can potentially be used to generate male sterility, which facilitates production of hybrid seeds. Additionally, they might be used to engineer apomixis (reproduction without fertilization) in crop plants, enabling the fixation of hybrid genotypes. This would enable the maintenance of hybrid vigor for many crop plants, without the need for expensive hybrid seed production. Seed formation is critically dependent upon the formation of the egg cell which will give rise to the embryo, as well as the growth of the endosperm after fertilization. We have identified genes that control specification of the egg cell. We have also discovered genetic loci that are required by the embryo sac for fertilization. Identification of gametophyte genes controlling embryo and endosperm is necessary for improving seed size and yield, and are therefore seed production, which is of agricultural importance. In our work on rice and maize, we have been able to identify the large set of regulatory small RNAs which can be used to manipulate gene expression. Rice is the only plant other than Arabidopsis for which the genome has been sequenced, and is the best model for the agronomically important cereal crops as well as an important crop in its own right in several states including California and Arkansas. Maize is the most important field crop in the USA. The project to determine small RNAs in the rice and maize genomes will lead to a more detailed understanding of these genomes and how they respond to adverse conditions. Such knowledge can be applied for breeding commercial varieties of rice and maize with improved stress tolerance and higher yields.

Publications

• Chen, Z., Tan, J., Inguoff, M., Sundaresan, V. , Berger, F. 2008. The Chromatin Assembly Factor 1 regulates the cell cycle but not the cell fate during male gametogenesis in Arabidopsis thaliana. Development, 135:65-73.
• Capron, A., Gourgues, M., Neiva, L.S., Faure, J.E., Berger, F., Pagnussat, G., Krishnan, A., Alvarez-Meija, C., Vielle-Calzada, J.-P., Lee, Y.-R., Liu, B., Sundaresan, V. 2008. Maternal control of male-gamete delivery in Arabidopsis involves a putative GPI-anchored protein encoded by the LORELEI gene. The Plant Cell, 20: 3038-3049.

Progress 01/01/07 to 12/31/07

Outputs
In a collaborative project with Dr. Sheila McCormick of USDA (PGEC Albany), to characterize genes required for the gametophytic phase of plant reproduction. We have used the model plant Arabidopsis, to complete an initial characterization of 130 mutants in which the female gametophyte (embryo sac) is defective, representing the largest such collection in the world. The mutant seeds have been sent to the Arabidopsis Research Stock Center at Ohio State University for public distribution. We also identified over 220 genes expressed specifically in the embryo sac. The gene sequences and the mutant phenotypes are available through a publicly accessible website at http://www-plb.ucdavis.edu/Labs/sundar/GameDev.htm We are also continuing a collaborative project with Dr. Vicki Vance and Dr. Lew Bowman of the University of South Carolina to decipher the small RNA component of the rice and maize genomes. Small RNAs are newly discovered regulatory RNAs that are very important to the control of gene expression after transcription, as well as the silencing of chromatin. Currently, we have molecularly and bioinformatically identified greater than100,000 unique small RNAs in these two crop plants, to establish the Cereal Small RNA Database (CSRDB), which is publicly accessible at http://csrdb.ucdavis.edu

Impacts
The above projects are expected to have long term benefits for agriculture. Genes that control female gametophyte development can potentially be used to engineer apomixis (reproduction without fertilization) in crop plants, enabling the fixation of hybrid genotypes. This would enable the maintenance of hybrid vigor for many crop plants, without the need for expensive hybrid seed production. Such an advance would benefit the farmer by reducing the costs required to get higher yields. Seed formation is critically dependent upon the formation of the egg cell which will give rise to the embryo, as well as the growth of the endosperm after fertilization. We have identified genes that control specification of the egg cell, and mutations that lead to the formation of multiple egg cells. We have also discovered genetic loci that are required by the embryo sac for the development of the endosperm after fertilization. Identification of gametophyte genes controlling embryo and endosperm is necessary for improving seed size and yield, and are therefore seed production, which is of agricultural importance. In our work on rice, we have also discovered that the frequency at which small RNAs arise from the rice genome rises markedly at flowering, indicating a close relationship between flowering and production of small RNAs which is tied to chromatin silencing. Rice is the only plant other than Arabidopsis for which the genome has been sequenced, and is the best model for the agronomically important cereal crops as well as an important crop in its own right in several states including California and Arkansas. Maize is the most important field crop in the USA. The project to determine small RNAs in the rice and maize genomes will lead to a more detailed understanding of these genomes and how they respond to adverse conditions. Such knowledge can be applied for breeding commercial varieties of rice and maize with improved stress tolerance and higher yields.

Publications

• Johnson, C., Sundaresan, V. 2007. Regulatory small RNAs in plants. In Plant Systems Biology Birkhauser, Springer-Verlag, Switzerland. pp. 99-113. Griffith, M.E., Mayer, U., Capron, A., Ngo, Q., Surendrarao A., McClinton, R., Jurgens, G., Sundaresan, V. 2007. The TORMOZ gene encodes a nucleolar protein required for regulated division planes and embryo development in Arabidopsis. The Plant Cell 19: 2246-2263. Dewitte, W., Scofield, S., Alcasabas, A.A., Maughan, S.C., Menges, M., Braun, N., Collins, C., Nieuwland, J., Prinsen, E., Sundaresan, V., Murray, J.A.H. 2007. Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses Proc. Natl. Acad. Sci. USA, 104:14537-14542. Ngo, Q.A., Moore, J.M., Baskar R., Grossniklaus, U., and Sundaresan, V. 2007. The Arabidopsis glauce mutant reveals a novel regulatory function of the female gametophyte in autonomous endosperm development and expression of paternal alleles during early embryogenesis. Developmen

Progress 01/01/06 to 12/31/06

Outputs
1. Control of plant reproduction: Gametophyte genes This project is a collaborative project with Dr. Sheila McCormick of USDA (PGEC Albany), to characterize genes required for the gametophytic phase of plant reproduction. We have previously (Pagnussat et al,. 2005) used the model plant Arabidopsis, to complete an initial characterization of 130 mutants in which the female gametophyte (embryo sac) is defective, representing the largest such collection in the world. Now, we have embarked on a more detailed characterization of these mutants. Analysis of one of these novel mutants called eostre, which makes two egg cells instead of one, has led us to discover that specification of the egg cell requires the correct positioning of the gametophytic nuclei after fertilization, a process which can be disrupted by mis-expression of a homeodomain transcription factor called BLH1. Yet another mutant called ned1 (no endosperm 1) has led us to uncover a novel pathway required for activation of endosperm growth and development. 2. Insertional mutagenesis of rice Rice is the only plant other than Arabidopsis for which the genome has been sequenced, and is the best model for the agronomically important cereal crops as well as an important crop in its own right in several states including California and Arkansas. Our approach relies on the random insertion of transposons into the rice genome, resulting in disruptions of individual genes on a large scale. These gene disruptions (or "knockouts") can be analyzed to determine the functions of the genes. We have used modified transposable elements from maize (Ac-Ds and Spm/En) in combination with fluorescent markers to generate transposon insertion lines. In collaboration with Dr. Rod Wing (U. Arizona) we identify the genomic locations of these insertions by DNA sequencing. Previously we have demonstrated for the first time that the En/Spm system is an effective insertional mutagen in rice (Kumar et al, 2005). We have now shown that it is superior to Ac-Ds in terms of efficiency of insertions as well as specificity. We are currently the only laboratory in the world that has been able to use this efficient transposon system, En/Spn, for the mutagenesis of the rice genome. We have now generated over 15,000 independent insertion lines, and determined the flanking sequences for over 10,000 of these insertions. In the coming years, we will be generating additional numbers of insertions and sequences, to generate a valuable genetic resource for the US crop genetics community. 3. small RNAs in rice and maize We have also embarked on a collaborative project with Dr. Vicki Vance and Dr. Lew Bowman of the University of South Carolina to decipher the small RNA component of the rice and maize genomes. Small RNAs are newly discovered regulatory RNAs that are very important to the control of gene expression after transcription, as well as the silencing of chromatin. Currently, we have molecularly and bioinformatically identified about 100,000 small RNA loci in these two crop plants, and displayed them on a publicly accessible genome browser (Johnson et al. 2006).

Impacts
The above projects are expected to have long term benefits for agriculture. Genes that control female gametophyte development can potentially be used to engineer apomixis (reproduction without fertilization) in crop plants, enabling the fixation of hybrid genotypes. This would enable the maintenance of hybrid vigor for many crop plants, without the need for expensive hybrid seed production. Such an advance would benefit the farmer by reducing the costs required to get higher yields. Seed formation is critically dependent upon the formation of the egg cell which will give rise to the embryo, as well as the growth of the endosperm after fertilization. Identification of gametophyte genes controlling embryo and endosperm is necessary for improving seed size and yield, and are therefore seed production, which is of agricultural importance . The project to determine functions of sequenced genes and small RNAs in the rice genome will identify genes for important traits. Knowledge of these genes will be extremely useful for rice breeders as well as other commercially important cereals including maize and wheat.

Publications

• Jiang, L.X., Yang, S.L, Xie, L.F., Puah, C.S., Zhang. X., Yang, W.C., Sundaresan, V., Ye, D., 2005. VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract. The Plant Cell 17: 584-596.
• Shi, D.Q., Liu, J., Xiang, Y.H., Sreenivasan R., Ye, D., Sundaresan, V., Yang, W.C., 2005. SLOW WALKER1, Essential for Gametogenesis in Arabidopsis, Encodes a WD40 Protein Involved in 18S Ribosomal RNA Biogenesis. The Plant Cell 17:2340-54.
• Johnson C, Bowman L, Adai AT, Vance V, Sundaresan V. 2006. CSRDB: a small RNA integrated database and browser resource for cereals. Nucleic Acids Res. 2006 Dec 14; [Epub ahead of print]
• Adai A., Johnson, C., Mlotshwa, S., Archer-Evans, S., Manocha, V., Vance, V., Sundaresan, V., 2005 Computational prediction of miRNAs in Arabidopsis thaliana. Genome Research 15:78-91.

Progress 01/01/05 to 12/31/05

Outputs
1. Control of plant reproduction: Gametophyte genes Last year, we used the model plant Arabidopsis, to complete an initial characterization of 130 mutants in which the female gametophyte (embryo sac) is defective, representing the largest such collection in the world. This past year, we have embarked on a more detailed characterization of these mutants. We have identified a large group of mutants that have a "maternal effect", i.e. genes encoding products required for embryo development that are exclusively contributed by the embryo sac. We have also focused on mutants that are required for the correct specification of the egg cell, for the proper delivery of the sperm cells after fertilization, and for the growth of the endosperm. These are unique mutants, not previously described in the literature, and we are in the process of characterizing the relevant genes in order to identify their molecular functions. In addition, we used expression profiling with DNA chips to identify more than two hundred Arabidopsis genes that are specifically expressed in the embryo sac. This past year, genetic studies to describe the exact biological functions of these genes were undertaken; these studies should be completed this coming year. 2. Insertional mutagenesis of rice Rice is the only plant other than Arabidopsis for which the genome has been sequenced, and is the best model for the agronomically important cereal crops as well as an important crop in its own right in several states including California and Arkansas. Our approach relies on the random insertion of transposons into the rice genome, resulting in disruptions of individual genes on a large scale. These gene disruptions (or "knockouts") can be analyzed to determine the functions of the genes. We have used modified transposable elements from maize (Ac-Ds and Spm/En) in combination with fluorescent markers to isolate a collection of transposon insertion lines. We then sequence the DNA flanking the insertions so that researchers can easily identify lines carrying knockouts in genes of interest. During this past year, we have demonstrated for the first time that the En/Spm system is an effective insertional mutagen in rice. We also find that it is superior to Ac-Ds, and we have now switched over to mainly using the En/Spm transposons. We have now generated over 9,000 independent insertion lines, and determined the flanking sequences for over 5000 of these insertions. In the coming years, we will be generating additional numbers of insertions and sequences, to generate a valuable genetic resource for the US crop genetics community.

Impacts
The above projects are expected to have long term benefits for agriculture. Genes that control female gametophyte development can potentially be used to engineer apomixis (reproduction without fertilization) in crop plants, enabling the fixation of hybrid genotypes. This would enable the maintenance of hybrid vigor for many crop plants, without the need for expensive hybrid seed production. Such an advance would benefit the farmer by reducing the costs required to get higher yields. The project to determine functions of sequenced genes in the rice genome will identify genes for important traits. Knowledge of these genes will be extremely useful for rice breeders as well as other commercially important cereals including maize and wheat.

Publications

• Nishal, B., Tantikanjana, T., Sundaresan, V., 2005. An Inducible Targeted Tagging System for Localized Saturation Mutagenesis in Arabidopsis. Plant Physiology 137:3-12. (cover article)
• Kumar, C.S., Wing, R.A., Sundaresan, V. 2005. Efficient insertional mutagenesis in rice using the maize En/Spm elements. The Plant Journal 44: 879-892.
• Yu, H.-J., Hogan, P.S., Sundaresan, V. 2005. Analysis of the female gametophyte transcriptome of Arabidopsis by comparative expression profiling. . Plant Physiology 139: 1853-1869.
• Sundaresan, V. 2005. Control of seed size in plants. Proc. Natl. Acad. Sci. USA 50: 17887 - 17888.
• Pagnussat, G., Yu, H.-J., Ngo, Q., Rajani, S., Mayalagu, S., Johnson, C., Capron, A., Xie, L.-F., Ye, D., Sundaresan, V., 2005. Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis. Development 132:603-614.

Progress 01/01/04 to 12/31/04

Outputs
1. Control of plant reproduction: Gametophyte genes Over the past year, we have made very good progress in our studies of genes that are required for the formation of the gametophyte, which control a critical step in plant reproduction. Using the model plant Arabidopsis, we have completed initial characterization of 130 mutants in which the female gametophyte (embryo sac) is defective. These mutants were generated using a transposon tag, allowing us to identify the corresponding genes that are required to form a functional embryo sac. To our knowledge, this analysis represents the largest such effort in the world. A further 70 gene disruptions that primarily affect the male gametophyte (pollen) are being studied in collaboration with the laboratory of Sheila McCormick at PGEC (USDA-Albany). By a different strategy, using expression profiling with DNA chips, we have been able to identify another 300 Arabidopsis genes that are specifically expressed in the embryo sac. Further analysis of these genes is underway. 2. Insertional mutagenesis of rice Rice is the only plant other than Arabidopsis for which the genome has been sequenced, and is the best model for the agronomically important cereal crops as well as an important crop in its own right in several states including California and Arkansas. Our approach relies on the random insertion of transposons into the rice genome, resulting in disruptions of individual genes on a large scale. These gene disruptions (or "knockouts") can be analyzed to determine the functions of the genes. We have used modified transposable elements from maize (Ac-Ds and Spm/En) in combination with fluorescent markers to isolate a collection of transposon insertion lines. We have also begun to sequence the DNA flanking the insertions so that researchers can easily identify lines carrying knockouts in genes of interest. We have now generated over 4000 independent insertion lines, and determined the flanking sequences for 1000 of these insertions. In the coming year, we will be generating even larger numbers of insertions and sequences, to generate a valuable genetic resource for the US crop genetics community.

Impacts
The above projects are expected to have long term benefits for agriculture. Genes that control female gametophyte development can potentially be used to engineer apomixis (reproduction without fertilization) in crop plants, enabling the fixation of hybrid genotypes. This would enable the maintenance of hybrid vigor for many crop plants, without the need for expensive hybrid seed production. Such an advance would benefit the farmer by reducing the costs required to get higher yields. The project to determine functions of sequenced genes in the rice genome will identify genes for important traits. Knowledge of these genes will be extremely useful for rice breeders as well as other commercially important cereals including maize and wheat.

Publications

• Hirochika H, Guiderdoni E, An G, Hsing YI, Eun MY, Han CD, Upadhyaya N, Ramachandran S, Zhang Q, Pereira A, Sundaresan V, Leung H, 2004. Rice mutant resources for gene discovery. Plant Molecular Biology 54:325-334.
• Tantikanjana T, Mikkelsen MD, Hussain M, Halkier BA, Sundaresan V, 2004. Functional analysis of the tandem-duplicated P450 genes SPS/BUS/CYP79F1 and CYP79F2 in glucosinolate biosynthesis and plant development by Ds transposition-generated double mutants. Plant Physiology. 135:840-848.
• Kolesnik, T., Szeverenyi, I., Bachmann , D., Chellian, S.K., Jiang, S., Rengasamy, R., Cai, M., Ma, Z., Sundaresan, V., Ramachandran, S. , 2004. Establishing an Efficient Ac/Ds Tagging System in Rice: Large-Scale Analysis of Ds Flanking Sequences. The Plant Journal 37:301-314.

Progress 01/01/03 to 12/31/03

Outputs
We have continued our studies on the model plant Arabidopsis, the first plant species whose genome has been sequenced, to identify genes that are required for normal reproduction in plants. Our approach relies on a genetic screen applied to a population of transposon-tagged plants, and we have isolated 200 mutants in which gametophyte function is impaired. More detailed studies of the mutants are underway. We have been able to identify genes that are required for the different steps involved in the formation of the gametophyte, as well as for fertilization and embryogenesis. A second project is the development of an efficient system for insertional mutagenesis in rice. Rice is the only plant other than Arabidopsis for which the genome has been sequenced, and is the best model for the agronomically important cereal crops as well as an important crop in its own right in several states including California and Arkansas. Our approach relies on the random insertion of transposons into the rice genome, resulting in disruptions of individual genes on a large scale. These gene disruptions (or 'knockouts') can be analyzed to determine the functions of the genes. We have used modified transposable elements from maize (Ac-Ds and Spm/En) in combination with fluorescent markers to isolate a collection of transposon insertion lines. We have also begun to sequence the DNA flanking the insertions so that researchers can easily identify lines carrying knockouts in genes of interest. These efforts will be scaled up in the years ahead to provide a resource for both forward and reverse genetics for cereal crop researchers in USA.

Impacts
Genes that control female gametophyte development can potentially be used to engineer apomixis (reproduction without fertilization) in crop plants, enabling the fixation of hybrid genotypes and eliminating hybrid seed production. The project to determine functions of sequenced genes in the rice genome will identify genes for important traits, which will be very useful for rice breeders as well as other commercially important cereals including maize and wheat.

Publications

• Buell, C.R., and Sundaresan, V., 2003. A workshop on establishing a rice resource center in the United States. Plant Physiology 132:1137.
• Reddy, T.V., Agashe, B., Kaur, J., Sundaresan, V. and Siddiqi, I., 2003. The DUET Gene is Necessary for Chromosome Organization and Progression During Male Meiosis in Arabidopsis and Encodes a PHD Finger Protein. Development 130:5975-87.
• Kwee, H.S., and Sundaresan, V. , 2003. The NOMEGA gene required for female gametophyte development encodes the putative APC6/CDC16 component of the Anaphase Promoting Complex in Arabidopsis. The Plant Journal 36:853-866.

Progress 01/01/02 to 12/31/02

Outputs
Using the model plant Arabidopsis (the first plant species whose genome has been sequenced) we have identified a large number of genetic loci that are required for normal reproduction. These include about 200 mutants in which gametophyte function is impaired. We have begun the molecular characterization of some of these genes. We have shown that two genes called FILAMENTOUS FLOWER and YABBY3 which affect flower development, are required both for the specification of polarity in leaves and flowers, as well as the repression of genes that promote formation of the shoot meristem. We have also identified several genes which are involved in gametophyte or embryo development. These include the TORMOZ gene which we have found to be important for proper orientation of cell divisions within the embryo, and several genes that are required for gametophyte function either in the male (pollen) or the female (embryo sac) or both. A second project is the establishment of an effective system for insertional mutagenesis in rice. Rice is the only plant other than Arabidopsis for which the genome has been sequenced. It is currently the best model for the agronomically important cereals. We have shown that transposable elements from maize (Ac-Ds and Spm/En) can transpose at useful frequencies in rice. We have engineered vectors using fluorescent markers to permit efficient detection of transposition events. We plan to scale up our experiments for large-scale transposon mutagenesis in the coming years, and provide a resource for both forward and reverse genetics in rice.

Impacts
The studies on Arabidopsis should will identify genes important for plant reproduction that can be used for the control of reproduction in crop plants. The studies on rice will help elucidate the functions of the sequenced genes in the genome of rice, which has twice the number of genes as Arabidopsis. The information that we generate will be useful not only for rice breeders, but also for research on other cereals such as maize and wheat.

Publications

• Rajani, S. and Sundaresan, V., 2001. The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence. Current Biology (cover article) 11:1914-1922
• Kumaran M., Bowman, J.L., and Sundaresan, V., 2002. YABBY Polarity Genes Mediate Repression of KNOX Homeobox Genes in Arabidopsis. The Plant Cell 14: 2761-2770