Source: COLD SPRING HARBOR LABORATORY ASSOCIATION, INC submitted to
MAIZE EAR DEVELOPMENT, A NEW PATHWAY ACTING THROUGH FASCIATED EAR3
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1008540
Grant No.
2016-67013-24572
Project No.
NY.W-2015-06319
Proposal No.
2015-06319
Multistate No.
(N/A)
Program Code
A1101
Project Start Date
Dec 15, 2015
Project End Date
Dec 14, 2019
Grant Year
2016
Project Director
Jackson, D. P.
Recipient Organization
COLD SPRING HARBOR LABORATORY ASSOCIATION, INC
1 BUNGTOWN RD
COLD SPRING HARBOR,NY 11724-2209
Performing Department
Plant Biology
Non Technical Summary
Cereal crops produce the grain that constitutes the majority of our food and feed, therefore a fundamental understanding of their development has potential to improve agricultural yields. Maize in particular is the most cultivated and productive crop worldwide, and work from many groups has shown that changes in developmental control genes have been important for yield increases. This proposal is about the mechanisms by which plants grow and develop, using pools of stem cells called meristems. These structures are maintained in balance by a feedback loop between the CLAVATA and WUSCHEL genes. The proposal will use genetic and genomic approaches to study a newly identified control gene, FASCIATED EAR3 (FEA3), which codes for a cell surface receptor protein. FEA3 and its predicted ligand are expressed in specific groups of cells in the growing plant, suggesting a fundamentally new pathway in growth regulation in plants.Expected outcomes of this proposal are a deeper understanding of signaling during plant growth, and identification of new variants that could be used to enhance inflorescence size and seed productivity, important traits for food, feed and biomass production. The objectives target the program area priority of the NIFA Growth and Development program by using molecular, biochemical, and cellular approaches to improve plant productivity through studies of plant growth and developmental processes. The genes studied in the proposal could be applied in conventional breeding or in biotechnology approaches to improve crop yields and improve sustainability of US agriculture.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20615101050100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1510 - Corn;

Field Of Science
1050 - Developmental biology;
Goals / Objectives
This project will characterize a new plant developmental signaling pathway in maize. Previous work in our lab has identified a morphological mutant of maize called fasciated ear3, in which tassel and ear development is severely compromised. Both structures are enlarged, particularly at the growing tip, or meristem. We identified the underlying gene, and found that it encodes a trans-membrane receptor protein, similar to CLAVATA type receptors that function in stem cell signaling and maintenance. However, our preliminary data suggests that this new gene functions in a different pathway, and the proposed work will characterize this pathway, and understand its role in plant development. They project has three major goalsTo perform a detailed analysis of fea3 mutants, and mutants in its candidate receptor, called FCP1.To find proteins that interact with the FEA3 receptor, using proteomics, and candidate downstream factors, using gene expression profiling.To ask if FEA3 can improve maize seed yields. We have found preliminary data that a weak allele of FEA3 has bigger ears with additional rows of kernels. We will continue to characterize this effect, as well as searching for additional genetic modifiers of FEA3 function in natural populations of maize.
Project Methods
A number of different methods will be used to achieve the project goals, as follows:Aim 1. Detailed analysis of fea3 and Zmfcp1 - mutants and mechanism.We will complete the analysis of expression and genetic interactions. This will include histological and molecular analysis, as well as analysis of developmental analysis of double mutants. In addition, tissue specific gene expression using a trans-activation system will be used to test the new signaling pathway hypothesis.AIM 2. FEA3 interactors and networks. Here we will further investigate the mechanism of FEA3 action, by looking for interactors using proteomics, and mRNAseq transcriptome profiling to find genes that may function in a FEA3 network. These experiments should identify candidate downstream signal transduction components as well as additional genes that function in the control of ear development.Aim 3. Can FEA3 improve crop yield traits? Fasciated mutant ears, such as those produced by fea3 mutants, have low seed yield, because they make too many primordia that are very disorganized, and do not fill properly. However, we found that weak alleles of fea3 make more kernels. We will continue to investigate this phenomenon by breeding the weak alleles into different inbred and hybrid backgrounds and measuring kernel yields. We will also look for second site modifiers of the fea3 mutations by crossing to diverse maize germplasm and looking for enhanced phenotypes. Such enhancer loci will be mapped and molecularly isolated.

Progress 12/15/15 to 12/14/19

Outputs
Target Audience:The target audience for this project was academic researchers in the plant biology community, including Faculty, Postdoctoral Researchers, Graduate and Undergraduate students studying plant genetics, genomics and signaling. A second target audience was plant breeders or biotechnology experts working in industry or in academia, who will be able to use the knowledge or varieties produced in the project for developing new strains with enhanced yield traits. The work was shared in seminars by the project PI and post doc, and in training workshops at Cold Spring Harbor Laboratory, where it can inspire others to improve crop productivity through application of basic knowledge of plant development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher, Fang Xu, was trained in maize genetics and genomics and developmental biology under this award. She recently accepted an Assistant Professor Position at Shandong University, China. How have the results been disseminated to communities of interest?DJ presented results from this project in Conferences and seminars, as follows: Plant Stem Cell Symposium, Sendai, Japan May 11th- 13th, 2019, Huazhong Agricultural University Conference on Crop Genomics, Wuhan, China, June 2-5, 2019, Society for In Vitro Biology Meeting, Tampa, Florida, June 10- 12, 2019, FASEB Conference on Plant Development, New York, July 29- Aug 2, 2019, US- Mexico Plant Molecular Biology Meeting, Merida, Mexico, Oct 28- 31, 2019. 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.) What was accomplished under these goals? This project aimed to study a new plant developmental signaling pathway in maize discovered using a morphological mutant of maize called fasciated ear3 (fea3) in which tassel and ear development is severely compromised. Both structures are enlarged, particularly at the growing tip, or meristem. FEA3 encodes a trans-membrane receptor protein, similar to CLAVATA type receptors that function in stem cell signaling and maintenance. However, our preliminary data suggested that this new gene functions in a different pathway, and the proposed work will characterize this pathway, and understand its role in plant development. Theproject has three major goals. Progress: 1. We performed a detailed analysis of fea3 mutants, and mutants in its candidate ligand, called FCP1. We completed the analysis of fea3 mutants as well as mutants in its candidate ligand, FCP1. We found that FEA3 is expressed in a domain of the meristem below WUSCHEL, and FCP1 is expressed in developing primordia, suggesting a "new" CLAVATA pathway distinct from the canonical CLV3-CLV1 WUS pathway that has been described in Arabidopsis. Mutants in FCP1 were isolated from a transposon collection and generated by CRISPR/Cas9 genome editing. We collaborated with the group of Henrik Johnsson (Cambridge lab, UK) to produce a mathematical model to integrate the new CLV pathway with the canonical one. We tested this model using maize transgenic expression of FCP1. To understand if FEA3 related genes function redundantly in maize, we made a phylogeny of FEA3 related proteins, and several highly similar genes that are also expressed in inflorescences were identified and mutated by CRISPR/Cas9. We backcrossed the CRISPR mutants with B73 to control for genetic background and generated quadruple homozygous mutants. However, no obvious inflorescence development phenotype was observed, and surprisingly the quadruple mutant didn't enhance the inflorescence phenotype of fea3. In a newer version of the maize B73 genome, we identified an additional fea3 homolog, and have knocked out this gene by CRISPR/Cas9 to overcome genetic redundancy in this receptor family and are combining with the previously isolated mutants to screen for phenotypes. 2. To find proteins that interact with the FEA3 receptor, using proteomics, and candidate downstream factors, using gene expression profiling. To further understand FEA3 function, we asked if it can bind FCP1 directly, in a collaboration with the Matsubayashi lab (Univ. Nagoya, Japan). We found that FEA3 does not bind FCP1, and therefore it may have a co-receptor. However, the Arabidopsis BARELY ANY MERISTEM (AtBAM1) protein, a leucine-rich repeat receptor kinase related to CLV1, binds FCP1, suggesting it could be a receptor (Jackson and Matsubayashi, unpublished). In situ hybridization experiments by another lab member, Lei Liu, revealed a similar expression pattern of ZmBAM1d and FEA3 in inflorescence meristems. We performed co-IP experiment in N.benthamiana to show that ZmBAM1d interacts with FEA3. These data together suggest that ZmBAM1d is a candidate coreceptor for FEA3. We generated pFEA3::RFP-FEA3 and pUbi::ZmBAM1d-YFP double transgenic plants to validate their interaction in vivo by Co-IP. Future work will perform FCP1 peptide response assays in bam1d mutants (available in our lab as part of a different project) and by checking the binding of FCP1 peptide by ZmBAM1d using isothermal titration calorimetry (ITC), in collaboration with the Yin lab (HZAU, China). We generated tagged FEA3 transgenic lines and crossed them to a proliferative meristem background (branched silkless; Tunicate (bdTu)) to collect large amounts of meristems. We performed IP-mass spectrometry (MS) analysis and found candidate interactors for FEA3, including a candidate downstream signaling protein, protein phosphatase 2A (PP2A) We validated this interaction by co-IPs in N.benthamiana, and have started to make CRISPR/ Cas9 knockout lines to search for phenotypes. However, we did not detect ZmBAM1d or other candidate co-receptor proteins. We will continue to optimize the IP process to search for a FEA3 coreceptor. We also carried out mRNAseq analysis of fea3 mutants, and over 700 genes were differentially expressed, including many known meristem development related genes. For instance, ZmWUS1, FCP1 and ZmCLE7 were significantly upregulated, as predicted. Gene ontology (GO) analysis revealed biological processes affected in fea3 mutants, including gene expression regulation, intracellular signal transduction, response to auxin and superoxide, cytokinin activated signaling and trehalose metabolic process. 3. To ask if FEA3 can improve maize seed yields. A weak allele of FEA3 has bigger ears with additional rows of kernels, and we found up to 50% increased yield in maize ears in our own (low density) field growing conditions. We continue to characterize this effect in field yield trials, in collaboration with Corteva. Excitingly, they have successfully introduced fea3 weak alleles into elite germplasm using CRISPR/Cas9 genome editing. To find fea3 modifiers, we previously isolated a transposon insertion line for the maize ortholog of CORYNE (ZmCRN), which functions in meristem control in Arabidopsis. Zmcrn mutants had enlarged shoot and inflorescence meristems. By mapping a new EMS fasciated mutant, fea148, we identified a second allele of Zmcrn, which contains an early stop codon mutation. Double mutant and transgenic analysis indicated that ZmCRN acts in parallel with FEA3, but in contrast it interacts and functions together with FEA2. Further detailed genetic analysis and peptide assays show that FEA2 transmits signaling from 2 distinct CLE peptides, the maize CLV3 ortholog ZmCLE7 and ZmFCP1, through the alpha subunit of the maize heterotrimeric G protein COMPACT PLANT2 (CT2), and ZmCRN respectively. In addition, we found that the ZmCRN locus is significantly associated with kernel row number (KRN) in a maize GWAS analysis, revealing ZmCRN as a good candidate for fine-manipulation to improve maize yield. Another new discovery that we made in collaboration with the group of Jianbing Yan, Huazhong Agricultural University, China, was that our candidate FEA3 co-receptor, ZmBAM1d, acts as a QTL to control seed size in maize. This is a very striking finding that may allow us to understand the tradeoff between seed number and size in grain crops.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Yang, N., Liu, J., Gao, Q., Gui, S., Chen, L., Yang, L., Huang, J., Deng, T., Luo, J., He, L., Wang, Y., Xu, P., Peng, Y., Shi, Z., Lan, L., Ma, Z., Yang, X., Zhang, Q., Bai, M., Li, S., Li, W., Liu, L., Jackson, D., Yan, J. (2019) Genome assembly of a tropical maize inbred line provides insights into structural variation and crop improvement. Nature Genetics. 51 (6). pp. 1052-1059.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Kitagawa, M., Jackson, D. (2019) Control of meristem size. Annu. Rev. Plant Biol. 70. pp. 269-291.


Progress 12/15/17 to 12/14/18

Outputs
Target Audience:The target audience for this project will be academic scientists, who are interested in crop genetics, and yield traits, as well as industry scientists and breeders working to improve productivity in maize and other crops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher, Fang Xu, is being trained in maize genetics and genomics and developmental biology under this award. How have the results been disseminated to communities of interest?Results from this project have been presented in seminars and posters, as follows: Fang Xu presented her data in a short talk at the 4th Chinese Maize Meeting, Qingdao, China, April 2018. Jackson presented work from the project in Departmental seminars and conferences including Univ Toronto,Canada, Univ Lausanne, Switzerland, Gatersleben Institute, Germany, The Agriculture Genetics Institute, Ha Noi, Viet Nam, Molecular Plant Conference, Xian, China, The 29th International Conference on Arabidopsis Research (ICAR) 2018, Turku, Finland, The Max Plank Institute, Golm, Germany, IV European Workshop on Plant Peptides & Receptors, Antequera (Málaga), Spain, The John Innes Institute, Norwich, UK, Univ. North Texas, and U Mass, Amherst. Byoung Il Je and Fang Xu presented their data in posters at the 59th Annual Maize Genetics Conference, St. Louis, Missouri, USA, March 2017. Fang Xu also presented her data in a poster at the 3rd Chinese Maize Meeting, Anhui, April 2017 and at Plant Genomes &Biotechnology: From Genes to Networks, Cold Spring Harbor, NY, December 2017. Meetings / Seminars (PI): Results from this project have been presented in invited talks at: MU Pioneer Symposium, Indiana Univ., Japan Society for Plant Physiology, CRISPR conference, HZAU, Wuhan, China, VIB Conference 'At the forefront of Plant Biology', International Botanical Congress, Shenzhen, and at Inari Ag, Boston, MA. Jackson also organized a Cereal Genomics workshop at CSHL in 2016, and 16 postdoctoral scholars or graduate students were trained in theory and practice of cereal genomes. The workshop was partially supported with a separate award from NIFA. What do you plan to do during the next reporting period to accomplish the goals?The project goals are progressing according to the original plan, no major changes are anticipated.

Impacts
What was accomplished under these goals? This project aims to study a new plant developmental signaling pathway in maize discovered using a morphological mutant of maize called fasciated ear3 (fea3) in which tassel and ear development is severely compromised. Both structures are enlarged, particularly at the growing tip, or meristem. FEA3 encodes a trans-membrane receptor protein, similar to CLAVATA type receptors that function in stem cell signaling and maintenance. However, our preliminary data suggested that this new gene functions in a different pathway, and the proposed work will characterize this pathway, and understand its role in plant development. They project has three major goals. Progress: 1. To perform a detailed analysis of fea3 mutants, and mutants in its candidate ligand, called FCP1. We have completed our analysis of fea3 mutants as well as mutants in its candidate ligand, FCP1. FEA3 is expressed in a domain of the meristem below WUSCHEL, and FCP1 is expressed in developing primordia, suggesting a "new" CLAVATA pathway distinct from the canonical CLV3-CLV1 WUS pathway that has been described in Arabidopsis. Mutants in FCP1 were isolated from a transposon collection and by using CRISPR/Cas9 genome editing. We collaborated with the group of Henrik Johnsson (Cambridge lab, UK) to produce a mathematical model to integrate the new CLV pathway with the canonical one. We tested this model using maize transgenic expression of FCP1. To understand if FEA3 related genes function redundantly in maize, we made a phylogeny of FEA3 related proteins, and 4 highly similar genes that are also expressed in inflorescences were identified and mutated by CRISPR/Cas9. We backcrossed the CRISPR mutants with B73 to control for genetic background and generated quadruple homozygous mutants. However, no obvious inflorescence development phenotype was observed, and surprisingly the quadruple mutant didn't enhance the inflorescence phenotype of fea3. In the past year, we re-analyzed the phylogeny based on new annotations in the maize B73 v4 genome, we identified an additional fea3 homolog, which was not annotated in maize v3. We knocked out this gene by CRISPR/Cas9 to overcome genetic redundancy in this receptor family and are combining with the previously isolated mutants to screen for phenotypes. 2. To find proteins that interact with the FEA3 receptor, using proteomics, and candidate downstream factors, using gene expression profiling. To further understand FEA3 function, we asked if it can bind FCP1 directly, in a collaboration with the Matsubayashi lab (Univ. Nagoya, Japan). We found that FEA3 does not bind FCP1, and therefore it may have a co-receptor. However, the Arabidopsis BARELY ANY MERISTEM (AtBAM1) protein, a leucine-rich repeat receptor kinase related to CLV1, binds FCP1, suggesting it could be a receptor. In situ hybridization experiments by another lab member, Lei Liu, revealed a similar expression pattern of ZmBAM1d and FEA3 in inflorescence meristems. We performed co-IP experiment in N.benthamiana to show that ZmBAM1d interacts with FEA3. These data together suggest that ZmBAM1d is a candidate coreceptor for FEA3. We are generating pFEA3::RFP-FEA3 and pUbi::ZmBAM1d-YFP double transgenic plants to validate their interaction in vivo by Co-IP. We are further testing the hypothesis by carrying out FCP1 peptide response assays in bam1d mutants (available in our lab as part of a different project) and by checking the binding of FCP1 peptide by ZmBAM1d using isothermal titration calorimetry (ITC), in collaboration with the Yin lab (HZAU, China). In addition, we have backcrossed pFEA3::RFP-FEA3 and tandem affinity protein (TAP-) tagged FEA3 transgenic lines to a proliferative meristem background (branched silkless; Tunicate (bdTu)) to collect large amounts of meristems. We performed IP-mass spectrometry (MS) analysis and found candidate interactors for FEA3. We are now validating these interactions by co-IPs in N.benthamiana. However, we did not detect ZmBAM1d or other candidate co-receptor proteins. We will continue to optimize the IP process to search for FEA3 coreceptor. We also carried out mRNAseq analysis of fea3 mutants, and over 700 genes were differentially expressed in fea3 mutants, including many known meristem development related genes. For instance, ZmWUS1, FCP1 and ZmCLE7 were significantly upregulated, as predicted. Gene ontology (GO) analysis revealed biological processes affected in fea3 mutants, including gene expression regulation, intracellular signal transduction, response to auxin and superoxidase, cytokinin activated signaling and trehalose metabolic process. 3. To ask if FEA3 can improve maize seed yields. A weak allele of FEA3 has bigger ears with additional rows of kernels, and we found up to 50% increased yield in maize ears in our own (low density) field growing conditions. We are characterizing this effect in field yield trials, in collaboration with Dupont Pioneer. Excitingly, they have successfully introduced fea3 weak alleles into elite germplasm using CRISPR/Cas9 genome editing. To find fea3 modifiers, we previously isolated a transposon insertion line for the maize ortholog of CORYNE (ZmCRN), which functions in meristem control in Arabidopsis. Zmcrn mutants had enlarged shoot and inflorescence meristems. By mapping a new EMS fasciated mutant, fea148, we identified a second allele of Zmcrn, which contains an early stop codon mutation. Double mutant and transgenic analysis indicated that ZmCRN acts in parallel with FEA3, but in contrast it interacts and functions together with FEA2. Further detailed genetic analysis and peptide assays show that FEA2 transmits signaling from 2 distinct CLE peptides, the maize CLV3 ortholog ZmCLE7 and ZmFCP1, through the alpha subunit of the maize heterotrimeric G protein COMPACT PLANT2 (CT2), and ZmCRN respectively. In addition, we found that the ZmCRN locus is significantly associated with kernel row number (KRN) in a maize GWAS analysis, revealing ZmCRN as a good candidate for fine-manipulation to improve maize yield. A paper describing these findings was published in eLife.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wu, Q., Xu, F., Jackson, D. (2018) All together now, a magical mystery tour of the maize shoot meristem. Current Opinion in Plant Biology. 45: 26-35.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Xu, F., Jackson, D. (2018) Learning from CIK plants. Nature Plants. 4(4):195.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Je, BI., Xu, F., Wu, Q., Liu, L., Meeley, R., Gallagher, J.P., Corcilius, L., Payne, R.J., Bartlett, M.E., Jackson, D. (2018) The CLAVATA receptor FASCIATED EAR2 responds to distinct CLE peptides by signaling through downstream effectors. Elife. 7:e35673


Progress 12/15/16 to 12/14/17

Outputs
Target Audience:The target audience for this project will be academic scientists, who are interested in crop genetics, and yield traits, as well as industry scientists and breeders working to improve productivity in maize and other crops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher, Byoung Il Je, is supported by this award and has been trained in maize genetics, biochemistry, and imaging. He recently got a faculty position at Pusan National University in Republic of Korea. In addition, a second post doc, Fang Xu, is being trained in maize genetics and genomics and developmental biology. How have the results been disseminated to communities of interest?Results from this project have been presented in seminars and posters, as follows: Byoung Il Je and Fang Xu presented their data in posters at the 59th Annual Maize Genetics Conference, March 9-12, 2017, St. Louis, Missouri, USA. Fang Xu also presented her data in a poster at the Chinese Maize Meeting, Anhui, April 2017 and at Plant Genomes & Biotechnology: From Genes to Networks, Cold Spring Harbor, NY, December 2017 Meetings / Seminars (PI): Results from this project have been presented in invited talks at: MU Pioneer Symposium, Indiana Univ., Japan Society for Plant Physiology, CRISPR conference, HZAU, Wuhan, China, VIB Conference 'At the forefront of Plant Biology', International Botanical Congress, Shenzhen, and at Inari Ag, Boston, MA. Jackson also organized a Cereal Genomics workshop at CSHL in 2016, and 16 postdoctoral scholars or graduate students were trained in theory and practice of cereal genomes. The workshop was partially supported with a separate award from NIFA. What do you plan to do during the next reporting period to accomplish the goals?The project goals are progressing according to the original plan, no major changes are anticipated.

Impacts
What was accomplished under these goals? This project aims to study a new plant developmental signaling pathway in maize discovered using a morphological mutant of maize called fasciated ear3 (fea3) in which tassel and ear development is severely compromised. Both structures are enlarged, particularly at the growing tip, or meristem. FEA3 encodes a trans-membrane receptor protein, similar to CLAVATA type receptors that function in stem cell signaling and maintenance. However, our preliminary data suggested that this new gene functions in a different pathway, and the proposed work will characterize this pathway, and understand its role in plant development. The project has three major goals. Progress: 1. To perform a detailed analysis of fea3 mutants, and mutants in its candidate ligand, called FCP1. In previous year, we completed our analysis of fea3 mutants as well as mutants in its candidate ligand, FCP1. FEA3 is expressed in a domain of the meristem below WUSCHEL, and FCP1 is expressed in developing primordia, suggesting a "new" CLAVATA pathway distinct from the canonical CLV3-CLV1 WUS pathway that has been described in Arabidopsis. Mutants in FCO1 were isolated from a transposon collection and by using CRISPR/Cas9 genome editing. We collaborated with the group of Henrik Johnsson (Cambridge lab, UK) to produce a mathematical model to integrate the new CLV pathway with the canonical one. We tested this model using maize transgenic expression of FCP1. To understand if FEA3 related genes function redundantly in maize, we made a phylogeny of FEA3 related proteins, and 4 highly similar genes that are also expressed in inflorescences were identified and mutated by CRISPR/Cas9. We backcrossed the CRISPR mutants with B73 to control for genetic background and generated quadruple homozygous mutants. However, no obvious inflorescence development phenotype was observed, and surprisingly the quadruple mutant didnt enhance the inflorescence phenotype of fea3. By re-analyzing the phylogeny based on new annotations in the maize B73 v4 genome, we identified two additional fea3 homologs, which were not annotated in maize v3. We will continue to knockout these genes to overcome genetic redundancy in this receptor family and look for phenotypes. 2. To find proteins that interact with the FEA3 receptor, using proteomics, and candidate downstream factors, using gene expression profiling. To further understand FEA3 function, we asked if it can bind FCP1 directly, in a collaboration with the Matsubayashi lab (Univ. Nagoya, Japan). Initial results suggest that FEA3 does not bind FCP1, and therefore it may have a co-receptor. However, control experiments indicated that the Arabidopsis BARELY ANY MERISTEM (AtBAM1) protein, a leucine-rich repeat receptor kinase related to CLV1, binds FCP1, suggesting it could be a receptor. We also carried out mRNAseq analysis of fea3 mutants and segregating wild type sibs in a 1:1 segregating population. About 700 genes were differentially expressed in fea3 mutants, including genes involved in auxin and gibberellin pathways. In particular, ZmBAM1d, a homolog to AtBAM1, was up-regulated in fea3. in situ hybridization experiments by another lab member, Lei Liu, revealed a similar expression pattern of ZmBAM1d and FEA3 in inflorescence meristems. These data together suggest that ZmBAM1d is a candidate co-receptor for FEA3, and we are planning to test this hypothesis by assaying the physical interaction between ZmBAM1d and FEA3 by in vivo co-IP, and carrying out FCP1 peptide response assays in bam1d mutants (available in our lab as part of a different project) and by checking the binding of FCP1 peptide by ZmBAM1d. In addition, we have made and backcrossed tandem affinity protein (TAP-) tagged FEA3 transgenic plants to fea3 mutants as well as to a proliferative meristem background (branched silkless; Tunicate (bdTu)) to enable collection of large amounts of meristems, and plan to perform in vivo IP-mass spectrometry soon, so that we can confirm the BAM1d interaction and identify other candidate co-receptors. 3. To ask if FEA3 can improve maize seed yields. We confirmed that a weak allele of FEA3 has bigger ears with additional rows of kernels, and found up to 50% increased yield in maize ears in our own (low density) field growing conditions. We are continuing to characterize this effect by initiating proper field yield trials, in collaboration with Dupont Pioneer. Excitingly, they have successfully introduced fea3 weak alleles into elite germplasm using CRISPR/Cas9 genome editing. We are also searching for genetic modifiers of FEA3 to find other players in this new developmental pathway. We have crossed fea3 to each of the diverse maize NAM founder lines, and screened F2 populations for modified phenotypes. Preliminary results suggest at least 2 lines enhance and one line suppresses fea3, and mapping populations will now be created as a first step to identify the modifier loci. As an alternative approach, we also mutagenized fea3 using EMS. However, no candidate suppressors were confirmed from over 600 M2 families. A much bigger M2 population will need to be screened in the future to find the candidate suppressors. As an alternative approach to find fea3 modifiers, we previously isolated a transposon insertion line for the maize ortholog of CORYNE (ZmCRN), which functions in meristem control in Arabidopsis. Zmcrn mutants had enlarged shoot and inflorescence meristems. By mapping a new EMS fasciated mutant, fea148, we identified a second allele of Zmcrn, which contains an early stop codon mutation. Double mutant and transgenic analysis indicated that ZmCRN acts in parallel with FEA3, but in contrast it interacts and functions together with FEA2. Further detailed genetic analysis and peptide assays show that FEA2 transmits signaling from 2 distinct CLE peptides, the maize CLV3 ortholog ZmCLE7 and ZmFCP1, through the alpha subunit of the maize heterotrimeric G protein COMPACT PLANT2 (CT2), and ZmCRN respectively. In addition, we found that the ZmCRN locus is significantly associated with kernel row number (KRN) in a maize GWAS analysis, revealing ZmCRN as a good candidate for fine-manipulation to improve maize yield. A paper describing these findings is in press in eLife. We also continued to propagate and map additional fea mutants. We have mapped another fasciated mutant, tr183, which contains an amino acid change in a conserved residue of the beta subunit of the maize heterotrimeric G protein, ZmGB1.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Je, BI., Xu, F., Wu, Q., Liu, L., Meeley, R., Gallagher, JP., Corcilius, L., Payne, RJ., Bartlett, ME., Jackson, D. (2018) The CLAVATA receptor FASCIATED EAR2 responds to distinct CLE peptides by signaling through two downstream effectors. eLife. In Press.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Xu, F., Jackson, D. Learning from CIK plants. Nature Plants. In Press.


Progress 12/15/15 to 12/14/16

Outputs
Target Audience:The target audience for this project will be academic scientists, who are interested in crop genetics, and yield traits, as well as industry scientists and breeders is working to improve productivity in maize and other crops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher, Byoung Il Je, is supported by this award and has been trained in maize genetics, biochemistry, and imaging. In addition, a second post doc, Fang Xu, joined our lab last year following her PhD in an Arabidopsis disease resistance lab. Fang is being trained in maize genetics and genomics and developmental biology. How have the results been disseminated to communities of interest?Results from this project have been presented in seminars and posters, as follows: Both post docs presented their data in posters at the Maize Genetic Conference, March 2016. Meetings / Seminars (PI): Results from this project have been presented in invited talks at: The European Maize Conference, Hamburg, Germany (Plenary Speaker), The Arabidopsis Conference, Korea, ComBio, Brisbane, Australia (Plenary Speaker), CSH Asia Conference, Awaji, Japan, Pioneer Conference, Univ. Missouri (Plenary Speaker), and Univ. Indiana, Bloomington. Jackson also organized a Cereal Genomics workshop at CSHL in 2016, and 16 postdoctoral scholars or graduate students were trained in theory and practice of cereal genomes. The workshop was partially supported with a separate award from NIFA. What do you plan to do during the next reporting period to accomplish the goals?The project goals are progressing according to the original plan, no major changes are anticipated.

Impacts
What was accomplished under these goals? a. A comparison of actual accomplishments with the goals established for the reporting period (where the output of the project can be expressed readily in numbers, a computation of the cost per unit of output should be submitted if the information is considered useful); This project aims to study a new plant developmental signaling pathway in maize discovered using a morphological mutant of maize called fasciated ear3 (fea3) in which tassel and ear development is severely compromised. Both structures are enlarged, particularly at the growing tip, or meristem. FEA3 encodes a trans-membrane receptor protein, similar to CLAVATA type receptors that function in stem cell signaling and maintenance. However, our preliminary data suggested that this new gene functions in a different pathway, and the proposed work will characterize this pathway, and understand its role in plant development. They project has three major goals. Progress: 1. To perform a detailed analysis of fea3 mutants, and mutants in its candidate receptor, called FCP1. We have completed our analysis of fea3 mutants as well as mutants in its candidate ligand, FCP1. FEA3 is expressed in a domain of the meristem below WUSCHEL, and FCP1 is expressed in developing primordia suggesting a "new" CLAVATA pathway distinct from the canonical CLV3-CLV1 WUS pathway that has been described in Arabidopsis. We collaborated with the group of Henrik Johnsson (Cambridge lab, UK) to produce a mathematical model to integrate the new CLV pathway with the canonical one. We tested this model using maize transgenic expression of FCP1. We also found that weak fea3 alleles can enhance maize yields, by increasing kernel row number. A paper describing these findings was published in Nature Genetics. We also made a phylogeny of FEA3 related proteins, and found 4 highly similar genes that are also expressed in inflorescences. We have knocked out these genes using CRISPR/Cas9, and will produce homozygous lines to look for phenotypes. 2. To find proteins that interact with the FEA3 receptor, using proteomics, and candidate downstream factors, using gene expression profiling. To further understand FEA3 function, we asked if it can bind FCP1 directly, in a collaboration with the Matsubayashi lab (Univ. Nagoya, Japan). Initial results suggest that FEA3 does not bind FCP1, and therefore it may have a co-receptor. To identify this, as well as other interacting proteins, we made tandem affinity protein (TAP-) tagged versions of FEA3 and transformed into maize. We have confirmed expression of the tagged protein and are backcrossing to fea3 mutants as well as to a proliferative meristem background (branched silkless; Tunicate (bdTu)) for collecting large amounts of meristems. 3. To ask if FEA3 can improve maize seed yields. We confirmed that a weak allele of FEA3 has bigger ears with additional rows of kernels, and found up to 50% increased yield in maize ears in our own (low density) field growing conditions. We are continuing to characterize this effect by initiating proper field yield trials, in collaboration with Dupont Pioneer. We are also searching for genetic modifiers of FEA3 to find other players in this new developmental pathway. We have crossed fea3 to each of the diverse maize NAM founder lines, and screened F2 populations for modified phenotypes. Preliminary results suggest at least 2 lines enhance and one line suppresses fea3, and mapping populations will now be created as a first step to identify the modifier loci. As an alternative approach, we also mutagenized fea3 using EMS, and in a preliminary screen of ~ 300 M2 lines this winter we found two putative modifiers that fully suppress the fea3 phenotype. Additional activity has been to characterize another new maize fasciated mutant, called coryne (crn). This was identified as a transposon insertion in a candidate gene, and we are performing genetic analyses to place it into known fasciated pathways. We also continue to propagate and map additional mutants from EMS screens. b. The reasons for slippage if established goals were not met; So far all project goals are progressing as expected. c. Additional pertinent information including, when appropriate, analysis and explanation of cost overruns or unexpectedly high unit costs. The project is progressing extremely well and there are no issues in meeting the goals in the funding period or with funding.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Somssich, M., Je, BI., Simon, R., Jackson, D. (2016) CLAVATA-WUSCHEL signaling in the shoot meristem. Development. 143(18): 3238-48.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Altpeter, F., Springer, NM., Bartley, LE., Blechl, AE., Brutnell, TP., Citovsky, V., Conrad, LJ., Gelvin, SB., Jackson, D., Kausch, AP., Lemaux, PG., Medford, JI., Orozco-C�rdenas, ML., Tricoli, DM., Van, Eck J., Voytas, DF., Walbot, V., Wang, K., Zhang, ZJ., Stewart, CN Jr. (2016) Advancing crop transformation in the era of genome editing. The Plant Cell, 28 (7): 1510-1520.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Je, BI., Gruel, J., Lee, YK., Bommert, P., Arevalo, ED., Eveland, AL., Wu, Q., Goldshmidt, A., Meeley, R., Bartlett, M., Komatsu, M., Sakai, H., J�nsson, H., Jackson, D. (2016) Signaling from maize organ primordia via FASCIATED EAR3 regulates stem cell proliferation and yield traits. Nature Genetics 48(7):785-91.