CLARIFYING SIGNALING MECHANISMS IN THE FLOWERING GENE NETWORKS IN ARABIDOPSIS AND SOYBEAN

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004571
• Project Start Date: Oct 29, 2014
• Project End Date: Dec 31, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
Crop Sciences

Non Technical Summary
Flowering is a major developmental transition from a vegetative growth phase to a reproductive phase to produce progeny. The flowering transition is central to reproductive success in plants and for determining environmental fitness. In the case of crop species, flowering-related traits are major targets in the domestication process by human selection. The flowering process involves extensive rearrangement of plant architecture and is metabolically costly for both annual and perennial plants. Therefore it is important to achieve tight and precise control of the timing of flowering to synchronize with suitable environments (i.e. availability of pollinators or seasonal timing that allows seed maturation) or with agricultural production practices. Understanding molecular mechanisms of the flowering process will provide genetic and genomic tools to modify flowering time in a variety of plant species. Although flowering-gene pathways are known to be one of the best studied molecular networks in plants, applications of flowering genes for agricultural use have been limited. First, most of our current knowledge has been focused on perception and response to external signals, rather than the internal signaling mechanisms that may allow molecular switches to be turned on/off easily and directly by artificial modulation. Secondly, most of present efforts are restricted to model species, and studies in commercially important crop species are less advanced.In this project, our first aim is to clarify the core flower signaling mechanisms by antagonistic regulators FT and TFL1, a major florigen and anti-florigen in a model plant Arabidopsis thaliana and in the crop plant, soybean. FT and TFL1 provide excellent tools to dissect the processes of flower signaling. Elucidation of the molecular actions of FT and TFL1 and examination of evolutionary relationships through species comparisons will provide us tools to control the actions of florigen and anti-florigen activity in economically important crop plants. Furthermore, our second aim, identification of novel flowering loci in soybean, will expand our information in soybean flowering control and allow agricultural application of flowering genes for developing superior crop species that are highly adaptive to diverse and changing environments by genetic modification or breeding techniques.

Animal Health Component

20%

Research Effort Categories

Basic

60%

Applied

20%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1820	1081	35%
201	1820	1080	35%
201	2420	1081	15%
201	2420	1080	15%

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

Subject Of Investigation
1820 - Soybean; 2420 - Noncrop plant research;

Field Of Science
1081 - Breeding; 1080 - Genetics;

Keywords

arabidopsis thaliana

soybean

flowering gene

inflorescences

flower signaling mechanisms

regulatory gene network

photoperiod

temperature

abiotic stress

Goals / Objectives
Objective I: Clarify the signaling mechanisms of FT and TFL1 in flowering control in Arabidopsis and soybean.Objective II: Identify novel flowering loci in soybean.

Project Methods
Objective 1) Clarify the signaling mechanisms of FT and TFL1 in flowering control in Arabidopsis and soybean.We will determine whether the inositol phosphatase TIL3/5PTase13 uses flowering proteins as its substrate in vitro and in vivo. TFL1, FT and their mutant proteins, as well as other TFL1 interacting proteins and known flowering proteins such as AP1 and FD will be also tested.Protein-protein interaction between TFL1 and the sucrose nonfermenting-1-related kinase (SnRK1) interaction will be tested using the bimolecular fluorescence complementation system (BiFC) and pull-down assay in tobacco. We will then examine whether SnRK1 phosphorylates TFL1 in vitro and in vivo, and identify phospho-sites by by LC-MS/MS to identify modification sites using procedures established in the lab. Identified phosphosites will be examined by site-directed mutagenesis and sequence- and modification-specific antibodies.We will obtain/create snrk1 knockout mutants and SnRK1-overexpressing transgenic plants co-expressed with TFL1-GFP or FT-GFP, and phosphorylation of TFL1-GFP or FT-GFP will be examined. Of particular interest is whether phosphorylation status and/or protein interactions affect the membrane or nuclear localization of these proteins as well as their function in vivo.Objective 2) Identify novel flowering loci in soybean. We will focus on soybean wild ancestor G. soja accessions for association mapping. By exploring the publicly available soy 50K SNPs, first we have selected 96 G. soja accessions that showed relatively small population structure while well diversified (Sedivy and Hanzawa, unpublished). We are currently observing agronomically-important phenotypes of these G. soja accessions in controlled greenhouse conditions under different photoperiods and temperature conditions with two replication experiments to identify genetic loci associated with photo-thermal effects of soybean by association mapping.For QTL mapping, we will use selected 6 G. soja accessions as parents of crosses with the G. max standard accession Williams 82. We will obtain 2,000-3,000 F2 seeds from 30-50 F1 seeds. The obtained F2 seeds will be grown under high and low temperature conditions and agronomically important traits will be measured with two repeated experiments using two sets of F2 seeds to identify genetic loci associated with temperature effects of soybean.To aid clarification of temperature-regulated flowering mechanisms, we will mutagenize soybean seeds of Williams 82 with NMU (N-Nitroso-N-methylurea, an alkalyting agent) and screen mutants that show early or late flowering phenotype under certain temperature conditions.

Progress 10/29/14 to 12/31/16

Outputs
Target Audience:The target audience includes plant and animal biologists, as well as plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunities for postdoctoral, graduate, undergraduate, and high school studentstraining in laboratoryresearch and public presentations. In addition, this project provided a training opportunity to an international intern student. How have the results been disseminated to communities of interest?The obtained results were published in peer-reviewed journals andpresented at scientific conferences andinvited seminars. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective I: Clarify the signaling mechanisms of FT and TFL1 in flowering control in Arabidopsis and soybean. The key flowering regulators FLOWERING LOCUS T (FT) and TERMINALFLOWER 1 (TFL1) are homologous, but their functions in flowering control are opposite. We have characterized the functions of thenovel factors that are involved in the signaling mechanisms and evolution ofFT and TFL1. Objective II: Identify novel flowering loci in soybean. Our GWAS approaches using a panel of G. maxand G. soja accessions identifiedloci that showsignificant association withtemperature response.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wickland, D.P. and Hanzawa, Y. 2015. The flowering locus T/terminal flower 1 gene family: Functional evolution and molecular mechanisms. Molecular Plant. 8(7):983-997.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Baumann K., Venail, J., Berbel, A., Domenech, A.J., Money, T., Conti, L., Hanzawa, Y., Madueno, F. and Bradley, D. 2015. Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture. Journal of Experimental Botany. 66(15):4769-4780.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Serrano-Mislata, A., Fern�ndez-Nohales, P., Dom�nech, M.J., Hanzawa, Y., Bradley, D. and Madue�o, F. 2016. Separate elements of the terminal flower 1 cis-regulatory region integrate pathways to control flowering time and shoot meristem identity. Development. 143(18):3315-3327.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Serrata, A., Haider, W., Mei, Y., Emad, A, Gregis, V., Kamater, M., Bradley, D., Alon, U., Milenkovic, O., Madue�o, F. and Hanzawa, Y. 2015. Paradoxical feedback circuits fine-tune the flowering gene network. Workshop on Mechanisms Controlling Flower Development, Aiguablava, Spain.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Sedivy, E.J., Mei, Y., Donahue, J., Nakamura, Y., Teo, N.Z.W., Wenk, M.R., Ito, T., Bradley, D., Gillaspy, G. and Hanzawa, Y. 2015. Phospholipid signaling modulates flowering time controlled by TFL1 in Arabidopsis. Workshop on Mechanisms Controlling Flower Development, Aiguablava, Spain.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Wu, F., Sedivy, E.J., Wickland, D.P., Price, W.B., Haider, W. and Hanzawa, Y. 2016. The evolution of FT homologs and its roles in soybean domestication. Soy2016: Molecular and Cellular Biology of the Soybean 16th Biennial Conference. Columbus, Ohio.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Sedivy, E., Nasarudin, N.S., Maniero, R., Han, M. and Hanzawa, Y. 2016. Association mapping of temperature response in agronomically important traits in soybean. Soy2016: Molecular and Cellular Biology of the Soybean 16th Biennial Conference. Columbus, Ohio.

Progress 10/29/14 to 09/30/15

Outputs
Target Audience:The target audience includes plant and animal biologists, as well as plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunities for postdoctoral, graduate,undergraduate and high school students training in laboratory research and public presentations. In addition, this project provided a training opportunity to an international intern student. How have the results been disseminated to communities of interest?The obtained results were presented at a scientific conference and an invited seminar. What do you plan to do during the next reporting period to accomplish the goals?Protein-protein interaction and cellularlocalization of flowering proteins will be examined in planta, and genetic analyses will be conducted to characterize their roles in flowering regulation. For higher accuracy of our GWAS approaches, we will conduct a genotype-by-sequencing approach to obtain a higher number of genome-wide polymorphisms.

Impacts
What was accomplished under these goals? Objective I: Clarify the signaling mechanisms of FT and TFL1 in flowering control in Arabidopsis and soybean. Deeply conserved across flowering plants, the key flowering regulators FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1) are homologous, but their functions in flowering control are opposite. FT, known as a mobile florigen, promotes flowering transition, while TFL1 represses. Both FT and TFL1 bind the bZIP transcription factor FD in the shoot apical meristem to modulate a set of floral meristem identity genes, but the molecular bases of their opposing functions remain unknown. We have obtained novel factors that may help clarify the evolution of the opposite functions of FT and TFL1, and are currently dissecting the roles of these factors in the FT and TFL1 pathway. Among several classes of TFL1 interacting proteins identified by yeast 2-hybrid, we currently focus on TFL1 IN LOVE3 (TIL3) and PLC2, that are involved inphospholipid signaling.BiFC tests confirmed the interaction between TFL1 and TIL3/5PTase13 in the nucleus and ER, as well as the interaction between TFL1 and PLC2 in plasma membranes in tobacco and Arabidopsis. Objective II: Identify novel flowering loci in soybean. To identify loci that control soybean temperature response, we are conducting GWAS approaches using 250 G. max accessions and 192 G. soja accessions grown under 20C and 30C in greenhouse. Diverse adaptive traits including germination, flowering and maturation time, numbers of trifoliates, flowers and pods are measured and loci that show significant association with phenotypic variation and temperature response have been identified using the SoySNP50K. We have identifed several previously identified loci as wel as novel loci.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Antonio Serrano-Mislata, Yu Mei, Waseem Haider, Amin Emad, Veronica Gregis, Carlos Gim�nez, Maria Jose Domenech, Martin M Kater, Olgica Milenkovic, Uri Alon, Desmond Bradley, Francisco Madue�o and Yoshie Hanzawa. 2015. Paradoxical feedback circuits fine-tune the flowering gene network. The 7th Workshop on Mechanisms Controlling Flower Development. Aiguablava, Spain.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Eric J. Sedivy, Yu Mei, Janet Donahue, Yuki Nakamura, Norman Z.W. Teo, Markus R. Wenk, Toshiro Ito, Glenda Gillaspy and Yoshie Hanzawa. 2015. Phospholipid signaling modulates flowering time controlled by TFL1. The 7th Workshop on Mechanisms Controlling Flower Development. Aiguablava, Spain.