Molecular characterization of signaling mechanisms in flowering transition in arabidopsis and association mapping of temperature response in floral development within glycine soja

Flowering is a major developmental event that occurs in the life cycle of all angiosperms. It is a finely tuned and highly elaborate set of mechanisms that combines internal and external stimuli to facilitate the transition from vegetative to reproductive growth. The timing of flowering has a significant impact on the fitness and reproductive efficacy of plants. To better understand floral control we address two major gaps in our knowledge. First, much of the molecular and biochemical mechanisms behind FLOWERING LOCUS T (FT), a flowering inducer, and TERMINAL FLOWER 1 (TFL1), a flowering repressor, are still largely unknown. Second, there is limited information about the mechanisms of flowering control in economically important crop species. In this study, we aim to further understand floral regulation using two approaches. 1) We will explore the role of inositol polyphosphate 5-phosphatase13 (5PTase13) in floral regulation mediated by TFL1 and FT. 2) We will take a broader view of floral regulation by examining the genome-wide effects of temperature response in flowering and maturity of Glycine Soja, the wild progenitor of cultivated soybean In the first approach Yeast Two-Hybrid assay reveals 5PTase13, an inositol polyphosphate 5-phosphatase involved in phospholipid signaling, interacts with TFL1 at a key functional residue. Both overexpressed TFL1 and mutant tfl1-1 are revealed to have increased sensitivity to root gravitropism similar to 5PTase13. While 5ptase single and double mutants do not exhibit a flowering phenotype, mutations in 5ptase13-1 and its homologs 5ptase14-1 and 5ptase12-1 significantly reduce the late flowering phenotype of overexpressed TFL1. Moreover, the 5PTases mitigate the late and early flowering phenotypes of ft-1 mutants and overexpressed FT respectively. Mutations in PHOSPHOLIPASE C2 (PLC2) an enzyme upstream of 5PTase13 in the phospholipid signaling pathway, is observed to enhance the early flowering phenotype of tfl1-1 mutants, whereas overexpression of PLC2 mitigates the early and late flowering phenotypes of tfl1-1 mutants and overexpressed TFL1 respectively. Fluorescent microscopy reveals that TFL1, FT and 5PTase13 localize in the nucleus and associate with the endoplasmic reticulum (ER). Bimolecular complementation experiments in Nicotiana tabacum and Arabidopsis further support our in vitro results, demonstrating that TFL1 and 5PTase13, and FT and 5PTase13 physically interact in both the nucleus and ER of live cells. Furthermore it is revealed the major sperm protein (MSP) domain within 5PTase13 is necessary and sufficient for TFL1 interaction. Similarly, changing the TFL1 histidine-88 residue to a tyrosine inhibits 5PTase13 interaction, whereas changing the residue to phenylalanine has very little impact on 5PTase13 association. We observed that transiently expressed 5PTase13 in N. tabacum and Arabidopsis shifted localization from the ER to the nucleus upon application of the vesicle trafficking inhibitor, brefeldin A. Moreover, 5PTase13 experiences a similar relocation to the nucleus when co-infiltrated with TFL1. We propose that in addition to the well-documented actions in lipid signaling, 5PTase13 also plays an important role in floral regulation through interaction with TFL1. In the second approach we observe two panels of 96 (P1) and 192 (P2) Glycine soja accessions grown at 20°C and 30°C to examine soybean ambient temperature response. Measured traits include R1 flowering, R5 seed-filling, R7-pod maturity, height, lodging, branching, vining, pod number, trifoliate number, bud number, and internode length. Using genotypic information from the SoySNP50K, both STRUCTURE and principle component analysis revealed that accessions clustered into three subpopulations that were correlated with their geographic origins. In general, plants grown at 30°C exhibited accelerated R1 flowering, and increased branching, height, and vining compared to 20°C, with accession maturity group having a large effect on accession phenotype across all traits. The G. soja accessions that were both most and least sensitive to temperature originated from Northeast China, Russia, Japan, and South Korea. A genome wide association study was conducted using the relative mean difference between 20°C and 30°C as a measure of temperature response. In P1, 592 and 188 SNPs were observed to be significant at an fdr of 0.20 and 0.05, respectively, across 13 traits. In P2, 1371 and 324 SNPs were observed to be significant at an fdr of 0.20, and 0.05, respectively, across 11 traits. 1778 and 837 genes were identified nearby these significant SNPs in P1 and P2 respectively, with 31 of those genes being previously characterized in plant temperature response. Notable examples are HEAT SHOCK PROTEIN 90, HEAT SHOCK FACTOR A4A, and HEAT SHOCK FACTOR A1A. Given the limited information on soybean temperature regulation, these candidates may set the framework for what genes are involved in soybean ambient temperature response