Separate elements of the TERMINAL FLOWER 1 cis-regulatory region integrate pathways to control flowering time and shoot meristem identity.

TERMINAL FLOWER 1 (TFL1) is a key regulator of Arabidopsis plant architecture that responds to developmental and environmental signals to control flowering time and the fate of shoot meristems. TFL1 expression is dynamic, being found in all shoot meristems, but not in floral meristems, with the level and distribution changing throughout development. Using a variety of experimental approaches we have analysed the TFL1 promoter to elucidate its functional structure. TFL1 expression is based on distinct cis-regulatory regions, the most important being located 3' of the coding sequence. Our results indicate that TFL1 expression in the shoot apical versus lateral inflorescence meristems is controlled through distinct cis-regulatory elements, suggesting that different signals control expression in these meristem types. Moreover, we identified a cis-regulatory region necessary for TFL1 expression in the vegetative shoot and required for a wild-type flowering time, supporting that TFL1 expression in the vegetative meristem controls flowering time. Our study provides a model for the functional organisation of TFL1 cis-regulatory regions, contributing to our understanding of how developmental pathways are integrated at the genomic level of a key regulator to control plant architecture.

Evolutionary trajectories of duplicated FT homologues and their roles in soybean domestication.

To clarify the molecular bases of flowering time evolution in crop domestication, here we investigate the evolutionary fates of a set of four recently duplicated genes in soybean: FT2a, FT2b, FT2c and FT2d that are homologues of the floral inducer FLOWERING LOCUS T (FT). While FT2a maintained the flowering inducer function, other genes went through contrasting evolutionary paths. FT2b evolved attenuated expression potentially associated with a transposon insertion in the upstream intergenic region, while FT2c and FT2d obtained a transposon insertion and structural rearrangement, respectively. In contrast to FT2b and FT2d whose mutational events occurred before the separation of G. max and G. soja, the evolution of FT2c is a G. max lineage specific event. The FT2c allele carrying a transposon insertion is nearly fixed in soybean landraces and differentiates domesticated soybean from wild soybean, indicating that this allele spread at the early stage of soybean domestication. The domesticated allele causes later flowering than the wild allele under short day and exhibits a signature of selection. These findings suggest that FT2c may have underpinned the evolution of photoperiodic flowering regulation in soybean domestication and highlight the evolutionary dynamics of this agronomically important gene family.

Soybean domestication: the origin, genetic architecture and molecular bases.

Domestication provides an important model for the study of evolution, and information learned from domestication research aids in the continued improvement of crop species. Recent progress in de novo assembly and whole-genome resequencing of wild and cultivated soybean genomes, in addition to new archeological discoveries, sheds light on the origin of this important crop and provides a clearer view on the modes of artificial selection that drove soybean domestication and diversification. This novel genomic information enables the search for polymorphisms that underlie variation in agronomic traits and highlights genes that exhibit a signature of selection, leading to the identification of a number of candidate genes that may have played important roles in soybean domestication, diversification and improvement. These discoveries provide a novel point of comparison on the evolutionary bases of important agronomic traits among different crop species.

Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture.

Models for the control of above-ground plant architectures show how meristems can be programmed to be either shoots or flowers. Molecular, genetic, transgenic, and mathematical studies have greatly refined these models, suggesting that the phase of the shoot reflects different genes contributing to its repression of flowering, its vegetativeness ('veg'), before activators promote flower development. Key elements of how the repressor of flowering and shoot meristem gene TFL1 acts have now been tested, by changing its spatiotemporal pattern. It is shown that TFL1 can act outside of its normal expression domain in leaf primordia or floral meristems to repress flower identity. These data show how the timing and spatial pattern of TFL1 expression affect overall plant architecture. This reveals that the underlying pattern of TFL1 interactors is complex and that they may be spatially more widespread than TFL1 itself, which is confined to shoots. However, the data show that while TFL1 and floral genes can both act and compete in the same meristem, it appears that the main shoot meristem is more sensitive to TFL1 rather than floral genes. This spatial analysis therefore reveals how a difference in response helps maintain the 'veg' state of the shoot meristem.

Clarifying the Temporal Dynamics of the Circadian Clock and Flowering Gene Network Using Overexpression and Targeted Mutagenesis of Soybean EARLY FLOWERING 3-1 ( GmELF3-1 ).

With progressing climate fluctuations, an understanding of the molecular mechanisms of crop plants that regulate their flowering responses to environments is crucial. To achieve this goal, we aimed at clarifying the gene regulatory networks among the circadian clock and flowering genes in soybean ( Glycine max ). Based on our network inference approach , we hypothesize that GmELF3-1 , one of the Evening Complex (EC) gene homologs in soybean's circadian clock, may have an integrative role in transcriptional regulation of the circadian clock and flowering gene network. In this study, we verify GmELF3-1 ' s regulatory roles in its potential downstream genes by modulating the activity of GmELF3-1 using overexpression and CRISPR-Cas9 in soybean protoplasts. Our results indicate that GmELF3-1 may control the expression of the PRR genes in the circadian clock and the flowering gene GmCOL1a .

Elucidating the Temporal Patterns of Gene Expression in the Inferred Regulatory Interactions of GmCOL1b in Glycine max.

The CONSTANS ( CO ) gene in Arabidopsis thaliana has a central role in photoperiodic regulation of flowering. However, the roles of CO genes in mediating flowering in soybeans ( Glycine max ) remain uncertain. We previously inferred regulatory interactions of a soybean CO homolog, GmCOL1b , using in-house RNA-seq data and the network inference algorithm package CausNet. Here, we identify potential GmCOL1b downstream genes and experimentally verify them by expressing GmCOL1b in soybean protoplast cells. Temporal expression patterns of these genes indicate the regulatory effects of GmCOL1b on the expression of the circadian clock genes GmLCL1 and GmLCL4 and the flowering regulator GmTEM1a .

Experimental Verification of Inferred Regulatory Interactions of EARLY FLOWERING 3 ( GmELF3-1 ) in Glycine max.

Understanding the roles of evening complex (EC) genes in the circadian clock of plants can inform how diurnal transcriptional loops in the clock gene network function to regulate key physiological and developmental events, including flowering transition. Gene regulatory interactions among soybean's circadian clock and flowering genes were inferred using time-series RNA-seq data and the network inference algorithmic package CausNet. In this study, we seek to clarify the inferred regulatory interactions of the EC gene GmELF3-1. A gene expression analysis using soybean protoplasts as a transient model indicated regulatory roles of GmELF3-1 in expression of selected flowering genes.

Population genomics of the Arabidopsis thaliana flowering time gene network.

The time to flowering is a key component of the life-history strategy of the model plant Arabidopsis thaliana that varies quantitatively among genotypes. A significant problem for evolutionary and ecological genetics is to understand how natural selection may operate on this ecologically significant trait. Here, we conduct a population genomic study of resequencing data from 52 genes in the flowering time network. McDonald-Kreitman tests of neutrality suggested a strong excess of amino acid polymorphism when pooling across loci. This excess of replacement polymorphism across the flowering time network and a skewed derived frequency spectrum toward rare alleles for both replacement and noncoding polymorphisms relative to synonymous changes is consistent with a large class of deleterious polymorphisms segregating in these genes. Assuming selective neutrality of synonymous changes, we estimate that approximately 30% of amino acid polymorphisms are deleterious. Evidence of adaptive substitution is less prominent in our analysis. The photoperiod regulatory gene, CO, and a gibberellic acid transcription factor, AtMYB33, show evidence of adaptive fixation of amino acid mutations. A test for extended haplotypes revealed no examples of flowering time alleles with haplotypes comparable in length to those associated with the null fri(Col) allele reported previously. This suggests that the FRI gene likely has a uniquely intense or recent history of selection among the flowering time genes considered here. Although there is some evidence for adaptive evolution in these life-history genes, it appears that slightly deleterious polymorphisms are a major component of natural molecular variation in the flowering time network of A. thaliana.

From graph topology to ODE models for gene regulatory networks.

A gene regulatory network can be described at a high level by a directed graph with signed edges, and at a more detailed level by a system of ordinary differential equations (ODEs). The former qualitatively models the causal regulatory interactions between ordered pairs of genes, while the latter quantitatively models the time-varying concentrations of mRNA and proteins. This paper clarifies the connection between the two types of models. We propose a property, called the constant sign property, for a general class of ODE models. The constant sign property characterizes the set of conditions (system parameters, external signals, or internal states) under which an ODE model is consistent with a signed, directed graph. If the constant sign property for an ODE model holds globally for all conditions, then the ODE model has a single signed, directed graph. If the constant sign property for an ODE model only holds locally, which may be more typical, then the ODE model corresponds to different graphs under different sets of conditions. In addition, two versions of constant sign property are given and a relationship between them is proved. As an example, the ODE models that capture the effect of cis-regulatory elements involving protein complex binding, based on the model in the GeneNetWeaver source code, are described in detail and shown to satisfy the global constant sign property with a unique consistent gene regulatory graph. Even a single gene regulatory graph is shown to have many ODE models of GeneNetWeaver type consistent with it due to combinatorial complexity and continuous parameters. Finally the question of how closely data generated by one ODE model can be fit by another ODE model is explored. It is observed that the fit is better if the two models come from the same graph.

LHY and CCA1 are partially redundant genes required to maintain circadian rhythms in Arabidopsis.

Several genes are known to regulate circadian rhythms in Arabidopsis, but the identity of the central oscillator has not been established. LHY and CCA1 are related MYB-like transcription factors proposed to be closely involved. Here we demonstrate that, as shown previously for CCA1, inactivation of LHY shortens the period of circadian rhythms in gene expression and leaf movements. By constructing lhy cca1-1 double mutants, we show that LHY and CCA1 are partially redundant and essential for the maintenance of circadian rhythms in constant light. Under light/dark cycles the lhy cca1-1 plants show dramatically earlier phases of expression of GI and TOC1, genes associated with the generation of circadian rhythms and the promotion of LHY and CCA1 expression. We conclude that LHY and CCA1 appear to be negative regulatory elements required for central oscillator function.

Time series experimental design under one-shot sampling: The importance of condition diversity.

Many biological data sets are prepared using one-shot sampling, in which each individual organism is sampled at most once. Time series therefore do not follow trajectories of individuals over time. However, samples collected at different times from individuals grown under the same conditions share the same perturbations of the biological processes, and hence behave as surrogates for multiple samples from a single individual at different times. This implies the importance of growing individuals under multiple conditions if one-shot sampling is used. This paper models the condition effect explicitly by using condition-dependent nominal mRNA production amounts for each gene, it quantifies the performance of network structure estimators both analytically and numerically, and it illustrates the difficulty in network reconstruction under one-shot sampling when the condition effect is absent. A case study of an Arabidopsis circadian clock network model is also included.

Transcriptome-Enabled Network Inference Revealed the GmCOL1 Feed-Forward Loop and Its Roles in Photoperiodic Flowering of Soybean.

Photoperiodic flowering, a plant response to seasonal photoperiod changes in the control of reproductive transition, is an important agronomic trait that has been a central target of crop domestication and modern breeding programs. However, our understanding about the molecular mechanisms of photoperiodic flowering regulation in crop species is lagging behind. To better understand the regulatory gene networks controlling photoperiodic flowering of soybeans, we elucidated global gene expression patterns under different photoperiod regimes using the near isogenic lines (NILs) of maturity loci (E loci). Transcriptome signatures identified the unique roles of the E loci in photoperiodic flowering and a set of genes controlled by these loci. To elucidate the regulatory gene networks underlying photoperiodic flowering regulation, we developed the network inference algorithmic package CausNet that integrates sparse linear regression and Granger causality heuristics, with Gaussian approximation of bootstrapping to provide reliability scores for predicted regulatory interactions. Using the transcriptome data, CausNet inferred regulatory interactions among soybean flowering genes. Published reports in the literature provided empirical verification for several of CausNet's inferred regulatory interactions. We further confirmed the inferred regulatory roles of the flowering suppressors GmCOL1a and GmCOL1b using GmCOL1 RNAi transgenic soybean plants. Combinations of the alleles of GmCOL1 and the major maturity locus E1 demonstrated positive interaction between these genes, leading to enhanced suppression of flowering transition. Our work provides novel insights and testable hypotheses in the complex molecular mechanisms of photoperiodic flowering control in soybean and lays a framework for de novo prediction of biological networks controlling important agronomic traits in crops.

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

The FLOWERING LOCUS T/TERMINAL FLOWER 1 Gene Family: Functional Evolution and Molecular Mechanisms.

In plant development, the flowering transition and inflorescence architecture are modulated by two homologous proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1). The florigen FT promotes the transition to reproductive development and flowering, while TFL1 represses this transition. Despite their importance to plant adaptation and crop improvement and their extensive study by the plant community, the molecular mechanisms controlling the opposing actions of FT and TFL1 have remained mysterious. Recent studies in multiple species have unveiled diverse roles of the FT/TFL1 gene family in developmental processes other than flowering regulation. In addition, the striking evolution of FT homologs into flowering repressors has occurred independently in several species during the evolution of flowering plants. These reports indicate that the FT/TFL1 gene family is a major target of evolution in nature. Here, we comprehensively survey the conserved and diverse functions of the FT/TFL1 gene family throughout the plant kingdom, summarize new findings regarding the unique evolution of FT in multiple species, and highlight recent work elucidating the molecular mechanisms of these proteins.

Characterization of the spermidine synthase-related gene family in Arabidopsis thaliana.

The Arabidopsis genome contains four genes that encode proteins similar to both spermidine synthase and spermine synthase of other organisms. Our previous study revealed that one of these genes, designated ACAULIS5 (ACL5), encodes spermine synthase and that its null mutation results in a severe defect in the elongation of stem internodes. Here we report the characterization of the other three genes, designated SPDS1, SPDS2 and SPDS3. Our results showed that SPDS1 and SPDS2 possess spermidine synthase activity in yeast spermidine synthase-deficient mutants, but the enzyme activity of SPDS3 remained to be determined. RNA gel blot analysis revealed that all of these genes are expressed in all plant organs but show different responses to exogenous plant hormones, suggesting that they are involved in different aspects of growth by modulating the contents of polyamines in plant cells.

Functional and evolutionary characterization of the CONSTANS gene family in short-day photoperiodic flowering in soybean.

CONSTANS (CO) plays a central role in photoperiodic flowering control of plants. However, much remains unknown about the function of the CO gene family in soybean and the molecular mechanisms underlying short-day photoperiodic flowering of soybean. We identified 26 CO homologs (GmCOLs) in the soybean genome, many of them previously unreported. Phylogenic analysis classified GmCOLs into three clades conserved among flowering plants. Two homeologous pairs in Clade I, GmCOL1a/GmCOL1b and GmCOL2a/GmCOL2b, showed the highest sequence similarity to Arabidopsis CO. The mRNA abundance of GmCOL1a and GmCOL1b exhibited a strong diurnal rhythm under flowering-inductive short days and peaked at dawn, which coincided with the rise of GmFT5a expression. In contrast, the mRNA abundance of GmCOL2a and GmCOL2b was extremely low. Our transgenic study demonstrated that GmCOL1a, GmCOL1b, GmCOL2a and GmCOL2b fully complemented the late flowering effect of the co-1 mutant in Arabidopsis. Together, these results indicate that GmCOL1a and GmCOL1b are potential inducers of flowering in soybean. Our data also indicate rapid regulatory divergence between GmCOL1a/GmCOL1b and GmCOL2a/GmCOL2b but conservation of their protein function. Dynamic evolution of GmCOL regulatory mechanisms may underlie the evolution of photoperiodic signaling in soybean.

Variation in Arabidopsis flowering time associated with cis-regulatory variation in CONSTANS.

The onset of flowering, the change from vegetative to reproductive development, is a major life history transition in flowering plants. Recent work suggests that mutations in cis-regulatory mutations should play critical roles in the evolution of this (as well as other) important adaptive traits, but thus far there has been little evidence that directly links regulatory mutations to evolutionary change at the species level. While several genes have previously been shown to affect natural variation in flowering time in Arabidopsis thaliana, most either show protein-coding changes and/or are found at low frequency (<5%). Here we identify and characterize natural variation in the cis-regulatory sequence in the transcription factor CONSTANS that underlies flowering time diversity in Arabidopsis. Mutation in this regulatory motif evolved recently and has spread to high frequency in Arabidopsis natural accessions, suggesting a role for these cis-regulatory changes in adaptive variation of flowering time.

Candidate gene association mapping of Arabidopsis flowering time.

The pathways responsible for flowering time in Arabidopsis thaliana comprise one of the best characterized genetic networks in plants. We harness this extensive molecular genetic knowledge to identify potential flowering time quantitative trait genes (QTGs) through candidate gene association mapping using 51 flowering time loci. We genotyped common single nucleotide polymorphisms (SNPs) at these genes in 275 A. thaliana accessions that were also phenotyped for flowering time and rosette leaf number in long and short days. Using structured association techniques, we find that haplotype-tagging SNPs in 27 flowering time genes show significant associations in various trait/environment combinations. After correction for multiple testing, between 2 and 10 genes remain significantly associated with flowering time, with CO arguably possessing the most promising associations. We also genotyped a subset of these flowering time gene SNPs in an independent recombinant inbred line population derived from the intercrossing of 19 accessions. Approximately one-third of significant polymorphisms that were associated with flowering time in the accessions and genotyped in the outbred population were replicated in both mapping populations, including SNPs at the CO, FLC, VIN3, PHYD, and GA1 loci, and coding region deletions at the FRI gene. We conservatively estimate that approximately 4-14% of known flowering time genes may harbor common alleles that contribute to natural variation in this life history trait.

The dwarf phenotype of the Arabidopsis acl5 mutant is suppressed by a mutation in an upstream ORF of a bHLH gene.

Loss-of-function mutants of the Arabidopsis thaliana ACAULIS 5 (ACL5) gene, which encodes spermine synthase, exhibit a severe dwarf phenotype. To elucidate the ACL5-mediated regulatory pathways of stem internode elongation, we isolated four suppressor of acaulis (sac) mutants that reverse the acl5 dwarf phenotype. Because these mutants do not rescue the dwarfism of known phytohormone-related mutants, the SAC genes appear to act specifically on the ACL5 pathways. We identify the gene responsible for the dominant sac51-d mutant, which almost completely suppresses the acl5 phenotype. sac51-d disrupts a short upstream open reading frame (uORF) of SAC51, which encodes a bHLH-type transcription factor. Our results indicate that premature termination of the uORF in sac51-d results in an increase in its own transcript level, probably as a result of an increased translation of the main ORF. We suggest a model in which ACL5 plays a role in the translational activation of SAC51, which may lead to the expression of a subset of genes required for stem elongation.

A single amino acid converts a repressor to an activator of flowering.

Homologous proteins occurring through gene duplication may give rise to novel functions through mutations affecting protein sequence or expression. Comparison of such homologues allows insight into how morphological traits evolve. However, it is often unclear which changes are key to determining new functions. To address these ideas, we have studied a system where two homologues have evolved clear and opposite functions in controlling a major developmental switch. In plants, flowering is a major developmental transition that is critical to reproductive success. Arabidopsis phosphatidylethanolamine-binding protein homologues TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) are key controllers of flowering, determining when and where flowers are made, but as opposing functions: TFL1 is a repressor, FT is an activator. We have uncovered a striking molecular basis for how these homologous proteins have diverged. Although <60% identical, we have shown that swapping a single amino acid is sufficient to convert TFL1 to FT function and vice versa. Therefore, these key residues may have strongly contributed to the selection of these important functions over plant evolution. Further, our results suggest that TFL1 and FT are highly conserved in biochemical function and that they act as repressors or activators of flowering through discrimination of structurally related interactors by a single residue.