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.

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.

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.

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.

Identification of a soybean MOTHER OF FT AND TFL1 homolog involved in regulation of seed germination.

Seed germination is an important event in the life cycle of seed plants, and is controlled by complex and coordinated genetic networks. Many genes involved in the regulation of this process have been identified in different plant species so far. Recent studies in both Arabidopsis and wheat have uncovered a new role of MOTHER OF FT AND TFL1 (MFT) in seed germination. Here, we reported a homolog of MFT in soybean (GmMFT) which strongly expressed in seeds. Detailed expression analysis showed that the mRNA level of GmMFT increased with seed development but declined during seed germination. The transcription of GmMFT also responded to exogenous application of ABA and GA3. Ectopic expression of GmMFT CDS in Arabidopsis moderately inhibited seed germination. All these evidences suggest that GmMFT may be a negative regulator of seed germination.

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.

Identification and molecular characterization of FKF1 and GI homologous genes in soybean.

In Arabidopsis, FKF1 (FLAVIN BINDING, KELCH REPEAT, F-BOX1) and GI (GIGANTEA) play important roles in flowering pathway through regulating daytime CO (CONSTANS) expression, and such a function is conserved across plants studied. But related reports are limited for soybean. In this study, we cloned FKF1 and GI homologs in soybean, and named as GmFKF1, GmFKF2, GmGI1, GmGI2, and GmGI3, respectively. GmGI1 had two alternative splicing forms, GmGI1α and GmGI1ß. GmFKF1/2 transcripts were diurnally regulated, with a peak at zeitgeber time 12 (ZT12) in long days and at ZT10 in short days. The diurnal phases between GmGIs transcript levels greatly differed. GmGI2 expression was regulated by both the circadian clock and photoperiod. But the rhythmic phases of GmGI1 and GmGI3 expression levels were mainly conferred by long days. GmFKFs shared similar spatio-temporal expression profiles with GmGIs in all of the tissue/organs in different developmental stages in both LD and SD. Both GmFKF and GmGI proteins were targeted to the nucleus. Yeast two hybrid assays showed GmFKF1/GmFKF2 interacted with GmGI1/GmGI2/GmCDF1 (CYCLING DOF FACTOR CDF1 homolog in soybean); and the LOV (Light, Oxygen, or Voltage) domain in GmFKF1/GmFKF2 played an important role in these interactions. N-terminus of GmGI2 was sufficient to mediate its interaction with GmCDF1. Interestingly, N-terminus not full of GmGI3 interacted with GmFKF1/GmFKF2/GmCDF1. Ectopic over-expression of the GmFKF1 or GmFKF2 in Arabidopsis enhanced flowering in SD. Collectively, GmFKF and GmGI in soybean had conserved functional domains at DNA sequence level, but specific characters at function level with their homologs in other plants.

The phytochrome gene family in soybean and a dominant negative effect of a soybean PHYA transgene on endogenous Arabidopsis PHYA.

KEY MESSAGE: The evolutionary origin of the phytochrome genes in soybean was analyzed. The expression profiles of PHYA paralogs were characterized. The heterologous expression of GmPHYA1 in Arabidopsis resulted in longer hypocotyls. The phytochromes (PHY) are a small family of red/far-red light photoreceptors which regulate a number of important developmental responses in plants. So far, the members of the PHY gene family in soybean (Glycine max) remain unclear and an understanding of each member's physiological functions is limited. Our present in silico analysis revealed that the soybean genome harbors four PHYA, two PHYB and two PHYE, totally four pairs of eight PHY loci. The phylogenetic analysis suggested that the four PHY paralogous pairs originated from the latest round of genome duplication (~13 million years ago) and the four copies of PHYA were remnants of the two rounds of genome duplication (~58 and ~13 million years ago). A possible evolutionary history of PHYA homologs in the three legume species (soybean, Medicago truncatula, and Lotus japonicus) was proposed and the fate of duplicate soybean PHYA genes following polyploidization was discussed. The expression profiles of a soybean PHYA paralogous pair (GmPHYA1 and GmPHYA2) showed that the transcript abundance was highest in the aerial organs of young plants. The physiological role of GmPHYA1 was explored by observing the de-etiolation phenotype of transgenic Arabidopsis plants constitutively expressing GmPHYA1. The GmPHYA1 protein interfered with the function of endogenous PHYA with respect to de-etiolation in a dominant negative manner when exogenously expressed in Arabidopsis. The elucidation of the PHY gene family members in soybean provide us with a general description and understanding of the photoreceptor gene family in this important crop plant.

Ectopic expression reveals a conserved PHYB homolog in soybean.

Phytochromes sense red/far-red light and trigger a cascade of physiological responses in plant. Here, a phytochrome B homolog, GmPHYB1, was amplified from the soybean genome, and its expression profiles were obtained for various parts of the plant and at various developmental stages. The gene was ectopically expressed in Arabidopsis thaliana, driven by CaMV 35S promoter, to study the physiological functions of the gene product. The overexpressors of GmPHYB1 behaved similarly to those of AtPHYB, but with some subtle differences with respect to the acceleration of flowering under short day conditions and the growth of the hypocotyl under certain light fluence rate. The results suggested that this soybean PHYB homolog was well conserved both at the level of sequence and physiological function.