The alleles at the E1 locus impact the expression pattern of two soybean FT-like genes shown to induce flowering in Arabidopsis

A small gene family of phosphatidyl ethanolamine-binding proteins (PEBP) has been shown to function as key regulators in flowering; in Arabidopsis thaliana the FT protein promotes flowering whilst the closely related TFL1 protein represses flowering. Control of flowering time in soybean [Glycine max (L.) Merrill] is important for geographic adaptation and maximizing yield. Soybean breeders have identified a series of loci, the E-genes, that control photoperiod-mediated flowering time, yet how these loci control flowering is poorly understood. The objectives of this study were to evaluate the expression of GmFT-like genes in the E1 near-isogenic line (NIL) background. Of the 20 closely related PEBP proteins in the soybean genome, ten are similar to the Arabidopsis FT protein. Expression analysis of these ten GmFT-like genes confirmed that only two are detectable in the conditions tested. Further analysis of these two genes in the E1 NILs grown under short-day (SD) and long-day (LD) conditions showed a diurnal expression and tissue specificity expression commensurate with soybean flowering time under SD and LD conditions, suggesting that these were good candidates for flowering induction in soybean. Arabidopsis ft mutant lines flowered early when transformed with the two soybean genes, suggesting that the soybean genes can complement the Arabidopsis FT function. Flowering time in E1 NILs is consistent with the differential expression of the two GmFT-like genes under SD and LD conditions, suggesting that the E1 locus, at least in part, impacts time to flowering through the regulation of soybean FT expression.     

Soybean adaption to high-latitude regions is associated with natural variations of GmFT2b,an ortholog of FLOWERING LOCUS T

Day length has an important influence on flowering and growth habit in many plant species. In crops such as soybean, photoperiod sensitivity determines the geographical range over which a given cultivar can grow and flower. The soybean genome contains ~10 genes homologous to FT, a central regulator of flowering from Arabidopsis thaliana. However, the precise roles of these soybean FTs are not clearly. Here we show that one such gene, GmFT2b, promotes flowering under long-days (LDs). Overexpression of GmFT2b upregulates expression of flowering-related genes which are important in regulating flowering time. We propose a ‘weight’ model for soybean flowering under short-day (SD) and LD conditions. Furthermore, we examine GmFT2b sequences in 195 soybean cultivars, as well as flowering phenotypes, geographical distributions and maturity groups. We found that Hap3, a major GmFT2b haplotype, is associated with significantly earlier flowering at higher latitudes. We anticipate our assay to provide important resources for the genetic improvement of soybean, including new germplasm for soybean breeding, and also increase our understanding of functional diversity in the soybean FT gene family.     

Diurnal Expression Pattern,Allelic Variation,and Association Analysis Reveal Functional Features of the E1 Gene in Control of Photoperiodic Flowering in Soybean

Although four maturity genes, E1 to E4, in soybean have been successfully cloned, their functional mechanisms and the regulatory network of photoperiodic flowering remain to be elucidated. In this study, we investigated how the diurnal expression pattern of the E1 gene is related to photoperiodic length; and to what extent allelic variation in the B3-like domain of the E1 gene is associated with flowering time phenotype. The bimodal expression of the E1 gene peaked first at around 2 hours after dawn in long-day condition. The basal expression level of E1 was enhanced by the long light phase, and decreased by duration of dark. We identified a 5bp (3 SNP and 2-bp deletion) mutation, referred to an e1-b3a, which occurs in the middle of B3 domain of the E1 gene in the early flowering cultivar Yanhuang 3. Subcellular localization analysis showed that the putative truncated e1-b3a protein was predominately distributed in nuclei, indicating the distribution pattern of e1-b3a was similar to that of E1, but not to that of e1-as. Furthermore, genetic analysis demonstrated allelic variations at the E1 locus significantly underlay flowering time in three F₂ populations. Taken together, we can conclude the legume specific E1 gene confers some special features in photoperiodic control of flowering in soybean. Further characterization of the E1 gene will extend our understanding of the soybean flowering pathway in soybean.     

Natural variation in GmGBP1 promoter affects photoperiod control of flowering time and maturity in soybean

The present study screened for polymorphisms in coding and non‐coding regions of the GmGBP1 gene in 278 soybean accessions with variable maturity and growth habit characteristics under natural field conditions in three different latitudes in China. The results showed that the promoter region was highly diversified compared with the coding sequence of GmGBP1. Five polymorphisms and four haplotypes were closely related to soybean flowering time and maturity through association and linkage disequilibrium analyses. Varieties with the polymorphisms SNP_‐796G, SNP_‐770G, SNP_‐307T, InDel_‐242normal, SNP_353A, or haplotypes Hap‐3 and Hap‐4 showed earlier flowering time and maturity in different environments. The shorter growth period might be largely due to higher GmGBP1 expression levels in soybean that were caused by the TCT‐motif with SNP_‐796G in the promoter. In contrast, the lower expression level of GmGBP1 in soybean caused by RNAi interference of GmGBP1 resulted in a longer growth period under different day lengths. Furthermore, the gene interference of GmGBP1 also caused a reduction in photoperiod response sensitivity (PRS) before flowering in soybean. RNA‐seq analysis on GmGBP1 underexpression in soybean showed that 94 and 30 predicted genes were significantly upregulated and downregulated, respectively. Of these, the diurnal photoperiod‐specific expression pattern of three significant flowering time genes GmFT2a, GmFT5a, and GmFULc also showed constantly lower mRNA levels in GmGBP1‐i soybean than in wild type, especially under short day conditions. Together, the results showed that GmGBP1 functioned as a positive regulator upstream of GmFT2a and GmFT5a to activate the expression of GmFULc to promote flowering on short days.     

A map-based cloning strategy employing a residual heterozygous line reveals that the GIGANTEA gene is involved in soybean maturity and flowering

Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In soybean (Glycine max), a flowering quantitative trait locus, FT2, corresponding to the maturity locus E2, was detected in recombinant inbred lines (RILs) derived from the varieties "Misuzudaizu" (ft2/ft2; JP28856) and "Moshidou Gong 503" (FT2/FT2; JP27603). A map-based cloning strategy using the progeny of a residual heterozygous line (RHL) from the RIL was employed to isolate the gene responsible for this quantitative trait locus. A GIGANTEA ortholog, GmGIa (Glyma10g36600), was identified as a candidate gene. A common premature stop codon at the 10th exon was present in the Misuzudaizu allele and in other near isogenic lines (NILs) originating from Harosoy (e2/e2; PI548573). Furthermore, a mutant line harboring another premature stop codon showed an earlier flowering phenotype than the original variety, Bay (E2/E2; PI553043). The e2/e2 genotype exhibited elevated expression of GmFT2a, one of the florigen genes that leads to early flowering. The effects of the E2 allele on flowering time were similar among NILs and constant under high (43°N) and middle (36°N) latitudinal regions in Japan. These results indicate that GmGIa is the gene responsible for the E2 locus and that a null mutation in GmGIa may contribute to the geographic adaptation of soybean.     

Overexpression of the Gm<Emphasis Type="Italic">GAL2</Emphasis> Gene Accelerates Flowering in <Emphasis Type="BoldItalic">Arabidopsis</Emphasis>

A soybean MADS box gene GmGAL2 (Glycine max AGAMOUS Like 2), a homolog of AGL11/STK, was investigated in transgenic Arabidopsis lines. Ectopic expression of GmGAL2 in Arabidopsis enhanced flowering, under both long-day and short-day conditions, by promoting expression of key flowering genes, CONSTANS (CO) and FLOWERING LOCUS T (FT), and lowering expression of floral inhibiter FLOWERING LOCUS C (FLC). Moreover, frequency of silique pod set was also lower in transgenic compared to control Arabidopsis plants. RT-PCR results revealed that GmGAL2 was primarily expressed in the flowers and pods of soybean plants, GmGAL2 expressed higher in SD than LD in soybean.     

The differential expression of HvCO9, a member of the CONSTANS-like gene family, contributes to the control of flowering under short-day conditions in barley

HvCO9 was characterized to elucidate the barley flowering control mechanisms and to investigate the functional diversification of the barley CONSTANS-like (CO-like) genes in flowering. HvCO9 was located on the same chromosome, 1HL, as Ppd-H2 (HvFT3), which is a positive regulator of short-day (SD) flowering. A phylogenetic analysis showed that HvCO9 was located on the same branch of the CO-like gene tree as rice Ghd7 and the barley and wheat VRN2 genes, which are all negative regulators of flowering. High level HvCO9 expressions were observed under SD conditions, whereas its expression levels were quite low under long-day (LD) conditions. HvCO9 expression correlated with HvFT1 and HvFT2 expression under SD conditions, although no clear effect of HvCO9 on HvFT3 expression, or vice versa, under SD conditions was observed. The over-expression of HvCO9 in rice plants produced a remarkable delay in flowering. In transgenic rice, the expression levels of the flowering-related Ehd1 gene, which is a target gene of Ghd7, and its downstream genes were suppressed, causing a delay in flowering. These results suggest that HvCO9 may act as a negative regulator of flowering under non-inductive SD conditions in barley; this activity is similar to that of rice Ghd7 under non-inductive LD conditions, but the functional targets of these genes may be different. Our results indicate that barley has developed its own pathways to control flowering by using homologous genes with modifications for the timing of expression. Further, it is hypothesized that each pathway may target different genes after gene duplication or species diversification.     

Flowering and dwarfism induced by DNA demethylation in Pharbitis nil

Flowering and dwarfism induced by 5‐azacytidine and zebularine, which both cause DNA demethylation, were studied in a short‐day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non‐inductive long‐day (LD) conditions. The flower‐inducing effect of 5‐azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non‐inductive photoperiodic conditions. These results suggest that the flowering‐related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A₃ but not by t‐zeatin or kinetin. The change in the genome‐wide methylation state was examined by methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD‐specific fragments were extracted from the gel of the MS‐AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins.     

Natural variation and CRISPR/Cas9‐mediated mutation in GmPRR37 affect photoperiodic flowering and contribute to regional adaptation of soybean

Flowering time is a critical determinant of the geographic distribution and regional adaptability of soybean (Glycine max) and is strongly regulated by photoperiod and temperature. In this study, quantitative trait locus (QTL) mapping and subsequent candidate gene analysis revealed that GmPRR37, encoding a pseudo‐response regulator protein, is responsible for the major QTL qFT12‐2, which was identified from a population of 308 recombinant inbred lines (RILs) derived from a cross between a very late‐flowering soybean cultivar, ‘Zigongdongdou (ZGDD)’, and an extremely early‐flowering cultivar, ‘Heihe27 (HH27)’, in multiple environments. Comparative analysis of parental sequencing data confirmed that HH27 contains a non‐sense mutation that causes the loss of the CCT domain in the GmPRR37 protein. CRISPR/Cas9‐induced Gmprr37‐ZGDD mutants in soybean exhibited early flowering under natural long‐day (NLD) conditions. Overexpression of GmPRR37 significantly delayed the flowering of transgenic soybean plants compared with wild‐type under long photoperiod conditions. In addition, both the knockout and overexpression of GmPRR37 in soybean showed no significant phenotypic alterations in flowering time under short‐day (SD) conditions. Furthermore, GmPRR37 down‐regulated the expression of the flowering‐promoting FT homologues GmFT2a and GmFT5a, and up‐regulated flowering‐inhibiting FT homologue GmFT1a expression under long‐day (LD) conditions. We analysed haplotypes of GmPRR37 among 180 cultivars collected across China and found natural Gmprr37 mutants flower earlier and enable soybean to be cultivated at higher latitudes. This study demonstrates that GmPRR37 controls soybean photoperiodic flowering and provides opportunities to breed optimized cultivars with adaptation to specific regions and farming systems.     

Regulation of flowering time by Arabidopsis MSI1

The transition to flowering is tightly controlled by endogenous programs and environmental signals. We found that MSI1 is a novel flowering-time gene in Arabidopsis. Both partially complemented msi1 mutants and MSI1 antisense plants were late flowering, whereas ectopic expression of MSI1 accelerated flowering. Physiological experiments revealed that MSI1 is similar to genes from the autonomous promotion of flowering pathway. Expression of most known flowering-time genes did not depend on MSI1, but the induction of SOC1 was delayed in partially complemented msi1 mutants. Delayed activation of SOC1 is often caused by increased expression of the floral repressor FLC. However, MSI1 function is independent of FLC. MSI1 is needed to establish epigenetic H3K4 di-methylation and H3K9 acetylation marks in SOC1 chromatin. The presence of these modifications correlates with the high levels of SOC1 expression that induce flowering in Arabidopsis. Together, the control of flowering time depends on epigenetic mechanisms for the correct expression of not only the floral repressor FLC, but also the floral activator SOC1.     

In situ expression of the GmNMH7 gene is photoperiod-dependent in a unique soybean (Glycine max [L.] Merr.) flowering reversion system

Soybean is a short-day plant and its flowering process can be reversed when switching from short-day to long-day conditions. Flowering reversion provides a useful system to study the flowering process in both forward and backward directions. In this study, we optimized a soybean flowering reversion system using a photoperiod-sensitive cultivar Zigongdongdou. Three types of terminal structures were found during flowering reversion: reversed terminal raceme (RTR), short terminal raceme (STR), and vegetative terminal (VT). The relative frequency of these terminal structures during flowering reversion under long day was dependent on the duration of the prior short day (SD) pretreatment. This process is phytochrome dependent and young plants were more susceptible to flowering reversion. Leaf removal increased the minimal SD period needed for the induction of STR. To demonstrate the application of this system, we studied the patterns of in situ expression of the GmNMH7 gene during flowering development and reversion. NMH7 family members encode MADS-box proteins and are unique in legume families since their expression can be detected in both developing flowers and nodules. In situ hybridization experiments using plants grown under different photoperiod cycles provided several lines of evidence supporting a close relationship between GmNMH7 gene expression and floral development in soybean. Furthermore, it seems that GmNMH7 may participate in flower development at different stages. Interestingly, the expression pattern of GmNMH7 in root nodules was also found to be regulated by photoperiod. These results support the notion that the photoperiod sensitive GmNMH7 gene may play multiple roles in growth and development in soybean.C. Wu and Q. Ma contributed equally to this work.     

The circadian clock‐associated gene gigantea1 affects maize developmental transitions

The circadian clock is an internal timing mechanism that allows plants to make developmental decisions in accordance with environmental conditions. In model plants, circadian clock‐associated gigantea (gi) genes are directly involved in control of growth and developmental transitions. The maize gigantea1 (gi1) gene is the more highly expressed of the two gi homeologs, and its function is uncharacterized. To understand the role of gi1 in the regulatory networks of the maize circadian clock system, gi1 mutants were evaluated for changes in flowering time, phase change and growth control. When grown in long‐day (LD) photoperiods, gi1 mutants flowered earlier than non‐mutant plants, but this difference was not apparent in short‐day (SD) photoperiods. Therefore, gi1 participates in a pathway that suppresses flowering in LD photoperiods, but not in SD. Part of the underlying cause of early flowering was up‐regulated expression of the FT‐like floral activator gene zea mays centroradialis8 (zcn8) and the CONSTANS‐like flowering regulatory gene constans of zea mays1 (conz1). gi1 mutants also underwent vegetative phase change earlier and grew taller than non‐mutant plants. These findings indicate gi1 has a repressive function in multiple regulatory pathways that govern maize growth and development.     

Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas9 contributes for expanding the regional adaptability of soybean

Flowering time is a key agronomic trait that directly influences the successful adaptation of soybean (Glycine max) to diverse latitudes and farming systems. GmFT2a and GmFT5a have been extensively identified as flowering activators and integrators in soybean. Here, we identified two quantitative trait loci (QTLs) regions harbouring GmFT2a and GmFT5a, respectively, associated with different genetic effects on flowering under different photoperiods. We analysed the flowering time of transgenic plants overexpressing GmFT2a or GmFT5a, ft2a mutants, ft5a mutants and ft2aft5a double mutants under long‐day (LD) and short‐day (SD) conditions. We confirmed that GmFT2a and GmFT5a are not redundant, they collectively regulate flowering time, and the effect of GmFT2a is more prominent than that of GmFT5a under SD conditions whereas GmFT5a has more significant effects than GmFT2a under LD conditions. GmFT5a, not GmFT2a, was essential for soybean to adapt to high latitude regions. The ft2aft5a double mutants showed late flowering by about 31.3 days under SD conditions and produced significantly increased numbers of pods and seeds per plant compared to the wild type. We speculate that these mutants may have enormous yield potential for the tropics. In addition, we examined the sequences of these two loci in 202 soybean accessions and investigated the flowering phenotypes, geographical distributions and maturity groups within major haplotypes. These results will contribute to soybean breeding and regional adaptability.