The nuclear pore Y-complex functions as a platform for transcriptional regulation of FLOWERING LOCUS C in Arabidopsis.

The nuclear pore complex (NPC) has multiple functions beyond the nucleo-cytoplasmic transport of large molecules. Subnuclear compartmentalization of chromatin is critical for gene expression in animals and yeast. However, the mechanism by which the NPC regulates gene expression is poorly understood in plants. Here we report that the Y-complex (Nup107-160 complex, a subcomplex of the NPC) self-maintains its nucleoporin homeostasis and modulates FLOWERING LOCUS C (FLC) transcription via changing histone modifications at this locus. We show that Y-complex nucleoporins are intimately associated with FLC chromatin through their interactions with histone H2A at the nuclear membrane. Fluorescence in situ hybridization assays revealed that Nup96, a Y-complex nucleoporin, enhances FLC positioning at the nuclear periphery. Nup96 interacted with HISTONE DEACETYLASE 6 (HDA6), a key repressor of FLC expression via histone modification, at the nuclear membrane to attenuate HDA6-catalyzed deposition at the FLC locus and change histone modifications. Moreover, we demonstrate that Y-complex nucleoporins interact with RNA polymerase II to increase its occupancy at the FLC locus, facilitating transcription. Collectively, our findings identify an attractive mechanism for the Y-complex in regulating FLC expression via tethering the locus at the nuclear periphery and altering its histone modification.

Photoperiod-responsive changes in chromatin accessibility in phloem companion and epidermis cells of Arabidopsis leaves.

Photoperiod plays a key role in controlling the phase transition from vegetative to reproductive growth in flowering plants. Leaves are the major organs perceiving day-length signals, but how specific leaf cell types respond to photoperiod remains unknown. We integrated photoperiod-responsive chromatin accessibility and transcriptome data in leaf epidermis and vascular companion cells of Arabidopsis thaliana by combining isolation of nuclei tagged in specific cell/tissue types with assay for transposase-accessible chromatin using sequencing and RNA-sequencing. Despite a large overlap, vasculature and epidermis cells responded differently. Long-day predominantly induced accessible chromatin regions (ACRs); in the vasculature, more ACRs were induced and these were located at more distal gene regions, compared with the epidermis. Vascular ACRs induced by long days were highly enriched in binding sites for flowering-related transcription factors. Among the highly ranked genes (based on chromatin and expression signatures in the vasculature), we identified TREHALOSE-PHOSPHATASE/SYNTHASE 9 (TPS9) as a flowering activator, as shown by the late flowering phenotypes of T-DNA insertion mutants and transgenic lines with phloem-specific knockdown of TPS9. Our cell-type-specific analysis sheds light on how the long-day photoperiod stimulus impacts chromatin accessibility in a tissue-specific manner to regulate plant development.

Nup96 and HOS1 Are Mutually Stabilized and Gate CONSTANS Protein Level, Conferring Long-Day Photoperiodic Flowering Regulation in Arabidopsis.

The nuclear pore complex profoundly affects the timing of flowering; however, the underlying mechanisms are poorly understood. Here, we report that Nucleoporin96 (Nup96) acts as a negative regulator of long-day photoperiodic flowering in Arabidopsis (Arabidopsis thaliana). Through multiple approaches, we identified the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (HOS1) and demonstrated its interaction in vivo with Nup96. Nup96 and HOS1 mainly localize and interact on the nuclear membrane. Loss of function of Nup96 leads to destruction of HOS1 proteins without a change in their mRNA abundance, which results in overaccumulation of the key activator of long-day photoperiodic flowering, CONSTANS (CO) proteins, as previously reported in hos1 mutants. Unexpectedly, mutation of HOS1 strikingly diminishes Nup96 protein level, suggesting that Nup96 and HOS1 are mutually stabilized and thus form a novel repressive module that regulates CO protein turnover. Therefore, the nup96 and hos1 single and nup96 hos1 double mutants have highly similar early-flowering phenotypes and overlapping transcriptome changes. Together, this study reveals a repression mechanism in which the Nup96-HOS1 repressive module gates the level of CO proteins and thereby prevents precocious flowering in long-day conditions.

Novel Seed Size: A Novel Seed-Developing Gene in Glycine max.

Soybean-seed development is controlled in multiple ways, as in many known regulating genes. Here, we identify a novel gene, Novel Seed Size (NSS), involved in seed development, by analyzing a T-DNA mutant (S006). The S006 mutant is a random mutant of the GmFTL4pro:GUS transgenic line, with phenotypes with small and brown seed coats. An analysis of the metabolomics and transcriptome combined with RT-qPCR in the S006 seeds revealed that the brown coat may result from the increased expression of chalcone synthase 7/8 genes, while the down-regulated expression of NSS leads to small seed size. The seed phenotypes and a microscopic observation of the seed-coat integument cells in a CRISPR/Cas9-edited mutant nss1 confirmed that the NSS gene conferred small phenotypes of the S006 seeds. As mentioned in an annotation on the Phytozome website, NSS encodes a potential DNA helicase RuvA subunit, and no such genes were previously reported to be involved in seed development. Therefore, we identify a novel gene in a new pathway controlling seed development in soybeans.

An Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method in a Soybean Root Biology Study.

The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by Agrobacterium rhizogenes is widely applied in studying root-specific processes, nodulation, biochemical and molecular functions of genes of interest, gene editing efficiency of CRISPR/Cas9, and biological reactors and producers. Therefore, many laboratories have developed unique protocols to obtain hairy roots in composite plants composed of transgenic roots and wild-type shoots. However, these protocols still suffer from the shortcomings of low efficiency and time, space, and cost consumption. To address this issue, we developed a new protocol efficient regeneration and transformation of hairy roots (eR&T) in soybean, by integrating and optimizing the main current methods to achieve high efficiency in both hairy root regeneration and transformation within a shorter period and using less space. By this eR&T method, we obtained 100% regeneration of hairy roots for all explants, with an average 63.7% of transformation frequency, which promoted the simultaneous and comparative analysis of the function of several genes. The eR&T was experimentally verified Promoter:GUS reporters, protein subcellular localization, and CRISPR/Cas9 gene editing experiments. Employing this approach, we identified several novel potential regulators of nodulation, and nucleoporins of the Nup107-160 sub-complex, which showed development-dependent and tissue-dependent expression patterns, indicating their important roles in nodulation in soybean. Thus, the new eR&T method is an efficient and economical approach for investigating not only root and nodule biology, but also gene function.

Spatial Divergence of PHR-PHT1 Modules Maintains Phosphorus Homeostasis in Soybean Nodules.

Maintaining phosphorus (Pi) homeostasis in nodules is the key to nodule development and nitrogen fixation, an important source of nitrogen for agriculture and ecosystems. PHOSPHATE-TRANSPORTER1 (PHT1) and its regulator PHOSPHATE-STARVATION-RESPONSE1 (PHR1), which constitute the PHR1-PHT1 module, play important roles in maintaining Pi homeostasis in different organs. However, the PHR1-PHT1 module and its functions in nodules remain unknown. We identified one PHT1 (GmPHT1;11) and four PHR1 (GmPHR1) homologs in soybean (Glycine max) plants, which displayed specific expression patterns in different tissues in nodules, similar to previously reported GmPHT1;1 and GmPHT1;4 Through the integration of different approaches, GmPHR-GmPHT1 modules were confirmed. Combining our results and previous reports, we established multiple GmPHR-GmPHT1 modules acting in the infected or noninfected tissues in nodules. A single GmPHR had more than one GmPHT1 target, and vice versa. Therefore, overlapping and cross-talking modules monitored the wave of available Pi to maintain Pi homeostasis in nodules, which sequentially regulated nodule initiation and development. High levels of GmPHT1;11 enhanced Pi accumulation in nodules, increased nodule size, but decreased nodule number. Nitrogenase activity was also enhanced by GmPHT1;11 Our findings uncover GmPHR-GmPHT1 modules in nodules, which expands our understanding of the mechanism of maintaining Pi homeostasis in soybean plants.

Two Nucleoporin98 homologous genes jointly participate in the regulation of starch degradation to repress senescence in Arabidopsis.

BACKGROUND: Starch is synthesized during daylight for temporary storage in leaves and then degraded during the subsequent night to support plant growth and development. Impairment of starch degradation leads to stunted growth, even senescence and death. The nuclear pore complex is involved in many cellular processes, but its relationship with starch degradation has been unclear until now. We previously identified that two Nucleoporin98 genes (Nup98a and Nup98b) redundantly regulate flowering via the CONSTANS (CO)-independent pathway in Arabidopsis thaliana. The double mutant also shows severe senescence phenotypes. RESULTS: We find that Nucleoporin 98 participates in the regulation of sugar metabolism in leaves and is also involved in senescence regulation in Arabidopsis. We show that Nup98a and Nup98b function redundantly at different stages of starch degradation. The nup98a-1 nup98b-1 double mutant accumulates more starch, showing a severe early senescence phenotype compared to wild type plants. The expression of marker genes related to starch degradation is impaired in the nup98a-1 nup98b-1 double mutant, and marker genes of carbon starvation and senescence express their products earlier and in higher abundance than in wild type plants, suggesting that abnormalities in energy metabolism are the main cause of senescence in the double mutant. Addition of sucrose to the growth medium rescues early senescence phenotypes of the nup98a-1 nup98b-1 mutant. CONCLUSIONS: Our results provide evidence for a novel role of the nuclear pore complex in energy metabolism related to growth and development, in which Nup98 functions in starch degradation to control growth regulation in Arabidopsis.

Light- and temperature-entrainable circadian clock in soybean development.

In plants, the spatiotemporal expression of circadian oscillators provides adaptive advantages in diverse species. However, the molecular basis of circadian clock in soybean is not known. In this study, we used soybean hairy roots expression system to monitor endogenous circadian rhythms and the sensitivity of circadian clock to environmental stimuli. We discovered in experiments with constant light and temperature conditions that the promoters of clock genes GmLCLb2 and GmPRR9b1 drive a self-sustained, robust oscillation of about 24-h in soybean hairy roots. Moreover, we demonstrate that circadian clock is entrainable by ambient light/dark or temperature cycles. Specifically, we show that light and cold temperature pulses can induce phase shifts of circadian rhythm, and we found that the magnitude and direction of phase responses depends on the specific time of these two zeitgeber stimuli. We obtained a quadruple mutant lacking the soybean gene GmLCLa1, LCLa2, LCLb1, and LCLb2 using CRISPR, and found that loss-of-function of these four GmLCL orthologs leads to an extreme short-period circadian rhythm and late-flowering phenotype in transgenic soybean. Our study establishes that the morning-phased GmLCLs genes act constitutively to maintain circadian rhythmicity and demonstrates that their absence delays the transition from vegetative growth to reproductive development.

Correction to: Nucleoporin Nup98 participates in flowering regulation in a CONSTANS-independent mode.

The authors signal an error in Fig. 1b which does not show the correct set of plants and should be replaced with the included new figure.

Nucleoporin Nup98 participates in flowering regulation in a CONSTANS-independent mode.

KEY MESSAGE: Two redundant nucleoporin genes Nup98a and Nup98b bypass the CO-check point in photoperiodic signaling and integrated signals from multiple pathways to directly target FT for flowering control in Arabidopsis. Flowering regulation is an important and widely studied plant development event. Even though nucleoporin Nup98 has been proven to play pivotal roles in the growth and development of mammalian cells and yeast, it is still unknown if Nup98 participates in flowering control in plants. In this study, we investigated the function of two Nup98 homologs, Nup98a and Nup98b, in flowering regulation in Arabidopsis. The results showed that Nup98a and Nup98b redundantly inhibit flowering through multiple pathways including clock, photoperiod, and age pathways. Single mutants of nup98a and nup98b do not show any obvious abnormal phenotypes compared to wild-type plants; however, the nup98a1 nup98b1 double mutant displays early flowering. Significantly, Nup98a/Nup98b gate flowering in a CONSTANS (CO)-independent mode. Therefore, Nup98a/Nup98b bypasses the CO checkpoint in photoperiodic signaling and integrated signals from multiple pathways to directly target FLOWERING LOCUS T (FT) for flowering control. In addition, our results provide a line of genetic evidence for uncoupling the mechanism of flowering and senescence at Nup98a/Nup98b genes in Arabidopsis, which are classically recognized as two coupled developmental events.

GmILPA1, Encoding an APC8-like Protein, Controls Leaf Petiole Angle in Soybean.

Leaf petiole angle (LPA) is an important plant architectural trait that affects canopy coverage, photosynthetic efficiency, and ultimately productivity in many legume crops. However, the genetic basis underlying this trait remains unclear. Here, we report the identification, isolation, and functional characterization of Glycine max Increased Leaf Petiole Angle1 (GmILPA1), a gene encoding an APC8-like protein, which is a subunit of the anaphase-promoting complex/cyclosome in soybean (Glycine max). A gamma ray-induced deletion of a fragment involving the fourth exon of GmILPA1 and its flanking sequences led to extension of the third exon and formation of, to our knowledge, a novel 3'UTR from intronic and intergenic sequences. Such changes are responsible for enlarged LPAs that are associated with reduced motor cell proliferation in the Gmilpa1 mutant. GmILPA1 is mainly expressed in the basal cells of leaf primordia and appears to function by promoting cell growth and division of the pulvinus that is critical for its establishment. GmILPA1 directly interacts with GmAPC13a as part of the putative anaphase-promoting complex. GmILPA1 exhibits variable expression levels among varieties with different degrees of LPAs, and expression levels are correlated with the degrees of the LPAs. Together, these observations revealed a genetic mechanism modulating the plant petiole angle that could pave the way for modifying soybean plant architecture with optimized petiole angles for enhanced yield potential.

Two SUMO Proteases SUMO PROTEASE RELATED TO FERTILITY1 and 2 Are Required for Fertility in Arabidopsis.

In plants, the posttranslational modification small ubiquitin-like modifier (SUMO) is involved in regulating several important developmental and cellular processes, including flowering time control and responses to biotic and abiotic stresses. Here, we report two proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2, that regulate male and female gamete and embryo development and remove SUMO from proteins in vitro and in vivo. spf1 mutants exhibit abnormal floral structures and embryo development, while spf2 mutants exhibit largely a wild-type phenotype. However, spf1 spf2 double mutants exhibit severe abnormalities in microgametogenesis, megagametogenesis, and embryo development, suggesting that the two genes are functionally redundant. Mutation of SPF1 and SPF2 genes also results in misexpression of generative- and embryo-specific genes. In vitro, SPF1 and SPF2 process SUMO1 precursors into a mature form, and as expected in vivo, spf1 and spf2 mutants accumulate SUMO conjugates. Using a yeast two-hybrid screen, we identified EMBRYO SAC DEVELOPMENT ARREST9 (EDA9) as an SPF1-interacting protein. In vivo, we demonstrate that EDA9 is sumolyated and that, in spf1 mutants, EDA9-SUMO conjugates increase in abundance, demonstrating that EDA9 is a substrate of SPF1. Together, our results demonstrate that SPF1 and SPF2 are two SUMO proteases important for plant development in Arabidopsis (Arabidopsis thaliana).

Conserved CO-FT regulons contribute to the photoperiod flowering control in soybean.

BACKGROUND: CO and FT orthologs, belonging to the BBX and PEBP family, respectively, have important and conserved roles in the photoperiod regulation of flowering time in plants. Soybean genome experienced at least three rounds of whole genome duplications (WGDs), which resulted in multiple copies of about 75% of genes. Subsequent subfunctionalization is the main fate for paralogous gene pairs during the evolutionary process. RESULTS: The phylogenic relationships revealed that CO orthologs were widespread in the plant kingdom while FT orthologs were present only in angiosperms. Twenty-eight CO homologous genes and twenty-four FT homologous genes were gained in the soybean genome. Based on the collinear relationship, the soybean ancestral CO ortholog experienced three WGD events, but only two paralogous gene pairs (GmCOL1/2 and GmCOL5/13) survived in the modern soybean. The paralogous gene pairs, GmCOL1/2 or GmCOL5/13, showed similar expression patterns in pair but different between pairs, indicating that they functionally diverged. GmFTL1 to 7 were derived from the same ancestor prior to the whole genome triplication (WGT) event, and after the Legume WGD event the ancestor diverged into two branches, GmFTL3/5/7 and GmFTL1/2/4/6. GmFTL7 were truncated in the N-terminus compared to other FT-lineage genes, but ubiquitously expressed. Expressions of GmFTL1 to 6 were higher in leaves at the flowering stage than that at the seedling stage. GmFTL3 was expressed at the highest level in all tissues except roots at the seedling stage, and its circadian pattern was different from the other five ones. The transcript of GmFTL6 was highly accumulated in seedling roots. The circadian rhythms of GmCOL5/13 and GmFT1/2/4/5/6 were synchronized in a day, demonstrating the complicate relationship of CO-FT regulons in soybean leaves. Over-expression of GmCOL2 did not rescue the flowering phenotype of the Arabidopsis co mutant. However, ectopic expression of GmCOL5 did rescue the co mutant phenotype. All GmFTL1 to 6 showed flower-promoting activities in Arabidopsis. CONCLUSIONS: After three recent rounds of whole genome duplications in the soybean, the paralogous genes of CO-FT regulons showed subfunctionalization through expression divergence. Then, only GmCOL5/13 kept flowering-promoting activities, while GmFTL1 to 6 contributed to flowering control. Additionally, GmCOL5/13 and GmFT1/2/3/4/5/6 showed similar circadian expression profiles. Therefore, our results suggested that GmCOL5/13 and GmFT1/2/3/4/5/6 formed the complicate CO-FT regulons in the photoperiod regulation of flowering time in soybean.

BioVector, a flexible system for gene specific-expression in plants.

BACKGROUND: Functional genomic research always needs to assemble different DNA fragments into a binary vector, so as to express genes with different tags from various promoters with different levels. The cloning systems available bear similar disadvantages, such as promoters/tags are fixed on a binary vector, which is generally with low cloning efficiency and limited for cloning sites if a novel promoter/tag is in need. Therefore, it is difficult both to assemble a gene and a promoter together and to modify the vectors in hand. Another disadvantage is that a long spacer from recombination sites, which may be detrimental to the protein function, exists between a gene and a tag. Multiple GATEWAY system only resolves former problem at the expense of very low efficiency and expensive for multiple LR reaction. RESULTS: To improve efficiency and flexibility for constructing expression vectors, we developed a platform, BioVector, by combining classical restriction enzyme/ligase strategy with modern Gateway DNA recombination system. This system included a series of vectors for gene cloning, promoter cloning, and binary vector construction to meet various needs for plant functional genomic study. CONCLUSION: This BioVector platform makes it easy to construct any vectors to express a target gene from a specific promoter with desired intensity, and it is also waiting to be freely modified by researchers themselves for ongoing demands. This idea can also be transferred to the different fields including animal or yeast study.

The pattern of Phosphate transporter 1 genes evolutionary divergence in Glycine max L.

BACKGROUND: The Phosphate transporter 1 (PHT1) gene family has crucial roles in phosphate uptake, translocation, remobilization, and optimization of metabolic processes using of Pi. Gene duplications expand the size of gene families, and subfunctionalization of paralog gene pairs is a predominant tendency after gene duplications. To date, experimental evidence for the evolutionary relationships among different paralog gene pairs of a given gene family in soybean is limited. RESULTS: All potential Phosphate transporter 1 genes in Glycine max L. (GmPHT1) were systematically analyzed using both bioinformatics and experimentation. The soybean PHT1 genes originated from four distinct ancestors prior to the Gamma WGT and formed 7 paralog gene pairs and a singleton gene. Six of the paralog gene pairs underwent subfunctionalization, and while GmPHT1;4 paralog gene experienced pseudogenization. Examination of long-term evolutionary changes, six GmPHT1 paralog gene pairs diverged at multiple levels, in aspects of spatio-temporal expression patterns and/or quanta, phosphates affinity properties, subcellular localization, and responses to phosphorus stress. CONCLUSIONS: These characterized divergences occurred in tissue- and/or development-specific modes, or conditional modes. Moreover, they have synergistically shaped the evolutionary rate of GmPHT1 family, as well as maintained phosphorus homeostasis at cells and in the whole plant.

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.

[Advances on SUMO substrates in Arabidopsis].

SUMO (Small ubiquitin-related modifier) modification, one of the essential posttranslational modifications in eukaryotes, plays an important role in various cellular processes. This review summarized the recent progresses on SUMO substrates in Arabidopsis. We firstly described the pathway of SUMO conjugation, and then focused on screening for SUMO substrates, including the methods of identification and SUMO substrates identified. Finally, we classified these substrates on the basis of their subcellular localization, functions, and biological processes. It is expected to provide the basis for better understanding the roles of sumoylation of proteins in plants.

[Expression and functional analysis of SUA41gene in Arabidopsis thaliana].

In plants, multiple floral induced-pathways including photoperiod signaling, vernalization signaling, autonomous pathway, gibberellin signaling, and thermosensory signaling are well known to mediate signaling from different cues to confer flowering regulation. SUA41 (SUMO substrate 41) is a SUMO (Small ubiquitin modifier) substrate screened out in our laboratory. Previous reports indicate that the SUA41 gene is involved in autonomous pathway to regulate flowering time of Arabidopsis, but its mechanism remains to be elucidated. In this study, the spatiotemporal expression pattern for SUA41, responses of its mutant to environmental factors, and its regulation of mechanism of flowering time were investigated. The sua41 mutant flowered earlier than Col-0 at both normal temperature (22℃) and low temperature (16℃) under long day (LD) or short day (SD) conditions. In addition, the flowering times of sua41 had no significant difference between 22℃ and 16℃ conditions. Over-expression of SUA41 rescued the early flowering phenotype of the sua41 mutant. Expression of SUA41 was at similar levels in seedlings, roots, stems, leaves, flowers, or the samples at all developmental stages examined, suggesting that SUA41 is a constitutive expression gene. Expression of SUA41 mRNA was not responsive to GA treatment, but highly induced by low temperature and inhibited in fve and fca mutants defective in the thermosensory pathway. Compared with Col-0, the expression levels of FT and SOC1 increased, whereas the expression level of FLC mRNA decreased and CO expression was not significantly altered in the sua41 mutant. The results showed that the SUA41 gene plays a role in not only the autonomous pathway but also the thermosensory pathway to regulate flowering time of Arabidopsis.

Molecular cloning and functional analysis of one ZEITLUPE homolog GmZTL3 in soybean.

ZEITLUPE (ZTL) plays an important role in the control of flowering time and photomorpogenesis in Arabidopsis and is highly conserved throughout the plant kingdom. Here, we report the characterization of a soybean ZTL homolog GmZTL3 (Glycine max ZTL 3). The absorption spectrum of the recombinant GmZTL3 proteins indicates that it may be a UV/blue photoreceptor. The GmZTL3 expression is independent of diurnal cycles and varies in different tissues along with developmental stages. Before the unifoliolates open fully, GmZTL3 transcripts concentrate in the roots and hypocotyls, while at flowering GmZTL3 accumulates at higher abundance in stems and petioles. Furthermore, the GmZTL3 mRNA accumulates in all kinds of leaves before flowering and concentrates in maturation seeds. In Arabidopsis, the ectopic expression of GmZTL3 delays flowering, implicating GmZTL3 is an inhibitor of flowering induction. Our data indicate that GmZTL3 probably functions as a photoreceptor and plays a role in multiple developmental processes, including the control of flowering time.

Validation of reference genes for real-time quantitative PCR normalization in soybean developmental and germinating seeds.

Most of traditional reference genes chosen for real-time quantitative PCR normalization were assumed to be ubiquitously and constitutively expressed in vegetative tissues. However, seeds show distinct transcriptomes compared with the vegetative tissues. Therefore, there is a need for re-validation of reference genes in samples of seed development and germination, especially for soybean seeds. In this study, we aimed at identifying reference genes suitable for the quantification of gene expression level in soybean seeds. In order to identify the best reference genes for soybean seeds, 18 putative reference genes were tested with various methods in different seed samples. We combined the outputs of both geNorm and NormFinder to assess the expression stability of these genes. The reference genes identified as optimums for seed development were TUA5 and UKN2, whereas for seed germination they were novel reference genes Glyma05g37470 and Glyma08g28550. Furthermore, for total seed samples it was necessary to combine four genes of Glyma05g37470, Glyma08g28550, Glyma18g04130 and UKN2 [corrected] for normalization. Key message We identified several reference genes that stably expressed in soybean seed developmental and germinating processes.