Molecular breeding for improvement of photothermal adaptability in soybean.

Soybean (Glycine max (L.) Merr.) is a typical short-day and temperate crop that is sensitive to photoperiod and temperature. Responses of soybean to photothermal conditions determine plant growth and development, which affect its architecture, yield formation, and capacity for geographic adaptation. Flowering time, maturity, and other traits associated with photothermal adaptability are controlled by multiple major-effect and minor-effect genes and genotype-by-environment interactions. Genetic studies have identified at least 11 loci (E1-E4, E6-E11, and J) that participate in photoperiodic regulation of flowering time and maturity in soybean. Molecular cloning and characterization of major-effect flowering genes have clarified the photoperiod-dependent flowering pathway, in which the photoreceptor gene phytochrome A, circadian evening complex (EC) components, central flowering repressor E1, and FLOWERING LOCUS T family genes play key roles in regulation of flowering time, maturity, and adaptability to photothermal conditions. Here, we provide an overview of recent progress in genetic and molecular analysis of traits associated with photothermal adaptability, summarizing advances in molecular breeding practices and tools for improving these traits. Furthermore, we discuss methods for breeding soybean varieties with better adaptability to specific ecological regions, with emphasis on a novel strategy, the Potalaization model, which allows breeding of widely adapted soybean varieties through the use of multiple molecular tools in existing elite widely adapted varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01406-z.

Genomic Dissection and Diurnal Expression Analysis Reveal the Essential Roles of the PRR Gene Family in Geographical Adaptation of Soybean.

Pseudo-response regulator (PRR) family members serve as key components of the core clock of the circadian clock, and play important roles in photoperiodic flowering, stress tolerance, growth, and the development of plants. In this study, 14 soybean PRR genes were identified, and classified into three groups according to phylogenetic analysis and structural characteristics. Real-time quantitative PCR analysis revealed that 13 GmPRRs exhibited obvious rhythmic expression under long-day (LD) and short-day (SD) conditions, and the expression of 12 GmPRRs was higher under LD in leaves. To evaluate the effects of natural variations in GmPRR alleles on soybean adaptation, we examined the sequences of GmPRRs among 207 varieties collected across China and the US, investigated the flowering phenotypes in six environments, and analyzed the geographical distributions of the major haplotypes. The results showed that a majority of non-synonymous mutations in the coding region were associated with flowering time, and we found that the nonsense mutations resulting in deletion of the CCT domain were related to early flowering. Haplotype analysis demonstrated that the haplotypes associated with early flowering were mostly distributed in Northeast China, while the haplotypes associated with late flowering were mostly cultivated in the lower latitudes of China. Our study of PRR family genes in soybean provides not only an important guide for characterizing the circadian clock-controlled flowering pathway but also a theoretical basis and opportunities to breed varieties with adaptation to specific regions and farming systems.

GmFT3a fine-tunes flowering time and improves adaptation of soybean to higher latitudes.

Onset of flowering of plants is precisely controlled by extensive environmental factors and internal molecular networks, in which FLOWERING LOCUS T (FT) is a key flowering integrator. In soybean, a typical short-day plant, 11 FT homologues are found in its genome, of which several homologues are functionally diversified in flowering pathways and the others including GmFT3a are yet unknown. In the current study, we characterized GmFT3a, which is located on the same chromosome as the flowering promoters GmFT2a and GmFT5a. Overexpression of GmFT3a significantly promoted flowering of Arabidopsis under the inductive long-day (LD) photoperiod. GmFT3a over-expressed soybean also flowered earlier than the control under LD, but they were not significantly different under inductive short-day (SD) conditions, indicating that GmFT3a acts as a flowering promoter in the non-inductive photoperiod in soybean. Compared with other GmFT homologues, GmFT3a exhibited a slighter effect in flowering promotion than GmFT2a, GmFT5a and GmFT2b under LD conditions. GmFT3a promoted flowering by regulating the expression of downstream flowering-related genes and also affected the expression of other GmFTs. According to the re-sequencing data, the regional distributions of two major haplotypes in 176 soybean varieties were analyzed. The varieties with GmFT3a-Hap2 haplotype matured relatively early, and relative higher expression of GmFT3a was detected in early maturing varieties, implying that Hap2 variation may contribute to the adaptation of soybean to higher latitude regions by increasing expression level of genes in metabolism and signaling pathways. The early flowering germplasm generated by overexpression of GmFT3a has potential to be planted at higher latitudes where non-inductive long day is dominant in the growing season, and GmFT3a can be used to fine-tune soybean flowering and maturity time and improve the geographical adaptation.

CONSTANS Polymorphism Modulates Flowering Time and Maturity in Soybean.

CONSTANS (CO) plays a critical role in the photoperiodic flowering pathway. However, the function of soybean CO orthologs and the molecular mechanisms in regulating flowering remain largely unknown. This study characterized the natural variations in CO family genes and their association with flowering time and maturity in soybeans. A total of 21 soybean CO family genes (GmCOLs) were cloned and sequenced in 128 varieties covering 14 known maturity groups (MG 0000-MG X from earliest to latest maturity). Regarding the whole genomic region involving these genes, GmCOL1, GmCOL3, GmCOL8, GmCOL9, GmCOL10, and GmCOL13 were conserved, and the remaining 15 genes showed genetic variation that was brought about by mutation, namely, all single-nucleotide polymorphisms (SNPs) and insertions-deletions (InDels). In addition, a few genes showed some strong linkage disequilibrium. Point mutations were found in 15 GmCOL genes, which can lead to changes in the potential protein structure. Early flowering and maturation were related to eight genes (GmCOL1/3/4/8/13/15/16/19). For flowering and maturation, 11 genes (GmCOL2/5/6/14/20/22/23/24/25/26/28) expressed divergent physiognomy. Haplotype analysis indicated that the haplotypes of GmCOL5-Hap2, GmCOL13-Hap2/3, and GmCOL28-Hap2 were associated with flowering dates and soybean maturity. This study helps address the role of GmCOL family genes in adapting to diverse environments, particularly when it is necessary to regulate soybean flowering dates and maturity.

Establishment of a novel experimental system for studying the photoperiodic response of short-day dicots using soybean 'cotyledon-only plant' as material.

Soybean is an important model crop for photoperiodic response studies in plants and contributes significantly to the study of plant development and physiology in the past century. Because soybean plant is much bigger in size and longer in life cycle than Arabidopsis, it needs much more space for growth and time for investigation, which significantly hamper the efficiency of research. In the current study, we tested the photoperiodic response of a distinctive artificially-made cotyledon-only plant (COP) using a photoperiod-sensitive soybean variety Zigongdongdou (ZGDD) and other varieties with diverse sensitivity to photoperiod. ZGDD COPs flowered 39.4 ± 2.5 d after emergence under short-day conditions but maintained vegetative growth under long-day and night break conditions, which is similar to the case in the intact ZGDD plants. The COPs of early-maturing and medium-maturing soybean varieties also grew and flowered normally under natural day-length conditions. At the molecular level, the key genes in the photoperiodic pathway such as E1, GmFT1a, GmFT2a, and GmFT5a in the COPs also showed the same photoperiod sensitivity as in the intact plants. In addition, a simpler material of COP with only one cotyledon and root was generated and found to be sensitive to photoperiod as well. Notably, the COPs are only one-fifth the height of intact plants and one-third the maximum diameter of the intact plants grown in chambers 30 d after emergence. Based on COPs, we established a novel experimental system characterized by an entire photoperiodic response and longer longevity of cotyledons in addition to small plant size, ensuring the consistency, reliability, and stability of plant materials. COPs have the potential to be a novel model material for studies of the developmental biology of soybean and other dicots.

Cotyledons facilitate the adaptation of early-maturing soybean varieties to high-latitude long-day environments.

Soybean (Glycine max), a typical short-day plant (SDP) domesticated in temperate regions, has expanded to high latitudes where daylengths are long from soybean emergence to bloom, but rapidly decrease from seed filling to maturity. Cotyledons are well known as the major storage organs in seeds, but it is unclear whether developing cotyledons store flowering substances at filling stage in SD for upcoming seedlings, or instead respond to photoperiod for floral induction after emergence of matured seeds in long-day (LD). Here, we report that cotyledons accelerate flowering of early-maturing varieties not resulting from stored floral stimuli but by perceiving photoperiod after emergence. We found that light signal is indispensable to activate cotyledons for floral induction, and flowering promoting gene GmFT2a is required for cotyledon-dependent floral induction via upregulation of floral identity gene GmAP1. Interestingly, cotyledons are competent to support the entire life cycle of a cotyledon-only plant to produce seeds, underlying a new photoperiod study system in soybean and other dicots. Taken together, these results demonstrate a substantial role for cotyledons in flowering process, whereby we propose a 'cotyledon-based self-reliance' model highlighting floral induction from emergence as a key ecological adaptation for rapid flowering of SDPs grown in LD environments at high latitudes.

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.

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.

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.

A phyB-PIF1-SPA1 kinase regulatory complex promotes photomorphogenesis in Arabidopsis.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) is a highly conserved E3 ubiquitin ligase from plants to animals and acts as a central repressor of photomorphogenesis in plants. SUPPRESSOR OF PHYA-105 1 family members (SPA1-SPA4) directly interact with COP1 and enhance COP1 activity. Despite the presence of a kinase domain at the N-terminus, no COP1-independent role of SPA proteins has been reported. Here we show that SPA1 acts as a serine/threonine kinase and directly phosphorylates PIF1 in vitro and in vivo. SPAs are necessary for the light-induced phosphorylation, ubiquitination and subsequent degradation of PIF1. Moreover, the red/far-red light photoreceptor phyB interacts with SPA1 through its C-terminus and enhances the recruitment of PIF1 for phosphorylation. These data provide a mechanistic view on how the COP1-SPA complexes serve as an example of a cognate kinase-E3 ligase complex that selectively triggers rapid phosphorylation and removal of its substrates, and how phyB modulates this process to promote photomorphogenesis.

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.

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).

Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions.

The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth.

DWARF 53 acts as a repressor of strigolactone signalling in rice.

Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCF(D3) ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCF(D3) ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex.

Casein kinase II α subunits affect multiple developmental and stress-responsive pathways in Arabidopsis.

Casein kinase II (formerly known as CK2), a ubiquitous Ser/Thr kinase, plays critical roles in all higher organisms including plants. The CK2 holoenzyme consists of two catalytic α subunits and two regulatory ß subunits. The Arabidopsis genome has four α subunit and four ß subunit genes, and members of both the α and ß subunit families have been shown to be localized in the cytoplasm, nucleus and also in chloroplasts. However, the biological roles of CK2 subunits have not been fully characterized yet. Here we identified T-DNA insertion mutants in three α subunit genes (α1, α2 and α3) and made double and triple mutants. The CK2 α1α2α3 triple mutants displayed reduced CK2 activity compared with wild-type seedlings. Phenotypic characterization showed that CK2 α1α2α3 triple mutants are late flowering under both long- and short-day conditions. Genes encoding floral integrators are differentially regulated in the triple mutant compared with the wild-type plants. CK2 α1α2α3 triple mutants also displayed reduced hypocotyl growth, smaller cotyledon size and a reduced number of lateral roots compared with wild-type seedlings under light. Abscisic acid-induced blockage of seed germination and cotyledon greening is reduced in CK2 α subunit mutants in an additive manner. Moreover, CK2 α subunit mutants are also hyposensitive to a NaCl-induced blockage of seed germination. Taken together, these data suggest that CK2 α subunits affect diverse developmental and stress responsive pathways in Arabidopsis.

Dimerization and blue light regulation of PIF1 interacting bHLH proteins in Arabidopsis.

Phytochrome Interacting Factor 1 (PIF1), a basic helix-loop-helix (bHLH) protein, functions as a negative regulator of various facets of photomorphogenesis. To indentify PIF1-interacting proteins, we performed yeast two-hybrid screening using PIF1 as a bait and identified a group of proteins including PIF1 itself, PIF3 and long hypocotyl in far-red 1 (HFR1), an atypical HLH protein. Directed yeast two-hybrid interaction assays showed that PIF1 can form heterodimers with all other PIFs as well as with HFR1. PIF1 and PIF3 interacted with each other in both in vitro and in vivo co-immunoprecipitation assays. PIF1-PIF3 heterodimer also bound to a G-box DNA sequence element in vitro. To understand the biological significance of these interactions, a pif1pif3 double mutant was obtained and characterized. Analyses of the single and double mutants showed that PIF3 plays a prominent role in repressing photomorphogenesis under continuous blue light conditions. pif1 and pif3 showed additive phenotypes more prominently under discontinuous blue light conditions. Similar to PIF1, PIF3 was also rapidly phosphorylated, poly-ubiquitylated and degraded in response to blue light. PIF3 also interacted with phytochromes in response to blue light. A PIF3 mutant defective in interaction with both phyA and phyB displayed reduced degradation under blue light, suggesting that phy-interaction was necessary for the blue light-induced degradation of PIF3. Taken together, these data suggest a combinatorial control of photomorphogenesis by bHLH proteins in response to light in Arabidopsis.

Over-expression of an AT-hook gene, AHL22, delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana.

The Arabidopsis genome encodes 29 AHL (AT-hook motif nuclear localized) proteins, but the function for most of them remains unknown. We report here a study of the AHL22 gene, which was originally identified as a gain-of-function allele that enhanced the phenotype of the cry1 cry2 mutant. AHL22 is a nuclear protein with the binding activity for an AT-rich DNA sequence. AHL22 overexpression delayed flowering and caused a constitutive photomorphogenic phenotype. The loss-of-function AHL22 mutant showed no clear phenotype on flowering, but slightly longer hypocotyls. However, silencing four AHL genes (AHL22, AHL18, AHL27, and AHL29) resulted in early flowering and enhanced ahl22-1 mutant phenotype on the growth of hypocotyls, suggesting genetic redundancy of AHL22 with other AHL genes on these plant developmental events. Further analysis showed that AHL22 controlled flowering and hypocotyl elongation might result from primarily the regulation of FT and PIF4 expression, respectively.

Association of the circadian rhythmic expression of GmCRY1a with a latitudinal cline in photoperiodic flowering of soybean.

Photoperiodic control of flowering time is believed to affect latitudinal distribution of plants. The blue light receptor CRY2 regulates photoperiodic flowering in the experimental model plant Arabidopsis thaliana. However, it is unclear whether genetic variations affecting cryptochrome activity or expression is broadly associated with latitudinal distribution of plants. We report here an investigation of the function and expression of two cryptochromes in soybean, GmCRY1a and GmCRY2a. Soybean is a short-day (SD) crop commonly cultivated according to the photoperiodic sensitivity of cultivars. Both cultivated soybean (Glycine max) and its wild relative (G. soja) exhibit a strong latitudinal cline in photoperiodic flowering. Similar to their Arabidopsis counterparts, both GmCRY1a and GmCRY2a affected blue light inhibition of cell elongation, but only GmCRY2a underwent blue light- and 26S proteasome-dependent degradation. However, in contrast to Arabidopsis cryptochromes, soybean GmCRY1a, but not GmCRY2a, exhibited a strong activity promoting floral initiation, and the level of protein expression of GmCRY1a, but not GmCRY2a, oscillated with a circadian rhythm that has different phase characteristics in different photoperiods. Consistent with the hypothesis that GmCRY1a is a major regulator of photoperiodic flowering in soybean, the photoperiod-dependent circadian rhythmic expression of the GmCRY1a protein correlates with photoperiodic flowering and latitudinal distribution of soybean cultivars. We propose that genes affecting protein expression of the GmCRY1a protein play an important role in determining latitudinal distribution of soybeans.