Origin, variation, and selection of natural alleles controlling flowering and adaptation in wild and cultivated soybean.

Soybean (Glycine max) is an economically important crop worldwide, serving as a major source of oil and protein for human consumption and animal feed. Cultivated soybean was domesticated from wild soybean (Glycine soja) which both species are highly sensitive to photoperiod and can grow over a wide geographical range. The extensive ecological adaptation of wild and cultivated soybean has been facilitated by a series of genes represented as quantitative trait loci (QTLs) that control photoperiodic flowering and maturation. Here, we review the molecular and genetic basis underlying the regulation of photoperiodic flowering in soybean. Soybean has experienced both natural and artificial selection during adaptation to different latitudes, resulting in differential molecular and evolutionary mechanisms between wild and cultivated soybean. The in-depth study of natural and artificial selection for the photoperiodic adaptability of wild and cultivated soybean provides an important theoretical and practical basis for enhancing soybean adaptability and yield via molecular breeding. In addition, we discuss the possible origin of wild soybean, current challenges, and future research directions in this important topic.

Agronomical selection on loss-of-function of GIGANTEA simultaneously facilitates soybean salt tolerance and early maturity.

Salt stress and flowering time are major factors limiting geographic adaptation and yield productivity in soybean (Glycine max). Although improving crop salt tolerance and latitude adaptation are essential for efficient agricultural production, whether and how these two traits are integrated remains largely unknown. Here, we used a genome-wide association study to identify a major salt-tolerance locus controlled by E2, an ortholog of Arabidopsis thaliana GIGANTEA (GI). Loss of E2 function not only shortened flowering time and maturity, but also enhanced salt-tolerance in soybean. E2 delayed soybean flowering by enhancing the transcription of the core flowering suppressor gene E1, thereby repressing Flowering Locus T (FT) expression. An E2 knockout mutant e2CR displayed reduced accumulation of reactive oxygen species (ROS) during the response to salt stress by releasing peroxidase, which functions in ROS scavenging to avoid cytotoxicity. Evolutionary and population genetic analyses also suggested that loss-of-function e2 alleles have been artificially selected during breeding for soybean adaptation to high-latitude regions with greater salt stress. Our findings provide insights into the coupled selection for adaptation to both latitude and salt stress in soybean; and offer an ideal target for molecular breeding of early-maturing and salt-tolerant cultivars.

The legume-specific transcription factor E1 controls leaf morphology in soybean.

BACKGROUND: The leaf is a determinate organ essential for photosynthesis, whose size and shape determine plant architecture and strongly affect agronomic traits. In soybean, the molecular mechanism of leaf development is not well understood. The flowering repressor gene E1, which encodes a legume-specific B3-like protein, is known to be the gene with the largest influence on soybean flowering and maturity. However, knowledge of its potential other functions remains poor. RESULTS: Here, we identified a novel function of E1 protein in leaf development. Unifoliolate leaves of E1-overexpression (E1-OE) lines were smaller and curlier than those of wild type DongNong 50 (DN50) and Williams 82 (W82). Transverse histological sections showed disorganized cells and significantly elevated palisade tissue number, spongy tissue number, and bulliform cell number in E1-OE lines. Our results indicate that E1 binds to the promoters of the leaf- development-related CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor genes to negatively regulate their expression. CONCLUSIONS: Our findings identify E1 as an important new factor in soybean leaf development.

CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean.

BACKGROUND: Soybean (Glycine max) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants. RESULTS: Here, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL (LHY) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using Agrobacterium rhizogenes strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of GmLHY displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in Gmlhy1a1b2a2b was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of GmLHY were significantly decreased in comparison to the WT. These results suggest that GmLHY encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies. CONCLUSIONS: Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLHY genes reduces soybean plant height and shortens internodes from 20 to 35 days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.

The genetic basis of high-latitude adaptation in wild soybean.

In many plants, flowering time is influenced by daylength as an adaptive response. In soybean (Glycine max) cultivars, however, photoperiodic flowering reduces crop yield and quality in high-latitude regions. Understanding the genetic basis of wild soybean (Glycine soja) adaptation to high latitudes could aid breeding of improved cultivars. Here, we identify the Tof4 (Time of flowering 4) locus, which encodes by an E1-like protein, E1La, that represses flowering and enhances adaptation to high latitudes in wild soybean. Moreover, we found that Tof4 physically associates with the promoters of two important FLOWERING LOCUS T (FT2a and FT5a) and with Tof5 to inhibit their transcription under long photoperiods. The effect of Tof4 on flowering and maturity is mediated by FT2a and FT5a proteins. Intriguingly, Tof4 and the key flowering repressor E1 independently but additively regulate flowering time, maturity, and grain yield in soybean. We determined that weak alleles of Tof4 have undergone natural selection, facilitating adaptation to high latitudes in wild soybean. Notably, over 71.5% of wild soybean accessions harbor the mutated alleles of Tof4 or a previously reported gain-of-function allele Tof5H2, suggesting that these two loci are the genetic basis of wild soybean adaptation to high latitudes. Almost no cultivated soybean carries the mutated tof4 allele. Introgression of the tof4-1 and Tof5H2 alleles into modern soybean or editing E1 family genes thus represents promising avenues to obtain early-maturity soybean, thereby improving productivity in high latitudes.

Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9.

Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding. Nevertheless, a significant portion of soybean cultivars are still pod shattering-susceptible, limiting their regional and climatic adaptabilities. Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar, Huachun6 (HC6), and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1 (PDH1) gene, which exists in a significant portion of soybean cultivars. We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). In T2 progenies, several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits. The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma. Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.

Genome-Wide Analysis of DREB Genes Identifies a Novel Salt Tolerance Gene in Wild Soybean (Glycine soja).

Salt stress is a major factor limiting the growth and yield of soybean (Glycine max). Wild soybeans (Glycine soja) contain high allelic diversity and beneficial alleles that can be re-introduced into domesticated soybeans to improve adaption to the environment. However, very few beneficial alleles have been identified from wild soybean. Here, we demonstrate that wild soybean is more salt tolerant than cultivated soybean and examine dehydration responsive element-binding (DREB) family transcription factor genes to look for advantageous alleles that might improve drought tolerance in cultivated soybean. Our genome-wide analysis identified 103 DREB genes from the Glycine max genome. By combined RNA-sequencing and population genetics of wild, landrace, and cultivated soybean accessions, we show that the natural variation in DREB3a and DREB3b is related to differences in salt tolerance in soybean accessions. Interestingly, DREB3b, but not DREB3a, appears to have undergone artificial selection. Soybean plants carrying the wild soybean DREB3b allele (DREB3b39Del ) are more salt tolerant than those containing the reference genome allele (DREB3bRef ). Together, our results suggest that the loss of the DREB3b39Del allele through domestication of cultivated soybean may be associated with a reduction in salt tolerance. Our findings provide crucial information for improving salt tolerance in soybean through molecular breeding.

Parallel selection of distinct Tof5 alleles drove the adaptation of cultivated and wild soybean to high latitudes.

Photoperiod responsiveness is a key factor limiting the geographic distribution of cultivated soybean and its wild ancestor. In particular, the genetic basis of the adaptation in wild soybean remains poorly understood. In this study, by combining whole-genome resequencing and genome-wide association studies we identified a novel locus, Time of Flowering 5 (Tof5), which promotes flowering and enhances adaptation to high latitudes in both wild and cultivated soybean. By genomic, genetic and transgenic analyses we showed that Tof5 encodes a homolog of Arabidopsis thaliana FRUITFULL (FUL). Importantly, further analyses suggested that different alleles of Tof5 have undergone parallel selection. The Tof5H1 allele was strongly selected by humans after the early domestication of cultivated soybean, while Tof5H2 allele was naturally selected in wild soybean, and in each case facilitating adaptation to high latitudes. Moreover, we found that the key flowering repressor E1 suppresses the transcription of Tof5 by binding to its promoter. In turn, Tof5 physically associates with the promoters of two important FLOWERING LOCUS T (FT), FT2a and FT5a, to upregulate their transcription and promote flowering under long photoperiods. Collectively, our findings provide insights into how wild soybean adapted to high latitudes through natural selection and indicate that cultivated soybean underwent changes in the same gene but evolved a distinct allele that was artificially selected after domestication.

A functionally divergent SOC1 homolog improves soybean yield and latitudinal adaptation.

Soybean (Glycine max) grows in a wide range of latitudes, but it is extremely sensitive to photoperiod, which reduces its yield and ability to adapt to different environments. Therefore, understanding of the genetic basis of soybean adaptation is of great significance for breeding and improvement. Here, we characterized Tof18 (SOC1a) that conditions early flowering and growth habit under both short-day and long-day conditions. Molecular analysis confirmed that the two SOC1 homologs present in soybeans (SOC1a and SOC1b) underwent evolutionary functional divergence, with SOC1a having stronger effects on flowering time and stem node number than SOC1b due to transcriptional differences. soc1a soc1b double mutants showed stronger functional effects than either of the single mutants, perhaps due to the formation of SOC1a and SOC1b homodimers or heterodimers. Additionally, Tof18/SOC1a improves the latitudinal adaptation of cultivated soybeans, highlighting the functional importance of SOC1a. The Tof18G allele facilitates adaptation to high latitudes, whereas Tof18A facilitates adaptation to low latitudes. We demonstrated that SOC1s contribute to floral induction in both leaves and shoot apex through inter-regulation with FTs. The SOC1a-SOC1b-Dt2 complex plays essential roles in stem growth habit by directly binding to the regulatory sequence of Dt1, making the genes encoding these proteins potential targets for genome editing to improve soybean yield via molecular breeding. Since the natural Tof18A allele increases node number, introgressing this allele into modern cultivars could improve yields, which would help optimize land use for food production in the face of population growth and global warming.

Genetic basis and adaptation trajectory of soybean from its temperate origin to tropics.

Soybean (Glycine max) serves as a major source of protein and edible oils worldwide. The genetic and genomic bases of the adaptation of soybean to tropical regions remain largely unclear. Here, we identify the novel locus Time of Flowering 16 (Tof16), which confers delay flowering and improve yield at low latitudes and determines that it harbors the soybean homolog of LATE ELONGATED HYPOCOTYL (LHY). Tof16 and the previously identified J locus genetically additively but independently control yield under short-day conditions. More than 80% accessions in low latitude harbor the mutations of tof16 and j, which suggests that loss of functions of Tof16 and J are the major genetic basis of soybean adaptation into tropics. We suggest that maturity and yield traits can be quantitatively improved by modulating the genetic complexity of various alleles of the LHY homologs, J and E1. Our findings uncover the adaptation trajectory of soybean from its temperate origin to the tropics.

The Soybean Gene J Contributes to Salt Stress Tolerance by Up-Regulating Salt-Responsive Genes.

Soybean [Glycine max (L.) Merr.] is an important crop for oil and protein resources worldwide, and its farming is impacted by increasing soil salinity levels. In Arabidopsis the gene EARLY FLOWERING 3 (ELF3), increased salt tolerance by suppressing salt stress response pathways. J is the ortholog of AtELF3 in soybean, and loss-of-function J-alleles greatly prolong soybean maturity and enhance grain yield. The exact role of J in abiotic stress response in soybean, however, remains unclear. In this study, we showed that J expression was induced by NaCl treatment and that the J protein was located in the nucleus. Compared to NIL-J, tolerance to NaCl was significantly lower in the NIL-j mutant. We also demonstrated that overexpression of J increased NaCl tolerance in transgenic soybean hairy roots. J positively regulated expression of downstream salt stress response genes, including GmWRKY12, GmWRKY27, GmWRKY54, GmNAC, and GmSIN1. Our study disclosed a mechanism in soybean for regulation of the salt stress response. Manipulation of these genes should facilitate improvements in salt tolerance in soybean.

Effect of paclitaxel-loaded nanoparticles on the viability of human hepatocellular carcinoma HepG2 cells.

OBJECTIVE: To explore effects of paclitaxel-loaded poly lactic-co-glycolic acid (PLGA) particles on the viability of human hepatocellular carcinoma (HCC) HepG2 cells. MATERIALS AND METHODS: The viability of HepG2 cells was assessed using MTT under different concentrations of prepared paclitaxel-loaded particles and paclitaxel (6.25, 12.5, 25, 50, and 100 mg/L), and apoptosis was analyzed using Hochest33342/Annexin V-FITC/PI combined with an IN Cell Analyzer 2000. RESULTS: Paxlitaxel-loaded nanoparticles were characterized by narrow particle size distribution (158.6 nm average particle size). The survival rate of HepG2 cells exposed to paclitaxel-loaded PLGA particles decreased with the increase of concentration and time period (P<0.01 or P<0.05), the dose- and time-dependence indicating sustained release (P<0.05). Moreover, apoptosis of HepG2 cells was induced, again with an obvious dose- and time-effect relationship (P<0.05). CONCLUSIONS: Paclitaxel-loaded PLGA particles can inhibit the proliferation and induce the apoptosis of HCC HepG2 cells. This new-type of paclitaxel carrier body is easily made and has low cost, good nanoparticle characterization and sustained release. Hence, paclitaxel- loaded PLGA particles deserve to be widely popularized in the clinic.

Transcellular transport characteristics of huperzine alone or in combination with ginkgolide B across Caco-2 and Madin-Darby canine kidney cell monolayer.

OBJECTIVE: To study the various processes involved in transcellular transport (TT) of huperzine A alone or in combination with ginkgolide B in Caco-2 and Madin-Darby canine renal (MDCK) cell monolayer. METHODS: The transepithelial passage was assayed in the apical-to-basolateral (AP to BL) direction and opposite direction (BL to AP) in both cell lines. The determination of huperzine A and ginkgolide B were performed by high performance liquid chromatography (HPLC). The passage rates of huperzine A and ginkgolide B were calculated. Bi-directional TT (absorption and secretion) were taken in huperzine A and ginkgolide B in Caco-2 and MDCK cell monolayer. RESULTS: TT absorption and secretion kinetics of huperzine A and ginkgolide B across two cells existed at the same time. The passage rates of huperzine A were increased significantly with adding different concentrations of ginkgolide B. CONCLUSIONS: The compound preparations of HA in combination with GB for dementia caused by cerebral ischemic have synergistic effects on the pharmacodynamics, and improve the bioavailability through BBB.

[Preparation of monoclonal antibody against human c-erbB2 and identification of its specificity].

AIM: To prepare monoclonal antibody(mAb) against human c-erbB2 and identify its specificity. METHODS: The epitope of human c-erbB2 antigen was analyzed by using computer software and a immunodominant epitope at the carboxyl-terminal was selected. A peptide consisting of 13 amino acids was synthesized and coupled with keyholelimpet hemocyanin (KLH), and then it was used to immunize BLAB/c mice. The splenocytes of the immunized mice were fused with Sp2/0 cells routinely and the hybridoma cells were selected by HAT selected culture, indirect ELISA, and immunohistochemical staining, and cloned by limiting dilution. The specificity of the mAb was identified by cross-reaction test and blocking test. RESULTS: A hybridoma cell line SC8C1, stably secreting anti-c-erbB2 mAb was obtained. The mAb SC8C1 could react to breast cancer tissue expressing c-erbB2 molecule but did not react to other c-erbB2-negative cells. The mAb will lose the activity after being blocked with synthesized 13 peptide. CONCLUSION: A anti-c-erbB2 mAb SC8C1 is prepared successfully using synthesized 13 peptide as immunogen.