Baseline oxygen consumption decreases with cortical depth.

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase-the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than reflecting baseline energy needs. Our study provides to our knowledge the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards better understanding of brain energy metabolism.

Novel and multifaceted regulations of photoperiodic flowering by phytochrome A in soybean.

Photoperiod is an important environmental cue. Plants can distinguish the seasons and flower at the right time through sensing the photoperiod. Soybean is a sensitive short-day crop, and the timing of flowering varies greatly at different latitudes, thus affecting yields. Soybean cultivars in high latitudes adapt to the long day by the impairment of two phytochrome genes, PHYA3 and PHYA2, and the legume-specific flowering suppressor, E1. However, the regulating mechanism underlying phyA and E1 in soybean remains largely unknown. Here, we classified the regulation of the E1 family by phyA2 and phyA3 at the transcriptional and posttranscriptional levels, revealing that phyA2 and phyA3 regulate E1 by directly binding to LUX proteins, the critical component of the evening complex, to regulate the stability of LUX proteins. In addition, phyA2 and phyA3 can also directly associate with E1 and its homologs to stabilize the E1 proteins. Therefore, phyA homologs control the core flowering suppressor E1 at both the transcriptional and posttranscriptional levels, to double ensure the E1 activity. Thus, our results disclose a photoperiod flowering mechanism in plants by which the phytochrome A regulates LUX and E1 activity.

A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation.

Photoperiod sensitivity is a key factor in plant adaptation and crop production. In the short-day plant soybean, adaptation to low latitude environments is provided by mutations at the J locus, which confer extended flowering phase and thereby improve yield. The identity of J as an ortholog of Arabidopsis ELF3, a component of the circadian evening complex (EC), implies that orthologs of other EC components may have similar roles. Here we show that the two soybean homeologs of LUX ARRYTHMO interact with J to form a soybean EC. Characterization of mutants reveals that these genes are highly redundant in function but together are critical for flowering under short day, where the lux1 lux2 double mutant shows extremely late flowering and a massively extended flowering phase. This phenotype exceeds that of any soybean flowering mutant reported to date, and is strongly reminiscent of the "Maryland Mammoth" tobacco mutant that featured in the seminal 1920 study of plant photoperiodism by Garner and Allard [W. W. Garner, H. A. Allard, J. Agric. Res. 18, 553-606 (1920)]. We further demonstrate that the J-LUX complex suppresses transcription of the key flowering repressor E1 and its two homologs via LUX binding sites in their promoters. These results indicate that the EC-E1 interaction has a central role in soybean photoperiod sensitivity, a phenomenon also first described by Garner and Allard. EC and E1 family genes may therefore constitute key targets for customized breeding of soybean varieties with precise flowering time adaptation, either by introgression of natural variation or generation of new mutants by gene editing.

Diverse flowering responses subjecting to ambient high temperature in soybean under short-day conditions.

Flowering time is one of important agronomic traits determining the crop yield and affected by high temperature. When facing high ambient temperature, plants often initiate early flowering as an adaptive strategy to escape the stress and ensure successful reproduction. However, here we find opposing ways in the short-day crop soybean to respond to different levels of high temperatures, in which flowering accelerates when temperature changes from 25 to 30 °C, but delays when temperature reaches 35 °C under short day. phyA-E1, possibly photoperiodic pathway, is crucial for 35 °C-mediated late flowering, however, does not contribute to promoting flowering at 30 °C. 30 °C-induced up-regulation of FT2a and FT5a leads to early flowering, independent of E1. Therefore, distinct responsive mechanisms are adopted by soybean when facing different levels of high temperatures for successful flowering and reproduction.

Natural variation of FKF1 controls flowering and adaptation during soybean domestication and improvement.

Soybean (Glycine max) is a major source of protein and edible oil world-wide and is cultivated in a wide range of latitudes. However, it is extremely sensitive to photoperiod, which influences flowering time, maturity, and yield, and severely limits soybean latitude adaptation. In this study, a genome-wide association study (GWAS) identified a novel locus in accessions harboring the E1 allele, called Time of flowering 8 (Tof8), which promotes flowering and enhances adaptation to high latitude in cultivated soybean. Gene functional analyses showed that Tof8 is an ortholog of Arabidopsis FKF1. We identified two FKF1 homologs in the soybean genome. Both FKF1 homologs are genetically dependent on E1 by binding to E1 promoter to activate E1 transcription, thus repressing FLOWERING LOCUS T 2a (FT2a) and FT5a transcription, which modulate flowering and maturity through the E1 pathway. We also demonstrate that the natural allele FKF1bH3 facilitated adaptation of soybean to high-latitude environments and was selected during domestication and improvement, leading to its rapid expansion in cultivated soybean. These findings provide novel insights into the roles of FKF1 in controlling flowering time and maturity in soybean and offer new means to fine-tune adaptation to high latitudes and increase grain yield.

Candidate loci for breeding compact plant-type soybean varieties.

Plant height and node number are important agronomic traits that influence yield in soybean (Glycine max L.). Here, to better understand the genetic basis of the traits, we used two recombinant inbred line (RIL) populations to detect quantitative trait loci (QTLs) associated with plant height and node number in different environments. This analysis detected 9 and 21 QTLs that control plant height and node number, respectively. Among them, we identified two genomic regions that overlap with Determinate stem 1 (Dt1) and Dt2, which are known to influence both plant height and node number. Furthermore, different combinations of Dt1 and Dt2 alleles were enriched in distinct latitudes. In addition, we determined that the QTLs qPH-13-SE and qPH-13-DW in the two RIL populations overlap with genomic intervals associated with plant height and the QTL qNN-04-DW overlaps with an interval associated with node number. Combining the dwarf allele of qPH-13-SE/qPH-13-DW and the multiple-node allele of qNN-04-DW produced plants with ideal plant architecture, i.e., shorter main stems with more nodes. This plant type may help increase yield at high planting density. This study thus provides candidate loci for breeding elite soybean cultivars for plant height and node number. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01352-2.

A phase III randomized, double-blind, placebo-controlled trial of the denosumab biosimilar QL1206 in postmenopausal Chinese women with osteoporosis and high fracture risk.

The current study evaluated the efficacy and safety of a denosumab biosimilar, QL1206 (60 mg), compared to placebo in postmenopausal Chinese women with osteoporosis and high fracture risk. At 31 study centers in China, a total of 455 postmenopausal women with osteoporosis and high fracture risk were randomly assigned to receive QL1206 (60 mg subcutaneously every 6 months) or placebo. From baseline to the 12-month follow-up, the participants who received QL1206 showed significantly increased bone mineral density (BMD) values (mean difference and 95% CI) in the lumbar spine: 4.780% (3.880%, 5.681%), total hip :3.930% (3.136%, 4.725%), femoral neck 2.733% (1.877%, 3.589%) and trochanter: 4.058% (2.791%, 5.325%) compared with the participants who received the placebo. In addition, QL1206 injection significantly decreased the serum levels of C-terminal crosslinked telopeptides of type 1 collagen (CTX): -77.352% (-87.080%, -66.844%), and N-terminal procollagen of type l collagen (P1NP): -50.867% (-57.184%, -45.217%) compared with the placebo over the period from baseline to 12 months. No new or unexpected adverse events were observed. We concluded that compared with placebo, QL1206 effectively increased the BMD of the lumbar spine, total hip, femoral neck and trochanter in postmenopausal Chinese women with osteoporosis and rapidly decreased bone turnover markers. This study demonstrated that QL1206 has beneficial effects on postmenopausal Chinese women with osteoporosis and high fracture risk.

Risk factors of mortality and second fracture after elderly hip fracture surgery in Shanghai, China.

INTRODUCTION: Hip fracture is one of the leading causes of death and disability in the elderly. We analyzed the risk factors of mortality and second fracture within 2 years after hip fracture surgery in elderly Chinese patients. MATERIALS AND METHODS: A total of 613 elderly patients after hip fracture surgery were selected, including 181 males and 432 females, and the patients were followed for at least 24 months. Information about patients and surgery was collected from medical records. Information on death, secondary fracture, and postoperative activities of daily living (ADL) was obtained by telephone follow-up. Cox regression was performed to identify risk factors associated with mortality and second fracture, measured by hazard ratio (HR). RESULTS: The 1-year and 2-year mortality rates after hip fracture were 13.4% and 20.7%, respectively. The second fracture rate within 2 years was 9.5%. Male gender (HR 1.51, P = 0.035), increased age (HR 1.07, P < 0.001), preoperative hypoalbuminemia (HR 1.79, P = 0.004), preoperative pneumonia (HR 2.60, P = 0.005) and poor ADL (P = 0.048) were independent risk factors for 2-year mortality, while high preoperative hemoglobin (HR 0.98, P = 0.002), high preoperative eGFR (HR 0.99, P = 0.031), high preoperative LVEF (HR 0.92, P = 0.048) were protective factors for 2-year mortality. Poor ADL (P = 0.002) was the independent risk factor for second fracture within 2 years. CONCLUSIONS: The 2-year mortality rate and second fracture rate after hip fracture in elderly remained high, which was related to old age and complications exists. Postoperative rehabilitation and improving ADL were very important to reduce mortality and second fracture.

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.

Association between Serum Oxytocin, Bone Mineral Density and Body Composition in Chinese Adult Females.

Background and Objectives: Oxytocin (OT) is a neuropeptide hormone which is known for its classical effects in pregnancy and lactation. Recently, growing evidence demonstrated a close relation between OT and bone. The present study aimed to explore the relationship between OT, bone and osteoporosis risk in Chinese adult females. Materials and Methods: in total, 149 adult females were enrolled. The serum OT levels were measured using ELISA kits. Bone mineral density (BMD) and body composition were measured by dual-energy X-ray absorptiometry (DXA). The study subjects were divided into two groups according to their menopause status and then divided into tertiles based on their serum OT level. Results: Serum OT, serum estradiol and BMD at three skeletal sites were significantly higher in the premenopausal group than in the postmenopausal group (p < 0.001, p = 0.008 and p < 0.001, respectively). In the tertile analysis, relative to tertile 1, significant associations were found for tertile 3 for OT levels and higher BMD in the femoral neck and total hip, in both pre- and postmenopausal groups. Using logistic regression analysis, tertile 3 appeared less likely to have low-BMD osteoporosis than tertile 1 (OR = 0.257, 95% CI = 0.073, 0.910). In multivariate stepwise regression analysis, OT and total lean mass were two positive determinants of BMD in the femoral neck and total hip in the premenopausal group (adjusted R2 for the model = 0.232 and 0.199, respectively; both p < 0.001). Conclusion: Our study demonstrated positive associations between serum OT levels and BMD in a Chinese (non-Caucasian) population. OT appeared to be more strongly associated with hip BMD in premenopausal females. These results may suggest a protective role and potential therapeutic use of OT in osteoporosis, especially for premenopausal women.

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.

Two homologous LHY pairs negatively control soybean drought tolerance by repressing the abscisic acid responses.

The circadian clock plays essential roles in diverse plant biological processes, such as flowering, phytohormone biosynthesis and abiotic stress responses. The manner in which circadian clock genes regulate drought stress responses in model plants has been well established, but comparatively little is known in crop species, such as soybean, a major global crop. This paper reports that the core clock components GmLHYs, the orthologues of CCA1/LHY in Arabidopsis, negatively control drought tolerance in soybean. The expressions of four GmLHYs were all induced by drought, and the quadruple mutants of GmLHYs demonstrated significantly improved drought tolerance. Transcriptome profiling suggested that the abscisic acid (ABA) signaling pathway is regulated by GmLHYs to respond to drought tolerance. Genetic dissections showed that two homologous pairs of LHY1a and LHY1b redundantly control the drought response. Functional characterization of LHY1a and LHY1b in Arabidopsis and soybean further supported the notion that GmLHYs can maintain cellular homeostasis through the ABA signaling pathway under drought stress. This study improves our understanding of the underlying molecular mechanisms on soybean drought tolerance. Furthermore, the two homologues of LHY1a and LHY1b provide alternative targets for genome editing to rapidly generate mutant alleles in elite soybean cultivars to enhance their drought tolerance.

A flowering time locus dependent on E2 in soybean.

Soybean [Glycine max (L.) Merrill] is very sensitive to changes in photoperiod as a typical short-day plant. Photoperiodic flowering influences soybean latitudinal adaptability and yield to a considerable degree. Identifying new quantitative trait loci (QTLs) controlling flowering time is a powerful initial approach for elucidating the mechanisms underlying flowering time and adaptation to different latitudes in soybean. In this study, we developed a Recombinant Inbred Lines (RILs) population and recorded flowering time under natural long-day conditions. We also constructed a high-density genetic map by genotyping-by-sequencing and used it for QTL mapping. In total, we detected twelve QTLs, four of which are stable and named by qR1-2, qR1-4, qR1-6.1, and qR1-10, respectively. Among these four QTLs, qR1-4 and qR1-6.1 are novel. QTL mapping in two sub-populations classified by the genotype of the maturity locus E2, genetic interaction evaluation between E2 and qR1-2, and qRT-PCR indicated that E2 has an epistatic effect on qR1-2, and that causal gene of qR1-2 acts upstream of E2. We presumed the most likely candidate genes according to the resequencing data and briefly analyzed the geographic distributions of these genes. These findings will be beneficial for our understanding of the mechanisms underlying photoperiodic flowering in soybean, contribute to further investigate of E2, and provide genetic resources for molecular breeding of soybean. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01224-1.

Serum concentrations of oxytocin, DHEA and follistatin are associated with osteoporosis or sarcopenia in community-dwelling postmenopausal women.

BACKGROUND: Osteoporosis and sarcopenia are major health issues in postmenopausal women due to their high prevalence and association with several adverse outcomes. However, no biomarkers may be used for screening and diagnosis. The current study investigated potential biomarkers for osteoporosis and/or sarcopenia in postmenopausal women. METHODS: A cross-sectional study on 478 healthy community-dwelling postmenopausal women aged 50-90 years was performed. Osteoporosis and sarcopenia were defined according to the World Health Organization (WHO) and Asian Working Group for Sarcopenia (AWGS). RESULTS: Dehydroepiandrosterone (DHEA) was related to muscle strength (ß = 0.19, p = 0.041) and function (ß = 0.58, p = 0.004). Follistatin (ß = - 0.27, p = 0.01) was related to muscle mass. Oxytocin (ß = 0.59, p = 0.044) and DHEA (ß = 0.51, p = 0.017) were related to bone mass. After adjusting for age, oxytocin (odds ratio (OR) 0.75; 95% confidence intervals (CI) 0.63-0.98; p = 0.019) was associated with osteoporosis, and DHEA (OR 0.73; 95% CI 0.51-0.96; p = 0.032) and follistatin (OR 1.66; 95% CI 1.19-3.57; p = 0.022) were associated with sarcopenia. CONCLUSIONS: Postmenopausal women with sarcopenia were more likely to have lower DHEA levels and higher follistatin levels, and postmenopausal women with osteoporosis were more likely to have lower oxytocin levels.

A recent retrotransposon insertion of J caused E6 locus facilitating soybean adaptation into low latitude.

Soybean (Glycine max) is an important legume crop that was domesticated in temperate regions. Soybean varieties from these regions generally mature early and exhibit extremely low yield when grown under inductive short-day (SD) conditions at low latitudes. The long-juvenile (LJ) trait, which is characterized by delayed flowering and maturity, and improved yield under SD conditions, allowed the cultivation of soybean to expand to lower latitudes. Two major loci control the LJ trait: J and E6. In the current study, positional cloning, sequence analysis, and transgenic complementation confirmed that E6 is a novel allele of J, the ortholog of Arabidopsis thaliana EARLY FLOWERING 3 (ELF3). The mutant allele e6PG , which carries a Ty1/Copia-like retrotransposon insertion, does not suppress the legume-specific flowering repressor E1, allowing E1 to inhibit Flowering Locus T (FT) expression and thus delaying flowering and increasing yields under SD conditions. The e6PG allele is a rare allele that has not been incorporated into modern breeding programs. The dysfunction of J might have greatly facilitated the adaptation of soybean to low latitudes. Our findings increase our understanding of the molecular mechanisms underlying the LJ trait and provide valuable resources for soybean breeding.

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.

MTHFD1L as a folate cycle enzyme correlates with prognostic outcome and its knockdown impairs cell invasive behaviors in osteosarcoma via mediating the AKT/mTOR pathway.

Osteosarcoma (OS) is the most frequent primary malignancy initially in bone with multiple genomic aberrations. Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) is linked with the progression of diverse tumors. However, its function in OS is not understood completely. The expression pattern and prognostic significance of MTHFD1L in OS tissues were analyzed based on GEO database. The expression level of MTHFD1L in OS cell lines was explored by qRT-PCR. The cell proliferation, colony formation ability, invasion as well as migration in OS cells after MTHFD1L knockdown were determined using cell counting kit 8 (CCK-8) assay, colony formation and transwell methods. GSEA analysis was performed to predict the underlying mechanisms of MTHFD1L in OS development. Furthermore, the western blot was utilized to study the influence of MTHFD1L on AKT/mTOR pathway. Our results indicated that MTHFD1L expression was significantly up-regulated in OS tissues and cells compared with normal tissues and cells. High expression of MTHFD1L could lead to poor prognosis of OS patients. Cell proliferation, colony formation ability, migration and invasion were blocked because of reduced MTHFD1L in vitro. Moreover, cell cycle and AKT/mTOR pathway were all associated with MTHFD1L expression. In conclusion, the findings revealed that MTHFD1L might promote the development of OS via mediating cell cycle and AKT/mTOR pathway, indicating that MTHFD1L might act as a promising therapeutic target for OS treatment.

Mesenchymal stromal cells in adipose tissue of breast cancer patients: a cross-sectional study.

Mesenchymal stromal cells (MSCs) are important components of the tumor microenvironment and are believed to facilitate cancer growth affecting cancer cells. We investigated this issue in breast cancer patients. In this cross-sectional study biopsy or mastectomy specimens from breast cancer patients who underwent surgery were evaluated based on the presence of MSCs. Flow cytometric analysis was used to evaluate the expression of CD73, CD90, CD105, and absence of CD11b or CD14, CD79, CD45, and HLA-DR expression as the criteria of MSC presence. Patients were divided into MSC (+) or (-). SPSS software was used to evaluate the association of age, tumor type, tumor size, disease grade, stage, and metastasis with the presence of MSC cells in adipose tissue of specimens. 82 patients with a mean age of 44.43±9.2 years were included in the study. MSC (+) happened in 13 (15.83) patient's samples. A regression model was conducted to evaluate the association of patient characteristics and the MSC positivity. The patient's age was associated with MSC positivity (B=-0.194, p=0.006); and the disease stage was associated with MSC positivity (B=-0.734, p=0.019). Significantly lower age of patients was seen in MSC (+) patients vs. MSC (-) patients (p=0.002). While no difference in the case of tumor type, tumor size, disease grade, stage, and metastasis was seen between two groups (P>0.05). Our result indicated that Mesenchymal stromal cells are seen more in the tumor microenvironment of younger breast cancer patients. Also, patients having mesenchymal stromal cells may have milder disease and lower disease stage.

Soybean AP1 homologs control flowering time and plant height.

Flowering time and plant height are key agronomic traits that directly affect soybean (Glycine max) yield. APETALA1 (AP1) functions as a class A gene in the ABCE model for floral organ development, helping to specify carpel, stamen, petal, and sepal identities. There are four AP1 homologs in soybean, all of which are mainly expressed in the shoot apex. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 technology to generate a homozygous quadruple mutant, gmap1, with loss-of-function mutations in all four GmAP1 genes. Under short-day (SD) conditions, the gmap1 quadruple mutant exhibited delayed flowering, changes in flower morphology, and increased node number and internode length, resulting in plants that were taller than the wild type. Conversely, overexpression of GmAP1a resulted in early flowering and reduced plant height compared to the wild type under SD conditions. The gmap1 mutant and the overexpression lines also exhibited altered expression of several genes related to flowering and gibberellic acid metabolism, thereby providing insight into the role of GmAP1 in the regulatory networks controlling flowering time and plant height in soybean. Increased node number is the trait with the most promise for enhancing soybean pod number and grain yield. Therefore, the mutant alleles of the four AP1 homologs described here will be invaluable for molecular breeding of improved soybean yield.

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.