CONSTANS-LIKE 1a positively regulates salt and drought tolerance in soybean.

Salt and drought stresses are major factors limiting soybean (Glycine max [L.] Merr.) growth and development; thus, improving soybean stress tolerance is critical. In this study, both salt stress and drought stress induced mRNA levels of CONSTANS-like 1a (GmCOL1a) and stabilized the GmCOL1a protein. Transgenic 35S:GmCOL1a soybean plants exhibited enhanced salt and drought tolerance, with higher relative water content in leaves, greater proline content, lower malondialdehyde (MDA) content, and less reactive oxygen species (ROS) production compared with wild-type plants; the GmCOL1a knockout co-9 mutant showed opposite phenotypes. In addition, GmCOL1a promoted the expression of genes related to salt tolerance, effectively reducing the Na+/K+ ratio in soybean plants, especially in stems and leaves of 35S:GmCOL1a soybean. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis identified two potential direct targets of GmCOL1a, late embryogenesis abundant (GmLEA) and Δ1-pyrroline-5-carboxylate synthetase (GmP5CS) genes, which were verified by chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR), electrophoretic mobility shift assay (EMSA), and transient transcriptional activation assays. GmCOL1a bound directly to the Myc(bHLH)-binding and Che-binding motifs of GmLEA and GmP5CS promoters to stimulate mRNA expression. Analysis of transgenic hairy-root GmP5CS:GmP5CS soybean plants in wild type, co-9, and 35S:GmCOL1a backgrounds further revealed that GmCOL1a enhances salt and drought tolerance by promoting GmP5CS protein accumulation in transgenic soybean hairy roots. Therefore, we demonstrate that GmCOL1a plays an important role in tolerance to abiotic stress in soybean.

Growth Repressor GmRAV Binds to the GmGA3ox Promoter to Negatively Regulate Plant Height Development in Soybean.

Plant height is an important component of plant architecture, and significantly affects crop quality and yield. A soybean GmRAV (Related to ABI3/VP1) transcription factor containing both AP2 and B3 domains is a growth repressor. Three GmRAV-overexpressing (GmRAV-ox) transgenic lines displayed extremely shorter height and shortened internodes compared with control plants, whereas transgenic inhibition of GmRAV expression resulted in increased plant height. GmRAV-ox soybean plants showed a low active gibberellin level and the dwarf phenotype could be rescued by treatment with exogenous GA3 treatment. ChIP (Chromatin immunoprecipitation)-qPCR assay showed that GmRAV could directly regulate the expression of the GA4 biosynthetic genes GA3-oxidase (GmGA3ox) by binding two CAACA motifs in the GmGA3ox promoter. The GmGA3ox promoter was bound by GmRAV, whose expression levels in leaves were both elevated in GmRAV-i-3 and decreased in GmRAV-ox-7 soybean plants. Transient expression assay in N. benthamiana also showed that the proGmRAV:GmRAV-3F6H effector strongly repressed the expression of LUC reporter gene driven by GmGA3ox promoter containing two CAACA motifs. Together, our results suggested that GmRAV protein repressed the expression of GmGA3ox by directly binding to the two CAACA motifs in the promoter to limit soybean plant height.

GmRAV confers ecological adaptation through photoperiod control of flowering time and maturity in soybean.

Photoperiod strictly controls vegetative and reproductive growth stages in soybean (Glycine max). A soybean GmRAV (Related to ABI3/VP1) transcription factor containing both AP2 and B3 domains was shown to be a key component of this process. We identified six polymorphisms in the GmRAV promoter that showed significant association with flowering time and maturity of soybean in one or multiple environments. Soybean varieties with minor polymorphism exhibited a longer growth period contributing to soybean adaptation to lower latitudes. The cis-acting element GT1CONSENSUS motif of the GmRAV promoter controlled the growth period, and the major allele in this motif shortened duration of late reproductive stages by reducing GmRAV expression levels. Three GmRAV-overexpressing (GmRAV-ox) transgenic lines displayed later flowering time and maturity, shorter height and fewer numbers of leaves compared with control plants, whereas transgenic inhibition of GmRAV expression resulted in earlier flowering time and maturity and increased plant height. Combining DNA affinity purification sequencing and RNA sequencing analyses revealed 154 putative target genes directly bound and transcriptionally regulated by GmRAV. Two GmRAV binding motifs [C(A/G)AACAA(G/T)A(C/T)A(G/T)] and [C(T/A)A(C)C(T/G)CTG] were identified, and acting downstream of E3E4, GmRAV repressed GmFT5a transcriptional activity through binding a CAACA motif, thereby delaying soybean growth and extending both vegetative and reproductive phases.

DNA-encoded CH functionality via photoredox-mediated hydrogen atom transformation catalysis.

We described a mode of catalytic activation that accomplished the α-alkylation of N-Boc saturated heterocycles with DNA-linked acrylamide via photoredox-mediated hydrogen atom transfer (HAT) catalysis. This C(sp3)-C(sp3) bond formation reaction tolerated five-, six- and seven-membered cyclic substrates, substantially streamline synthetic efforts to functionalize the α-position of heterocycles with native CH functional handle. This photoredox catalyzed CH functionalization proceeded in mild DNA-compatible condition, and suited for the construction of DNA-encoded libraries.

Overexpression of GmGAMYB Accelerates the Transition to Flowering and Increases Plant Height in Soybean.

The flowering time and plant height of soybean are important agronomic characters, which control the adaptability and yield of soybean. R2R3 MYB transcription factor plays an important regulatory role in plant growth and development. In this study, soybean GmGAMYB gene of R2R3-MYB type was induced by long-days (LDs). GmGAMYB showed higher transcriptional levels in the flowers, leaves and pods of soybean. Overexpression of GmGAMYB in transgenic soybean showed earlier flowering time and maturity in LDs and short-days (SDs). GmGAMYB interacted with GmGBP1 and might promote flowering time by up-regulating the expression of GmFULc gene in soybean. Moreover, the expression level of GmGAMYB was also induced by gibberellins (GAs) and the plant height of GmGAMYB-ox plants was significantly increased, which was caused by the enlargement of internode cell in stem. Furthermore, GmGAMYB overexpression led to increased GA sensitivity in the hypocotyl of soybean seedlings compared with WT. GmGAMYB may be a positive regulator of GA response of promoting plant height by up-regulating the expression of GmGA20ox gene in soybean. Together, our studies preliminarily showed that the partial functions of GmGAMYB in regulating flowering time and GA pathway.

GmIDD Is Induced by Short Days in Soybean and May Accelerate Flowering When Overexpressed in Arabidopsis via Inhibiting AGAMOUS-LIKE 18.

Photoperiod is one of the main climatic factors that determine flowering time and yield. Some members of the INDETERMINATE DOMAIN (IDD) transcription factor family have been reported to be involved in regulation of flowering time in Arabidopsis, maize, and rice. In this study, the domain analysis showed that GmIDD had a typical ID domain and was a member of the soybean IDD transcription factor family. Quantitative real-time PCR analysis showed that GmIDD was induced by short day conditions in leaves and regulated by circadian clock. Under long day conditions, transgenic Arabidopsis overexpressing GmIDD flowered earlier than wild-type, and idd mutants flowered later, while the overexpression of GmIDD rescued the late-flowering phenotype of idd mutants. Chromatin immunoprecipitation sequencing assays of GmIDD binding sites in GmIDD-overexpression (GmIDD-ox) Arabidopsis further identified potential direct targets, including a transcription factor, AGAMOUS-like 18 (AGL18). GmIDD might inhibit the transcriptional activity of flower repressor AGL18 by binding to the TTTTGGTCC motif of AGL18 promoter. Furthermore, the results also showed that GmIDD overexpression increased the transcription levels of flowering time-related genes FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), LEAFY (LFY) and APETALA1 (AP1) in Arabidopsis. Taken together, GmIDD appeared to inhibit the transcriptional activity of AGL18 and induced the expression of FT gene to promote Arabidopsis flowering.

Visualization of arrestin recruitment by a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are critically regulated by ß-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ß2 adrenergic receptor (ß2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ß-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-ß-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ß2AR-ß-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ß2AR and ß-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ß-arrestin 1 to the ß2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ß-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ß-arrestin 1 when coupled to the ß2AR. A molecular model of the ß2AR-ß-arrestin signalling complex was made by docking activated ß-arrestin 1 and ß2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.