Cryo-EM structure and molecular mechanism of abscisic acid transporter ABCG25.

Abscisic acid (ABA) is one of the plant hormones that regulate various physiological processes, including stomatal closure, seed germination and development. ABA is synthesized mainly in vascular tissues and transported to distal sites to exert its physiological functions. Many ABA transporters have been identified, however, the molecular mechanism of ABA transport remains elusive. Here we report the cryogenic electron microscopy structure of the Arabidopsis thaliana adenosine triphosphate-binding cassette G subfamily ABA exporter ABCG25 (AtABCG25) in inward-facing apo conformation, ABA-bound pre-translocation conformation and outward-facing occluded conformation. Structural and biochemical analyses reveal that the ABA bound with ABCG25 adopts a similar configuration as that in ABA receptors and that the ABA-specific binding is dictated by residues from transmembrane helices TM1, TM2 and TM5a of each protomer at the transmembrane domain interface. Comparison of different conformational structures reveals conformational changes, especially those of transmembrane helices and residues constituting the substrate translocation pathway during the cross-membrane transport process. Based on the structural data, a 'gate-flipper' translocation model of ABCG25-mediated ABA cross-membrane transport is proposed. Our structural data on AtABCG25 provide new clues to the physiological study of ABA and shed light on its potential applications in plants and agriculture.

The Prmt5-Vasa module is essential for spermatogenesis in Bombyx mori.

In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, we engineered mutations in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, we found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl, implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.

Preparation, characterization and bioactivities of derivatives of an exopolysaccharide from Lachnum.

An exopolysaccharide, obtained previously LEP-2b from Lachnum YM405, was phosphated and sulfated successfully. The derivatives named PLEP-2b and SLEP-2b, respectively, and their respective degree of substitution were 0.174 and 0.431. Phosphate groups -PO3H2 substituted at C-6 of 1,4-ß-D-mannopyranose, C-5 of 2,6-ß-d-1-OMe-mannofuranoside, C-3 of 1,6-ß-D-galactopyranose, C-2 of 1-ß-D-glucopyranose, and C-6 of 1,2-α-D-rhampyranose, while sulfate groups SO3H were mainly at C-6 of 1,4-ß-D-Manp, C-6 of 1-ß-D-Glcp and C-6 of 1,2-α-D-Rhap. Compared with LEP-2b, the scavenging effects of the derivatives, on hydroxyl radical and superoxide anion were significantly increased after the modifications, except for reducing power. Meanwhile, phosphorylated and sulfated modifications remarkably strengthened the inhibiting effect of LEP-2b on the proliferation of CT-26 murine colon carcinoma, Lewis lung carcinoma and human hepatocellular carcinoma HepG2 cells. The derivatives significantly enhanced the antioxidant and antitumor activities in vitro. Compared with sulfation, phosphorylation improved the inhibitory effect more contraposingly on some specific tumor cells.

Carboxymethylation of an exopolysaccharide from Lachnum and effect of its derivatives on experimental chronic renal failure.

Carboxymethylated polysaccharide CLEP-1b was prepared from a single component (LEP-1b) of Lachnum YM281 exopolysaccharides by molecular modification with a degree of substitution (DS) of 0.286. Infrared result proved that the carboxymethylation of LEP-1b succeeded and (13)C NMR result showed that the carboxymethyl group (CH2COOH) was chemically linked to an oxygen (O) atom of the hydroxyl on C-3 of LEP-1b. LEP-1b could improve the histopathological status of kidney and significantly reduce the contents of serum creatinine (Scr) and blood urea nitrogen (BUN), and increase the contents of total protein and albumin. It could also enhance the activity of SOD, GSH-PX, CAT, GSH and decrease MDA contents in the nephridial and hepatic tissues. What's more, CLEP-1b showed more significant effects than LEP-1b at the same dosage. The research indicated that LEP-1b and CLEP-1b could mitigate the chronic renal failure of mice and the effects were closely associated with antioxidant activity.