Search

In this study, a polydopamine (PDA)-modified hollow fiber-immobilized xanthine oxidase (XOD) was prepared for screening potential XOD inhibitors from flavonoids. Several parameters for the preparation of PDA-modified hollow fiber-immobilized XOD, including the dopamine concentration, modification time, XOD concentration and immobilization time, were optimized. The results show that the optimal conditions for immobilized XOD activity were a dopamine concentration of 2.0 mg/mL in 10.0 mM Tris-HCl buffer (pH 8.5), a modification time of 3.0 h, an XOD concentration of 1000 μg/mL in 10.0 mM phosphate buffer (pH 7.5) and an immobilization time of 3.0 h. Subsequently, the enzymatic reaction conditions such as the pH value and temperature were investigated, and the enzyme kinetics and inhibition parameters were determined. The results indicate that the optimal pH value (7.5) and temperature (37 °C) of the PDA-modified hollow fiber-immobilized XOD were consistent with the free enzyme. Moreover, the PDA-modified hollow fiber-immobilized XOD could still maintain above 50% of its initial immobilized enzyme activity after seven consecutive cycles. The Michaelis–Menten constant (Km) and the half-maximal inhibitory concentration (IC50) of allopurinol on the immobilized XOD were determined as 0.25 mM and 23.2 μM, respectively. Furthermore, the PDA-modified hollow fiber-immobilized XOD was successfully applied to evaluate the inhibitory activity of eight flavonoids. Quercetin, apigenin, puerarin and epigallocatechin showed a good inhibition effect, and their percentages of inhibition were (79.86 ± 3.50)%, (80.98 ± 0.64)%, (61.15 ± 6.26)% and (54.92 ± 0.41)%, respectively. Finally, molecular docking analysis further verified that these four active compounds could bind to the amino acid residues in the XOD active site. In summary, the PDA-modified hollow fiber-immobilized XOD is an efficient method for the primary screening of XOD inhibitors from natural products.

Leaf angle is a key parameter that determines plant architecture and crop yield. Hormonal crosstalk involving brassinosteroid (BR) plays an essential role in leaf angle regulation in cereals. In this study, we investigated whether abscisic acid (ABA), an important stress-responsive hormone, co-regulates lamina joint inclination together with BR, and, if so, what the underlying mechanism is. Therefore, lamina joint inclination assay and RNA sequencing (RNA-Seq) analysis were performed here. ABA antagonizes the promotive effect of BR on leaf angle. Hundreds of genes responsive to both hormones that are involved in leaf-angle determination were identified by RNA-Seq and the expression of a gene subset was confirmed using quantitative real-time PCR (qRT-PCR). Results from analysis of rice mutants or transgenic lines affected in BR biosynthesis and signaling indicated that ABA antagonizes the effect of BR on lamina joint inclination by targeting the BR biosynthesis gene D11 and BR signaling genes GSK2 and DLT, thus forming a multi-level regulatory module that controls leaf angle in rice. Taken together, our findings demonstrate that BR and ABA antagonistically regulate lamina joint inclination in rice, thus contributing to the elucidation of the complex hormonal interaction network that optimizes leaf angle in rice.