ABSTRACT
The mechanisms of plant drought resistance are unclear but may involve membrane trafficking and metabolic reprogramming, including proline accumulation. Forward genetic screening using a proline dehydrogenase 1 (ProDH1) promoter:reporter identified a drought hypersensitive mutant with a single-amino acid substitution (P335L) in the nonphototrophic hypocotyl 3 (NPH3) domain of NPH3/root phototropism 2-like 5 (NRL5)/naked pins in Yucca 8 (NPY8). Further experiments found that NRL5 and other NPH3 domain proteins are guanosine triphosphatases (GTPases). NRL5, but not NRL5P335L, interacted with the RABE1c and RABH1b GTPases and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Vesicle-Associated Membrane Protein (VAMP)721/722. These proteins controlled NRL5 localization and connection to trafficking while also being genetically downstream of, and potentially regulated by, NRL5. These data demonstrate that NRL5-mediated restraint of proline catabolism is required for drought resistance and also reveal unexpected functions of the NPH3 domain such that the role of NPH3 domain proteins in signaling, trafficking, and cellular polarity can be critically reevaluated.
Subject(s)
Droughts , Protein Transport , Arabidopsis/genetics , Arabidopsis/metabolism , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Protein Domains , Stress, Physiological , Plant Proteins/metabolism , Plant Proteins/genetics , Mutation , Drought ResistanceABSTRACT
Isatis indigotica Fort. (family Cruciferae), is an herb widely used in traditional herbal medicine and its dried leave was named "ISATIDIS FOLIUM". Baphicacanthus cusia (Ness) Bremek. and Polygonum tinctorium Ait. are commonly misused as ISATIDIS FOLIUM in Chinese Medicine pharmacy. For the purpose of being not misused, specific primers based on the sequence difference of chloroplast trnH-psbA intergenic spacer were designed and multiplex polymerase chain reaction method (multiplex PCR) was developed. In this study, 29 original herbal materials were analyzed and our results show that DNA size after multiplex PCR was able to distinguish variations between three herbs. DNA fragments of 464, 297, 170 base pairs (bps) were represented for I. indigotica and B. cusia and P. tinctorium, respectively. In conclusion, our investigations demonstrate that molecular identification method provides more accurate results for medicinal plants detection and good quality control of ISATIDIS FOLIUM.
Subject(s)
Isatis , Plants, Medicinal , Isatis/genetics , Multiplex Polymerase Chain Reaction , Plant Leaves , Plants, Medicinal/genetics , Sequence Analysis, DNA , Species SpecificityABSTRACT
A simple gradient high-performance liquid chromatography with diode array detection (HPLC-DAD) method was used to simultaneously to analyze characteristics of six indicator compounds in the traditional Chinese medicine (TCM) formulation Wen-Qing-Yin (WQY). Separate optimization was performed using a Cosmosil C18 column gradient method with 0.1% formic acid in both mobile phases of aqueous and acetonitrile (ACN), at a flow rate, detection wavelength, and sample volume of 1.8 mL/min, 268 nm, and 10 µL, respectively. The linear regression of six active compounds berberine (BER), baicalin (BAI), ferulic acid (FER), geniposide (GEN), hydorxymethoxylfurfural (HMF), and paeoniflorin (PAE) was produced at the concentration range of 10-2000 µg/mL. The method validation revealed an acceptable precision (intra- and inter-day precision < 3.39% and 4.11%, respectively) and recovery (85.60-110.45% and 86.58-110.90%), a recovery range of 86.61-109.42%, and sensitivity (limit of detection [LOD] and limit of quantification [LOQ] values were in the range of 0.03-3.13, and 0.08-9.38 µg/mL, respectively) while the calibration curves were linear with a correlation coefficient (R2) ranging from 0.9966 to 0.9989. The qualitative and quantitative analyses were performed by direct comparison of the peaks of the WCY extract to retention times of reference standards. Additionally, principal component analysis (PCA) successfully discriminated four purchased commercial samples of all six indicator constituents, and the present results indicate their comprehensive potential usefulness for qualitative and quantitative analyses of the WQY decoction and its commercial products.