Cucumis sativus PHLOEM PROTEIN 2-A1 like gene positively regulates salt stress tolerance in cucumber seedlings.

PHLOEM PROTEIN 2-A1 like (PP2-A1) gene is a member of the PP2 multigene family, and the protein encoded by which has the function of stress defense. Based on our previous proteomic study of cucumber phloem sap, CsPP2-A1 protein expression was significantly enriched under salt stress. In this paper, we obtained CsPP2-A1 interfering (CsPP2-A1-RNAi) cucumber by Agrobacterium tumefaciens-mediated method. The phenotypic changes of wild-type (WT) cucumber, CsPP2-A1-overexpressing (OE) cucumber, and CsPP2-A1-RNAi cucumber under salt treatment were observed and compared. Furthermore, physiological indicators were measured in four aspects: osmoregulation, membrane permeability, antioxidant system, and photosynthetic system. The analysis of contribution and correlation for each variable were conducted by principal component analysis (PCA) and Pearson's correlation coefficient. The above results showed that CsPP2-A1-RNAi cucumber plants exhibited weaker salt tolerance compared to WT cucumber and CsPP2-A1-OE cucumber plants in terms of phenotype and physiological indicators in response to salt stress, while CsPP2-A1-OE cucumber always showed the robust salt tolerance. Together, these results indicated that CsPP2-A1 brought a salinity tolerance ability to cucumber through osmoregulation and reactive oxygen species (ROS) homeostasis. The results of the study provided evidence for the function of CsPP2-A1 in plant salt tolerance enhancement, and they will serve as a reference for future salt-tolerant cucumber genetic manipulation.

The phloem lectin PP2-A1 enhances aphid resistance by affecting aphid behavior and maintaining ROS homeostasis in cucumber plants.

Aphid (Aphis gossypii Glover) attack frequently results in a significant loss of output and deterioration of fruit quality in cucumber (Cucumis sativus L.). Phloem protein 2 (PP2) is conserved as a phloem lectin in plants, and few studies have been conducted on the regulatory mechanism of PP2. Based on our previous study of CsPP2-A1 in cucumber, to further investigate the biological function of CsPP2-A1, we compared the changes of selectivity, non-selectivity, colonization, reproductions of aphids, and the phenotype in wild type (WT), CsPP2-A1 overexpressing (CsPP2-A1-OE), and CsPP2-A1 interfering (CsPP2-A1-RNAi) cucumber plants after inoculation with aphids. We found that CsPP2-A1-OE cucumber plants generated resistance to aphids. The aphid colonization rate and number of reproductions of CsPP2-A1-OE cucumber plants were significantly lower than that of WT and CsPP2-A1-RNAi cucumber plants. Through Pearson's correlation and principal component analysis (PCA), it was found that CsPP2-A1 played a crucial role in the balance of reactive oxygen species (ROS) in plants. Overexpression of the CsPP2-A1 resulted in increased levels of antioxidant enzyme, eliminating ROS and preventing the damage by ROS in cucumber. Furthermore, nutritional imbalance for aphids and content of secondary metabolites were increased in overexpressed CsPP2-A1 cucumber plants, and thus preventing aphid attack. These together may improve cucumber resistance against aphids and the mechanism of CsPP2-A1 defense against aphids was preliminarily explored.

Prokaryotic expression, purification, physicochemical properties and antifungal activity analysis of phloem protein PP2-A1 from cucumber.

Phloem protein 2 (PP2) is a protein having lectin properties that can be isolated from the phloem sap. Based on our previous proteomic study of phloem sap of Cucumis sativus, it was found that the expression of PP2 A1-like was significantly up-regulated under salt stress, which may be a molecular mechanism of plant adaptation to stress. This paper carried out the expression and purification of the CsPP2-A1 gene in E. coli for further characteristic analysis. The results demonstrated that the CsPP2-A1 in shake flask cultures was mainly expressed in the soluble form at 15 °C or in inclusion bodies at 37 °C. Secondly, Ni-IDA affinity chromatography and SDS-PAGE were employed to yield highly purified CsPP2-A1 protein. The purified CsPP2-A1 was then subjected to Western blot and MALDI-TOF-MS analysis for protein identification. The biological activity analysis results showed that CsPP2-A1 had hemagglutinating activities to rabbit erythrocytes, and Chitotetraose may be the specific inhibitory sugar of CsPP2-A1. The optimal hemagglutination activity of CsPP2-A1 protein was achieved between pH 5-9, and between 20 and 60 °C. Moreover, CsPP2-A1 had significant inhibitory effects on Botrytis cinerea and Phytophthora infestans, and the inhibitory effect on B. cinerea was better than that on P. infestans.

Integrated pathological cell fishing and network pharmacology approach to investigate main active components of Er-Xian decotion for treating osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative damage to osteoblasts was a key factor in the development of osteoporosis. Er-Xian Decotion (EXD) is widely used in China for the treatment of osteoporosis, which has a variety of antioxidant active ingredients. EXD may be an important source of protection against oxidative damage in osteoblasts, but the anti-osteoporotic active components of EXD is currently unclear. AIM OF THE STUDY: This work established an effective and reliable drug screening method to find main active ingredients in EXD (M-EXD) that can protect osteoblasts against oxidative stress and achieve anti-osteoporosis effects. MATERIALS AND METHODS: H2O2-induced osteoblast cell fishing with UHPLC-QTOF/MS was firstly used to discover the potential active components from EXD. Afterword, the EXD compound-osteoporosis target network was constructed using network pharmacology, thus potentially anti-osteoporosis ingredients were founded, and their combination were defined as the M-EXD. Finally, pharmacology effects of M-EXD was evaluated by ovariectomized rats, prednisolone induced-zebrafish and H2O2-induced osteoblasts. RESULTS: 40 candidate active ingredients in EXD were initially screened out via pathological cell fishing. According to network pharmacology result, M-EXD consisted of 13 ingredients since they had a close relationship with 65 osteoporosis-related targets. Pharmacological evaluation showed that M-EXD significantly ameliorated oxidative stress in H2O2-induced osteoblast model, evidently reversed the activity of ALP, ROS, GSH-px, NO and MDA compared with the model group. M-EXD showed better anti-oxidative activities than individual ingredients, presenting obvious synergetic effects. In osteoporosis rat and zebrafish models, M-EXD also demonstrated good anti-osteoporotic properties by mitigating the osteoporosis bone loss and increasing serum bone morphogenetic protein 2, and reversing osteocalcin expression in bone tissue. It significantly ameliorated oxidative stress in the in-vivo models. Moreover, M-EXD and EXD showed similar anti-osteoporotic and anti-oxidative properties, while the rest components of EXD had no satisfactory anti-osteoporotic efficacy. CONCLUSIONS: Our work successfully identified the main active components in EXD, which could represent the efficacy of EXD on treating osteoporosis, and meanwhile, it also provided an effective strategy to investigate active ingredients from natural medicines, which might be helpful for drug development and application.