Alternative splicing of CsWRKY21 positively regulates cold response in tea plant.

Alternative splicing (AS) was an important post-transcriptional mechanism that involved in plant resistance to adversity stress. WRKY transcription factors function as transcriptional activators or repressors to modulate plant growth, development and stress response. However, the role of alternate splicing of WRKY in cold tolerance is poorly understood in tea plants. In this study, we found that the CsWRKY21 transcription factor, a member of the WRKY IId subfamily, was induced by low temperature. Subcellular localization and transcriptional activity assays showed that CsWRKY21 localized to the nucleus and had no transcriptional activation activity. Y1H and dual-luciferase reporter assays showed that CsWRKY21 suppressed expression of CsABA8H and CsUGT by binding with their promoters. Transient overexpression of CsABA8H and CsUGT reduced abscisic acid (ABA) content in tobacco leaves. Furthermore, we discovered that CsWRKY21 undergoes AS in the 5'UTR region. The AS transcript CsWRKY21-b was induced at low temperature, up to 6 folds compared to the control, while the full-length CsWRKY21-a transcript did not significantly change. Western blot analysis showed that the retention of introns in the 5'UTR region of CsWRKY21-b led to higher CsWRKY21 protein content. These results revealed that alternative splicing of CsWRKY21 involved in cold tolerance of tea plant by regulating the protein expression level and then regulating the content of ABA, and provide insights into molecular mechanisms of low temperature defense mediated by AS in tea plant.

Advanced researches of traditional uses, phytochemistry, pharmacology, and toxicology of medical Uncariae Ramulus Cum Uncis.

ETHNOPHARMACOLOGICAL RELEVANCE: Medical Uncariae Ramulus Cum Uncis consists of Uncaria rhynchophylla (Miq.) Miq. ex Havil, Uncaria macrophylla Wall, Uncaria sinensis (Oliv.) Havil, Uncaria hirsuta Havil, and Uncaria sessilifructus Roxb, which belongs to the species widely used in the genus Uncaria. These species resource widely distributed in China and abroad, and the hook-bearing stem is the primary constituent enrichment site. There are many different forms and architectures of chemicals, depending on the extraction site. Traditional remedies employing URCU had been used widely in antiquity and were first compiled in renowned ancient masterpiece 'Mingyi Bielu ()' written by Hongjing Tao. In modern pharmacological studies, both the total extracts and the phytoconstituents isolated from URCU have been shown to have neuroprotective, antioxidant, anti-inflammatory, anticancer, antibacterial, and autophagy-enhancer properties. AIM OF THE STUDY: This review concentrates on the traditional uses, phytochemistry, pharmacology, toxicology, and nanomaterials studies of URCU, with a perspective to assist with further research and advance. MATERIAL AND METHODS: The Chinese and English literature studies of this review are based on these database searches including Science Direct, CNKI, Wiley online library, Spring Link, Web of Science, PubMed, Medalink, Google scholar, Elsevier, ACS Publications, iPlant, Missouri Botanical Garden, Plant of the World Online. The pertinent data on URCU was gathered. RESULTS: Based on the examination of the genus Uncaria, 107 newly marked chemical compositions have been identified from URCU from 2015 to present, including alkaloids, terpenoids, flavonoids, steroids, and others. Pharmacological studies have demonstrated that URCU has a variety of benefits in diseases such as neurodegenerative diseases, cancer, cardiovascular diseases, diabetes, and migraine, due to its neuroprotective, anti-inflammatory, antioxidant, anti-tumor, anti-bacterial and anti-viral properties. According to metabolic and toxicological studies, the dosage, frequency, and interactions of the drugs that occur in vivo are of great significance for determining whether the organic bodies can perform efficacy or produce toxicity. The research on URCU-mediated nanomaterials is expanding and increasing in order to address the inadequacies of conventional Chinese medicine. The alkaloids in URCU have the capability to self-assemble with other classes of components in addition to being biologically active. CONCLUSION: URCU plants are widely distributed, abundant in chemical constituents, and widely used in both traditional and modern medicine for a variety of pharmacological effects. The utilization of herbal medicines can be raised by assessing the pharmacological distinctions among several species within the same genus and may accelerate the modernization of traditional Chinese medicine. Controlling the concentration of drug administration, monitoring metabolic markers, and inventing novel nanotechnologies are effective strategies for synergistic influence and detoxification to alleviate the main obstacles that toxicity, low bioavailability, and poor permeability. This review can assist further research and advances.

Mitochondria-anchoring self-assembled nanoparticles for multi-path energy depletion: A "nano bomb" in chemo-co-starvation therapy.

As the main systemic treatment for triple-negative breast cancer (TNBC), the bleak medical prognosis of chemotherapy resulted in impaired life quality by tumor recurrence and metastasis. The feasible cancer starvation therapy could inhibit tumor progression by blocking energy supplements, however, the mono-therapeutic modality showed limited curing efficacy due to heterogeneity and abnormal energy metabolism of TNBC. Thus, the development of a synergistic nano-therapeutic modality involving different anti-tumor mechanisms to simultaneously transport medicines to the organelle where metabolism took place, might remarkably improve curing efficacy, targeting ability, and bio-safety. Herein, the hybrid BLG@TPGS NPs were prepared by doping multi-path energy inhibitors Berberine (BBR) and Lonidamine (LND) as well as the chemotherapeutic agent Gambogic acid (GA). Our research indicated that Nanobomb-BLG@TPGS NPs inherited the mitochondria targeting ability from BBR to accumulate precisely at the "energy factory" mitochondria, and then induce starvation therapy to efficiently eradicated cancer cells by coordinately powered off tumor cells via a "three-prone strategy" to cut off mitochondrial respiration, glycolysis, and glutamine metabolism. The inhibition of tumor proliferation and migration was enlarged by the synergistic combination with chemotherapy. Besides, apoptosis via mitochondria pathway and mitochondria fragmentation supported the hypothesis that NPs eliminated MDA-MB-231 cells by violently attacking MDA-MB-231 cells and especially the mitochondria. In summary, this synergistic chemo-co-starvation nanomedicine proposed an innovative site-specific targeting strategy for improved tumor treatment and decreased toxicity to normal tissues, which provided an option for clinical TNBC-sensitive treatment.

Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses.

BACKGROUND: Laccase (LAC) is the pivotal enzyme responsible for the polymerization of monolignols and stress responses in plants. However, the roles of LAC genes in plant development and tolerance to diverse stresses are still largely unknown, especially in tea plant (Camellia sinensis), one of the most economically important crops worldwide. RESULTS: In total, 51 CsLAC genes were identified, they were unevenly distributed on different chromosomes and classified into six groups based on phylogenetic analysis. The CsLAC gene family had diverse intron-exon patterns and a highly conserved motif distribution. Cis-acting elements in the promoter demonstrated that promoter regions of CsLACs encode various elements associated with light, phytohormones, development and stresses. Collinearity analysis identified some orthologous gene pairs in C. sinensis and many paralogous gene pairs among C. sinensis, Arabidopsis and Populus. Tissue-specific expression profiles revealed that the majority of CsLACs had high expression in roots and stems and some members had specific expression patterns in other tissues, and the expression patterns of six genes by qRT‒PCR were highly consistent with the transcriptome data. Most CsLACs showed significant variation in their expression level under abiotic (cold and drought) and biotic (insect and fungus) stresses via transcriptome data. Among them, CsLAC3 was localized in the plasma membrane and its expression level increased significantly at 13 d under gray blight treatment. We found that 12 CsLACs were predicted to be targets of cs-miR397a, and most CsLACs showed opposite expression patterns compared to cs-miR397a under gray blight infection. Additionally, 18 highly polymorphic SSR markers were developed, these markers can be widely used for diverse genetic studies of tea plants. CONCLUSIONS: This study provides a comprehensive understanding of the classification, evolution, structure, tissue-specific profiles, and (a)biotic stress responses of CsLAC genes. It also provides valuable genetic resources for functional characterization towards enhancing tea plant tolerance to multiple (a)biotic stresses.

Identification and Functional Analysis of Two Alcohol Dehydrogenase Genes Involved in Catalyzing the Reduction of (Z)-3-Hexenal into (Z)-3-Hexenol in Tea Plants (Camellia sinensis).

Alcohol dehydrogenase (ADH) is a vital enzyme in the biosynthesis pathway of six-carbon volatiles in plants. However, little is known about its functions in tea plants. Here, we identified two ADH genes (CsADH1 and CsADH2). An in vitro protein expression assay showed that both CsADH1 and CsADH2 proteins can catalyze the reduction of (Z)-3-hexenal into (Z)-3-hexenol. Subcellular localization revealed that both CsADH1 and CsADH2 proteins were predominantly localized in the nucleus and cytosol. CsADH1 had high transcripts in young stems in autumn, while CsADH2 showed extremely high expression levels in stems and roots. The expression of CsADH2 was mainly downregulated under ABA treatment, while CsADH1 and CsADH2 transcripts were significantly lower under MeJA treatment at 12 and 24 h. Under cold treatment, CsADH1 transcripts first decreased and then increased, while CsADH2 demonstrated an almost opposite expression pattern. Notably, CsADH2 was significantly upregulated under simulated Ectropis obliqua invasion. Gene suppression by antisense oligonucleotides (AsODNs) demonstrated that AsODN_ADH2 treatment significantly reduced CsADH2 transcripts and the abundance of (Z)-3-hexenol products. The results indicate that the two CsADH genes may play an important role in response to (a)biotic stresses and in the process of (Z)-3-hexenol biosynthesis.

Fe(II) enhances simultaneous phosphorus removal and denitrification in heterotrophic denitrification by chemical precipitation and stimulating denitrifiers activity.

Using Fe(II) salt as the precipitant in heterotrophic denitrification achieves improved TP removal, and enhancement in denitrification was often observed. This study aimed to obtain a better understanding of Fe(II)-enhanced denitrification with sufficient carbon source supply. Laboratory-scale experiments were conducted in SBRs with or without Fe(II) addition. Remarkably improved TP removal was experienced. TP removal efficiency in Fe(II) adding reactor was 85.8 ± 3.4%; whereas, that in the reactor without Fe(II) addition was 31.1 ± 2.8%. Besides improved TP removal, better TN removal efficiency (94.1 ± 1.1%) were recorded when Fe(II) was added, and that in the reactor without Fe(II) addition was 89 ± 0.8%. The specific denitrification rate were observed increase by 12.6% when Fe(II) was added. Further microbial analyses revealed increases in the abundances of typical denitrifiers (i.e. Niastella, Opitutus, Dechloromonas, Ignavibacterium, Anaeromyxobacter, Pedosphaera, and Myxococcus). Their associated denitrifying genes, narG, nirS, norB, and nosZ, were observed had 14.2%, 19.4%, 21.6%, and 9.9% elevation, respectively. Such enhancement in denitrification shall not be due to nitrate-dependent ferrous oxidation, which prevails in organic-deficient environments. In an environment with a continuous supply of Fe(II) and plenty of carbon sources, a cycle of denitrifying enzyme activity enhancement in the presence of Fe(II) facilitating nitrogen substrate utilization, stimulating denitrifier metabolism and growth, elevating denitrifying genes abundance, and increasing denitrifying enzymes expression were thought to be responsible for the Fe(II)-enhanced heterotrophic denitrification. Fe(II) salt is often a less expensive precipitant and has recently become attractive for TP removal in wastewater. The findings of this study solidify previous observation of enhancement of both TP and TN removal by adding Fe(II) in denitrification, and would be helpful for developing cost-effective pollutant removal processes.