Engineering Antimicrobial Metal-Phenolic Network Nanoparticles with High Biocompatibility for Wound Healing.

Antibiotic-resistant bacteria pose a global health threat by causing persistent and recurrent microbial infections. To address this issue, antimicrobial nanoparticles (NPs) with low drug resistance but potent bactericidal effects have been developed. However, many of the developed NPs display poor biosafety and their synthesis often involves complex procedures and the antimicrobial modes of action are unclear. Herein, a simple strategy is reported for designing antimicrobial metal-phenolic network (am-MPN) NPs through the one-step assembly of a seeding agent (diethyldithiocarbamate), natural polyphenols, and metal ions (e.g., Cu2+ ) in aqueous solution. The Cu2+ -based am-MPN NPs display lower Cu2+ antimicrobial concentrations (by 10-1000 times) lower than most reported nanomaterials and negligible toxicity across various models, including, cells, blood, zebrafish, and mice. Multiple antimicrobial modes of the NPs have been identified, including bacterial wall disruption, reactive oxygen species production, and quinoprotein formation, with the latter being a distinct pathway identified for the antimicrobial activity of the polyphenol-based am-MPN NPs. The NPs exhibit excellent performance against multidrug-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus (MRSA)), efficiently inhibit and destroy bacterial biofilms, and promote the healing of MRSA-infected skin wounds. This study provides insights on the antimicrobial properties of metal-phenolic materials and the rational design of antimicrobial metal-organic materials.

Al-induced CsUGT84J2 enhances flavonol and auxin accumulation to promote root growth in tea plants.

Although Al is not necessary or even toxic to most plants, it is beneficial for the growth of tea plants. However, the mechanism through which Al promotes root growth in tea plants remains unclear. In the present study, we found that flavonol glycoside levels in tea roots increased following Al treatment, and the Al-induced UDP glycosyltransferase CsUGT84J2 was involved in this mechanism. Enzyme activity assays revealed that rCsUGT84J2 exhibited catalytic activity on multiple types of substrates, including phenolic acids, flavonols, and auxins in vitro. Furthermore, metabolic analysis with UPLC-QqQ-MS/MS revealed significantly increased flavonol and auxin glycoside accumulation in CsUGT84J2-overexpressing Arabidopsis thaliana. In addition, the expression of genes involved in the flavonol pathway as well as in the auxin metabolism, transport, and signaling pathways was remarkably enhanced. Additionally, lateral root growth and exogenous Al stress tolerance were significantly improved in transgenic A. thaliana. Moreover, gene expression and metabolic accumulation related to phenolic acids, flavonols, and auxin were upregulated in CsUGT84J2-overexpressing tea plants but downregulated in CsUGT84J2-silenced tea plants. In conclusion, Al treatment induced CsUGT84J2 expression, mediated flavonol and auxin glycosylation, and regulated endogenous auxin homeostasis in tea roots, thereby promoting the growth of tea plants. Our findings lay the foundation for studying the precise mechanisms through which Al promotes the growth of tea plants.

Flavonol-Aluminum Complex Formation: Enhancing Aluminum Accumulation in Tea Plants.

Polyphenol-rich tea plants are aluminum (Al) accumulators. Whether an association exists between polyphenols and Al accumulation in tea plants remains unclear. This study revealed that the accumulation of the total Al and bound Al contents were both higher in tea samples with high flavonol content than in low, and Al accumulation in tea plants was significantly and positively correlated with their flavonol content. Furthermore, the capability of flavonols combined with Al was higher than that of epigallocatechin gallate (EGCG) and root proanthocyanidins (PAs) under identical conditions. Flavonol-Al complexes signals (94 ppm) were detected in the tender roots and old leaves of tea plants through solid-state 27Al nuclear magnetic resonance (NMR) imaging, and the strength of the signals in the high flavonol content tea samples was considerably stronger than that in the low flavonol content tea samples. This study provides a new perspective for studying Al accumulation in different tea varieties.

Aluminum-tolerant, growth-promoting endophytic bacteria as contributors in promoting tea plant growth and alleviating aluminum stress.

Unlike that of other crops, the growth of tea plants can be promoted by aluminum, but its regulation mechanism remains unclear. Some endophytes can also promote growth of plant hosts. In this paper, tea roots treated with aluminum were used to study the growth-promoting traits and aluminum tolerance of endophytes. Meta-16S rDNA analysis revealed that Burkholderia was enriched in tea roots after aluminum treatment, and it was the dominant strain for hydroponic tea roots and field tea roots. Actinomycetes constituted the dominant strains in hydroponic tea seedlings treated with aluminum. Sixteen endophytic bacteria, including 12 strains of Firmicutes, 2 strains of Proteobacteria and 2 strains of Actinomycetes, were isolated and identified from hydroponic tea roots treated with different aluminum concentrations. Growth-promoting activity analysis showed that the isolated endophytic bacteria all had more than one plant growth-promoting trait. Among them, B4 (Bacillus nealsonii), B8 (Brevibacterium frigoritolerans) and A2 (Nocardia nova) bacteria each had three growth-promoting traits. Aluminum tolerance ability analysis indicated that endophyte A1 (Leifsonia shinshuensis) had the strongest aluminum tolerance ability, up to 200 mg l-1 aluminum. Plant-bacteria interactions showed that endophytes A1, A2 and B4 and their synthetic community all had a growth-promoting effect on the growth of wheat lateral roots. Moreover, endophytes A1 and B4 alleviated aluminum stress in wheat. Endophyte A1 also promoted the growth of tea cuttings, especially lateral roots, with/without aluminum. Taken together, aluminum enhanced the distribution of aluminum-tolerant and growth-promoting bacteria, thereby promoting the growth of tea roots. This study provides a new aspect for research on the mechanism by which aluminum promotes tea plant growth.

Functional Analyses of Flavonol Synthase Genes From Camellia sinensis Reveal Their Roles in Anther Development.

Flavonoids, including flavonol derivatives, are the main astringent compounds of tea and are beneficial to human health. Many researches have been conducted to comprehensively identify and characterize the phenolic compounds in the tea plant. However, the biological function of tea flavonoids is not yet understood, especially those accumulated in floral organs. In this study, the metabolic characteristics of phenolic compounds in different developmental stages of flower buds and various parts of the tea flower were investigated by using metabolomic and transcriptomic analyses. Targeted metabolomic analysis revealed varying accumulation patterns of different phenolic polyphenol compounds during flowering; moreover, the content of flavonol compounds gradually increased as the flowers opened. Petals and stamens were the main sites of flavone and flavonol accumulation. Compared with those of fertile flowers, the content of certain flavonols, such as kaempferol derivatives, in anthers of hybrid sterile flowers was significantly low. Transcriptomic analysis revealed different expression patterns of genes in the same gene family in tea flowers. The CsFLSb gene was significantly increased during flowering and was highly expressed in anthers. Compared with fertile flowers, CsFLSb was significantly downregulated in sterile flowers. Further functional verification of the three CsFLS genes indicated that CsFLSb caused an increase in flavonol content in transgenic tobacco flowers and that CsFLSa acted in leaves. Taken together, this study highlighted the metabolic properties of phenolic compounds in tea flowers and determined how the three CsFLS genes have different functions in the vegetative and reproductive organs of tea plants. Furthermore, CsFLSb could regulated flavonol biosynthesis in tea flowers, thus influencing fertility. This research is of great significance for balancing the reproductive growth and vegetative growth of tea plants.

CsHCT-Mediated Lignin Synthesis Pathway Involved in the Response of Tea Plants to Biotic and Abiotic Stresses.

Many studies have shown that phenolic compounds such as lignin and flavonoids enhance plant resistance. Tea plants are rich in flavonoid compounds. Whether these compounds are related to tea plant resistance is unclear. In this study, an interesting conclusion was drawn on the basis of experimental results: in response to abiotic stress (except for sucrose treatment), gene expression was increased in the phenylpropanoid and lignin pathways and was reduced in the flavonoid pathway in tea plants. CsHCTs, the genes located at the branch point of the lignin and flavonoid pathways, are most suitable for regulating the ratio of carbon flow in the lignin pathway and flavonoid synthesis. Enzymatic and genetic modification experiments proved that CsHCTs encode hydroxycinnamoyl-coenzyme A:shikimate/quinate hydroxycinnamoyl transferase in vitro and in vivo. Furthermore, the genetic modification results showed that the contents of phenolic acids and lignin were increased in tobacco and Arabidopsis plants overexpressing CsHCTs, whereas the content of flavonol glycosides was decreased. Both types of transgenic plants showed resistance to many abiotic stresses and bacterial infections. We speculate that CsHCTs participate in regulation of the metabolic flow of carbon from the flavonoid pathway to the chlorogenic acid, caffeoylshikimic acid, and lignin pathways to increase resistance to biotic and abiotic stresses.

Functional characterization of three flavonol synthase genes from Camellia sinensis: Roles in flavonol accumulation.

Flavonol derivatives are a group of flavonoids benefiting human health. Their abundant presence in tea is associated with astringent taste. To date, mechanism pertaining to the biosynthesis of flavonols in tea plants remains unknown. In this study, we used bioinformatic analysis mining the tea genome and obtained three cDNAs that were annotated to encode flavonol synthases (FLS). Three cDNAs, namely CsFLSa, b, and c, were heterogenously expressed in E. coli to induce recombinant proteins, which were further used to incubate with three substrates, dihydrokampferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM). The resulting data showed that three rCsFLSs preferred to catalyze (DHK). Overexpression of each cDNA in tobacco led to the increase of kampferol and the reduction of anthocyanins in flowers. Further metabolic profiling of flavan-3-ols in young tea shoots characterized that kaempferol derivatives were the most abundant, followed by quercetin and then myricetin derivatives. Taken together, these data characterized the key step committed to the biosynthesis of flavonols in tea leaves. Moreover, these data enhance understanding the metabolic accumulation relevance between flavonols and other main flavonoids such as flavan-3-ols in tea leaves.

Proanthocyanidin-Aluminum Complexes Improve Aluminum Resistance and Detoxification of Camellia sinensis.

Aluminum (Al) influences crop yield in acidic soil. The tea plant (Camellia sinensis) has high Al tolerance with abundant monomeric catechins in its leaves, especially epigallocatechin gallate (EGCG), and polymeric proanthocyanidins in its roots (rPA). The role of these polyphenols in the Al resistance of tea plants is unclear. In this study, we observed that these polyphenols could form complexes with Al in vitro, and complexation capacity was positively influenced by high solution pH (pH 5.8), polyphenol type (rPA and EGCG), and high Al concentration. In the 27Al nuclear magnetic resonance (NMR) experiment, rPA-Al and EGCG-Al complex signals could be detected both in vitro and in vivo. The rPA-Al and EGCG-Al complexes were detected in roots and old leaves, respectively, of both greenhouse seedlings and tea garden plants. Furthermore, in seedlings, Al accumulated in roots and old leaves and mostly existed in the apoplast in binding form. These results indicate that the formation of complexes with tea polyphenols in vivo plays a vital role in Al resistance in the tea plant.

A Sucrose-Induced MYB (SIMYB) Transcription Factor Promoting Proanthocyanidin Accumulation in the Tea Plant ( Camellia sinensis).

Proanthocyanidins (PAs, also called condensed tannins), are an important class of secondary metabolites and exist widely in plants. Tea ( Camellia sinensis) is rich in PAs and their precursors, (-)-epicatechin (EC) and (+)-catechin (C). The biosynthesis of PAs is constantly regulated by many different MBW complexes, consisting of MYB transcription factors (TFs), basic-helix-loop-helix (bHLH) TFs, and WD-repeat (WDR) proteins. These regulatory factors can be environmentally affected, such as by biotic and abiotic stresses. In this study, we revalidated the effect of sucrose treatment on tea branches, and a sucrose-induced MYB (SIMYB) TF was screened and studied. Phylogenetic analysis indicted that this SIMYB TF belonged to MYB subgroup 5, named CsMYB5b. Heterologous expression of CsMYB5b in tobacco strongly induced PA accumulation, through up-regulating the key target genes LAR or ANRs. In addition, CsMYB5b restored PA production in the seed coat of A. thaliana tt2 mutant and rescued its phenotype. Yeast two-hybrid assay demonstrated CsMYB5b can interact directly with CsTT8 (an AtTT8 ortholog) and CsWD40 protein. Linking to the expression profiling of CsMYB5b and the PA accumulation pattern in tea plants suggest that the CsMYB5b acts as an important switch for the synthesis of monomeric catechins and PAs. Therefore, these data provide insight into the regulatory mechanisms controlling the biosynthesis of PAs.

Four flavonoid glycosyltransferases present in tea overexpressed in model plants Arabidopsis thaliana and Nicotiana tabacum for functional identification.

Tea possesses a distinctive flavor profile and can have health benefits owing to the high levels of flavonoids in its leaves. However, the mechanism of the flavonoid glycosylation hasn't been well studied in tea plants, especially glycosylation at the 7-OH site has rarely been reported. In this study, four UGT genes CsUGT73A20, CsUGT75L12, CsUGT78A14 and CsUGT78A15 were isolated from tea leaves and overexpressed in the model plants Arabidopsis thaliana and Nicotiana tabacum for the functional identification of genes in vivo. In order to characterize the CsUGT functions in model plants, flavonoids in seeds of Arabidopsis and the flowers of tobacco were identified first. In CsUGT73A20-overexpressing Arabidopsis and tobacco, the level of certain flavonol glycosides involved in glycosylation reactions at the 3-OH and 7-OH sites increased considerably, but the level of flavan-3-ols decreased. In CsUGT75L12 transgenic Arabidopsis, the level of flavonol glycosides exhibiting glucosyltransferase activity at the 7-OH position increased markedly, but the concentrations of quercetin and kaempferol and flavan-3-ols decreased. In both transgenic Arabidopsis and tobacco, CsUGT78A14 promoted the synthesis of more flavonol glucosides with UDP-glucose as a sugar donor at the 3-OH glycosylation site. In CsUGT78A15 transgenic plants, flavonol galactosides at the 3-OH glycosylation site with UDP-galactose as a sugar donor were increased. In the tea plant, the corresponding flavonoid glycosides such as kaempferol­3­O­ß­d­glucosides, kaempferol­3­O­ß­d­galactosides, kaempferol­7­O­ß­d­glucoside, and luteolin­7­O­ß­d­glucoside were identified. And it could be possible that they were products of CsUGT78A14, CsUGT78A15, CsUGT73A20 and CsUGT75L12, respectively.