Establishment of an efficient transformation system and its application in regulatory mechanism analysis of biological macromolecules in tea plants.

Tea (Camellia sinensis), one of the most important beverage crops originated from China and is now cultivated worldwide, provides numerous secondary metabolites that account for its health benefits and rich flavor. However, the lack of an efficient and reliable genetic transformation system has seriously hindered the gene function investigation and precise breeding of C. sinensis. In this study, we established a highly efficient, labor-saving, and cost-effective Agrobacterium rhizogenes-mediated hairy roots genetic transformation system for C. sinensis, which can be used for gene overexpression and genome editing. The established transformation system was simple to operate, bypassing tissue culture and antibiotic screening, and only took two months to complete. We used this system to conduct function analysis of transcription factor CsMYB73 and found that CsMYB73 negatively regulates L-theanine synthesis in tea plant. Additionally, callus formation was successfully induced using transgenic roots, and the transgenic callus exhibited normal chlorophyll production, enabling the study of the corresponding biological functions. Furthermore, this genetic transformation system was effective for multiple C. sinensis varieties and other woody plant species. By overcoming technical obstacles such as low efficiency, long experimental periods, and high costs, this genetic transformation will be a valuable tool for routine gene investigation and precise breeding in tea plants.

Machine learning uncovers accumulation mechanism of flavonoid compounds in Polygonatum cyrtonema Hua.

The compositions and yield of flavonoid compounds of Polygonatum cyrtonema Hua (PC) are important indices of the quality of medicinal materials. However, the flavonoids compositions and accumulation mechanism are still unclear in PC. Here, we identified 22 flavonoids using widely-targeted metabolome analysis in 15 genotypes of PC. Then weighted gene co-expression network analysis based on 45 transcriptome samples was performed to construct 12 co-expressed modules, in which blue module highly correlated with flavonoids was identified. Furthermore, 4 feature genes including PcCHS1, PcCHI, PcCHS2 and PcCHR5 were identified from 94 hub genes in blue module via machine learning methods support vector machine-recursive feature elimination (SVM-RFE) and random forest (RF), and their functions on metabolic flux of flavonoids pathway were confirmed by tobacco transient expression system. Our findings identified representative flavonoids and key enzymes in PC that provided new insight for elite breeding rich in flavonoids, and thus will be beneficial for rapid development of great potential economic and medicinal value of PC.

Specific Enriched Acinetobacter in Camellia Weevil Gut Facilitate the Degradation of Tea Saponin: Inferred from Bacterial Genomic and Transcriptomic Analyses.

Beneficial gut bacteria can enhance herbivorous arthropod adaptation to plant secondary compounds (PSMs), and specialist herbivores provide excellent examples of this. Tea saponin (TS) of Camellia oleifera is triterpenoids toxic to seed-feeding weevil pest, Curculio chinensis (CW). Previous studies disclosed that Acinetobacter, which was specific enriched in the CW's gut, was involved in helping CW evade TS toxicity of C. oleifera. However, it is still not clear whether Acinetobacter is associated with other anti-insect compounds, and the molecular mechanism of Acinetobacter degradation of TS has not been clarified. To address these questions, we explored the relationship between host plant toxin content and Acinetobacter of CW gut bacteria. Results demonstrated that TS content significantly affected the CW gut microbiome structure and enriched bacteria functional for TS degradation. We further isolated Acinetobacter strain and conducted its genome and transcriptome analyses for bacterial characterization and investigation on its role in TS degradation. Biological tests were carried out to verify the ability of the functional bacterium within CW larvae to detoxify TS. Our results showed that TS-degrading bacteria strain (Acinetobacter sp. AS23) genome contains 47 genes relating to triterpenoids degradation. The AS23 strain improved the survival rate of CW larvae, and the steroid degradation pathway could be the key one for AS23 to degrade TS. This study provides the direct evidence that gut bacteria mediate adaptation of herbivorous insects to phytochemical resistance. IMPORTANCE Microorganism is directly exposed to the plant toxin environment and play a crucial third party in herbivores gut. Although previous studies have proved the existence of gut bacteria that help CWs degrade TS, the specific core flora and its function have not been explored. In this study, we investigated the correlation between the larva gut microbiome and plant secondary metabolites. Acinetobacter genus was the target flora related to TS degradation. There were many terpenoids genes in Acinetobacter sp. AS23 genome. Results of transcriptome analysis and biological tests suggested that steroid degradation pathway be the key pathway of AS23 to degrade TS. This study not only provides direct evidence that gut microbes mediate the rapid adaptation of herbivorous insects to phytochemical resistance, but also provides a theoretical basis for further research on the molecular mechanism of intestinal bacteria cooperating with pests to adapt to plant toxins.

Histone Methylation Is Required for Virulence, Conidiation, and Multi-Stress Resistance of Alternaria alternata.

Histone methylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs). This study determined the function of 5 HMTs (AaDot1, AaHMT1, AaHnrnp, AaSet1, and AaSet2) and 1 HDMs (AaGhd2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. The vegetative growth, conidiation, and pathogenicity of ∆AaSet1 and ∆AaSet2 were severely inhibited indicating that AaSet1 and AaSet2 play critical roles in cell development in A. alternata. Multiple stresses analysis revealed that both AaSet1 and AaSet2 were involved in the adaptation to cell wall interference agents and osmotic stress. Meanwhile, ∆AaSet1 and ∆AaSet2 displayed serious vegetative growth defects in sole carbon source medium, indicating that AaSet1 and AaSet2 play an important role in carbon source utilization. In addition, ∆AaSet2 colony displayed white in color, while the wild-type colony was dark brown, indicating AaSet2 is an essential gene for melanin biosynthesis in A. alternata. AaSet2 was required for the resistance to oxidative stress. On the other hand, all of ∆AaDot1, ∆AaHMT1, and ∆AaGhd2 mutants displayed wild-type phenotype in vegetative growth, multi-stress resistance, pathogenicity, carbon source utilization, and melanin biosynthesis. To explore the regulatory mechanism of AaSet1 and AaSet2, RNA-seq of these mutants and wild-type strain was performed. Phenotypes mentioned above correlated well with the differentially expressed genes in ∆AaSet1 and ∆AaSet2 according to the KEGG and GO enrichment results. Overall, our study provides genetic evidence that defines the central role of HMTs and HDMs in the pathological and biological functions of A. alternata.

Highly efficient hairy root genetic transformation and applications in citrus.

Highly efficient genetic transformation technology is greatly beneficial for crop gene function analysis and precision breeding. However, the most commonly used genetic transformation technology for woody plants, mediated by Agrobacterium tumefaciens, is time-consuming and inefficient, which limits its utility for gene function analysis. In this study, a simple, universal, and highly efficient genetic transformation technology mediated by A. rhizogenes K599 is described. This technology can be applied to multiple citrus genotypes, and only 2-8 weeks were required for the entire workflow. Genome-editing experiments were simultaneously conducted using 11 plasmids targeting different genomic positions and all corresponding transformants with the target knocked out were obtained, indicating that A. rhizogenes-mediated genome editing was highly efficient. In addition, the technology is advantageous for investigation of specific genes (such as ACD2) for obtaining "hard-to-get" transgenic root tissue. Furthermore, A. rhizogenes can be used for direct viral vector inoculation on citrus bypassing the requirement for virion enrichment in tobacco, which facilitates virus-induced gene silencing and virus-mediated gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus that can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by reducing the cost, required workload, experimental period, and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.

Modified insoluble dietary fibers in okara affect body composition, serum metabolic properties, and fatty acid profiles in mice fed high-fat diets: an NMR investigation.

The potential health benefit of dietary fiber has attracted considerable attention in recent decades. In this study, the effects of modified dietary fibers (MDF) derived from okara on body composition, fat distribution, serum metabolomic parameters, and fatty acid profiles in mice fed high-fat diets (HFD) were evaluated by nuclear magnetic resonance (NMR)-based metabolic approach. HFD-induced C57BL mice were fed with a diet containing 100 g/kg MDF for 12 weeks. Compared with control mice, MDF-fed mice exhibited less fat and lower body weights, altered serum metabolomic profiles, and distinct fatty acid profiles. The levels of choline, phosphatidylcholine, glycerophosphorylcholine, glucose, lysine, scyllo-inositol, and glutamate for MDF group were higher than those for both CONT and HFD groups. A remarkable reduction of total cholesterol, total triglycerides, ω-6 fatty acids, alanine, citrate, creatine, or succinate was also observable for MDF group compared with HFD group. These findings demonstrated that the intake of MDF derived from okara clearly ameliorated some of the HFD-induced adverse metabolic effects and prevented adipose tissue accumulation.