In this study, deep eutectic solvents (DESs) were used for the ultrasound-assisted extraction (UAE) of valuable bioactive compounds from Chinese wild rice (Zizania spp.). To this end, 7 different choline chloride (CC)-based DESs were tested as green extraction solvents. Choline chloride/1,4-butanediol (DES-2) exhibited the best extraction efficiency in terms of parameters such as the total flavonoid content (TFC), total phenolic content (TPC), and free radical scavenging capacity (DPPH^● and ABTS^●+). Subsequently, the UAE procedure using 76.6% DES-2 was also optimized: An extraction temperature of 51.2 °C and a solid–liquid ratio of 37.0 mg/mL were considered optimal by a Box–Behnken experiment. The optimized extraction procedure proved efficient for the extraction of 9 phenolic and 3 flavonoid compounds from Chinese wild rice as determined by quantification based on ultra-performance liquid chromatography–triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS). This work, thus, demonstrates the possibility of customizing green solvents that offer greater extraction capacity than that of organic solvents.

Cembranoids are a group of natural diterpenoid compounds with pharmaceutical potentials, and the cembratriene-diols produced by Nicotiana (tobacco) species display activities in anti-nicotine addiction and neuron protection. Although the enzymes catalyzing cembratriene-diols’ formation in tobacco have been investigated, the regulatory mechanism underlying this physiological process remains unknown. This study has investigated the roles of phytohormone jasmonic acid (JA) in regulating cembratriene-diol formation in N. tabacum cv. TN90 and found that JA and COI1, the receptor protein of the bioactive derivative of JA (i.e., JA-Ile), display critical roles in regulating cembratriene-diols’ formation and the expression of cembranoid synthetic genes CBTS, P450 and NtLTP1. Further studies showed that over-expressing either the gene encoding bHLH transcription factor MYC2a or that encoding MYB transcription factor MYB305 could upregulate the cembranoid synthetic genes and enhance the cembranoid production in plants with dysfunction of COI1. Further studies suggest that COI1 and its downstream regulators MYC2a and MYB305 also modulate the trichome secretion, which is correlated with cembranoid formation. Taken together, this study has demonstrated a critical role of JA-signaling components in governing the cembratriene-diol formation and the transcription of cembratriene-diol synthetic genes in tobacco. Findings in this study are of great importance to reveal the molecular regulatory mechanism underlying cembranoid synthesis.

The symbiont endophytic fungi in tobacco are highly diverse and difficult to classify. Here, we sequenced the genomes of Curvularia trifolii and Leptosphaerulina chartarum isolated from tobacco plants. Finally, 41.68 Mb and 37.95 Mb nuclear genomes were sequenced for C. trifolii and L. chartarum with the scaffold N50, accounting for 638.94 Kb and 284.12 Kb, respectively. Meanwhile, we obtained 68,926 bp and 59,100 bp for their mitochondrial genomes. To more accurately classify C. trifolii and L. chartarum, we extracted seven nuclear genes and 12 mitochondrial genes from these two genomes and their closely related species. The genes were then used for calculation of evolutionary rates and for phylogenetic analysis. Results showed that it was difficult to achieve consistent results using a single gene due to their different evolutionary rates, while the phylogenetic trees obtained by combining datasets showed stable topologies. It is, therefore, more accurate to construct phylogenetic relationships for endophytic fungi based on multi-gene datasets. This study provides new insights into the distribution and characteristics of endophytic fungi in tobacco.