Liquid-liquid extraction and solid phase extraction followed by high-performance liquid chromatography (HPLC) connected with ultraviolet (UV) detection were used for the determination of phytohormones. The parameters influencing the performance of the HPLC-UV method, including composition of the mobile phase for gradient elution, column temperature, flow rate, and detection wavelength, were optimized. This method can simultaneously determine 11 phytohormones, including nine cytokinins, indole-3-acetic acid, and abscisic acid. The limit of detection of this method is 0.22 to 1.1 µg L⁻¹, and the coefficient factors of linear regression are >0.998. The recoveries of the target phytohormones ranged between 62.1~109.4%, and the relative standard deviations were <10%. This method is suitable for determining phytohormones, especially cytokinins, in young panicles, roots, and xylem sap of rice plants.

Illicium difengpi (Schisandraceae), an endangered medicinal plant endemic to karst areas, is highly tolerant to drought and thus can be used as an ideal material for investigating adaptive mechanism to drought stress. The understanding of the drought tolerance of I. difengpi, especially at the molecular level, is lacking. In the present study, we aimed to clarify the molecular mechanism underlying drought tolerance in endemic I. difengpi plant in karst regions. The response characteristics of transcripts and changes in metabolite abundance of I. difengpi subjected to drought and rehydration were analyzed, the genes and key metabolites responsive to drought and rehydration were screened, and some important biosynthetic and secondary metabolic pathways were identified. A total of 231,784 genes and 632 metabolites were obtained from transcriptome and metabolome analyses, and most of the physiological metabolism in drought-treated I. difengpi plants recovered after rehydration. There were more upregulated genes than downregulated genes under drought and rehydration treatments, and rehydration treatment induced stable expression of 65.25% of genes, indicating that rehydration alleviated drought stress to some extent. Drought and rehydration treatment generated flavonoids, phenolic acids, flavonols, amino acids and their derivatives, as well as metabolites such as saccharides and alcohols in the leaves of I. difengpi plants, which alleviated the injury caused by excessive reactive oxygen species. The integration of transcriptome and metabolome analyses showed that, under drought stress, I. difengpi increased glutathione, flavonoids, polyamines, soluble sugars and amino acids, contributing to cell osmotic potential and antioxidant activity. The results show that the high drought tolerance and recovery after rehydration are the reasons for the normal growth of I. difengpi in karst mountain areas.

Illicium difengpi (Schisandraceae), which is an endemic, medicinal, and endangered species found in small and isolated populations that inhabit karst mountain areas, has evolved strategies to adapt to arid environments and is thus an excellent material for exploring the mechanisms of tolerance to severe drought. In experiment I, I. difengpi plants were subjected to three soil watering treatments (CK, well-watered treatment at 50% of the dry soil weight for 18 days; DS, drought stress treatment at 10% of the dry soil weight for 18 days; DS-R, drought-rehydration treatment at 10% of the dry soil weight for 15 days followed by rewatering to 50% of the dry soil weight for another 3 days). The effects of the drought and rehydration treatments on leaf succulence, phytohormones, and phytohormonal signal transduction in I. difengpi plants were investigated. In experiment II, exogenous abscisic acid (ABA, 60 mg L⁻¹) and zeatin riboside (ZR, 60 mg L⁻¹) were sprayed onto DS-treated plants to verify the roles of exogenous phytohormones in alleviating drought injury. Leaf succulence showed marked changes in response to the DS and DS-R treatments. The relative concentrations of ABA, methyl jasmonate (MeJA), salicylic acid glucoside (SAG), and cis-zeatin riboside (cZR) were highly correlated with relative leaf succulence. The leaf succulence of drought-treated I. difengpi plants recovered to that observed with the CK treatment after exogenous application of ABA or ZR. Differentially expressed genes involved in biosynthesis and signal transduction of phytohormones (ABA and JA) in response to drought stress were identified by transcriptomic profiling. The current study suggested that the phytohormones ABA, JA, and ZR may play important roles in the response to severe drought and provides a preliminary understanding of the physiological mechanisms involved in phytohormonal regulation in I. difengpi, an endemic, medicinal, and highly drought-tolerant plant found in extremely small populations in the karst region of South China.

Illicium difengpi Kib and Kim, an endangered plant unique to karst areas in China, has evolved an extremely high tolerance to arid environments. To elucidate the molecular mechanisms of the response to drought stress in I. difengpi, physiological index determination and transcriptome sequencing experiments were conducted in biennial seedlings grown under different soil moisture conditions (70~80%, 40~50% and 10~20%). With increasing drought stress, the leaf chlorophyll content decreased, while the proline (Pro), soluble sugar (SS) and malondialdehyde (MDA) contents increased; superoxide dismutase (SOD) and peroxidase (POD) activities also increased. Transcriptome sequencing and pairwise comparisons of the treatments revealed 2489, 4451 and 753 differentially expressed genes (DEGs) in CK70~80 vs. XP40~50, CK70~80 vs. XP10~20 and XP40~50 vs. XP10~20, respectively. These DEGs were divided into seven clusters according to their expression trends, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment results of different clusters indicated that genes in the hormone signal transduction and osmotic regulation pathways were greatly activated under mild drought stress. When drought stress increased, the DEGs related to membrane system and protein modification and folding were all upregulated; simultaneously, chitin catabolism- and glycolysis/gluconeogenesis-related genes were continuously upregulated throughout drought stress, while the genes involved in photosynthesis were downregulated. Here, 244 transcription factors derived from 10 families were also identified. These results lay a foundation for further research on the adaptation of I. difengpi to arid environments in karst areas and the establishment of a core regulatory relationship in its drought resistance mechanism.

Illicium difengpi K.I.B. et K.I.M. is a member of the Illiciaceae family with a yet not fully explored utilization value. To provide references for the systematic understanding of I. difengpi (Illiciaceae), the morphological and structural characteristics, wild resource distribution, chemical compounds, pharmacological effects, utilization, and protective measures of this species are reviewed. We conclude that (i) I. difengpi (Illiciaceae) is an endemic and indigenous medicinal species that has been used to treat rheumatoid arthritis and traumatic injury in China; (ii) I. difengpi (Illiciaceae) can endure various abiotic stresses, especially extreme drought, and thus has scientific value for exploring adaptive mechanisms of tolerance to extreme drought and in the ecological restoration of karst rocky desertification areas; and (iii) the beautiful tree shapes of I. difengpi (Illiciaceae) give it potential ornamental value. However, the wild resources of I. difengpi (Illiciaceae) have rapidly decreased, and there is an urgent need to protect this endangered species to maintain its diversity. Protection measures include the protection of wild germplasm resources, the establishment of an I. difengpi (Illiciaceae) germplasm resource bank, and the development of large-scale ecological planting techniques. In further research, the medicinal and scientific value of I. difengpi (Illiciaceae) should be systematically explored to clarify the plant’s effective pharmaceutical value, clinical applications, mechanisms of drought adaptation, and genetic diversity.