An effective method for separating and purifying critical saponins (polyphyllin II and polyphyllin VII) from a Paris polyphylla var. yunnanensis extract was developed in this study which was environmentally friendly and economical. Static adsorption kinetics, thermodynamics, and the dynamic adsorption-desorption of macroporous resins were investigated, and then the conditions of purification and separation were optimized by fitting with an adsorption thermodynamics equation and a kinetic equation. Effective NKA-9 resin from seven macroporous resins was screened out to separate and purify the two saponins. The static adsorption and dynamic adsorption were chemical and physical adsorption dual-processes on the NKA-9 resin. Under the optimum parameters, the contents of polyphyllin II and polyphyllin VII in the product were 17.3-fold and 28.6-fold those in plant extracts, respectively. The total yields of the two saponins were 93.16%. This research thus provides a theoretical foundation for the large-scale industrial production of the natural drugs polyphyllin II and polyphyllin VII.

PEAR proteins are a type of plant-specific DNA binding with one finger (Dof) transcription factors that play a key role in the regulation of plant growth, especially during phloem cell growth and seed germination in Arabidopsis. However, the identification, characteristics and function of PEAR proteins, particularly in woody plants, need to be further studied. In the present study, 43 candidate PEAR proteins harboring the conserved Zf-Dof domain were obtained in Populus yunnanensis. Based on phylogenetic and structural analysis, 10 representative PEAR candidates were selected, belonging to different phylogenetic groups. The functions of PEAR proteins in the stress response, signal transduction, and growth regulation of stem cambium and roots undergoing vigorous cell division in Arabidopsis were revealed based on their expression patterns as characterized by qRT-PCR analysis, in accordance with the results of cis-element analysis. In vitro experiments showed that the interaction of transcription factor (E2F) and cyclin indirectly reflects the growth regulation function of PEAR through light signaling and cell-cycle regulation. Therefore, our results provide new insight into the identity of PEAR proteins and their function in stress resistance and vigorous cell division regulation of tissues in P. yunnanensis, which may serve as a basis for further investigation of the functions and characteristics of PEAR proteins in other plants.

Iron walnut (Juglans sigillata Dode) is a native species in southwestern China that exhibits variation in both fruit morphology and shell thickness. However, the underlying molecular processes controlling hardened endocarp development in walnut has not yet been reported. Here, we generated transcriptional profiles of iron walnut endocarp at three developmental stages using “Dapao”, the most common commercial variety. Using pairwise comparisons between these three stages, a total of 8555 non-redundant differentially expressed genes (DEGs) were identified, and more than one-half of the total DEGs exhibited significant differential expression in stage I as compared with stage II or stage III, suggesting that the first stage may ultimately determine the final characteristics of the mature walnut shell. Furthermore, in the clustering analysis of the above DEGs, 3682, 2349, and 2388 genes exhibited the highest expression in stages I, II, and III, respectively. GO enrichment analysis demonstrated that the major transcriptional variation among the three developmental stages was caused by differences in cell growth, plant hormones, metabolic process, and phenylpropanoid metabolism. Namely, using the tissue-specific expression analysis and a gene co-expression network, we identified MADS-box transcription factor JsiFBP2 and bHLH transcription factor JsibHLH94 as candidate regulators of endocarp formation in the early stage, and JsiNAC56 and JsiMYB78 might play key roles in regulating the lignification process of endocarp in the late stage. This study provides useful information for further research to dissect the molecular mechanisms governing the shell formation and development of iron walnut.

C2H2 zinc finger (C2H2-ZF) transcription factors participate in various aspects of normal plant growth regulation and stress responses. C1-2i C2H2-ZFs are a special subclass of conserved proteins that contain two ZnF-C2H2 domains. Some C1-2i C2H2-ZFs in Arabidopsis (ZAT) are involved in stress resistance and other functions. However, there is limited information on C1-2i C2H2-ZFs in Populus trichocarpa (PtriZATs). To analyze the function and evolution of C1-2i C2H2-ZFs, eleven PtriZATs were identified in P. trichocarpa, which can be classified into two subgroups. The protein structure, conserved ZnF-C2H2 domains and QALGGH motifs, showed high conservation during the evolution of PtriZATs in P. trichocarpa. The spacing between two ZnF-C2H2 domains, chromosomal locations and cis-elements implied the original proteins and function of PtriZATs. Furthermore, the gene expression of different tissues and stress treatment showed the functional differentiation of PtriZATs subgroups and their stress response function. The analysis of C1-2i C2H2-ZFs in different Populus species and plants implied their evolution and differentiation, especially in terms of stress resistance. Cis-elements and expression pattern analysis of interaction proteins implied the function of PtriZATs through binding with stress-related genes, which are involved in gene regulation by via epigenetic modification through histone regulation, DNA methylation, ubiquitination, etc. Our results for the origin and evolution of PtriZATs will contribute to understanding the functional differentiation of C1-2i C2H2-ZFs in P. trichocarpa. The interaction and expression results will lay a foundation for the further functional investigation of their roles and biological processes in Populus.

Paris polyphylla var. yunnanensis is one of the famous Chinese herbs, in which two saponins (polyphyllin II and polyphyllin VII) have anticancer effects. The endangerment of Paris polyphylla var. yunnanensis, makes the study of optimizing the extraction of polyphyllin II and polyphyllin VII from the leaves of Paris polyphylla var. yunnanensis more important. The study established and optimized the process of ultrasound-assisted extraction for polyphyllin II and polyphyllin VII using the Box Behnken Design method of response surface methodology. The results showed that the optimum extraction conditions for polyphyllin II and polyphyllin VII are ethanol concentration of 73 and 70%, extraction temperature of 43 and 50°C, and number of extraction 3, respectively. Under the above conditions, the contents of polyphyllin II and polyphyllin VII were measured to be 6.427 and 19.015 mg/g (DW). The results showed that the experimental model fitted well, and the response surface methodology (RSM) was feasible to optimize the extraction process of polyphyllin II and polyphyllin VII from Paris polyphylla var. yunnanensis leaves. This method provides an effective approach for the comprehensive development and utilization of non-medicinal parts of Paris polyphylla var. yunnanensis.

The process of extracting polyphyllin II and polyphyllin VII by water-assisted extraction was established and optimized in this study. Response surface methodology was used to establish a prediction model to optimize the extraction conditions. Based on the one-way test, the Box–Behnken design with three factors and three levels was used for the experimental program, and the composition analysis was carried out by high-performance liquid chromatography (HPLC). The optimal extraction conditions for polyphyllin II and polyphyllin VII were as follows: extraction time of 57 and 21 min, extraction temperature of 36 and 32 °C, solid-to-liquid ratio of 1:10 and 1:5 g/mL, respectively, and the yields of polyphyllin II and polyphyllin VII were 1.895 and 5.010%, which was similar to the predicted value of 1.835 and 4.979%. The results of the ANOVA showed that the model fit was good, and the Box–Behnken response surface method could optimize the water-assisted extraction of saponins from the leaves of Paris polyphylla var. yunnanensis. This study provides a theoretical basis for the application of polyphyllin II and polyphyllin VII in pharmaceutical production.