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.

Castor (Ricinus communis L.) seeds produce abundant ricinoleic acid during seed maturation, which is important for plant development and human demands. Ricinoleic acid, as a unique hydroxy fatty acid (HFA), possesses a distinct bond structure that could be used as a substitute for fossil fuels. Here, we identified all homologous genes related to glycolysis, hydroxy fatty acid biosynthesis, and triacylglycerol (TAG) accumulation in castor seeds. Furthermore, we investigated their expression patterns globally during five seed development stages. We characterized a total of 66 genes involved in the glycolysis pathway, with the majority exhibiting higher expression levels during the early stage of castor bean seed development. This metabolic process provided abundant acetyl-CoA for fatty acid (FA) biosynthesis. Subsequently, we identified 82 genes involved in the processes of de novo FA biosynthesis and TAG assembly, with the majority exhibiting high expression levels during the middle or late stages. In addition, we examined the expression patterns of the transcription factors involved in carbohydrate and oil metabolism. For instance, RcMYB73 and RcERF72 exhibited high expression levels during the early stage, whereas RcWRI1, RcABI3, and RcbZIP67 showed relatively higher expression levels during the middle and late stages, indicating their crucial roles in seed development and oil accumulation. Our study suggests that the high HFA production in castor seeds is attributed to the interaction of multiple genes from sugar transportation to lipid droplet packaging. Therefore, this research comprehensively characterizes all the genes related to glycolysis, fatty acid biosynthesis, and triacylglycerol (TAG) accumulation in the castor and provides novel insight into exploring the genetic mechanisms underlying seed oil accumulation in the endosperm of castor beans.

Oil-tea (Camellia oleifera), a typical oilseed tree, produces high-quality edible vegetable oils that contain rich unsaturated fatty acids and diverse lipid-soluble active compounds such as squalene. Although squalene biosynthesis and its molecular regulation have been studied in several plants, the molecular mechanisms underlying squalene biosynthesis in oil-tea seeds remain uncertain. We investigated and determined squalene accumulation with seed development. We conducted comparative transcriptomic analyses using the RNA-seq technique at the early, fast biosynthesis, and late stages of squalene accumulation with oil-tea seed development and identified 13 squalene biosynthesis key enzyme genes (such as CoHMGR_4, CoAACT_2, CoFPS_1, and CoFPS_2) in developing oil-tea seeds. According to whether the expressions of key enzyme genes were associated with squalene accumulation we found that the precursor IPP of squalene biosynthesis obtained via the MVA pathway was dominant with oil-tea seed development. Based on the gene co-expression analyses, we identified multiple transcription factors potentially involved in regulating squalene biosynthesis such as CoMYC2, CoREM39, CobZIP5, CoERF and CoWRKY. Using yeast one-hybrid and dual-luciferase assay experiments we demonstrated that the transcription factor CoMYC2 could activate the expression of a key enzyme gene CoHMGR_4, suggesting that CoMYC2 might be a critical regulator during squalene biosynthesis in oil-tea seed development. This study gives not only insights into understanding the molecular basis of squalene biosynthesis in oil-tea developing seeds but also provides gene resources for developing genetically improved varieties with higher content of squalene in oil-tea.