Chromatin architecture is an essential factor regulating gene transcription in different cell types and developmental phases. However, studies on chromatin architecture in perennial woody plants and on the function of chromatin organization in sex determination have not been reported. Here, we produced a chromosome-scale de novo genome assembly of the woody plant Jatropha curcas with a total length of 379.5 Mb and a scaffold N50 of 30.7 Mb using Pacific Biosciences long reads combined with genome-wide chromosome conformation capture (Hi-C) technology. Based on this high-quality reference genome, we detected chromatin architecture differences between monoecious and gynoecious inflorescence buds of Jatropha. Differentially expressed genes were significantly enriched in the changed A/B compartments and topologically associated domain regions and occurred preferentially in differential contact regions between monoecious and gynoecious inflorescence buds. Twelve differentially expressed genes related to flower development or hormone synthesis displayed significantly different genomic interaction patterns in monoecious and gynoecious inflorescence buds. These results demonstrate that chromatin organization participates in the regulation of gene transcription during the process of sex differentiation in Jatropha. We have revealed the features of chromatin architecture in perennial woody plants and investigated the possible function of chromatin organization in Jatropha sex differentiation. These findings will facilitate understanding of the regulatory mechanisms of sex determination in higher plants.

Diacylglycerol acyltransferase (DGAT) is the only enzyme that catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol (DAG) to form triacylglycerol (TAG). The two main types of DGAT enzymes in the woody perennial biofuel plant Jatropha curcas, JcDGAT1 and JcDGAT2, were previously characterized only in heterologous systems. In this study, we investigated the functions of JcDGAT1 and JcDGAT2 in J. curcas.JcDGAT1 and JcDGAT2 were found to be predominantly expressed during the late stages of J. curcas seed development, in which large amounts of oil accumulated. As expected, overexpression of JcDGAT1 or JcDGAT2 under the control of the CaMV35S promoter gave rise to an increase in seed kernel oil production, reaching a content of 53.7% and 55.7% of the seed kernel dry weight, respectively, which were respectively 25% and 29.6% higher than that of control plants. The increase in seed oil content was accompanied by decreases in the contents of protein and soluble sugars in the seeds. Simultaneously, there was a two- to four-fold higher leaf TAG content in transgenic plants than in control plants. Moreover, by analysis of the fatty acid (FA) profiles, we found that JcDGAT1 and JcDGAT2 had the same substrate specificity with preferences for C18:2 in seed TAGs, and C16:0, C18:0, and C18:1 in leaf TAGs. Therefore, our study confirms the important role of JcDGAT1 and JcDGAT2 in regulating oil production in J. curcas.

DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1), a phospholipase A1, utilizes galactolipids (18:3) to generate α-linolenic acid (ALA) in the initial step of jasmonic acid (JA) biosynthesis in Arabidopsis thaliana. In this study, we isolated the JcDAD1 gene, an ortholog of Arabidopsis DAD1 in Jatropha curcas, and found that it is mainly expressed in the stems, roots, and male flowers of Jatropha. JcDAD1-RNAi transgenic plants with low endogenous jasmonate levels in inflorescences exhibited more and larger flowers, as well as a few abortive female flowers, although anther and pollen development were normal. In addition, fruit number was increased and the seed size, weight, and oil contents were reduced in the transgenic Jatropha plants. These results indicate that JcDAD1 regulates the development of flowers and fruits through the JA biosynthesis pathway, but does not alter androecium development in Jatropha. These findings strengthen our understanding of the roles of JA and DAD1 in the regulation of floral development in woody perennial plants.

Diosgenin is a bioactive steroidal natural product extraced from plants and serves as an important precursor for the industrial production of steroidal hormone drugs. Despite its pharmacological significance, the biosynthetic and regulatory mechanisms underlying diosgenin production in the medicinal plant T. foenum-graecum remain poorly understood. In this study, we identified TfWRKY40, a WRKY transcription factor from T. foenum-graecum, whose expression strongly correlates with diosgenin accumulation. Using RNA interference and overexpression strategies combined with transcriptomic analysis and targeted metabolite quantification, we demonstrated that silencing of TfWRKY40 led to a 67.60% reduction in diosgenin content, which was accompanied by downregulation of key biosynthetic genes or transcript variants including ACAT1, HMGR1, PMK1, MVD, FPS, SQE2, CAS1, SMO3-1, SMO3-2, 8,7-SI, SMO4-3, CYP90B50, and CYP82J17 in the transgenic hairy roots. Conversely, overexpression of TfWRKY40 resulted in a 59.25% increase in diosgenin levels, along with upregulation of these biosynthetic genes or transcript variants. Taken together, these findings suggest that TfWRKY40 acts as a positive regulator of diosgenin biosynthesis in T. foenum-graecum, likely by activating the transcription of critical pathway genes, particularly CAS1, HMGR1, and CYP90B50. This work highlights TfWRKY40 as a promising target for metabolic engineering strategies aimed at enhancing diosgenin production and facilitating the development of diosgenin-derived steroidal therapeutics.

Trigonelline is a bioactive pyridine alkaloid in Trigonella foenum-graecum, which is a legume species, and is known for its hypoglycemic and hypolipidemic activities. Phytosulfokine-δ (PSK-δ), a recently identified legume-specific phytosulfokine peptide, has been implicated in the regulation of nodulation and root development. However, whether PSK-δ participates in the regulation of secondary metabolite biosynthesis remains unclear. In this study, exogenous PSK-δ treatment significantly promoted trigonelline accumulation in T. foenum-graecum seedlings, leading to a 93% increase in trigonelline content after 5 days relative to the scrambled-pentapeptide-treated control, whereas a slight downward trend was observed at 15 days. Comparative transcriptomic analyses between PSK-δ-treated seedlings and the corresponding controls at 5 and 15 days identified 15 candidate genes associated with trigonelline biosynthesis and PSK signaling, whose expression patterns were consistent with trigonelline accumulation dynamics. Co-expression network analysis between these 15 candidate genes and 831 transcription factor-encoding genes identified candidate transcription factors potentially involved in the coordinated regulation of trigonelline biosynthesis and PSK signaling. These findings suggest that PSK-δ may serve as a potential peptide-based biostimulant for regulating alkaloid accumulation in medicinal legume cultivation and provide insights into peptide hormone-mediated regulation of plant metabolism.