RNAi of Sterol Δ24-Isomerase Implicated Its Involvement in Physalin Biosynthesis in Physalis angulata L.

Abstract

Physalis angulata is a renowned traditional Chinese medicine for the treatment of various conditions. Physalin is the major type of bioactive constituents conferring medicinal properties of P. angulata. Despite the medicinal importance, the pathways leading to physalin are largely unknown. In this study, we employed a transcriptomic approach to identify a Pa24ISO gene from P. angulata. Through heterologous expression in yeast, Pa24ISO was revealed to catalyze an isomerization reaction in converting 24-methylenecholesterol to 24-methyldesmosterol. Real-time PCR analysis showed that the abundance of Pa24ISO transcripts correlated with the accumulation pattern of physalin B in different tissues of P. angulata. A direct role of Pa24ISO in channeling of 24-methylenecholesterol for physalin B biosynthesis was illustrated by suppressing the gene in P. angulata via the VIGS approach. Down-regulation of Pa24ISO led to reduced levels of 24-methyldesmosterol and physalin B, accompanied with an increase of campesterol content in P. angulata. The results supported that 24ISO is involved in physalin biosynthesis in plants.

Identification of P450 Candidates Associated with the Biosynthesis of Physalin-Class Compounds in Physalis angulata

“…To confirm the VIGS system previously developed for P. angulata [ 10 ], the PaPDS (phytoene desaturase) gene was cloned from P. angulata leaf, and used as a reporter gene (see the PaPDS cDNA sequence in Figure S1 ). The VIGS constructs of the genes of interest (GOIs) were prepared using the pTRV2 vector [ 10 ]. The primers used for preparation of the VIGS constructs are listed in Table S3 .…”

“…The primers used for preparation of the VIGS constructs are listed in Table S3 . The VIGS infection of P. angulata leaves was performed as previously described [ 10 ] with small modifications. Briefly, the Agrobacterium culture suspension of the pTRV1 and either pTRV2-GOIs, or the empty vector pTRV2 as a control, was mixed together at a 1:1 ratio, and the culture mixture was then infiltrated into the leaves of four to six-leaf-stage seedlings by a 1 mL syringe.…”

“…The conversion of 24-methylenecholesterol to 24-methyldesmosterol is catalyzed by a sterol delta-24-isomerase (24ISO) [9]. The 24ISO gene was recently cloned from P. angulata and its involvement in physalin biosynthesis was demonstrated by suppressing its expression in P. angulata [10]. In contrast, at present, enzymes involved in converting 24-methyldesmosterol to physalins have applied this up to-date technique to the pooled mRNA samples extracted from the roots, stems, leaves and calyxes P. angulata [11], revealing the full-length transcripts of this species.…”

Meroterpenoids? A historical and critical review of this biogenetic determinant

“…3). [21][22][23] It is likely that this derogation to Cornforth's proposal may be because the modication of one carbon atom at a time is considered to be of minor importance. This ought not to be the case with cardiac glycosides, such as digitoxin (12) from the plant Digitalis, which are also side chain-modied steroids (Fig.…”

Discovery of physalin biosynthesis and structure modification of physalins in Physalis alkekengi L. var. Franchetii

“…The first reported study on biosynthesis of physalins was carried out by Fukushima et al (2016) , where 24-methyldesmosterol was designated as a key intermediate in physalin biosynthesis ( Fukushima et al, 2016 ). Relevant studies were continually advanced, withanolide were designated as the new key intermediate physalin biosynthesis ( Knoch et al, 2018 ; Yang et al, 2022b ). However, the key steps for progress from 24-methyldesmosterol to physalins is still unexplored ( Figure 2 ).…”