Virus-induced gene silencing of the two squalene synthase isoforms of apple tree (Malus × domestica L.) negatively impacts phytosterol biosynthesis, plastid pigmentation and leaf growth

Abstract

The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.

Functional characterization of squalene synthase and squalene epoxidase in Taraxacum koksaghyz

“…Interestingly, TkSQS1 and TkSQS2 did not cluster together in the same branch of the SQS phylogenetic tree, suggesting that gene duplication may have predated speciation, hinting at a specialized tissue‐specific role or sub‐functionalization (Figure ). Similar results have been obtained for SQS proteins from G. glabra in phylogenetic analyses (Hayashi et al., ; Navarro Gallón et al., ). However, this contrasts with the M. domestica MdSQS1 and MdSQS2 isoforms, which show higher intraspecific identities and may indicate more recent gene duplication events (Navarro Gallón et al., ).…”

“…Similar results have been obtained for SQS proteins from G. glabra in phylogenetic analyses (Hayashi et al., ; Navarro Gallón et al., ). However, this contrasts with the M. domestica MdSQS1 and MdSQS2 isoforms, which show higher intraspecific identities and may indicate more recent gene duplication events (Navarro Gallón et al., ).…”

“…As such, SQS represents a rate‐limiting step in triterpene biosynthesis and is associated with the overall yield of these secondary metabolites. This has been demonstrated in several plant species: the overexpression of SQS in P. ginseng and Eleutherococcus senticosus increased the overall SQS activity and caused the accumulation of sterols and triterpenes (Lee et al., ; Seo et al., ), whereas the virus‐induced gene silencing of SQS in Withania somnifera and M. domestica caused the sterol levels to decline (Navarro Gallón et al., ; Singh et al., ). Various RNA interference (RNAi) methods have also been used to silence the SQS gene and reduce sterol levels and this also caused the accumulation of artemisinin in Artemisia annua (Zhang et al., ).…”

“…Only a single SQS gene is found in yeast and humans (Jennings, Tsay, Fisch, & Robinson, ; Robinson, Tsay, Kienzle, Smith‐Monroy, & Bishop, ), whereas there are 1–3 SQS genes in plants. Thus far, single SQS genes have been reported among others in Oryza sativa (Hata et al., ), Lotus japonicus (Akamine et al., ), Taxus cuspidata (Huang et al., ), and Euphorbia tirucalli (Uchida et al., ), whereas two paralogs are found in Nicotiana tabacum (Devarenne, Shin, Back, Yin, & Chappell, ), Glycyrrhiza glabra (Hayashi, Hirota, Hiraoka, & Ikeshiro, ), Glycine max (Nguyen et al., ), Malus domestica (Navarro Gallón et al., ), and Arabidopsis thaliana , although in the last case, one copy was shown to be a pseudogene (Busquets et al., ). Panax ginseng possesses three SQS paralogs (Kim, Han, Huh, & Choi, ).…”

An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)-based vector

“…In these years, various viral vectors have been successfully used in VIGS, such as tobacco mosaic virus (TMV) [13], satellite virus-induced silencing system (SVISS) [14], potato virus X (PVX) [15], barley stripe mosaic virus (BSMV) [16], Cotton leaf crumple virus (CLCrV) [17], African cassava mosaic virus (ACMV) [18], tobacco rattle virus (TRV) [19], cabbage leaf curl virus (CaLCuV) [20][21], and turnip yellow mosaic virus (TYMV) [22]. Among them, TRV has the widest host range and induces mild viral symptoms after infecting plants such as dicotyledons and monocotyledons [22,[27][28][29][30][31][32]. Each viral vector has a certain host range, and the effect of inducing silencing is different.…”