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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.

BackgroundIn nanomedicine, gold nanoparticles (AuNPs) have demonstrated versatile therapeutic efficiencies and, in particular, have been developed for the treatment of various cancers due to their high selectivity in killing cancer, not healthy, cells.MethodsIn this study, AuNPs were conjugated with the cell-penetrating peptide Cys-(Arg)8-Asp-Ser (CRRRRRRRRGDS) by direct cross-linking of the cysteine’s thiol group to the gold surface and a fibronectin-derived RGD group was also used due to its efficacy toward cancer cell targeting and possible promotion of healthy fibroblast functions.ResultsUltraviolet–visible absorbance spectrum and transmission electron microscope images of the synthesized peptide-capped AuNPs (PEP-AuNPs) validated the formation of AuNP aggregates. The presence of peptides on AuNPs was confirmed by Fourier transform infrared spectroscopy and quantified by a bicinchoninic acid assay. After being modified with the arginine-rich peptide, the AuNPs possessed a positive charge, as their zeta potential increased from −23.81±8.43 mV to 8 mV on average. In this manner, an easy method to conjugate AuNPs was shown here. Further, MTS assays were performed using healthy human dermal fibroblasts. After 24 hours of treatment with PEP-AuNPs, the cell density increased dramatically to around 25,000 cells/cm2. Results further showed a very high half-maximal inhibitory concentration of 69.2 µM for the PEP-AuNPs (indicating low toxicity).ConclusionThe results showed for the first time the ability of PEP-AuNPs to promote human dermal fibroblast cell viability, which after further investigation, may show an ability to replace cancerous tissue with healthy soft tissue.

Candida guilliermondii is an ascomycetous yeast widely studied due to its clinical importance, biotechnological interest, and biological control potential. During a series of preliminary experiments aiming at optimizing the electroporation procedure of C. guilliermondii cells, we observed that the efficiency of transformation of an ura5 recipient strain with the corresponding dominant marker URA5 was more than a thousand fold higher as compared with the transformation of an ura3 strain with the URA3 wild type allele. This result allowed the identification of an autonomously replicating sequence (ARS) within an A/T rich region located upstream of the URA5 open reading frame (ORF). Interestingly, linear double strand DNAs (dsDNAs) containing this putative ARS are circularized and then autonomously replicated in C. guilliermondii transformed cells. We demonstrated that the C. guilliermondii Lig4p ligase, involved in the canonical non-homologous end-joining (NHEJ) pathway, was responsible for this phenomenon since a lig4 mutant was unable to circularize and to autonomously maintain transforming dsDNAs containing the putative ARS. Finally, a functional dissection of the C. guilliermondii A/T rich region located upstream of the URA5 ORF revealed the presence of a 60 bp-length sequence essential and sufficient to confer ARS properties to shuttle plasmid and linear dsDNAs.