Movahedi, A. | Search bioRxiv

bioRxiv2020

Cross-talk between the methylerythritol phosphate and mevalonic acid pathways of isoprenoid biosynthesis in poplar

The isoprenoids found in plants are extremely important to survive with various human applications, such as flavoring, fragrance, dye, pharmaceuticals, and biomass used for biofuels. Methylerythritol phosphate (MEP) and mevalonic acid (MVA) pathways are critical in plants, responsible for isoprenoid biosynthesis. 1-deoxy-D-xylulose5-phosphate synthase (DXS) and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) catalyze the rate-limiting steps in the MEP pathway, while 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the rate-limiting step in the MVA pathway. Here, we showed while PtHMGR overexpressors (OEs) exhibited different MEP- and MVA-related gene expressions compared with non-transgenic poplars (NT), the PtDXR-OEs revealed upregulated MEP-related and downregulated MVA-related gene expressions. PtDXR and PtHMGR overexpressions caused changes in MVA-derived trans-zeatin-riboside, isopentenyl adenosine, castasterone, and 6-deoxocastasterone well as MEP-derived carotenoids and gibberellins. In PtHMGR-OEs, the accumulated geranyl diphosphate synthase (GPS) and geranyl pyrophosphate synthase (GPPS) transcript levels in the MEP pathway led to an accumulation of MEP-derived isoprenoids. In contrast, upregulation of farnesyl diphosphate synthase (FPS) expression in the MVA pathway contributed to increased levels of MVA-derived isoprenoids. In addition, PtHMGR-OEs increased MEP-related GPS and GPPS transcript levels, expanded MEP-derived isoprenoid levels, changed FPS transcript levels, and affected MVA-derived isoprenoid yields. These results demonstrate the contribution of MVA and MEP pathways regulating isoprenoid biosynthesis in poplars.

molecular biology↗

bioRxiv2020

CRISPR-Cas9 integrates exogeneous sorting new recombinant DNA in the model tree Populus trichocarpa

CRISPR-mediated genome editing has become a powerful tool for genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve the HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP Kinase gene to generate a BleoR-MKK2 fusion protein. Based on exogenous BleoR expression, the HDR-mediated knock-in efficiency was up to [~]40-fold greater when using a XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared to no HDR enhancement or NHEJ silencing. Furthermore, this corporation of HDR enhancer overexpression and NHEJ repression also resulted in 7-fold fewer CRISPR-induced Insertions and Deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammalians for improving CRISPR-mediated gene knock-in efficiency.

plant biology↗