An engineered extraplastidial pathway for carotenoid biofortification of leaves

Abstract

Carotenoids are lipophilic plastidial isoprenoids highly valued as nutrients and natural pigments. A correct balance of chlorophylls and carotenoids is required for photosynthesis and therefore highly regulated, making carotenoid enrichment of green tissues challenging. Here we show that leaf carotenoid levels can be boosted through engineering their biosynthesis outside the chloroplast. Transient expression experiments in Nicotiana benthamiana leaves indicated that high extraplastidial production of carotenoids requires an enhanced supply of their isoprenoid precursors in the cytosol, which was achieved using a deregulated form of the main ratedetermining enzyme of the mevalonic acid (MVA) pathway. Constructs encoding bacterial enzymes were used to convert these MVA-derived precursors into carotenoid biosynthetic intermediates that do not normally accumulate in leaves, such as phytoene and lycopene. Cytosolic versions of these enzymes produced extraplastidial carotenoids at levels similar to those of total endogenous (i.e. chloroplast) carotenoids. Strategies to enhance the development of endomembrane structures and lipid bodies as potential extraplastidial carotenoid storage systems were not successful to further increase carotenoid contents. Phytoene was found to be more bioaccessible when accumulated outside plastids, whereas lycopene formed cytosolic crystalloids very similar to those found in the chromoplasts of ripe tomatoes. This extraplastidial production of phytoene and lycopene led to an increased antioxidant capacity of leaves. Finally, we demonstrate that our system can be adapted for the biofortification of leafy vegetables such as lettuce.

Novel insights on the contribution of plastoglobules and reactive oxygen species to chromoplast differentiation

“…Agroinfiltration assays were carried out as described (Andersen et al, 2021) using cells from fresh Agrobacterium tumefaciens GV3101 cultures transformed with the appropriate constructs at an optical density at 600 nm of 0.5 in infiltration buffer (10 mM MES, 10 mM MgCl 2 , 150 µM acetosyringone, pH=5.5). Cultures were mixed in identical proportions when agroinfiltrating several constructs.…”

“…Plant material, growth conditions and photosynthetic measurements. Nicotiana benthamiana and Arabidopsis thaliana plants were grown as described previously (Llorente et al, 2016;Majer et al, 2017;Andersen et al, 2021). For the intense light experiments, plants were grown under standard conditions and then moved to an ARALAB 600 growth chamber where they were maintained for three days in two different light conditions: 50 μmol photons•m −2 •s −1 (named W50) and 500 μmol photons•m −2 •s −1 (W500) under a long day (16h light and 8h dark) photoperiod.…”

Fruit ripening: dynamics and integrated analysis of carotenoids and anthocyanins

“…Furthermore, the transfer of carotenoid biosynthesis to the cytosol can be brought about by the activity of crtB in plastids and its combination with cytosolic bacterial enzymes to produce carotenoids in extraplastidial sites [ 178 , 179 ] and accumulation of lycopene in the cytosol of tobacco [ 179 ]. The isoprenoid precursors are transferred to the cytosol via the MVA (mevalonic acid) pathway.…”

Allosteric feedback inhibition of deoxy-D-xylulose-5-phosphate synthase involves monomerization of the active dimer

“…The culture was centrifuged to collect cells and the pellet was resuspended in agroinfiltration buffer (10 mM MgCl 2 , 10 mM MES, 150 μl acetosyringone, pH=5.6) to achieve OD 600 =0.8. The mixture was incubated at 28°C and 200 rpm for 1.5h and then mixed with a similar culture harboring vector pGWB702-HCProWMV to prevent silencing (43). The 9:1 (DXS:HcProWMV) mix was split in three and the individual fractions were supplemented with either fosmidomycin (200 μM), norflurazon (40 μM) or water before infiltration of leaves from 4-week-old N. benthamiana plants as described (43).…”

“…The mixture was incubated at 28°C and 200 rpm for 1.5h and then mixed with a similar culture harboring vector pGWB702-HCProWMV to prevent silencing (43). The 9:1 (DXS:HcProWMV) mix was split in three and the individual fractions were supplemented with either fosmidomycin (200 μM), norflurazon (40 μM) or water before infiltration of leaves from 4-week-old N. benthamiana plants as described (43). Leaf samples agroinfiltrated with the pGWB420-SlDXS1 construct were collected at 5 dpi, snap-frozen in liquid nitrogen, and stored at −80°C.…”