Boosting pro-vitamin A content and bioaccessibility in leaves by combining engineered biosynthesis and storage pathways with high-light treatments.

Biofortification of green leafy vegetables with pro-vitamin A carotenoids, such as ß-carotene, has remained challenging to date. Here, we combined two strategies to achieve this goal. One of them involves producing ß-carotene in the cytosol of leaf cells to avoid the negative impacts on photosynthesis derived from changing the balance of carotenoids and chlorophylls in chloroplasts. The second approach involves the conversion of chloroplasts into non-photosynthetic, carotenoid-overaccumulating chromoplasts in leaves agroinfiltrated or infected with constructs encoding the bacterial phytoene synthase crtB, leaving other non-engineered leaves of the plant to sustain normal growth. A combination of these two strategies, referred to as strategy C (for cytosolic production) and strategy P (for plastid conversion mediated by crtB), resulted in a 5-fold increase in the amount of ß-carotene in Nicotiana benthamiana leaves. Following several attempts to further improve ß-carotene leaf contents by metabolic engineering, hormone treatments and genetic screenings, it was found that promoting the proliferation of plastoglobules with increased light-intensity treatments not only improved ß-carotene accumulation but it also resulted in a much higher bioaccessibility. The combination of strategies C and P together with a more intense light treatment increased the levels of accessible ß-carotene 30-fold compared to controls. We further demonstrated that stimulating plastoglobule proliferation with strategy P, but also with a higher-light treatment alone, also improved ß-carotene contents and bioaccessibility in edible lettuce (Lactuca sativa) leaves.

Advances in the Modulation of Potato Tuber Dormancy and Sprouting.

The post-harvest phase of potato tuber dormancy and sprouting are essential in determining the economic value. The intricate transition from dormancy to active growth is influenced by multiple factors, including environmental factors, carbohydrate metabolism, and hormonal regulation. Well-established environmental factors such as temperature, humidity, and light play pivotal roles in these processes. However, recent research has expanded our understanding to encompass other novel influences such as magnetic fields, cold plasma treatment, and UV-C irradiation. Hormones like abscisic acid (ABA), gibberellic acid (GA), cytokinins (CK), auxin, and ethylene (ETH) act as crucial messengers, while brassinosteroids (BRs) have emerged as key modulators of potato tuber sprouting. In addition, jasmonates (JAs), strigolactones (SLs), and salicylic acid (SA) also regulate potato dormancy and sprouting. This review article delves into the intricate study of potato dormancy and sprouting, emphasizing the impact of environmental conditions, carbohydrate metabolism, and hormonal regulation. It explores how various environmental factors affect dormancy and sprouting processes. Additionally, it highlights the role of carbohydrates in potato tuber sprouting and the intricate hormonal interplay, particularly the role of BRs. This review underscores the complexity of these interactions and their importance in optimizing potato dormancy and sprouting for agricultural practices.

Exploring the Deoxy-D-xylulose-5-phosphate Synthase Gene Family in Tomato (Solanum lycopersicum).

Isoprenoids are a wide family of metabolites including high-value chemicals, flavors, pigments, and drugs. Isoprenoids are particularly abundant and diverse in plants. The methyl-D-erythritol 4-phosphate (MEP) pathway produces the universal isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate in plant plastids for the downstream production of monoterpenes, diterpenes, and photosynthesis-related isoprenoids such as carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. The enzyme deoxy-D-xylulose 5-phosphate synthase (DXS) is the first and main rate-determining enzyme of the MEP pathway. In tomato (Solanum lycopersicum), a plant with an active isoprenoid metabolism in several tissues, three genes encode DXS-like proteins (SlDXS1 to 3). Here, we show that the expression patterns of the three genes suggest distinct physiological roles without excluding that they might function together in some tissues. We also confirm that SlDXS1 and 2 are true DXS enzymes, whereas SlDXS3 lacks DXS activity. We further show that SlDXS1 and 2 co-localize in plastidial speckles and that they can be immunoprecipitated together, suggesting that they might form heterodimers in vivo in at least some tissues. These results provide novel insights for the biotechnological use of DXS isoforms in metabolic engineering strategies to up-regulate the MEP pathway flux.

MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply.

Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase (DXS), a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway. Here we provide experimental insights into the underlying mechanism. Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro, causing a size shift. In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer. Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells. Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply, whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.