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Summary 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 rate‐determining 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.

Disruption of protein homeostasis in chloroplasts impairs the correct functioning of essential metabolic pathways, including the methylerythritol 4-phosphate (MEP) pathway for the production of plastidial isoprenoids involved in photosynthesis and growth. We previously found that misfolded and aggregated forms of the first enzyme of the MEP pathway are degraded by the Clp protease with the involvement of Hsp70 and Hsp100/ClpC1 chaperones in Arabidopsis thaliana. By contrast, the combined unfolding and disaggregating actions of Hsp70 and Hsp100/ClpB3 chaperones allow solubilization and hence reactivation of the enzyme. The repair pathway is promoted when the levels of ClpB3 proteins increase upon reduction of Clp protease activity in mutants or wild-type plants treated with the chloroplast protein synthesis inhibitor lincomycin (LIN). Here we show that LIN treatment rapidly increases the levels of aggregated proteins in the chloroplast, unleashing a specific retrograde signaling pathway that up-regulates expression of ClpB3 and other nuclear genes encoding plastidial chaperones. As a consequence, folding capacity is increased to restore protein homeostasis. This sort of chloroplast unfolded protein response (cpUPR) mechanism appears to be mediated by the heat shock transcription factor HsfA2. Expression of HsfA2 and cpUPR-related target genes is independent of GUN1, a central integrator of retrograde signaling pathways. However, double mutants defective in both GUN1 and plastome gene expression (or Clp protease activity) are seedling lethal, confirming that the GUN1 protein is essential for protein homeostasis in chloroplasts.

Carotenoids such as β-carotene (pro-vitamin A) and lycopene accumulate at high levels during tomato ( Solanum lycopersicum L.) fruit ripening, contributing to the characteristic color and nutritional quality of ripe tomatoes. Besides their role as pigments in chromoplast-harboring tissues such as ripe fruits, carotenoids are important for photosynthesis and photoprotection in the chloroplasts of photosynthetic tissues. Interestingly, recent work in Arabidopsis thaliana (L.) Heynh. has unveiled a critical role of chloroplast protein quality control components in the regulation of carotenoid biosynthesis. The accumulation (i.e. degradation rate) and activity (i.e. folding status) of phytoene synthase (PSY) and other Arabidopsis biosynthetic enzymes is modulated by chaperones such as Orange (OR) and Hsp70 in coordination with the stromal Clp protease complex. OR and Clp protease were recently shown to also influence PSY stability and carotenoid accumulation in tomato. Here we show how manipulating the levels of plastid-localized Hsp70 in transgenic tomato plants can also impact the accumulation of carotenoids in ripe fruit. The resulting carotenoid profile and chromoplast ultrastructure, however, are different from those obtained in tomatoes from transgenic lines with increased OR activity. These results suggest that different chaperone families target different processes related to carotenoid metabolism and accumulation during tomato ripening. We further discuss other possible targets for future manipulation in tomato based on the knowledge acquired in Arabidopsis .

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.

Background The soil bacterium Pseudomonas putida is a promising platform for the production of industrially valuable natural compounds. In the case of isoprenoids, the availability of biosynthetic precursors is a major limiting factor. In P. putida and most other bacteria, these precursors are produced from pyruvate and glyceraldehyde 3-phosphate by the methylerythritol 4-phosphate (MEP) pathway, whereas other bacteria synthesize the same precursors from acetyl-CoA using the unrelated mevalonate (MVA) pathway. Results Here we explored different strategies to increase the supply of isoprenoid precursors in P. putida cells using lycopene as a read-out. Because we were not aiming at producing high isoprenoid titers but were primarily interested in finding ways to enhance the metabolic flux to isoprenoids, we engineered the well-characterized P. putida strain KT2440 to produce low but detectable levels of lycopene under conditions in which MEP pathway steps were not saturated. Then, we compared lycopene production in cells expressing the Myxococcus xanthus MVA pathway genes or endogenous MEP pathway genes ( dxs , dxr , idi ) under the control of IPTG-induced and stress-regulated promoters. We also tested a shunt pathway producing isoprenoid precursors from ribulose 5-phosphate using a mutant version of the Escherichia coli ribB gene. Conclusions The most successful combination led to a 50-fold increase in lycopene levels, indicating that P. putida can be successfully engineered to substantially increase the supply of metabolic substrates for the production of industrially valuable isoprenoids.

Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts.

Because of the huge importance of carotenoids in plants and for human health, carotenoid metabolism and regulation have been studied extensively during the last 40 years. In this Research Topic, a review article describes our current understanding of the molecular and biochemical bases underlying OR-induced carotenoid accumulation and the potential of modifying the gene and chromoplast sink toward the development of healthy food for a growing population (Osorio). Investigation of three differently spliced transcripts of cauliflower BoOR suggests the requirement of OR dimerization for OR protein stability in planta, the need of simultaneous presence of PSY interaction-domains for the holdase function of OR, and the possible formation of various BoOR heterodimers for an enhanced activity on carotenoid accumulation (Welsch et al.). Besides OR, other chaperones and the Clp protease complex are known to influence PSY proteostasis and carotenoid accumulation. A study proposes that the MEP-independent mevalonate (MVA) pathway, which is not localized in plastids but in the cytosol, might provide GGPP isoprenoid intermediate which is then transported into plastids for carotenoids and chloroplast biogenesis in Arabidopsis embryos (Vranová et al.). Besides the well-known carotenoid-derived phytohormones (i.e., ABA and strigolactones) and signaling molecules (e.g., beta-cyclocitral), novel apocarotenoids are being identified to function as biologically-active regulators.

A wide diversity of isoprenoids is produced in different plant compartments. Most groups of isoprenoids synthesized in plastids, and some produced elsewhere in the plant cell derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. In Arabidopsis (Arabidopsis thaliana), five genes appear to encode GGPPS isoforms localized in plastids (two), the endoplasmic reticulum (two), and mitochondria (one). However, the loss of function of the plastid-targeted GGPPS11 isoform (referred to as G11) is sufficient to cause lethality. Here, we show that the absence of a strong transcription initiation site in the G11 gene results in the production of transcripts of different lengths. The longer transcripts encode an isoform with a functional plastid import sequence that produces GGPP for the major groups of photosynthesis-related plastidial isoprenoids. However, shorter transcripts are also produced that lack the first translation initiation codon and rely on a second in-frame ATG codon to produce an enzymatically active isoform lacking this N-terminal domain. This short enzyme localizes in the cytosol and is essential for embryo development. Our results confirm that the production of differentially targeted enzyme isoforms from the same gene is a central mechanism to control the biosynthesis of isoprenoid precursors in different plant cell compartments.

Apple is characterized by its high adaptation to diverse growing environments. However, little is still known about how different environments can regulate at the metabolic or molecular level specific apple quality traits such as the yellow fruit peel color. In this study, changes in carotenoids and chlorophylls, antioxidants as well as differences in the transcriptome were investigated by comparing the peel of “Golden Reinders” apples grown at different valley and mountain orchards. Mountain environment favored the development of yellow color, which was not caused by an enhanced accumulation of carotenoids but rather by a decrease in the chlorophyll content. The yellow phenotype was also associated to higher expression of genes related to chloroplast functions and oxidative stress. Time-course analysis over the last stages of apple development and ripening, in fruit from both locations, further revealed that the environment differentially modulated isoprenoids and phenylpropanoid metabolism and pointed out a key role for H 2 O 2 in triggering apple peel degreening. Overall, the results presented herein provide new insights into how different environmental conditions regulate pigment and antioxidant metabolism in apple leading to noticeable differences in the apple peel color.

Plants of several species, including crops, change their volatilome when exposed to a low ratio of red to far-red light (low R/FR) that informs about the presence of nearby plants (i.e., proximity shade). In particular, the volatile hormone ethylene was shown to be produced at higher levels in response to the low R/FR signal in shade-avoider plants. Here, we show that the shade-tolerant species Cardamine hirsuta produces more ethylene than shade avoiders such as Arabidopsis thaliana (a close relative of C. hirsuta) and tomato (Solanum lycopersicum) under white light (W). However, exposure to low R/FR (specifically to FR-supplemented W, referred to as W+FR or simulated shade) resulted in only a slight increase in ethylene emission in C. hirsuta compared to shade avoiders. Stimulation of ethylene production by growing plants in media supplemented with 1-aminocyclopropane-1-carboxylate (ACC) resulted in reduced hypocotyl growth under W+FR in both A. thaliana and C. hirsuta. ACC-dependent ethylene production also repressed hypocotyl elongation under low W and in the dark in C. hirsuta. By contrast, in A. thaliana, ACC supplementation inhibited hypocotyl elongation in the dark but stimulated it under W. Most interestingly, elongation of dark-grown A. thaliana seedlings was also repressed by exposure to the volatiles released by ACC-grown A. thaliana or tomato plants. This observation suggests that increased ethylene levels in the headspace can indeed impact the development of nearby plants. Although the amount of ethylene released by ACC-grown plants to their headspace was much higher than that released by exposure to low R/FR, our results support a contribution of this volatile hormone on the communication of proximity shade conditions to neighboring plants.