Metabolic crosstalk between hydroxylated monoterpenes and salicylic acid in tomato defense response against bacteria.

Hydroxylated monoterpenes (HMTPs) are differentially emitted by tomato (Solanum lycopersicum) plants resisting bacterial infection. We have studied the defensive role of these volatiles in the tomato response to bacteria, whose main entrance are through stomatal apertures. Treatments with some HMTPs resulted in stomatal closure and pathogenesis-related protein 1 (PR1) induction. Particularly, α-terpineol induced stomatal closure in a salicylic acid (SA) and abscisic acid-independent manner and conferred resistance to bacteria. Interestingly, transgenic tomato plants overexpressing or silencing the monoterpene synthase MTS1, which displayed alterations in the emission of HMTPs, exhibited changes in the stomatal aperture but not in plant resistance. Measures of both 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) and SA levels revealed competition for MEcPP by the methylerythritol phosphate (MEP) pathway and SA biosynthesis activation, thus explaining the absence of resistance in transgenic plants. These results were confirmed by chemical inhibition of the MEP pathway, which alters MEcPP levels. Treatments with BTH, a SA functional analogue, conferred enhanced resistance to transgenic tomato plants overexpressing MTS1. Additionally, these MTS1 overexpressors induced PR1 gene expression and stomatal closure in neighbouring plants. Our results confirm the role of HMTPs in both intra and inter-plant immune signalling and reveal a metabolic crosstalk between the MEP and SA pathways in tomato plants.

Arabidopsis FIBRILLIN6 influences carotenoid biosynthesis by directly promoting phytoene synthase activity.

Carotenoids are health-promoting plastidial isoprenoids with essential functions in plants as photoprotectants and photosynthetic pigments in chloroplasts. They also accumulate in specialized plastids named chromoplasts, providing color to non-photosynthetic tissues such as flower petals and ripe fruit. Carotenoid accumulation in chromoplasts requires specialized structures and proteins such as fibrillins (FBNs). The FBN family includes structural components of carotenoid sequestering structures in chromoplasts and members with metabolic roles in chloroplasts and other plastid types. However, the association of FBNs with carotenoids in plastids other than chromoplasts has remained unexplored. Here, we show that Arabidopsis (Arabidopsis thaliana) FBN6 interacts with phytoene synthase (PSY), the first enzyme of the carotenoid pathway. FBN6, but not FBN4 (a FBN that does not interact with PSY), enhances the activity of plant PSY (but not of the bacterial PSY crtB) in Escherichia coli cells. Overexpression of FBN6 in Nicotiana benthamiana leaves results in a higher production of phytoene, the product of PSY activity, whereas loss of FBN6 activity in Arabidopsis mutants dramatically reduces the production of carotenoids during seedling de-etiolation and after exposure to high light. Our work hence demonstrates that FBNs promote not only the accumulation of carotenoids in chromoplasts but also their biosynthesis in chloroplasts.

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.

In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation.

In humans, aggregation of polyglutamine repeat (polyQ) proteins causes disorders such as Huntington's disease. Although plants express hundreds of polyQ-containing proteins, no pathologies arising from polyQ aggregation have been reported. To investigate this phenomenon, we expressed an aggregation-prone fragment of human huntingtin (HTT) with an expanded polyQ stretch (Q69) in Arabidopsis thaliana plants. In contrast to animal models, we find that Arabidopsis sp. suppresses Q69 aggregation through chloroplast proteostasis. Inhibition of chloroplast proteostasis diminishes the capacity of plants to prevent cytosolic Q69 aggregation. Moreover, endogenous polyQ-containing proteins also aggregate on chloroplast dysfunction. We find that Q69 interacts with the chloroplast stromal processing peptidase (SPP). Synthetic Arabidopsis SPP prevents polyQ-expanded HTT aggregation in human cells. Likewise, ectopic SPP expression in Caenorhabditis elegans reduces neuronal Q67 aggregation and subsequent neurotoxicity. Our findings suggest that synthetic plant proteins, such as SPP, hold therapeutic potential for polyQ disorders and other age-related diseases involving protein aggregation.

MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Overlapping and specialized roles of tomato phytoene synthases in carotenoid and abscisic acid production.

Carotenoids are plastidial isoprenoids required for photoprotection and phytohormone production in all plants. In tomato (Solanum lycopersicum), carotenoids also provide color to flowers and ripe fruit. Phytoene synthase (PSY) catalyzes the first and main flux-controlling step of the carotenoid pathway. Three genes encoding PSY isoforms are present in tomato, PSY1 to PSY3. Mutants have shown that PSY1 is the isoform providing carotenoids for fruit pigmentation, but it is dispensable in photosynthetic tissues. No mutants are available for PSY2 or PSY3, but their expression profiles suggest a main role for PSY2 in leaves and PSY3 in roots. To further investigate isoform specialization with genetic tools, we created gene-edited lines defective in PSY1 and PSY2 in the MicroTom background. The albino phenotype of lines lacking both PSY1 and PSY2 confirmed that PSY3 does not contribute to carotenoid biosynthesis in shoot tissues. Our work further showed that carotenoid production in tomato shoots relies on both PSY1 and PSY2 but with different contributions in different tissues. PSY2 is the main isoform for carotenoid biosynthesis in leaf chloroplasts, but PSY1 is also important in response to high light. PSY2 also contributes to carotenoid production in flower petals and, to a lesser extent, fruit chromoplasts. Most interestingly, our results demonstrate that fruit growth is controlled by abscisic acid (ABA) specifically produced in the pericarp from PSY1-derived carotenoid precursors, whereas PSY2 is the main isoform associated with ABA synthesis in seeds and salt-stressed roots.

A quantitative method to measure geranylgeranyl diphosphate (GGPP) and geranylgeranyl monophosphate (GGP) in tomato (Solanum lycopersicum) fruit.

BACKGROUND: Isoprenoids are a very large class of metabolites playing a key role in plant physiological processes such as growth, stress resistance, fruit flavor, and color. In chloroplasts and chromoplasts, the diterpene compound geranylgeranyl diphosphate (GGPP) is the metabolic precursor required for the biosynthesis of tocopherols, plastoquinones, phylloquinone, chlorophylls, and carotenoids. Despite its key role for the plant metabolism, reports on GGPP physiological concentrations in planta have been extremely scarce. RESULTS: In this study, we developed a method to quantify GGPP and its hydrolysis product geranylgeranyl monophosphate (GGP) from tomato fruit, using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Quantification was done by external calibration and the method was validated in terms of specificity, precision, accuracy, and detection and quantitation limits. We further demonstrate the validity of our approach by analysing GGPP contents in the ripe fruits of wild-type tomatoes and mutants defective in GGPP production. Finally, we also show that the sample preparation is key to prevent GGPP hydrolysis and mitigate its conversion to GGP. CONCLUSION: Our study provides an efficient tool to investigate the metabolic fluxes required for GGPP supply and consumption in tomato fruit.

Molecular mechanisms of shade tolerance in plants.

Shade tolerance is an ecological concept used in a wide range of disciplines, from plant physiology to landscaping or gardening. It refers to the strategy of some plants to persist and even thrive in environments with low light levels because of the shade produced by the vegetation proximity (e.g. in the understory). Shade tolerance influences the organization, structure, functioning, and dynamics of plant communities. However, little is known about its molecular and genetic basis. By contrast, there is a good understanding on how plants deal with the proximity of other plants, a divergent strategy used by most crops to respond to vegetation proximity. While generally shade-avoiding species strongly elongate in response to the proximity of other plants, shade-tolerant species do not. Here we review the molecular mechanisms that control the regulation of hypocotyl elongation in shade-avoiding species as a reference framework to understand shade tolerance. Comparative studies indicate that shade tolerance is implemented by components also known to regulate hypocotyl elongation in shade-avoiding species. These components, however, show differential molecular properties that explain how, in response to the same stimulus, shade-avoiding species elongate while shade-tolerant ones do not.

Tomato geranylgeranyl diphosphate synthase isoform 1 is involved in the stress-triggered production of diterpenes in leaves and strigolactones in roots.

Carotenoids are photoprotectant pigments and precursors of hormones such as strigolactones (SL). Carotenoids are produced in plastids from geranylgeranyl diphosphate (GGPP), which is diverted to the carotenoid pathway by phytoene synthase (PSY). In tomato (Solanum lycopersicum), three genes encode plastid-targeted GGPP synthases (SlG1 to SlG3) and three genes encode PSY isoforms (PSY1 to PSY3). Here, we investigated the function of SlG1 by generating loss-of-function lines and combining their metabolic and physiological phenotyping with gene co-expression and co-immunoprecipitation analyses. Leaves and fruits of slg1 lines showed a wild-type phenotype in terms of carotenoid accumulation, photosynthesis, and development under normal growth conditions. In response to bacterial infection, however, slg1 leaves produced lower levels of defensive GGPP-derived diterpenoids. In roots, SlG1 was co-expressed with PSY3 and other genes involved in SL production, and slg1 lines grown under phosphate starvation exuded less SLs. However, slg1 plants did not display the branched shoot phenotype observed in other SL-defective mutants. At the protein level, SlG1 physically interacted with the root-specific PSY3 isoform but not with PSY1 and PSY2. Our results confirm specific roles for SlG1 in producing GGPP for defensive diterpenoids in leaves and carotenoid-derived SLs (in combination with PSY3) in roots.

Nutritional Enrichment of Plant Leaves by Combining Genes Promoting Tocopherol Biosynthesis and Storage.

The enrichment of plant tissues in tocochromanols (tocopherols and tocotrienols) is an important biotechnological goal due to their vitamin E and antioxidant properties. Improvements based on stimulating tocochromanol biosynthesis have repeatedly been achieved, however, enhancing sequestering and storage in plant plastids remains virtually unexplored. We previously showed that leaf chloroplasts can be converted into artificial chromoplasts with a proliferation of plastoglobules by overexpression of the bacterial crtB gene. Here we combined coexpression of crtB with genes involved in tocopherol biosynthesis to investigate the potential of artificial leaf chromoplasts for vitamin E accumulation in Nicotiana benthamiana leaves. We show that this combination improves tocopherol levels compared to controls without crtB and confirm that VTE1, VTE5, VTE6 and tyrA genes are useful to increase the total tocopherol levels, while VTE4 further leads to enrichment in α-tocopherol (the tocochromanol showing highest vitamin E activity). Additionally, we show that treatments that further promote plastoglobule formation (e.g., exposure to intense light or dark-induced senescence) result in even higher improvements in the tocopherol content of the leaves. An added advantage of our strategy is that it also results in increased levels of other related plastidial isoprenoids such as carotenoids (provitamin A) and phylloquinones (vitamin K1).

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

Plant tissues can be enriched in phytonutrients not only by stimulating their biosynthesis but also by providing appropriate sink structures for their sequestering and storage. In the case of carotenoids, they accumulate at high levels in chromoplasts naturally found in flowers and fruit. Chromoplasts can also be artificially differentiated from leaf chloroplasts by boosting carotenoid production with the bacterial protein crtB. Here we used electron and confocal microscopy together with subplastidial fractionation and transcript, protein and metabolite analyses to analyze the structural and biochemical changes occurring in crtB-induced artificial chromoplasts and their impact on the accumulation of health-related isoprenoids. We show that leaf chromoplasts develop plastoglobules (PG) harboring high levels of carotenoids (mainly phytoene and pro-vitamin A ß-carotene) but also other nutritionally relevant isoprenoids, such as tocopherols (vitamin E) and phylloquinone (vitamin K1). Further promoting PG proliferation by exposure to intense (high) light resulted in a higher accumulation of these health-related metabolites but also an acceleration of the chloroplast-to-chromoplast conversion. We further show that the production of reactive oxygen species (ROS) stimulates chromoplastogenesis. Our data suggest that carotenoid accumulation and ROS production are not just consequences but promoters of the chromoplast differentiation process.

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.

Open avenues for carotenoid biofortification of plant tissues.

Plant carotenoids are plastidial isoprenoids that function as photoprotectants, pigments, and precursors of apocarotenoids such as the hormones abscisic acid and strigolactones. Humans do not produce carotenoids but need to obtain them from their diet as precursors of retinoids, including vitamin A. Carotenoids also provide numerous other health benefits. Multiple attempts to improve the carotenoid profile of different crops have been carried out by manipulating carotenoid biosynthesis, degradation, and/or storage. Here, we will focus on open questions and emerging subjects related to the use of biotechnology for carotenoid biofortification. After impressive achievements, new efforts should be directed to extend the use of genome-editing technologies to overcome regulatory constraints and improve consumer acceptance of the carotenoid-enriched products. Another challenge is to prevent off-target effects like those resulting from altered hormone levels and metabolic homeostasis. Research on biofortification of green tissues should also look for new ways to deal with the negative impact that altered carotenoid contents may have on photosynthesis. Once a carotenoid-enriched product has been obtained, additional effort should be devoted to confirming that carotenoid intake from the engineered food is also improved. This work involves ensuring post-harvest stability and assessing bioaccessibility of the biofortified product to confirm that release of carotenoids from the food matrix has not been negatively affected. Successfully addressing these challenges will ensure new milestones in carotenoid biotechnology and biofortification.

Correction: Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

[This corrects the article DOI: 10.1371/journal.pone.0150539.].

A fast and simplified method to estimate bioaccessibility of carotenoids from plant tissues.

Carotenoids are lipophilic isoprenoids with roles in photosynthesis and signaling. Dietary carotenoids are nutritionally relevant as precursors of retinoids (including vitamin A). These pigments also provide health benefits as anti-oxidative, anti-inflammatory or anti-tumor agents, among other biological functions. Such health-related advantages have spurred a strong interest in the biofortification of food products with carotenoids. Most biotechnological approaches have been carried out in plants because dietary carotenoids are primarily obtained from fruits and vegetables. Successful examples abound in the literature but in most cases a critical aspect is neglected: bioaccessibility. A higher content of carotenoids in a given plant product does not necessarily mean an improved dietary intake because these lipophilic compounds must be released from the food and incorporated into intestinal micelles to reach the sites of action in the human body. Bioaccessibility refers to the percentage of the carotenoid that is released from the food matrix during digestion and incorporated into micelles in the gastrointestinal tract. Bioaccessibility substantially changes depending on the physicochemical context and subcellular environment where carotenoids accumulate within plant cells. Here, we present a fast, simplified, inexpensive and efficient in vitro method to estimate bioaccessibility that has been adapted to the requirements and equipment of typical plant molecular biology labs. The availability of this protocol should improve biotechnological efforts aimed at carotenoid biofortification by complementing compositional improvements with bioaccessibility data to better estimate the nutritional value of the newly generated functional food.

Environmentally driven transcriptomic and metabolic changes leading to color differences in "Golden Reinders" apples.

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 H2O2 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.

Correction: Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.pgen.1005824.].

Corrigendum: Evolutionary Recycling of Light Signaling Components in Fleshy Fruits: New Insights on the Role of Pigments to Monitor Ripening.

[This corrects the article DOI: 10.3389/fpls.2016.00263.].