Multiscale Metabolic Modeling: Dynamic Flux Balance Analysis on a Whole-Plant Scale

Plant metabolism is characterized by a unique complexity on the cellular, tissue, and organ levels. On a whole-plant scale, changing source and sink relations accompanying plant development add another level of complexity to metabolism. With the aim of achieving a spatiotemporal resolution of source-sink interactions in crop plant metabolism, a multiscale metabolic modeling (MMM) approach was applied that integrates static organ-specific models with a whole-plant dynamic model. Allowing for a dynamic flux balance analysis on a whole-plant scale, the MMM approach was used to decipher the metabolic behavior of source and sink organs during the generative phase of the barley (Hordeum vulgare) plant. It reveals a sink-to-source shift of the barley stem caused by the senescence-related decrease in leaf source capacity, which is not sufficient to meet the nutrient requirements of sink organs such as the growing seed. The MMM platform represents a novel approach for the in silico analysis of metabolism on a whole-plant level, allowing for a systemic, spatiotemporally resolved understanding of metabolic processes involved in carbon partitioning, thus providing a novel tool for studying yield stability and crop improvement.Plants are of vital significance as a source of food (Grusak and DellaPenna, 1999; Rogalski and Carrer, 2011), feed (Lu et al., 2011), energy (Tilman et al., 2006; Parmar et al., 2011), and feedstocks for the chemical industry (Metzger and Bornscheuer, 2006; Kinghorn et al., 2011). Given the close connection between plant metabolism and the usability of plant products, there is a growing interest in understanding and predicting the behavior and regulation of plant metabolic processes. In order to increase crop quality and yield, there is a need for methods guiding the rational redesign of the plant metabolic network (Schwender, 2009).Mathematical modeling of plant metabolism offers new approaches to understand, predict, and modify complex plant metabolic processes. In plant research, the issue of metabolic modeling is constantly gaining attention, and different modeling approaches applied to plant metabolism exist, ranging from highly detailed quantitative to less complex qualitative approaches (for review, see Giersch, 2000; Morgan and Rhodes, 2002; Poolman et al., 2004; Rios-Estepa and Lange, 2007).A widely used modeling approach is flux balance analysis (FBA), which allows the prediction of metabolic capabilities and steady-state fluxes under different environmental and genetic backgrounds using (non)linear optimization (Orth et al., 2010). Assuming steady-state conditions, FBA has the advantage of not requiring the knowledge of kinetic parameters and, therefore, can be applied to model detailed, large-scale systems. In recent years, the FBA approach has been applied to several different plant species, such as maize (Zea mays; Dal’Molin et al., 2010; Saha et al., 2011), barley (Hordeum vulgare; Grafahrend-Belau et al., 2009b; Melkus et al., 2011; Rolletschek et al., 2011), rice (Oryza sativa; Lakshmanan et al., 2013), Arabidopsis (Arabidopsis thaliana; Poolman et al., 2009; de Oliveira Dal’Molin et al., 2010; Radrich et al., 2010; Williams et al., 2010; Mintz-Oron et al., 2012; Cheung et al., 2013), and rapeseed (Brassica napus; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011), as well as algae (Boyle and Morgan, 2009; Cogne et al., 2011; Dal’Molin et al., 2011) and photoautotrophic bacteria (Knoop et al., 2010; Montagud et al., 2010; Boyle and Morgan, 2011). These models have been used to study different aspects of metabolism, including the prediction of optimal metabolic yields and energy efficiencies (Dal’Molin et al., 2010; Boyle and Morgan, 2011), changes in flux under different environmental and genetic backgrounds (Grafahrend-Belau et al., 2009b; Dal’Molin et al., 2010; Melkus et al., 2011), and nonintuitive metabolic pathways that merit subsequent experimental investigations (Poolman et al., 2009; Knoop et al., 2010; Rolletschek et al., 2011). Although FBA of plant metabolic models was shown to be capable of reproducing experimentally determined flux distributions (Williams et al., 2010; Hay and Schwender, 2011b) and generating new insights into metabolic behavior, capacities, and efficiencies (Sweetlove and Ratcliffe, 2011), challenges remain to advance the utility and predictive power of the models.Given that many plant metabolic functions are based on interactions between different subcellular compartments, cell types, tissues, and organs, the reconstruction of organ-specific models and the integration of these models into interacting multiorgan and/or whole-plant models is a prerequisite to get insight into complex plant metabolic processes organized on a whole-plant scale (e.g. source-sink interactions). Almost all FBA models of plant metabolism are restricted to one cell type (Boyle and Morgan, 2009; Knoop et al., 2010; Montagud et al., 2010; Cogne et al., 2011; Dal’Molin et al., 2011), one tissue or one organ (Grafahrend-Belau et al., 2009b; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011; Mintz-Oron et al., 2012), and only one model exists taking into account the interaction between two cell types by specifying the interaction between mesophyll and bundle sheath cells in C4 photosynthesis (Dal’Molin et al., 2010). So far, no model representing metabolism at the whole-plant scale exists.Considering whole-plant metabolism raises the problem of taking into account temporal and environmental changes in metabolism during plant development and growth. Although classical static FBA is unable to predict the dynamics of metabolic processes, as the network analysis is based on steady-state solutions, time-dependent processes can be taken into account by extending the classical static FBA to a dynamic flux balance analysis (dFBA), as proposed by Mahadevan et al. (2002). The static (SOA) and dynamic optimization approaches introduced in this work provide a framework for analyzing the transience of metabolism by integrating kinetic expressions to dynamically constrain exchange fluxes. Due to the requirement of knowing or estimating a large number of kinetic parameters, so far dFBA has only been applied to a plant metabolic model once, to study the photosynthetic metabolism in the chloroplasts of C3 plants by a simplified model of five biochemical reactions (Luo et al., 2009). Integrating a dynamic model into a static FBA model is an alternative approach to perform dFBA.In this study, a multiscale metabolic modeling (MMM) approach was applied with the aim of achieving a spatiotemporal resolution of cereal crop plant metabolism. To provide a framework for the in silico analysis of the metabolic dynamics of barley on a whole-plant scale, the MMM approach integrates a static multiorgan FBA model and a dynamic whole-plant multiscale functional plant model (FPM) to perform dFBA. The performance of the novel whole-plant MMM approach was tested by studying source-sink interactions during the seed developmental phase of barley plants.     

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.     

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen

Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.Isoprenoids constitute a versatile class of compounds fulfilling major physiological functions. They are formed by two pathways in plants, the mevalonate (MVA) pathway in the cytosol (Gershenzon and Croteau, 1993) and the 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids (Gershenzon and Croteau, 1993; Jomaa et al., 1999; Li and Sharkey, 2013b). The MVA pathway is primarily responsible for the synthesis of sesquiterpenes (C15), triterpenes (C30) including brassinosteroids, and even larger molecules such as dolichols (Bick and Lange, 2003; Li and Sharkey, 2013b; Rajabi Memari et al., 2013; Rosenkranz and Schnitzler, 2013). The DXP/MEP pathway is responsible for the synthesis of the simplest isoprenoids, isoprene and 2-methyl-3-buten-2-ol (C5), monoterpenes (C10), diterpenes (C20) including gibberellins and phytol residue of chlorophylls, and tetraterpenes (C40) including carotenoids (Rodríguez-Concepción and Boronat, 2002; Roberts, 2007).Given that in plants both pathways produce ultimately the same substrates, dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP), the pertinent question is to what extent the two pathways can exchange metabolites (Rodríguez-Concepción and Boronat, 2002). There is evidence of a certain exchange of IDP between cytosolic and plastidic compartments, although the contribution of IDP from one compartment to the pathway flux in the other seems to be relatively minor (Schwender et al., 2001; Rodríguez-Concepción and Boronat, 2002; Bick and Lange, 2003). Some studies have further demonstrated that the exchange of IDP is fully bidirectional (De-Eknamkul and Potduang, 2003; Rodríguez-Concepción, 2006), whereas other studies suggest that IDP export from plastids to cytosol operates with a greater efficiency than the opposite transport (Hemmerlin et al., 2003; Laule et al., 2003). However, although the overall intercompartmental exchange of isoprenoid substrates to pathway flux in the given compartment might seem minor under nonstressed conditions, the importance of cross talk among the pathways might increase under stress conditions that specifically inhibit isoprenoid synthesis in one pathway. In fact, the DXP/MEP pathway is strongly linked to photosynthetic metabolism, and therefore, inhibition of photosynthesis under stressful conditions such as heat stress or drought or photoinhibition could inhibit the synthesis of isoprenoids when they are most needed to fulfill their protective function (Loreto and Schnitzler, 2010; Niinemets, 2010; Possell and Loreto, 2013). There is some evidence demonstrating a certain cooperativity among the two isoprenoid synthesis pathways under conditions leading to a reduction of the activity of one of them (Piel et al., 1998; Jux et al., 2001; Page et al., 2004; Rodríguez-Concepción, 2006), but the capacity for such a replacement of function and regulation is poorly understood.Recent studies using genetically modified plants accumulating end products of the DXP/MEP pathway or using natural variation in product accumulation have demonstrated the existence of a potentially important feedback regulation of the DXP/MEP pathway flux by primary end products of the pathway (Banerjee et al., 2013; Ghirardo et al., 2014; Wright et al., 2014). In particular, binding of DMADP and perhaps IDP to DXP synthase, the first enzyme in the DXP/MEP pathway, leads to downregulation of the pathway flux when the end products cannot be used, such as under stress conditions. However, the strength of such a feedback regulation can be importantly modified by accumulation of phosphorylated intermediates of the pathway, such that DMADP and IDP do not accumulate. Previous studies have demonstrated that there is a certain pool of phosphorylated intermediates in vivo, and that this pool can strongly increase under certain conditions, including experimental and genetic modification of DXP/MEP pathway input and output (Li et al., 2011; Rasulov et al., 2011; Li and Sharkey, 2013a; Ghirardo et al., 2014; Wright et al., 2014).It has further been shown that 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (ME-cDP) is the metabolite accumulating in the plastids, and this accumulation can buffer DMADP and IDP changes in the case of varying DXP/MEP pathway input and consumption (Li and Sharkey, 2013a; Wright et al., 2014). A significant part of ME-cDP might even escape to cytosol, implying the existence of another interesting link between cytosolic and chloroplastic processes in isoprenoid synthesis (Wright et al., 2014). Furthermore, as ME-cDP is an important signaling molecule eliciting a number of gene expression responses (Xiao et al., 2012), accumulation of ME-cDP in plastids and flux to cytosol and further to the nucleus is particularly interesting from the perspective of long-term regulation of isoprenoid synthesis, and suggests a coordination of cellular stress responses by the plastidial isoprenoid synthesis pathway.Isoprene-emitting species constitute an exciting model system where a very large DXP/MEP pathway flux goes to isoprene synthesis under physiological conditions (Li and Sharkey, 2013b; Sharkey et al., 2013). In isoprene-emitting species, there is a concomitant use of the primary substrate DMADP between the plastidic synthesis of isoprene and isoprenoids with a larger molecular size, such as phytol residue of chlorophyll (C20) and carotenoids (C40; Ghirardo et al., 2014; Rasulov et al., 2014), and different from nonemitting species, isoprene emitters seem to support a much larger pool of DMADP without the onset of feedback inhibition (Ghirardo et al., 2014; Wright et al., 2014). However, it is poorly understood how inhibition of one branch of the pathway (isoprene versus larger isoprenoids) affects the other, to what extent it can lead to accumulation of phosphorylated intermediates, how it affects the overall pathway flux through the feedback regulation, and what is the possible role of cytosolic import and export of intermediates. These are all relevant questions to gain insight into the control of the partitioning of pathway flux between isoprene and larger isoprenoids and to understand the biological role of isoprene emission.Studies using metabolic inhibitors to deconvolute the factors involved in pathway regulation and understand the biological role of isoprene have so far used inhibitors that block the early steps of the corresponding pathways. In particular, fosmidomycin, a specific inhibitor of DXP reductoisomerase, the enzyme responsible for the synthesis of MEP from DXP, has been used to inhibit the DXP/MEP pathway (Loreto and Velikova, 2001; Sharkey et al., 2001; Loreto et al., 2004). In addition, lovastatin (mevinolin), the inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase that controls the MVA pathway flux, has been used to study the cooperativity of the two pathways (e.g. Laule et al., 2003; Mansouri and Salari, 2014). However, these inhibitors are not suitable to understand how end product accumulation can alter the pathway flux.Bisphosphonates constitute a promising class of inhibitors that could be particularly apt for studies on the effects of the inhibition of the end points of the pathway. They have been demonstrated to inhibit cytosolic farnesyl diphosphate (FDP) synthase activity (Oberhauser et al., 1998; Cromartie et al., 1999; van Beek et al., 1999; Bergstrom et al., 2000; Burke et al., 2004), аs well as geranyl diphosphate (GDP) and geranylgeranyl diphosphate (GGDP) synthase activities (Oberhauser et al., 1998; Cromartie et al., 1999; Kloer et al., 2006; No et al., 2012; Lindert et al., 2013). To our knowledge, bisphosphonates have not been used to study the effects of end product accumulation on the pathway flux in isoprene-emitting species, with the exception of one study that investigated the development of isoprene emission capacity through leaf ontogeny (Rasulov et al., 2014).A limitation with any inhibitor study could be a certain nonspecificity, inhibition of additional nondesired reactions, but so far there are no data on such nonspecificity of bisphosphonates. However, there is evidence that diphosphate and its analogs are inhibitors of any ferredoxin (Fd)-dependent reaction (Forti and Meyer, 1969; Bojko and Więckowski, 1999). This could be potentially relevant given that DXP/MEP pathway-reducing steps, at the level of 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate (HMBDP) synthase (HDS) and reductase (HDR), directly accept electrons from Fd in light (Eisenreich et al., 2001; Seemann et al., 2006; Li and Sharkey, 2013a). In addition to the DXP/MEP pathway, inhibition at the level of Fd could also affect photosynthetic reactions and thereby alter energy supply for the DXP/MEP pathway.In this study, we have investigated the effects of inhibition of the initial and final steps of the DXP/MEP pathway by fosmidomycin and bisphosphonate inhibitors alendronate and zoledronate in a strong isoprene emitter hybrid aspen (Populus tremula × Populus tremuloides). Alendronate is a highly specific inhibitor of GDP (Lange et al., 2001; Burke et al., 2004) and FDP synthases (Bergstrom et al., 2000; Burke et al., 2004), and a less specific inhibitor of GGDP synthase (Szabo et al., 2002). Zoledronate operates similarly to alendronate, but is a much stronger inhibitor, being operationally active in concentrations several orders of magnitude less than alendronate (Lange et al., 2001; Henneman et al., 2011; Wasko, 2011). A unique in vivo method was used to study dynamic changes in DMADP and phosphorylated intermediate pool sizes (Rasulov et al., 2009a, 2011; Li et al., 2011), and different inhibitors were applied alone or in sequence to study the regulation of the pathway flux in conditions when the flux out of the pathway or into the pathway is curbed and when both the input and the output are curbed. Dynamic model calculations were used to quantitatively evaluate the significance of the cytosolic intermediate input into chloroplastic isoprenoid synthesis under different conditions of the DXP/MEP pathway entrance and exit, and to evaluate the possible nonspecific inhibition of other steps controlling DXP/MEP pathway flux. The study demonstrates the important regulation of DXP/MEP pathway input and output under conditions of end product accumulation and partial cooperativity among chloroplastic and cytosolic isoprenoid synthesis pathways.