Alternative Carbon Sources for Isoprene Emission

Abstract

Isoprene and other plastidial isoprenoids are produced primarily from recently assimilated photosynthates via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, when environmental conditions limit photosynthesis, a fraction of carbon for MEP pathway can come from extrachloroplastic sources. The flow of extrachloroplastic carbon depends on the species and on leaf developmental and environmental conditions. The exchange of common phosphorylated intermediates between the MEP pathway and other metabolic pathways can occur via plastidic phosphate translocators. C₁ and C₂ carbon intermediates can contribute to chloroplastic metabolism, including photosynthesis and isoprenoid synthesis. Integration of these metabolic processes provide an example of metabolic flexibility, and results in the synthesis of primary metabolites for plant growth and secondary metabolites for plant defense, allowing effective use of environmental resources under multiple stresses.

Keywords: DMADP; MEP pathway; VOCs; energy control; plant stress.

Figure 1

Synthesis of major plant volatile isoprenoids, isoprene and monoterpenes, occurs via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in chloroplast, however other multiple biosynthetic pathways can contribute carbon intermediates to MEP pathway. Due to the compartmental nature of the chloroplast metabolism and cytosol, the intermediate exchange among different pathways is hampered. The key pathways interacting with the MEP pathway are Carbon 1 (C₁) metabolic pathway, oxidative pentose pathway, Calvin-Benson (CB) cycle, shikimate pathway, cytosolic glycolysis and mevalonate (MVA) pathway. Potential exchange of intermediates through the plastid membrane is also shown, as well as intermediate carbon partitioning. In addition, biosynthesis of different classes of isoprenoids formed from the same five carbon (C₅) precursors in both compartments is shown. Solid-line arrows indicate carbon flow from extra-chloroplast intermediate molecules and exchange of carbon intermediates between the compartments. The inhibitors of the MVA and MEP pathway are shown in red. Fosmidomycin (FMS) inhibits DXR, the enzyme responsible for the formation of MEP from DXP in the MEP pathway [30,89]. Bisphosphonates (BP) inhibitors (e.g. alendronate and zoledronate) are used to inhibit prenyltransferases reactions, suppressing the formation of isoprenoids formed from GDP in chloroplast and cytosol [44,88]. The lovastatin (LOV) (also referred to as mevinolin), is a specific inhibitor of HMGR in the MVA pathway [75,79,87]. Abbreviations: Metabolites: CDP-ME, 4-(cytidine 5’-diphospho)-2-C-methyl-D-erythritol; CDP-ME2P, 2-Phospho-4-(cytidine 5’-diphospho)-2-C-methyl-D-erythritol; DMADP, dimethylallyl diphosphate; E4-P; erythrose 4-phosphate; FDP, farnesyl diphosphate (FDP; C₁₅); GDP, geranyl diphosphate (GDP, C₁₀); geranylgeranyl diphosphate (GGDP, C₂₀); G6P, glucose 6-phosphate; G3-P, glyceraldehydes-3-phosphate; HCO₃^-, bicarbonate ion; HMBDP, 1-Hydroxy-2-methylbut-2-enyl-butenyl 4-diphosphate; HMG-CoA, 3-hydroxy-3-methyl-glutaryl-coenzyme A; IDP, isopentenyl diphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; ME-cDP, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; 2-PGA, 2- phosphoglycerate; 3-PGA, 3-phosphoglycerate; RuBP, ribulose-1,5-bisphosphate; Ru5-P, Ribulose 5-phosphate. Enzymes: CMK, 4-(cytidine 5’-diphospho)-2-C-methyl-D-erythritol kinase; DXS, 1-Deoxy-D xylulose 5-phosphate synthase; DXR, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase; HDR, 4-Hydroxy-3-methylbut-2-enyl-diphosphate reductase; HMGR, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; IDI, isopentenyl diphosphate isomerase; IspS, isoprene synthase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. Cofactors: ADP, adenosine diphosphate; ATP, adenosine triphosphate; CTP, cytidine triphosphate; NADH, nicotinamide adenine dinucleotide (form reduced); NAD⁺, nicotinamide adenine dinucleotide (form oxidized); NADPH, nicotinamide adenine dinucleotide phosphate (form reduced); NADP⁺, nicotinamide adenine dinucleotide phosphate (form oxidized). Additional abbreviations: Fd; ferredoxin-dependent reaction; PP_i; pyrophosphate.

Figure 2

A review of the current understanding of chloroplastic phosphoenolpyruvate (PEP) synthesis associated with pyruvate formation and specific transporters that mediate the transport of intermediates through the plastid membrane. The primary products of photosynthesis, triose phosphates, are exported via the triose phosphate/phosphate translocator (TPT), or metabolized in chloroplasts. The PEP, can be imported via PEP/phosphate translocator (PPT), or synthetized by plastidic enolase (2) or, to a lesser extent, by the pyruvate, orthophosphate dikinase (4) enzyme. PEP in the chloroplasts can enter the shikimic acid pathway or, after pyruvate (Pyr) formation, enter the MEP pathway. There are several possible sources for Pyr inside the chloroplasts: 1) export of cytosolic PEP through a PEP/phosphate translocator (PPT) [107] and its conversion to pyruvate via glycolysis involving phosphoglycerate mutase (PGL) and enolase (ENO) [148]; 2) synthesis from PEP by pyruvate kinase (PK) [149]; 3) via NADP-dependent malate dehydrogenase within chloroplast [150,151]; or 4) import from the cytosol via plastid Na⁺-coupled pyruvate transporter (PyT) [152]. The malate can be transported across the plastid inner envelope membrane through the so-called malate valve (or malate shunt) facilitated by 2-oxoglutarate (2-OG)-malate transporter (OMT) or via plastidial dicarboxylate transporter (DCT), which acts as an antiporter exchanging malate against glutamate (Glu) and aspartate (Asp) [119,151,153]. The product of starch and sucrose degradation, glucose 6-phosphate (G6P), is imported into the chloroplast by a hexose translocator (GPT). This specific metabolite is considered to be a positive allosteric effector of the PEP carboxylase enzyme (6). The figure shows a possible traffic of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-cDP) from plastid into cytosol and its transfer to the nucleus. In addition, pentose intermediates, D-xylulose and 1-deoxy-D-xylulose (DX), can be phosphorylated to and/or subsequently translocated into the MEP pathway from xylulose 5-phosphate (XPT) carrier. Metabolites: DXP, 1-Deoxy-D xylulose 5-phosphate; XU5P, xylulose 5-phosphate. Enzymes numbering: phosphoglycerate mutase (1); enolase (2); pyruvate kinase (3); pyruvate orthophosphate dikinase (4); NADP-malate dehydrogenase (5); PEP carboxylase (6); xylulose kinase (7). The questions marks indicate steps for which the enzymes involved in isoprenoid metabolism or transport activities (red circles) remain to be elucidated.

Figure 3

Schematic representation of the processes underlying the proposed hypothesis of the temperature and CO₂ effects on isoprene synthesis. (A) and (B) correspond to the sub-optimal temperature of photosynthesis, and (C) and (D) to the temperature above the thermal optimum of photosynthesis at low (A, C) and high CO₂ (B, D) concentrations. Under low [CO₂], the light-saturated photosynthesis is Rubisco limited (J_max>V_cmax; see glossary for definition), while under high [CO₂], photosynthesis it is limited by ribulose-1,5-bisphosphate (RuBP) regeneration or triose phosphate utilization (J_max<V_cmax), especially under low temperatures. The energy in excess to that used for CO₂ assimilation is available for isoprene production and photorespiration. The energy partitioning to CO₂ and O₂ reduction in the Calvin-Benson (CB) cycle, and MEP pathway is represented by white arrows. The P_i pool size is higher at low [CO₂] than at high [CO₂] and this could favor enhanced involvement of extrachloroplastic PEP sources via PEP/phosphate translocator (PPT). The cytosolic PEP pool size is lower at higher temperatures due to the greater activity of PEP carboxylase enzyme. However, heat stress can trigger enhanced starch and sucrose turnover and, thus, a greater net release of P_i from sugar phosphates. Under higher temperature and supra-optimum [CO₂], greater availability of carbon intermediates and photosynthetic energy can increase MEP pathway activity. This results in increases in DMADP availability and, together with the strong effect of temperature on isoprene synthase activity (IspS), this enhances the rate of isoprene synthesis. Note the thicker arrows indicate a higher flow of intermediaries (black arrows) and energy (white arrows) in response to the change in temperature and CO₂ concentration. Metabolites: 2-PG, 2-phosphoglycolate; F16BP, fructose 1,6-bisphosphate. Explanations of other abbreviations are given in the figures 1 and 2.