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

Bahtijor Rasulov; Eero Talts; Astrid Kännaste; Ülo Niinemets

doi:10.1104/pp.15.00470

. 2015 Apr 29;168(2):532–548. doi: 10.1104/pp.15.00470

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen^¹

Bahtijor Rasulov ^1,^2,³, Eero Talts ^1,^2,³, Astrid Kännaste ^1,^2,³, Ülo Niinemets ^1,^2,^3,^*

PMCID: PMC4453795 PMID: 25926480

The level of end products from the plastidic isoprenoid synthesis pathway is surprisingly constant because plastids can store large amounts of pathway intermediates.

Abstract

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.

RESULTS

Influence of Bisphosphonate Inhibitors on Isoprene Emission in Light and on Emission Dark Decay Kinetics

Feeding of leaves with bisphosphonate inhibitors, alendronate and zoledronate, resulted in significant changes in isoprene emission in light. In the case of alendronate, isoprene emission was initially somewhat enhanced during the first 15 to 20 min after the start of the feeding. Thereafter, the emission rate constantly decreased, stabilizing at approximately 75% of the rate in control leaves 40 min after the start of feeding (Fig. 1A; Table I). Different from alendronate, feeding by zoledronate resulted in a much smaller initial enhancement, if at all, followed by a monotonous decline of the emission rate (Fig. 1B). After 40 min of feeding, the emission rate stabilized at a similar value than in alendronate-fed leaves (Table II; the emission rates in alendronate- and zoledronate-fed leaves are not significantly different, P > 0.3).

Figure 1. — Illustration of the experimental protocol for measurements of steady-state isoprene emission rate in light and dark decay kinetics without inhibitors (0–3,000 s) and effects of application of bisphosphonate inhibitors (3,000–9,200 s) alendronate (A) and zoledronate (B) and subsequent determination of dark decay kinetics and light activation in leaves of hybrid aspen. The biphasic dark decay of isoprene emission was used to estimate the pool size of the immediate isoprene substrate DMADP and the pool size of upstream metabolites (dark pool; Li et al., 2011; Rasulov et al., 2011, 2014; Li and Sharkey, 2013a). The integral of the initial rapid decrease of isoprene emission rate for 200 to 400 s after switching off the light provided the estimate for DMADP pool size, whereas the secondary rise of isoprene emission between approximately 400 and 1,200 s after darkening was defined as the dark pool. The short time periods immediately before switching off the light (at approximately 600 s in A and B and at approximately 5,800 s in A and approximately 5,700 s in B) and before switching on the light (at approximately 7,300 s in A and at approximately 7,700 s in B) correspond to measurements of the reference line (no emission, and the background isoprene concentration is essentially zero).

Table I. Effects of alendronate and fosmidomycin treatments on isoprene emission rate, DMADP pool size, dark pool size, rate constant of isoprene synthase, and in vivo K_m, and maximum activity (V_max) of isoprene synthase in leaves of hybrid aspen.

The isoprene synthase rate constant is given as the initial slope of isoprene emission (I) versus DMADP pool size relationship, whereas in vivo K_m and V_max are calculated from the Hanes-Woolf plots (Fig. 3 for the sample plots). Both DMADP pool size and the dark pool size were determined from the biphasic dark decay of isoprene emission (Li et al., 2011; Rasulov et al., 2011, 2014; Li and Sharkey, 2013a). The initial rapid decay, 200 to 400 s after darkening, characterizes the pool of isoprene substrate (mainly DMADP and to some extent IDP that is rapidly converted to DMADP) that was present prior to darkening. In the darkness, there is a secondary rise of isoprene emission between approximately 400 and 1,200 s after darkening that results from conversion of phosphorylated intermediates to DMADP in the dark (dark pool; Li et al., 2011; Rasulov et al., 2011, 2014; Li and Sharkey, 2013a). This secondary rise occurs earlier in bisphosphonate-inhibited leaves due to a greater contribution of IDP and changed affinity of reducing enzymes for alternative electron donors (Figs. 1 and 2; see also “Discussion”). Two experimental protocols were used. The first protocol involved measurements without inhibitors (control), 40-min treatment with alendronate, and ultimately 30-min treatment with fosmidomycin. The second protocol consisted of measurements without inhibitors (control), 30-min treatment with fosmidomycin, and 40-min treatment with alendronate. Each value represents the mean ± se of five measurements in different plants. Means with different lowercase letters are significantly different at P < 0.05 according to Student's t tests (paired samples t tests for comparisons among treatments involving the same leaves and separate sample t tests for treatments with different leaves). NA, Not available.

Treatment	Isoprene Emission Rate	DMADP Pool Size	Dark Pool Size	Initial Slope	K_m	V_max
	nmol m⁻² s⁻¹	nmol m⁻²		s⁻¹	nmol m⁻²	nmol m⁻² s⁻¹
Control	25.6 ± 1.6a	1130 ± 120b	1340 ± 90d	0.0325 ± 0.0021a	2400 ± 180a	80.6 ± 4.8a
Alendronate (Ald, 40 min)	20.2 ± 1.8b	1680 ± 100a	5860 ± 680a	0.0205 ± 0.0013b	2800 ± 170a	58.2 ± 3.6b
Fosmidomycin (Fmd, 30 min)	1.63 ± 0.32d	61 ± 7e	793 ± 90e	0.0331 ± 0.0027a	NA	NA
Ald+Fmd (40 +30 min)	4.8 ± 0.5c	400 ± 70c	4800 ± 460b	0.0195 ± 0.0018b	2150 ± 190b	44 ± 6c
Fmd+Ald (30 +40 min)	2.46 ± 0.21d	126 ± 10d	3240 ± 330c	0.0202 ± 0.0014b	NA	NA

Open in a new tab

Table II. Effects of zoledronate and fosmidomycin on average (±se) isoprene emission rate, DMADP pool size, dark pool size, rate constant of isoprene synthase reaction, and K_m and V_max of isoprene synthase in vivo in leaves of hybrid aspen.

Definition of all characteristics, number of replicates, and statistical comparison of treatments are as in Table I. Two measurement protocols were used. In the case of the first protocol, measurements were started without inhibitors (control), followed by a 40-min treatment with zoledronate and a 30-min treatment with fosmidomycin. In the case of the second protocol, measurements were started without inhibitors (control) and were followed by a 30-min treatment with fosmidomycin and a 40-min treatment with zoledronate.

Treatment	Isoprene Emission Rate	DMADP Pool Size	Dark Pool Size	Initial Slope	K_m	V_max
	nmol m⁻² s⁻¹	nmol m⁻²		s⁻¹	nmol m⁻²	nmol m⁻² s⁻¹
Control	27.2 ± 1.7a	1160 ± 150b	1600 ± 150c	0.0331 ± 0.0022a	2370 ± 110ab	78 ± 7a
Zoledronate (Zld, 40 min)	19.3 ± 1.6b	1458 ± 90a	5900 ± 500a	0.0197 ± 0.0021b	2430 ± 120a	50 ± 5b
Fosmidomycin (Fmd, 30 min)	2.15 ± 0.31d	78 ± 4.5d	852 ± 60d	0.0341 ± 0.0018a	NA	NA
Zld+Fmd (40 +30 min)	10.5 ± 0.9c	615 ± 38c	6270 ± 70a	0.0212 ± 0.0012b	2280 ± 60b	47.1 ± 4.5b
Fmd+Zld (30 +40 min)	1.64 ± 0.23d	35 ± 4.8e	3960 ± 210b	0.0233 ± 0.0031b	NA	NA

Open in a new tab

Feeding leaves with bisphosphonate inhibitors also altered the dark decay kinetics of isoprene emission (Figs. 1 and 2). In particular, the dark emission of isoprene continued for a longer period, and the secondary rise of isoprene emission started earlier in bisphosphonate-fed leaves than in control leaves (Fig. 2). Different from the emissions in light, bisphosphonate inhibitors importantly enhanced the emissions in darkness during the secondary rise of isoprene emission (Figs. 1 and 2). In fact, in inhibited leaves, isoprene emissions continued in darkness for 15 to 20 min at a very high level of 20% to 50% of that observed during light in control leaves (Figs. 1 and 2), and dark emissions did not reach zero even after 3 h in darkness (data not shown).

Figure 2. — Comparison of light-dark transients in isoprene emission among noninhibited leaves and leaves inhibited by zoledronate for 20 and 40 min in hybrid aspen. Ref indicates measurement of the reference line before switching off the light, and the dashed lines denote the baselines used to separate the DMADP pool (first rapid decay in isoprene emission approximately 200–300 s after switching off the light) and the dark pool (second rise of dark isoprene emission between approximately 200 and 1,200 s after switching off the light).

Analysis of precursor pools indicated that both bisphosphonate inhibitors led to an elevation of the DMADP pool corresponding to the rapid dark decay of isoprene emission of approximately 200 to 300 s after switching off the light (Fig. 2; Tables I and II). However, the dark pool of isoprenoid precursors corresponding to the dark-activated secondary rise of isoprene emission between approximately 200 and 2,000 s since darkening was particularly dramatically increased, by approximately 4-fold in both alendronate- and zoledronate-inhibited leaves (Fig. 2; Tables I and II). The DMADP pool was somewhat more enhanced in alendronate-inhibited leaves than in zoledronate-inhibited leaves (P < 0.05), whereas the change in the dark pool size was similar among the two bisphosphonate inhibitors (P > 0.1).

Influence of Bisphosphonate Inhibitors on Isoprene Synthase Kinetics

The initial slope of isoprene emission versus DMADP pool size decreased in bisphosphonate-inhibited leaves by approximately 35% (Fig. 3A; Tables I and II), and the decrease was similar in alendronate- and zoledronate-inhibited leaves (P > 0.3 for the difference among the inhibitors). Further analysis of the DMADP dependence of isoprene emission using Hanes-Woolf plots (Fig. 3B for sample relationships) indicated that the in vivo K_m for isoprene synthase was not affected by bisphosphonate inhibition (Tables I and II), whereas the V_max of isoprene synthase was reduced by approximately 40% with alendronate and by approximately 60% with zoledronate treatment (Tables I and II; differences among the inhibitors were statistically significant at P < 0.001).

Figure 3. — Representative relationships of isoprene emission rate in relation to DMADP pool size in control, alendronate-inhibited, and zoledronate-inhibited leaves (A), and corresponding Hanes-Woolf plots used to estimate the in vivo K_m and V_max of isoprene synthase (B) in hybrid aspen. Both alendronate and zoledronate were applied for 40 min prior to start of the measurements. Paired values of DMADP pool size and isoprene emission rate were derived from the dark decay data of isoprene emission (for sample relationships, see Figs. 1 and 2).

Effects of Fosmidomycin on Isoprene Emission and Substrate Pool Size

Feeding leaves with fosmidomycin, the inhibitor of the chloroplastic DXP/MEP isoprenoid synthesis pathway, resulted in a strong reduction of isoprene emission to a level of approximately 6% to 8% of that in control leaves 30 min after the start of fosmidomycin feeding (Fig. 4A; Tables I and II). This was associated with a major reduction of the immediate release of isoprene in dark (Fig. 4B), reflecting a similar strong decrease, more than 90% of DMADP pool size. However, the isoprene synthase rate constant that characterizes the isoprene synthase activity was not affected by fosmidomycin treatment (Tables I and II). Different from DMADP pool size, the dark pool size responsible for the dark-activated isoprene release was much less affected. Compared with noninhibited leaves, the dark pool size was reduced by approximately 40% (Fig. 4B; Tables I and II). When light was switched on after a prolonged dark period of 25 to 40 min, there was a certain burst of isoprene emission that was of comparable size with the postillumination release of isoprene immediately after leaf darkening (Fig. 5A). Such an apparent burst was not observed in noninhibited leaves or in bisphosphonate-inhibited leaves, where switching on the light led to light-dependent activation of isoprene emission (Fig. 1).

Figure 4. — Representative time-dependent changes in isoprene emission after application of fosmidomycin to control and alendronate- and zoledronate-inhibited leaves in light (A), and corresponding light-dark decay kinetics in fosmidomycin-treated leaves, and in leaves treated with fosmidomycin followed by zoledronate application in hybrid aspen. A also demonstrates the integrals of isoprene emission after application of fosmidomycin until a steady-state isoprene emission rate was observed, and B and C demonstrate the dark pools (Table II).

Figure 5. — Comparison of the kinetics of light activation of isoprene emission in fosmidomycin-inhibited leaves (A) and in leaves treated first with fosmidomycin followed by zoledronate application in hybrid aspen (B). A, The light-dark decay kinetics are also shown (for corresponding kinetics in fosmidomycin + zoledronate-inhibited leaves, see Fig. 4C).

Influences of Fosmidomycin on Bisphosphonate-Inhibited Leaves

When bisphosphonate inhibitors were fed prior to application of fosmidomycin, the fosmidomycin-dependent reduction of isoprene emission rate was much slower, especially in zoledronate-treated leaves (Fig. 4A). After 30 min of fosmidomycin feeding to bisphosphonate-treated leaves, alendronate-treated leaves still maintained an emission rate of 20% of that in controls (Table I), whereas zoledronate-treated leaves maintained an even higher rate of 35% of that in control leaves (Table II; alendronate and zoledronate treatments are significantly different at P < 0.005). The reduction of isoprene emission in light under combined bisphosphonate/fosmidomycin treatments was associated with reduced DMADP pool size (65% reduction in alendronate-treated leaves and 50% reduction in zoledronate-treated leaves; Tables I and II). The treatment of bisphosphonate-inhibited leaves with fosmidomycin did not affect the rate constant of isoprene synthase in the case of both bisphosphonate inhibitors (Tables I and II). In vivo K_m and V_max for isoprene synthase in zoledronate-treated leaves also did not depend on fosmidomycin (Table II). However, fosmidomycin reduced both K_m and V_max of isoprene synthase in alendronate-treated leaves (Table I). Compared with bisphosphonate treatment alone, the dark pool size was reduced by approximately 20% in alendronate-inhibited leaves upon fosmidomycin treatment, but nevertheless, the pool remained very high compared with nonbisphosphonate-treated leaves (Table I). The dark pool size did not depend on fosmidomycin application in zoledronate-treated leaves, although the trend was decreasing (Table II).

Bisphosphonate Treatment following Fosmidomycin Application

Application of bisphosphonate inhibitors to fosmidomycin-treated leaves did not affect isoprene emissions at light compared with leaves treated with fosmidomycin only, and very low emission rates of approximately 10% of untreated controls were observed (Tables I and II). DMADP pool size was approximately 3-fold increased in alendronate-treated leaves (Table I) and approximately 2-fold reduced in zoledronate-treated leaves (Table II) compared with leaves treated only with fosmidomycin. Nevertheless, the DMADP pool sizes were still very low. Compared with nontreated leaves, the remaining DMADP pool size was only 13% in fosmidomycin- and alendronate-treated leaves and only 3% in fosmidomycin- and zoledronate-treated leaves (Tables I and II). The biggest difference among fosmidomycin treatment and fosmidomycin followed by bisphosphonate treatment was in dark-induced emission kinetics and extent (compare with Fig. 4, B and C). In particular, the dark-induced isoprene emission was induced earlier, lasted longer, and proceeded with a greater rate in bisphosphonate-treated leaves than in leaves treated only with fosmidomycin (compare with Fig. 4, A and B). This difference was associated with a much greater dark pool in alendronate-treated (2-fold greater; Table I) and zoledronate-treated (4.5-fold greater; Table II) leaves compared with leaves treated only with fosmidomycin. The isoprene synthase rate constant was inhibited by combined fosmidomycin/alendronate and fosmidomycin/zoledronate treatments similar to alendronate and zoledronate treatments alone (Tables I and II). The light burst pool observed immediately after switching on the light after prolonged darkness was much greater in combined fosmidomycin and bisphosphonate treatment compared with leaves treated only with fosmidomycin (Fig. 5B).

Effects of Bisphosphonate Inhibitors and Fosmidomycin on Foliage Photosynthetic Characteristics

The initial rapid response 150 to 600 s after the start of bisphosphonate treatment was an increase in stomatal conductance and concomitant increase in net assimilation rate (Fig. 6A). In contrast, long-term bisphosphonate inhibition significantly reduced foliage net assimilation rate, stomatal conductance, quantum yield of PSII (Fig. 6, A and B), postillumination CO₂ release (Fig. 6C), and quantum yield of net assimilation rate (Table III) over a time period of approximately 40 to 50 min. Long-term bisphosphonate-driven changes in net assimilation rate and stomatal conductance were proportional such that the intercellular CO₂ concentration was maintained at an almost constant value through the treatments (Fig. 6B).

Figure 6. — Representative time-dependent changes in effective quantum yield (QY) of PSII, net assimilation rate, and stomatal conductance to water vapor upon zoledronate application (A), and comparison of effects of treatments with fosmidomycin, alendronate, and zoledronate on these three characteristics (B and C), and dark respiration rate and postillumination CO₂ burst (C) in leaves of hybrid aspen. Data are averages ± se (n = 5). Averages with the same lowercase letter are not significantly different (P > 0.05; for the statistical analysis, see Table I). Values of intercellular CO₂ concentration (C_i) are also provided (none of the values was statistically different from others, P > 0.05). Postillumination CO₂ burst is primarily dependent on the rate of photorespiration (but see Sharkey, 1988 and “Materials and Methods”).

Table III. Effects of alendronate and zoledronate inhibitors on the initial quantum yields for isoprene emission and net assimilation rates and on the ratio of quantum yields in hybrid aspen leaves.

The initial quantum yields were calculated as the initial slopes of the light response curves (quantum flux density between 15 and 55 µmol m⁻² s⁻¹) of isoprene emission and net assimilation rate and are reported for an absorbed light. Each value corresponds to the average ± se of five measurements of different plants. Statistical comparison of data is as in Table I. Averages with the same lowercase letter are not significantly different.

Treatment	Initial Quantum Yield		Ratio
Treatment	Isoprene Emission	Net Assimilation	Ratio
	mmol mol⁻¹	mol mol⁻¹	mmol mol⁻¹
Control	0.0750 ± 0.0025a	0.0576 ± 0.0022a	1.280 ± 0.024a
Alendronate	0.0585 ± 0.0022b	0.0452 ± 0.0018b	1.27 ± 0.06a
Zoledronate	0.0522 ± 0.0019b	0.0394 ± 0.0013c	1.327 ± 0.033a

Open in a new tab

In general, the reduction in foliage photosynthetic characteristics was more pronounced for zoledronate than for alendronate treatment (Fig. 6; Table III). For instance, net assimilation rate and stomatal conductance decreased almost 3-fold in zoledronate-treated leaves and 2-fold in alendronate-treated leaves (Fig. 6).

The Relationships among Isoprene and Photosynthetic Characteristics through Bisphosphonate Treatments

As a result of photosynthetic inhibition, the percentage of photosynthetic carbon lost due to isoprene emission (calculated considering that six molecules of CO₂ need to be fixed for the release of one molecule isoprene) increased from an average (±se) of 1.22% ± 0.08% in control leaves to 1.71% ± 0.11% in alendronate-inhibited leaves and to 2.74% ± 0.16% in zoledronate-inhibited leaves (all means are significantly different at P < 0.001). The initial slope of the light response curve of isoprene emission (quantum yield) was also reduced by alendronate and zoledronate feeding, and the reduction was similar for both inhibitors (Table III). There was a strong correlation among the quantum yields for isoprene emission and net assimilation across the treatments (r² = 0.91, P < 0.001), and the ratio of the quantum yields for isoprene emission and net assimilation rate was unaffected by bisphosphonate treatment (Table III), indicating a proportional relationship. Analogously, at high light, a positive correlation was observed between the isoprene emission rate at high light and the effective quantum yield of PSII that provides a measure of the activity of photosynthetic electron transport in high light (Fig. 7).

Figure 7. — Correlation between the effective quantum yield (QY) of PSII and the isoprene emission rate through all of the treatments in hybrid aspen. Data were fitted by a linear regression (r² = 0.77, P < 0.001). The measurements were conducted at an incident quantum flux density of 650 μmol m⁻² s⁻¹.

Fosmidomycin treatments did not affect any of the foliage photosynthetic characteristics. Also, photosynthetic characteristics of bisphosphonate-inhibited leaves treated with fosmidomycin were similar to the leaves inhibited with bisphosphonates only (data not shown).

DISCUSSION

How Bisphosphonate Inhibition Affects Isoprene Emission: General Patterns

We have used a kinetic method based on biphasic postillumination isoprene release to separate the pool of DMADP responsible for the isoprene release in light and the pool of intermediates upstream of DMADP that does not immediately contribute to isoprene release in light (Li et al., 2011; Rasulov et al., 2011, 2014; Li and Sharkey, 2013a; Weise et al., 2013). The DMADP pool was estimated as the integral of the initial rapid dark release of isoprene emission, whereas the upstream metabolite pool was estimated from the secondary rise of isoprene emission. This secondary rise of isoprene emission in the dark has been suggested to result from dark conversion of intermediates of the DXP/MEP pathway upstream of DMADP synthesized in light; these intermediates are converted to DMADP as soon as ATP and NADPH become available in the dark (Li et al., 2011; Rasulov et al., 2011, 2014; Li and Sharkey, 2013a). The dark pool has been shown to mainly reflect the concentration of ME-cDP (Li and Sharkey, 2013a; Wright et al., 2014).

Given the evidence that bisphosphonates inhibit prenyltransferase activity, in particular, GDP and GGDP synthase activities in plastids and FDP and squalene synthase activity in cytosol (see introduction), we expected that bisphosphonate inhibition is associated with enhanced pool size of isoprene substrate, DMADP, and enhanced isoprene emissions (Fig. 8). Indeed, DMADP pool size was increased by bisphosphonate inhibition (Tables I and II), in agreement with past observations (Lange et al., 2001; Rasulov et al., 2014). However, there was only a minor initial enhancement of isoprene emission (approximately 0.5–1.5 nmol m⁻² s⁻¹), and sustained inhibition actually resulted in a reduction of isoprene emission rate (Figs. 1 and 2). On the other hand, there was a huge increase, 3- to 4-fold, in the dark pool size upon bisphosphonate inhibition (Tables I and II) that supported the dark emission of isoprene for a much longer time period and at a much higher rate than in noninhibited leaves (Figs. 1 and 2). The observed changes in isoprene light and dark emissions in our study are somewhat different from those in Rasulov et al. (2014). In the latter study, alendronate feeding more strongly, almost 2-fold, enhanced isoprene emission in developing leaves of hybrid aspen. We argue that the difference in the degree of initial enhancement of isoprene emission observed here and in the study by Rasulov et al. (2014) reflects the circumstance that in the developing leaves, the average rate of larger isoprenoid synthesis, in particular, carotenoids and phytol residue of chlorophylls, was much greater (severalfold greater rate of DMADP consumption than the rate of isoprene synthesis) than in the mature leaves studied here (approximately 4%–7% of the rate of isoprene emission; see also the following sections).

Figure 8. — Simplified diagrams of changes in the metabolite fluxes in cytosolic and chloroplastic isoprenoid synthesis pathways and in the importance of the cross talk between cytosolic and chloroplastic isoprenoid synthesis pathways as influenced by bisphosphonate (alendronate, zoledronate) inhibitors and fosmidomycin. Cytosolic MVA and chloroplastic DXP/MEP pathways of isoprenoid synthesis operate almost independently in control leaves with little cross talk among the two pathways. Both pathways produce DMADP) and IDP for production of isoprenoids. In chloroplasts, the bulk of the pathway flux is used for production of isoprene, and a lower proportion of the pathway flux goes to synthesis of larger isoprenoids formed from GDP (C10, e.g. monoterpenes) and GGDP (C20), and there is also a certain part of the flux used to build up the pool of phosphorylated intermediates such as ME-cDP. Bisphosphonates are strong inhibitors of prenyltransferases and suppress formation of higher molecular size isoprenoids formed from GDP in cytosol and chloroplasts, from FDP (C15) in cytosol, and from GGDP in chloroplasts. The figure shows the first prenyltransferase blocking reaction at the level of GDP. Fosmidomycin inhibits DXP reductoisomerase, the enzyme responsible for the synthesis of MEP from DXP, and thus the entire chloroplastic isoprenoid synthesis pathway. Apart from blocking prenyltransferase reactions, bisphosphonates noncompetitively inhibit isoprene synthase activity and reduce MEP pathway input as well as IDP conversion to DMADP (Fig. 9). This altogether results in a buildup of a phosphorylated intermediate pool and an IDP pool. In addition, the significance of cytosolic import of IDP and use for isoprene emission significantly increases. When fosmidomycin is applied prior to bisphosphonates, the bulk of the isoprene emitted (percentage of isoprene emission [Is Em]) is expected to result from cytosolic import of IDP. In the case of bisphosphonates applied alone, the percentage is calculated assuming a similar cytosolic flux rate. Red arrows indicate possible feedback inhibition of DXP/MEP pathway flux due to feedback inhibition of DXP synthase (Banerjee et al., 2013) by a mild buildup of DMADP and IDP in nonbisphosphonate-inhibited leaves (dashed lines) and by a major increase of the end products in bisphosphonate-inhibited leaves (solid lines). The thickness of arrows is approximately proportional to the magnitude of fluxes.

Both alendronate and zoledronate acted similarly, but a much lower concentration of zoledronate was necessary to obtain a given level of inhibition of isoprene emission and accumulation of intermediate pools. This is in agreement with the stronger binding of the larger aromatic molecule zoledronate to the active site of prenyltransferases, in particular, to the GGDP synthase active site (Lange et al., 2001; Henneman et al., 2011; Wasko, 2011), and stronger capacity to elicit conformational changes upon closing the active site of the enzyme inhibitor complex (No et al., 2012; Lindert et al., 2013). In addition, more hydrophobic zoledronate likely penetrates the membranes more feasibly than alendronate (Russell et al., 2008).

Changes in the DXP/MEP Pathway Flux under Bisphosphonate Treatment: Feedback Inhibition and Pathway Elasticity

The first reaction of the DXP/MEP pathway, condensation of pyruvate and glyceraldehyde 3-phosphate resulting in formation of DXP, is catalyzed by DXP synthase, a thiamin diphosphate (TDP)-dependent transketolase (Rohmer et al., 1996; Li and Sharkey, 2013b). As Banerjee et al. (2013) demonstrated, molecules with a diphosphate tail, IDP and DMADP, can compete for the TDP-binding site, leading to feedback inhibition of the DXP/MEP pathway in conditions of end product accumulation (see introduction; Figs. 8 and 9). However, there is a question about the strength of the feedback control at the level of DXP synthase. In vivo, isoprene-emitting species tolerate relatively high chloroplastic concentrations of DMADP, on the order of 1,400 to 1,600 nmol m⁻², without any signs of feedback inhibition (Rasulov et al., 2009a, 2011, 2014; Li et al., 2011; Ghirardo et al., 2014). This suggests that relatively high DMADP concentrations are needed for an operational feedback at least in vivo (i.e. the loop gain of the negative feedback is moderate; Fig. 9).

Figure 9. — Schematic representation of the interplay between cytosolic and chloroplastic processes in isoprenoid synthesis as influenced by bisphosphonate (alendronate and zoledronate) inhibitors and fosmidomycin. Isoprenoid precursors in chloroplasts are derived from recent photosynthates and partly from cytosolic phosphoenolpyruvate (PEP). Chloroplastic isoprenoid synthesis also requires energetic and reductive equivalents that in light are provided by photosynthetic electron transport chain. Bisphosphonate inhibition blocks synthesis of isoprenoids with larger molecular mass (≥C10), whereas fosmidomycin blocks formation of MEP from DXP. Bisphosphonate inhibition leads to a certain activation of transport of cytosolic isoprenoid pathway precursors into chloroplasts at the level of IDP. A number of secondary effects are induced by bisphosphonate inhibitors, including changes in isopentenyl diphosphate isomerase (IDI) activity, sharing of the product yield of HDR between IDP and DMADP, isoprene synthase (IspS), and chloroplastic and cytosolic transketolase activities. In addition, MEP pathway and photosynthetic reactions dependent on Fd, cytidine triphosphate (CTP), and TDP are also likely inhibited. Postulated main controls are shown by filled arrows and additional probable points of control with empty arrows, whereas the assumed strength of the control is shown by the width of the arrow. The green arrows surrounded by a red line highlight the processes that are inactivated in light but activated in darkness in bisphosphonate-fed leaves. The point of fosmidomycin inhibition of the DXP/MEP pathway, conversion of DXP to MEP by 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR; Kuzuyama et al., 1998), is denoted by ⊗. Accumulation of TDP analogs, DMADP and IDP, is expected to enhance the feedback regulation of the pathway flux at the level of 1-deoxy-d-xylulose 5-phosphate synthase DXS (Banerjee et al., 2013). Ac-CoA, Acetyl-coenzyme A; GAP, glyceraldehyde 3-phosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; MCT, 4-(cytidine 5-diphospho)-2-C-methyl-d-erythritol synthase; PGA, 3-phosphoglycerate; PPi, diphosphate; PYR, pyruvate; R5P, ribulose 5-phosphate; RuBP, ribulose-1,5-bisphosphate.

In our study, bisphosphonate inhibition did result in accumulation of DXP/MEP pathway intermediates, whereas the dark pool size was much more strongly enhanced than the DMADP pool size (Tables I and II). Maintenance of the DMADP pool size within relatively narrow limits compared with the intermediates upstream of DMADP is, in principle, consistent with the concept of a feedback regulation of DMADP pool size (Figs. 8 and 9). Considering both dynamic changes in isoprene emission rate and increases in the DMADP and dark pool sizes (see “Quantitative Separation of Fluxes Due to Different Substrate Sources in Inhibited Leaves” in “Materials and Methods”) makes it possible to determine the rate of intermediate accumulation in bisphosphonate-inhibited leaves, and ultimately analyze potential changes in carbon input into the DXP/MEP pathway. In addition to changes in isoprene emission, we considered inhibition of synthesis of isoprenoids other than isoprene due to blocking of prenyltransferase reactions in chloroplasts. Assuming a constant carbon input rate, alendronate-inhibited leaves were expected to accumulate 7.6 μmol m⁻² and zoledronate-inhibited leaves 10.7 μmol m⁻² C5 isoprenoid intermediates during the 40-min treatment due to inhibition of the isoprenoid synthesis rate (Fig. 1). However, the actual accumulation (sum of DMADP and dark pool size in inhibited leaves minus that in control leaves) was 5.1 μmol m⁻² in alendronate-inhibited and 5.3 μmol m⁻² in zoledronate-inhibited leaves. These differences suggest that the rate of carbon input was also reduced, on average 1.1 nmol m⁻² s⁻¹ in alendronate-inhibited and 2.2 nmol m⁻² s⁻¹ in zoledronate-inhibited leaves, during inhibitor application.

This evidence suggests that a moderate buildup of DMADP (and presumably IDP) in bisphosphonate-inhibited leaves could be at least partly responsible for decreases in the carbon input into the DXP/MEP pathway, consistent with feedback inhibition at the level of 1-deoxy-d-xylulose 5-phosphate synthase (Figs. 8 and 9). On the other hand, it is likely that bisphosphonate inhibitors can also interact with the TDP-binding site in a manner analogous to IDP and DMADP. In fact, several TDP analogs having diphosphate, phosphonate, and bisphosphonate functional groups have been shown to inhibit the DXP synthase activity (Erixon et al., 2008; Smith et al., 2014), suggesting that a direct inhibition of DXP synthase by bisphosphonates is plausible. Furthermore, the strong accumulation of pathway intermediates preceding DMADP suggests that maintenance of the DMADP pool size within relatively narrow limits cannot be explained by the regulation at the level of DXP synthase alone.

Why Is the Large Dark Pool of Isoprenoid Substrates Not Converted to DMADP in Inhibited Leaves?

The most exciting questions are what is the nature of the huge dark pool of isoprenoid precursors and how is it maintained in light when DMADP pool size moderately accumulates in bisphosphonate-inhibited leaves or is even strongly depleted in leaves inhibited by fosmidomycin or by combined inhibitor treatments. Liquid chromatographic analyses, although semiquantitative, have suggested that the bulk of the dark pool consists of ME-cDP in isoprene-emitting hybrid poplar (Populus tremula × P. alba; Li and Sharkey, 2013a). In addition, a large accumulation of ME-cDP has been demonstrated in a situation in which DMADP- and IDP-consuming reactions cannot keep up with the metabolic pressure generated by DXP synthase in Arabidopsis (Arabidopsis thaliana; Wright et al., 2014). In our study, the experiment in which light was switched on in fosmidomycin-inhibited or fosmidomycin and bisphosphonate-inhibited leaves, leading to a rapid light burst of isoprene emission as more NADPH and ATP were provided in light, demonstrates that the conversion of the dark pool to DMADP was at least partly energy dependent (Fig. 5, but see the next sections). This evidence provides strong support to the suggestion that the dark pool consists of phosphorylated intermediates, the conversion of which to DMADP is limited by energy supply.

The bisphosphonate inhibitors significantly reduced the net assimilation rate and stomatal conductance, but the intercellular CO₂ concentration was unaffected (Fig. 6). On the other hand, the initial quantum yield of photosynthesis and the effective PSII quantum yield and photorespiration rate in high light (Fig. 6; Table III) were also strongly reduced, conclusively demonstrating that energy-providing reactions of photosynthesis were impacted by bisphosphonates. We suggest that bisphosphonates can inhibit photosynthesis by two mechanisms (Fig. 9). First, TDP-dependent transketolase in the Calvin cycle can be inhibited by bisphosphonates analogously to DXP synthase. It has been demonstrated that inhibition of this enzyme by only 20% can dramatically reduce the rate of ribulose-1,5-bisphosphate regeneration (Henkes et al., 2001). Second, there is evidence that diphosphate and its analogs are inhibitors of any Fd-dependent reaction (Forti and Meyer, 1969; Bojko and Więckowski, 1999). Thus, the reduction in the rate of photosynthetic electron transport in bisphosphonate-inhibited leaves can result from the inhibition of Fd-NADP+ reductase (Fig. 9), leading to a buildup of oxidized electron transport carriers among the two photosystems and feedback inhibition of PSII. In addition, under such conditions, pseudocyclic electron flow from Fd to molecular oxygen (Mehler reaction) is activated (Allen, 2003; Foyer et al., 2012), generating superoxide radicals and H₂O₂ that, once not detoxified, can nonspecifically further inhibit photosynthetic reactions, and can also participate in a more severe oxidative burst upon darkening (Fig. 10). As there was a proportional relationship between the initial quantum yields of isoprene emission and photosynthesis (Table III) and among isoprene emission rate and PSII quantum yield at high light through the inhibitor treatments, a direct link between bisphosphonate inhibition of photosynthetic electron flow and limited conversion of the intermediates of the DXP/MEP pathway is plausible.

Figure 10. — Illustration of dark-release kinetics of C6 lipoxygenase (LOX) pathway volatiles in control and alendronate-inhibited leaves measured with proton transfer reaction-mass spectrometry (PTR-MS; A), and integrated pools of DMADP converted to isoprene and LOX volatiles during a dark period of 15 min in control and zoledronate-inhibited leaves measured by gas chromatography-mass spectrometry (GC-MS; B) in leaves of hybrid aspen.

Furthermore, both HDS and HDR directly accept electrons from Fd in light (Eisenreich et al., 2001; Seemann et al., 2006; Li and Sharkey, 2013a). Thus, not only are the overall chloroplastic energetic and reductive levels decreased, but bisphosphonates likely also directly block the conversion of phosphorylated DXP/MEP pathway intermediates upstream of HMBDP, explaining a buildup of such huge pools in conditions in which DXP synthase runs faster than the reactions consuming the pathway end products (Fig. 9). The situation is different in darkness when HDS and HDR accept electrons from NADPH that become presumably available through activation of the oxidative pentose phosphate cycle, leading to conversion of phosphorylated intermediates to DMADP and ultimately to isoprene (Li et al., 2011; Rasulov et al., 2011; Li and Sharkey, 2013a). So far, it is unclear how the switch between the electron donors occurs, but the faster conversion of the dark pool to DMADP in bisphosphonate-inhibited leaves (Fig. 2) could be indicative of improved affinity to NADPH.

Bisphosphonate Inhibition of End Reactions of the DXP/MEP Pathway at the Level of Isoprene Synthase

The enhancement of both the DMADP and the dark pool sizes suggests that the reduction in isoprene emission in bisphosphonate-inhibited leaves did not directly reflect the substrate level inhibition. In fact, bisphosphonate inhibition also resulted in the reduction of the rate constant and V_max of isoprene synthase (Fig. 3; Tables I and II), indicating a noncompetitive or irreversible inhibition. Existence of such a direct inhibition could be expected given that, in the case of inhibition of GDP, FDP, and GGDP synthases, alendronate and zoledronate bind to the three-Mg²⁺ cluster in the allyl-binding (DMADP-binding) pocket of the enzyme active site (Jahnke et al., 2010; No et al., 2012; Lindert et al., 2013; Liu et al., 2014). The active site of isoprene synthase also coordinates the diphosphate tail of DMADP by three Mg²⁺ atoms (Köksal et al., 2010), and is thus analogous to the allyl-binding active site in prenyltransferases. However, the active site for isoprene synthase is shallower and more rigid than the active site in prenyltransferases as indicated by lack of enzyme conformational changes upon DMADP binding by isoprene synthase (Köksal et al., 2010), which is different from prenyltransferases where DMADP and bisphosphonate binding results in enzyme conformational changes (Jahnke et al., 2010; Lindert et al., 2013; Liu et al., 2014). Steric limitations might explain the slow rate of bisphosphonate inhibition observed for isoprene synthase, and can explain the initial increase of emissions when the effect of enhanced DMADP availability was greater than the reduction of the activity of isoprene synthase (Fig. 1). On the other hand, the inhibitory constant for prenyltransferases is low, indicating a strong binding already at low concentrations (Jahnke et al., 2010; Lindert et al., 2013; Liu et al., 2014). A slow rate of inhibition of isoprene synthase does not rule out a strong binding once the inhibitor reaches the active site. Given further that K_m for DMADP of isoprene synthase is very high (Köksal et al., 2010; Rasulov et al., 2014), a low value of inhibitory constant would imply that bisphosphonate inhibition could be considered essentially irreversible over a physiological range of DMADP pool sizes, explaining the apparent reduction in isoprene synthase V_max values.

Interaction of Bisphosphonate Inhibitors with Fosmidomycin: Evidence of an Extra Precursor Source

Application of fosmidomycin, a specific inhibitor of the DXP reductoisomerase (Kuzuyama et al., 1998; Zeidler et al., 1998), resulted in strongly reduced isoprene emissions after 30-min application, in agreement with previous observations (Loreto et al., 2004; Rasulov et al., 2009a, 2011; Li and Sharkey, 2013a). The reduction of isoprene emission was associated with almost complete depletion of the DMADP pool (Fig. 4; Tables I and II). However, the dark pool size was much less reduced, and the overall reduction of DXP/MEP pathway intermediates (DMADP + dark pool) was less than 50% upon fosmidomycin inhibition (Tables I and II). Thus, in fosmidomycin-inhibited leaves, isoprene emission in light was not only limited by reduced input of intermediates by the DXP/MEP pathway, but also by conversion of intermediates to DMADP.

Combined application of fosmidomycin and bisphosphonates resulted in major differences in the isoprene emission kinetics (Fig. 4A). However, isoprene emission kinetics of combined bisphosphonate/fosmidomycin treatment critically depended on which inhibitor was applied first. When a bisphosphonate inhibitor was applied first, the pool of isoprene precursors was strongly elevated and fosmidomycin effect was much weaker than when fosmidomycin was applied alone (Fig. 4A; Tables I and II). In fact, alendronate-inhibited leaves still had an appreciable rate of isoprene emission for more than 2 h after application of fosmidomycin, and this time was even longer in zoledronate-inhibited leaves (Fig. 4A). It is tempting to suggest that such an enhanced emission in bisphosphonate- and fosmidomycin-inhibited leaves reflects greater pools of intermediates that have accumulated prior to fosmidomycin feeding. However, the total (DMADP + dark pool) reduction of isoprenoid precursor pools during the 30-min fosmidomycin feeding constituted only 10% of total isoprene emission in the case of leaves inhibited by alendronate first, and this percentage was 7% for zoledronate-inhibited leaves. In fact, bisphosphonate- and fosmidomycin-inhibited leaves preserved a significant DMADP pool, approximately 20% of that in alendronate-inhibited and 40% of that in zoledronate-inhibited leaves, whereas the dark pool was reduced only by approximately 20% compared with bisphosphonate only inhibition (Tables I and II).

This evidence suggests that either the fosmidomycin inhibition of DXP/MEP pathway flux was delayed in bisphosphonate-inhibited leaves or there was an extra-chloroplastic flux of isoprenoid precursors, or both. A delay in inhibition can be associated with a reduced transpiration rate in bisphosphonate-inhibited leaves (Fig. 6A), which may have curbed the rate of fosmidomycin delivery. Experiments in which fosmidomycin was applied first also clearly indicated that an additional carbon source contributed to the total isoprenoid precursor pool upon bisphosphonate application (Fig. 8). Addition of both bisphosphonates to fosmidomycin-inhibited leaves led to a major rise in the dark pool size (compare Fig. 4, B and C) and a greater DMADP pool and isoprene emission rate upon alendronate application (Table I). The overall contribution of this extra isoprenoid precursor source (change in emission + change in the total pool size) was estimated to be, on average, 2.1 nmol m⁻² s⁻¹ (approximately 85% of the rate of isoprene emission; Table I) in fosmidomycin- and alendronate-inhibited leaves, and 0.9 nmol m⁻² s⁻¹ (approximately 50%) in fosmidomycin- and zoledronate-inhibited leaves (Fig. 8).

The crucial question is, where do these extra isoprenoid precursors come from? As bisphosphonates inhibit prenyltransferases, one might suggest that an increase of the chloroplastic precursor pool size is associated with suppression of prenyltransferases that compete with isoprene synthase for DMADP. However, kinetic calculations through fosmidomycin-inhibition treatment (Fig. 4A) suggest that in fosmidomycin-only-inhibited leaves, prenyltransferases were responsible for a minor part, approximately 7% (0.2 nmol m⁻² s⁻¹) of total DMADP consumption, 30 min after the start of fosmidomycin inhibition. In addition, approximately 10% of isoprene emitted in light came from the existing intermediate pool (DMADP + conversion of the dark pool to DMADP), whereas the bulk of the remaining emission, 1.1 to 1.7 nmol m⁻² s⁻¹ (Tables I and II), was due to incomplete DXP/MEP pathway inhibition and/or extra-chloroplastic substrate sources. In fact, the lack of light activation of isoprene emission in fosmidomycin-inhibited leaves does indicate that DXP/MEP contribution to isoprene emission was minor (Fig. 5). Assuming that, upon continued fosmidomycin feeding for more than 1 h (Fig. 4A), the DXP/MEP pathway is almost completely blocked, an emission rate of approximately 0.9 nmol m⁻² s⁻¹ must be supported by an extra-chloroplastic source (approximately 0.8 nmol m⁻² s⁻¹), whereas the slow decay of the existing phosphorylated intermediate pool contributes only approximately 0.1 nmol m⁻² s⁻¹.

Given that bisphosphonates inhibit not only the chloroplastic prenyltransferases, but also cytosolic prenyltransferases, enhanced contribution of cytosolic isoprenoid precursors is a likely explanation for the enhancement of the total pool size in fosmidomycin- and bisphosphonate-inhibited leaves. As DMADP likely does not accumulate in cytosol (Weise et al., 2013) and does not well penetrate chloroplast envelope membranes (Schwender et al., 2001; Bick and Lange, 2003; Rodríguez-Concepción et al., 2004), cytosolic IDP (but see the following section for possible accumulation of ME-cDP) has been suggested to be the most likely candidate for such an exchange (Schwender et al., 2001; Bick and Lange, 2003; Rodríguez-Concepción et al., 2004). At any rate, quantitative calculations suggest that in inhibited leaves, the cytosolic supply can be large, up to 80% to 90% of the rate of isoprene emission (Fig. 8).

Changes in IDP:DMADP Equilibrium as a Possible Further Determinant Affecting DMADP Pool Size

Although the bulk of the experimental evidence seems to be consistent with the explanation of energy status as the factor controlling the pathway flux distribution between DMADP and phosphorylated intermediates, there is one remaining piece of evidence, accumulation of dark pool in fosmidomycin- and bisphosphonate-inhibited leaves, that seems to be in conflict with this explanation. If the accumulation of the dark pool indeed reflects a cytosolic source, and it is presumably IDP that is transported from the cytosol, then the question is why IDP is not readily converted to DMADP in fosmidomycin- and bisphosphonate-inhibited leaves in light. Obviously, the key issue is whether the activity of the IDI that reversibly converts IDP to DMADP (Nakamura et al., 2001) is affected by bisphosphonate treatment and by light-dark changes. In fact, the last enzyme of the DXP/MEP pathway, HDR, preferably produces IDP, with an IDP–to-DMADP ratio of approximately 6:1 (Rohdich et al., 2003). Due to high activity of IDI, the in vivo IDP:DMADP ratio is shifted much more strongly to DMADP (1:1–2:1) than the equilibrium concentration without IDI (Ramos-Valdivia et al., 1997; Brüggemann and Schnitzler, 2002b; Page et al., 2004; Weise et al., 2013; Zhou et al., 2013). However, bisphosphonates also can inhibit IDI (Cromartie et al., 1999; van Beek et al., 1999), suggesting that a buildup of IDP in bisphosphonate-inhibited leaves is plausible.

Yet, why is the possible IDP pool converted to DMADP in dark, but not in light? IDI has a very sharp pH optimum, with changes in pH by one unit reducing the activity of IDI by more than 50% (Holloway and Popják, 1968; Brüggemann and Schnitzler, 2002a). Thus, we suggest that light-dark changes in chloroplastic pH (pH = 8–8.2 in light and pH = approximately 7 in dark) can provide an explanation for changes in IDI activity. The optimum pH of IDI was 7 for oak (Quercus robur) chloroplasts (Brüggemann and Schnitzler, 2002a), and thus a drop in pH upon darkening brings the chloroplastic stroma pH close to the optimum value for IDI, resulting in an enhancement of IDP to DMADP conversion.

A general point from this reasoning is that the dark release of isoprene might be importantly driven by IDP conversion too, rather than by conversion of phosphorylated intermediates alone. In fact, in control leaves, there is a hiatus between the cessation of the isoprene emission after the DMADP pool responsible for isoprene emission in light is emptied upon switching off the light and activation of the second burst of dark release of isoprene emission. This hiatus is apparently missing in bisphosphonate-inhibited and in fosmidomycin-inhibited leaves and in leaves treated with both inhibitors (Figs. 1, 2, and 4). Although the two pools cannot be deconvoluted in these experiments, we suggest that the non-energy-dependent conversion that starts rapidly after switching on the light relies on IDP, whereas the energy-dependent conversion that is elicited later relies on phosphorylated intermediates (see also “Why Is the Large Dark Pool of Isoprenoid Substrates Not Converted to DMADP in Inhibited Leaves?” for a possible change in the affinity of different electron donors in bisphosphonate-inhibited leaves).

Although these two sources of intermediates responsible for the dark pool seem to be consistent with all of the experimental evidence, Wright et al. (2014) have observed the existence of a possible nonchloroplastic pool of ME-cDP with a slower turnover rate. A capacity of ME-cDP to penetrate the chloroplast envelope seems likely given that this metabolite participates in signaling in the nucleus (see introduction). If such a cytosolic pool of ME-cDP builds up in a sizeable way (e.g. in conditions favoring ME-cDP accumulation in chloroplasts in light), it is also likely that the cytosolic ME-cDP can reenter the chloroplast when the chloroplastic ME-cDP level decreases in dark, potentially further complicating the pool decay dynamics. We argue that further studies are needed to gain an insight into the nature and share of isoprenoid precursors and their exchange capacity among plastids and cytosol.

CONCLUSION

Our study highlights a complex interplay between changes in flux control between DXP/MEP pathway input and output, storage of intermediates, and exchange of intermediates with cytosol in bisphosphonate-inhibited leaves. The primary modification across inhibitor treatments was a strong accumulation of phosphorylated intermediates, and perhaps IDP, whereas the pool size of the isoprene substrate, DMADP, changed less. This suggests a significant feedback inhibition of DMADP formation, albeit the data suggest that this control cannot be achieved by feedback regulation of DXP synthase alone. Conversion of phosphorylated intermediates was constrained by limited energy supply, whereas IDP conversion was likely limited by inhibited IDP isomerase activity. As accumulation of the pathway intermediates does occur in vivo under different environmental conditions (Li et al., 2011; Rasulov et al., 2011, 2014; Li and Sharkey, 2013a), in dependence on leaf ontogeny (Rasulov et al., 2014), and across genotypes varying in constitutive activity of DXP synthase (Wright et al., 2014), we argue that accumulation of phosphorylated intermediates provides a key way for fine tuning of pathway flux by regulation of energy supply. Such a possibility for regulation of isoprene synthesis has been postulated (Niinemets et al., 1999; Rasulov et al., 2009b), but a physiological explanation has been lacking so far. In addition, the study also provides evidence of a certain cooperativity of the plastidic and cytosolic isoprenoid pathways. Under a strong metabolic pull when chloroplastic precursor pools are low, such as in growing leaves (Rasulov et al., 2014), and probably under severe environmental stress when synthesis of isoprene and larger carotenoids is most needed, a cytosolic supply of precursors could at least partly maintain the synthesis of the chloroplastic isoprenoids. We argue that use of bisphosphonate inhibitors can provide key insight into physiological controls on isoprene emission.

MATERIALS AND METHODS

Plant Material

The experiments were conducted with 1-year-old saplings of hybrid aspen (Populus tremula × Populus tremuloides) clone H200 grown in 4-L pots filled with sand and commercial potting soil under optimal supply of water and nutrients as in our previous studies (Rasulov et al., 2009b, 2011). The growth conditions were kept constant in a Percival plant growth chamber (CLF Plant Climatics GmbH). A quantum flux density of 650 μmol m⁻² s⁻¹ was provided for a 14-h photoperiod, air temperature was 25°С for day and 22°С for night, and air humidity was 65% to 70%. All experiments were carried out with fully mature 20- to 25-d-old leaves having maximum photosynthesis and isoprene emission rates. Detached leaves cut under water and fed with distilled water or with inhibitor solution through the petiole were used in all experiments. All experiments were replicated five times, and the data reported correspond to averages ± se.

Gas Exchange Measurements

Foliage net assimilation and transpiration rates were measured with an ultrafast gas exchange system that is particularly suitable for measurements of transient leaf physiological responses (system half time of approximately 0.15 s; Laisk et al., 2002; Rasulov et al., 2010). The system has a circular (cross-sectional area of 8.04 cm²) thermostatted clip on-type leaf cuvette. The upper leaf surface is glued to the leaf cuvette glass window by a starch paste to enhance the heat exchange, making it possible to maintain leaf temperature within 0.5°C of the temperature of the cuvette water jacket. The system has two identical gas lines where gas concentrations can be regulated independently. The lines can be switched between the sample and reference mode almost instantly (in less than 1 s) such that system delays due to stabilization of flows and gas concentrations are minimal. Synthetic air was prepared from purified gases using dynamic mixers, and the flow rate was fixed at 0.5 mmol s⁻¹ (Laisk et al., 2002). Measurement light was provided by a Schott KL 1500 halogen light source, and a heat-reflecting filter (Optical Coating Laboratory) was used to reduce the heat load on the sample leaf. CO₂ concentration was measured by an LI-6251 infrared gas analyzer (LI-Cor, Inc.), water vapor concentration by a custom-made micropsychrometer, and isoprene concentration (protonated parent ion with mass-to-charge ratio [m/z] of 69+) by PTR-MS (high-sensitivity version with a response time of approximately 0.1 s; Ionicon Analytik GmbH). Protonated ions corresponding to the main fragments of LOX pathway volatiles, hexenals (m/z = 81+), and other C6 volatiles (m/z = 83+, sum of hexenols, hexanal, and hexenyl acetates) and protonated parent ions corresponding to monoterpenes (m/z = 137+) were also recorded (Graus et al., 2004; Rasulov et al., 2014). A standard gas containing a spectrum of key volatiles (Ionimed GmbH) was used to calibrate PTR-MS.

The measurements were conducted at a leaf chamber CO₂ concentration of 360 μmol mol⁻¹, volumetric oxygen concentration of 21%, water vapor pressure deficit between the leaf and the atmosphere of 1.7 kPa, and leaf temperature of 30°С. After leaf enclosure, the leaf was stabilized at a quantum flux density of 650 μmol m⁻² s⁻¹ until net assimilation, transpiration, and isoprene emission rates reached a steady state. The steady-state values were recorded, light was switched off, and the kinetics of postillumination CO₂ and isoprene release were monitored until isoprene release reached the background level, typically in approximately 20 min in control leaves and 40 to 50 min in inhibitor-treated leaves (Fig. 1). At this time, light was switched on, and the steady-state conditions were established again. Isoprene dark decay kinetics were used to estimate the precursor pool sizes (DMADP pool size and dark pool size, see “Estimation of Isoprene Precursor Pools from Postillumination Isoprene Emission Decay Data”). Postillumination CO₂ release data were used to estimate the rate of postillumination CO₂ burst immediately after leaf darkening, and the rate of dark respiration after the dark CO₂ release had reached a steady state, in approximately 5 to 10 min since darkening. The postillumination CO₂ burst immediately after leaf darkening is primarily dependent on the rate of photorespiration (Peterson, 1983), but it also includes a component of mitochondrial respiration remaining in light and CO₂ uptake due to consumption of ribulose 1,5-bisphosphate formed in light (Sharkey, 1988).

Measurements of Chlorophyll Fluorescence and Initial Quantum Yields

The effective (light-adapted) quantum yield of PSII was determined with a PAM 101 fluorimeter (Walz GmbH) operated at 100 kHz. When the gas-exchange rates in light had reached a steady state, the steady-state fluorescence yield, F, was recorded and a saturated pulse of white light of 14,000 μmol m⁻² s⁻¹ for 2 s (provided by another Schott KL 1500 light source) was given to measure the maximum fluorescence yield (F_m′). The effective quantum yield of PSII was computed as (F_m′ − F)/F_m′.

Measurements of net assimilation and isoprene emission rates over the light range of 15 to 55 μmol m⁻² s⁻¹ were used to estimate initial quantum yields for CO₂ exchange and isoprene emission. Leaf absorptance was measured by an Ulbricht-type integrating sphere, and the quantum yields were calculated for an absorbed light basis.

Inhibitor Treatments

Two bisphosphonate inhibitors, alendronate (Fosamax; Merck Sharp and Dohme Corp.) and zoledronate (Zometa; Novartis International AG), were used to inhibit isoprenoid synthesis downstream of the immediate isoprene substrate, DMADP. The inhibitor concentrations in aqueous solution were 20 mm for alendronate, as in our previous study (Rasulov et al., 2014), and 74 μm for zoledronate. These concentrations were selected based on the evidence that almost full inhibition of GDP and GGDP synthase by alendronate is achieved by concentrations of this inhibitor of 10 to 20 mm (Lange et al., 2001), whereas analogous inhibition by zoledronate is achieved by concentrations of 25 to 100 μm (Henneman et al., 2011; Wasko, 2011). Experiments were carried out with different inhibitor application times (Fig. 2), and ultimately, a feeding time of 40 min that resulted in a quasi-stable isoprene emission rate (more than 90% of full response) was used, except when noted.

Fosmidomycin (Invitrogen), a known inhibitor of DXP reductoisomerase (Kuzuyama et al., 1998; Zeidler et al., 1998), was used at a concentration of 25 μm (Rasulov et al., 2011) to inhibit the chloroplastic DXP/MEP pathway upstream of DMADP. A feeding time of 30 min that resulted in more than 95% inhibition of isoprene emission was used (Fig. 4A).

In all cases, the experiments were started with noninhibited leaves fed with distilled water from a calibrated vial. After recording the steady-state values of photosynthetic and isoprene emission characteristics in light, the postillumination kinetics were measured, light was switched on again, and steady-state values were established again. After reaching the new steady state, an equal amount of inhibitor solution with twice higher than the target concentration was added to the vial with distilled water. The inhibitor was added with a syringe, ensuring turbulent mixing of the two solutions such that the required inhibitor concentration was established almost instantly. After the required period of feeding in light, postillumination isoprene release kinetics were measured again (Fig. 1).

In the case of double inhibitor treatments, bisphosphonates applied first followed by fosmidomycin, and fosmidomycin applied first followed by bisphosphonates, the second inhibitor was prepared in a separate vial, and the inhibitor change was achieved by rapidly switching the vials with two inhibitor solutions.

GC-MS Analyses

A light-dark transient is characteristically associated with an emission burst of LOX pathway products, in particular, emissions of C6 aldehydes (Graus et al., 2004). In addition, several C5 LOX products are emitted during some stresses such as wounding and leaf drying (Fall et al., 1999, 2001; Brilli et al., 2011). Parent ions of several C5 volatiles such as 1-penten-3-ol, 3-methylbutanal, and 2-methylbutanal have an m/z of 87+, but after water abstraction, they produce fragments with m/z of 69+ and are indistinguishable from isoprene in PTR-MS measurements. Although light-dark changes have not been associated with emissions of C5 LOX products (Graus et al., 2004), a previous study demonstrated that a light-dark-induced LOX emission burst is enhanced in bisphosphonate-inhibited leaves (Rasulov et al., 2014). To gain insight into the nature of dark LOX emission bursts in control, bisphosphonate-, and fosmidomycin-inhibited leaves, volatiles during light-dark transient were collected on multibed cartridges and analyzed with a combined Shimadzu TD20 automated cartridge desorber and Shimadzu 2010 plus GC-MS instrument according to the method of Kännaste et al. (2014).

We confirmed that bisphosphonate inhibition resulted in a more pronounced dark release of LOX volatiles. In noninhibited leaves, dark-induced LOX volatile burst continued for approximately 10 min, whereas the burst continued for approximately 20 min in bisphosphonate-inhibited leaves (Fig. 10A). GC-MS analyses further corroborated the enhancement of LOX emissions after bisphosphonate inhibition (Fig. 10B). The LOX emissions were dominated by 5-hexen-1-ol (on average [±se] 17% ± 5% of total LOX emissions) and 2-ethyl-1-hexanol (81% ± 6%) in both control and bisphosphonate-inhibited leaves. In addition, 3-Z-hexen-1-ol, 5-methyl-3-hexen-2-one, and 1-hepten-3-one were found in minor proportions. LOX emissions in fosmidomycin-inhibited leaves were similar to those in the noninhibited leaves. Possible monoterpene and C5 volatile emissions were below the detection limit. Thus, this analysis indicates that enhanced LOX emissions did not interfere with isoprene emission measurements with PTR-MS through all the treatments. In addition, this analysis also confirms an enhancement of the isoprene precursor dark pool in bisphosphonate-inhibited leaves (Fig. 10B; Tables I and II).

Estimation of Isoprene Precursor Pools from Postillumination Isoprene Emission Decay Data

The biphasic dark-release kinetics of isoprene emission (Fig. 1) were used to estimate the sizes of intermediate pools of chloroplastic isoprenoid synthesis (Rasulov et al., 2009a, 2011, 2014; Li et al., 2011; Li and Sharkey, 2013a). The first pool, defined as the DMADP pool, was obtained by integrating the initial rapid decay 200 to 400 s after darkening. It provides the immediate pool of isoprene substrate (mainly DMADP and to some extent IDP that is rapidly converted to DMADP) that was present prior to darkening (Rasulov et al., 2009a; Weise et al., 2013). In the darkness, a secondary process is activated providing DMADP that results in the rise of isoprene emission between approximately 400 and 1,200 s after darkening (Li et al., 2011; Rasulov et al., 2011, 2014; Li and Sharkey, 2013a). The amount of DMADP formed during the secondary isoprene burst was defined as the dark pool (Li et al., 2011; Rasulov et al., 2011, 2014; Li and Sharkey, 2013a).

Determination of in Vivo Isoprene Synthase Kinetic Characteristics

Data on fast postillumination decay of isoprene emission were used to estimate in vivo values of the K_m and V_max of isoprene synthase and isoprene synthase rate constant, as in Rasulov et al. (2011, 2014, 2015). Isoprene emission rate was integrated over different time periods since darkening, yielding paired values of isoprene emission rate at time t, I(t), and remaining DMADP pool size that supported the given rate of isoprene emission, C_DMADP(t). The isoprene synthase rate constant was given as the initial slope of I(t) versus C_DMADP(t), whereas in vivo K_m and V_max were calculated from the Hanes-Woolf plots (Fig. 3B).

Quantitative Separation of Fluxes Due to Different Substrate Sources in Inhibited Leaves

In chloroplasts, carbon entering into the isoprenoid synthesis pathway can be immediately converted to DMADP and IDP and used for synthesis of isoprene (C5) and larger isoprenoids (C10 and C20). However, a part of the carbon entering into the pathway can also accumulate in the form of phosphorylated intermediates such as ME-cDP (Li and Sharkey, 2013a; Wright et al., 2014). In addition, there can be a certain exchange of intermediates with cytosolic MVA-dependent isoprenoid synthesis pathway at the level of IDP. Fosmidomycin inhibits DXP reductoisomerase, and thus, blocks the initial step of the DXP/MEP pathway, whereas bisphosphonates block the chloroplastic consumption of DMADP and IDP for formation of isoprenoids other than isoprene. They also wholly inhibit the cytosolic isoprenoid synthesis (see introduction). Application of inhibitors is associated with changes in substrate pool sizes and isoprene and larger isoprenoid synthesis rates until a new steady state is reached. We have used experimentally estimated DMADP and dark pool sizes before application of inhibitors and after the application, the estimated kinetic characteristics of isoprene synthase (K_m and V_max) in inhibited and noninhibited leaves (Tables I and II), and isoprene emission rate before and through the inhibitor treatment to infer changes in the flux distribution for DMADP formation and isoprene emission, ME-cDP accumulation, larger isoprenoid synthesis, and cytosolic contribution of IDP. A set of differential equations describing changes in pool sizes and emission rates was constructed and solved iteratively as in Rasulov et al. (2014). In these calculations, we have used an effective in vivo K_m for larger chloroplastic isoprenoid synthesis of 260 nmol m⁻² estimated for hybrid aspen (Rasulov et al., 2014). We further assumed that the rate of synthesis of isoprenoids other than isoprene in noninhibited mature leaves is 1 nmol m⁻² s⁻¹ in C5 equivalents (Rasulov et al., 2014).

Acknowledgments

We thank Thomas D. Sharkey for insightful comments on the article and Kaia Kask for help with GC-MS analyses.

Glossary

MVA: mevalonate
DXP: 1-deoxy-d-xylulose 5-phosphate
MEP: 2-C-methyl-d-erythritol 4-phosphate
DMADP: dimethylallyl diphosphate
IDP: isopentenyl diphosphate
ME-cDP: 2-C-methyl-d-erythritol 2,4-cyclodiphosphate
FDP: farnesyl diphosphate
GDP: geranyl diphosphate
GGDP: geranylgeranyl diphosphate
Fd: ferredoxin
HMBDP: 4-hydroxy-3-methyl-2-(E)-butenyl-diphosphate
HDS: 4-hydroxy-3-methyl-2-(E)-butenyl-diphosphate synthase
HDR: 4-hydroxy-3-methyl-2-(E)-butenyl-diphosphate reductase
TDP: thiamin diphosphate
IDI: isopentenyl diphosphate isomerase
LOX: lipoxygenase
PTR: proton transfer reaction
PTR-MS: proton transfer reaction-mass spectrometry
GC-MS: gas chromatography-mass spectrometry
m/z: mass-to-charge ratio

Footnotes

This work was supported by the Estonian Ministry of Science and Education (institutional grant no. IUT–8–3), the Estonian Science Foundation (grant no. 9253), the European Commission through the European Regional Fund (the Center of Excellence in Environmental Adaptation), and the European Research Council (advanced grant no. 322603, Stress-Induced Plant Volatiles in Biosphere-Atmosphere System).

References

Allen JF. (2003) Cyclic, pseudocyclic and noncyclic photophosphorylation: new links in the chain. Trends Plant Sci 8: 15–19 [DOI] [PubMed] [Google Scholar]
Banerjee A, Wu Y, Banerjee R, Li Y, Yan H, Sharkey TD (2013) Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway. J Biol Chem 288: 16926–16936 [DOI] [PMC free article] [PubMed] [Google Scholar]
Bergstrom JD, Bostedor RG, Masarachia PJ, Reszka AA, Rodan G (2000) Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch Biochem Biophys 373: 231–241 [DOI] [PubMed] [Google Scholar]
Bick JA, Lange BM (2003) Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: unidirectional transport of intermediates across the chloroplast envelope membrane. Arch Biochem Biophys 415: 146–154 [DOI] [PubMed] [Google Scholar]
Bojko M, Więckowski S (1999) Effect of pyrophosphate on the ferredoxin:NADP+ oxidoreductase activity in vitro. In G Garab, ed, Photosynthesis: mechanisms and effects. Proceedings of the XIth International Congress on Photosynthesis, Budapest, Hungary, August 17–22, Vol I. Kluwer, Dordrecht, pp 667–670 [Google Scholar]
Brilli F, Ruuskanen TM, Schnitzhofer R, Müller M, Breitenlechner M, Bittner V, Wohlfahrt G, Loreto F, Hansel A (2011) Detection of plant volatiles after leaf wounding and darkening by proton transfer reaction “time-of-flight” mass spectrometry (PTR-TOF). PLoS ONE 6: e20419. [DOI] [PMC free article] [PubMed] [Google Scholar]
Brüggemann N, Schnitzler JP (2002a) Diurnal variation of dimethylallyl diphosphate concentrations in oak (Quercus robur) leaves. Physiol Plant 115: 190–196 [DOI] [PubMed] [Google Scholar]
Brüggemann N, Schnitzler JP (2002b) Relationship of isopentenyl diphosphate (IDP) isomerase activity to isoprene emission of oak leaves. Tree Physiol 22: 1011–1018 [DOI] [PubMed] [Google Scholar]
Burke C, Klettke K, Croteau R (2004) Heteromeric geranyl diphosphate synthase from mint: construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs. Arch Biochem Biophys 422: 52–60 [DOI] [PubMed] [Google Scholar]
Cromartie TH, Fisher KJ, Grossman JN (1999) The discovery of a novel site of action for herbicidal bisphosphonates. Pestic Biochem Physiol 63: 114–126 [Google Scholar]
De-Eknamkul W, Potduang B (2003) Biosynthesis of β-sitosterol and stigmasterol in Croton sublyratus proceeds via a mixed origin of isoprene units. Phytochemistry 62: 389–398 [DOI] [PubMed] [Google Scholar]
Eisenreich W, Rohdich F, Bacher A (2001) Deoxyxylulose phosphate pathway to terpenoids. Trends Plant Sci 6: 78–84 [DOI] [PubMed] [Google Scholar]
Erixon KM, Dabalos CL, Leeper FJ (2008) Synthesis and biological evaluation of pyrophosphate mimics of thiamine pyrophosphate based on a triazole scaffold. Org Biomol Chem 6: 3561–3572 [DOI] [PubMed] [Google Scholar]
Fall R, Karl T, Hansel A, Jordan A, Lindinger W (1999) Volatile organic compounds emitted after leaf wounding: on-line analysis by proton-transfer-reaction mass spectrometry. J Geophys Res 104: 15963–15974 [Google Scholar]
Fall R, Karl T, Jordan A, Lindinger W (2001) Biogenic C5 VOCs: release from leaves after freeze-thaw wounding and occurrence in air at a high mountain observatory. Atmos Environ 35: 3905–3916 [Google Scholar]
Forti G, Meyer EM (1969) Effect of pyrophosphate on photosynthetic electron transport reactions. Plant Physiol 44: 1511–1514 [DOI] [PMC free article] [PubMed] [Google Scholar]
Foyer CH, Neukermans J, Queval G, Noctor G, Harbinson J (2012) Photosynthetic control of electron transport and the regulation of gene expression. J Exp Bot 63: 1637–1661 [DOI] [PubMed] [Google Scholar]
Gershenzon J, Croteau RB (1993) Terpenoid biosynthesis: the basic pathway and formation of monoterpenes, sesquiterpenes, and diterpenes. In Moore TS, ed, Lipid metabolism in plants. CRC Press, Boca Raton - Ann Arbor - London -Tokyo, pp 339-388 [Google Scholar]
Ghirardo A, Wright LP, Bi Z, Rosenkranz M, Pulido P, Rodríguez-Concepción M, Niinemets Ü, Brüggemann N, Gershenzon J, Schnitzler JP (2014) Metabolic flux analysis of plastidic isoprenoid biosynthesis in poplar leaves emitting and nonemitting isoprene. Plant Physiol 165: 37–51 [DOI] [PMC free article] [PubMed] [Google Scholar]
Graus M, Schnitzler JP, Hansel A, Cojocariu C, Rennenberg H, Wisthaler A, Kreuzwieser J (2004) Transient release of oxygenated volatile organic compounds during light-dark transitions in grey poplar leaves. Plant Physiol 135: 1967–1975 [DOI] [PMC free article] [PubMed] [Google Scholar]
Hemmerlin A, Hoeffler JF, Meyer O, Tritsch D, Kagan IA, Grosdemange-Billiard C, Rohmer M, Bach TJ (2003) Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells. J Biol Chem 278: 26666–26676 [DOI] [PubMed] [Google Scholar]
Henkes S, Sonnewald U, Badur R, Flachmann R, Stitt M (2001) A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. Plant Cell 13: 535–551 [DOI] [PMC free article] [PubMed] [Google Scholar]
Henneman L, van Cruchten AG, Kulik W, Waterham HR (2011) Inhibition of the isoprenoid biosynthesis pathway: detection of intermediates by UPLC-MS/MS. Biochim Biophys Acta 1811: 227–233 [DOI] [PubMed] [Google Scholar]
Holloway PW, Popják G (1968) Isopentenyl pyrophosphate isomerase from liver. Biochem J 106: 835–840 [DOI] [PMC free article] [PubMed] [Google Scholar]
Jahnke W, Rondeau JM, Cotesta S, Marzinzik A, Pellé X, Geiser M, Strauss A, Götte M, Bitsch F, Hemmig R, et al. (2010) Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery. Nat Chem Biol 6: 660–666 [DOI] [PubMed] [Google Scholar]
Jomaa H, Wiesner J, Sanderbrand S, Altincicek B, Weidemeyer C, Hintz M, Türbachova I, Eberl M, Zeidler J, Lichtenthaler HK. , et al. (1999) Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285: 1573–1576 [DOI] [PubMed] [Google Scholar]
Jux A, Gleixner G, Boland W (2001) Classification of terpenoids according to the methylerythritolphosphate or the mevalonate pathway with natural ¹²C/¹³C isotope ratios: dynamic allocation of resources in induced plants. Angew Chem Int Ed Engl 40: 2091–2094 [DOI] [PubMed] [Google Scholar]
Kännaste A, Copolovici L, Niinemets Ü (2014) Gas chromatography mass-spectrometry method for determination of biogenic volatile organic compounds emitted by plants. In Rodríguez-Concepción M, ed, Plant isoprenoids: methods and protocols, Vol 1153 Humana Press, New York, pp 161-169 [DOI] [PubMed] [Google Scholar]
Kloer DP, Welsch R, Beyer P, Schulz GE (2006) Structure and reaction geometry of geranylgeranyl diphosphate synthase from Sinapis alba. Biochemistry 45: 15197–15204 [DOI] [PubMed] [Google Scholar]
Köksal M, Zimmer I, Schnitzler JP, Christianson DW (2010) Structure of isoprene synthase illuminates the chemical mechanism of teragram atmospheric carbon emission. J Mol Biol 402: 363–373 [DOI] [PMC free article] [PubMed] [Google Scholar]
Kuzuyama T, Shimizu T, Takashashi S, Seto H (1998) Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis. Tetrahedron Lett 39: 7913–7916 [Google Scholar]
Laisk A, Oja V, Rasulov B, Rämma H, Eichelmann H, Kasparova I, Pettai H, Padu E, Vapaavuori E (2002) A computer-operated routine of gas exchange and optical measurements to diagnose photosynthetic apparatus in leaves. Plant Cell Environ 25: 923–943 [Google Scholar]
Lange BM, Ketchum REB, Croteau RB (2001) Isoprenoid biosynthesis: metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis. Plant Physiol 127: 305–314 [DOI] [PMC free article] [PubMed] [Google Scholar]
Laule O, Fürholz A, Chang HS, Zhu T, Wang X, Heifetz PB, Gruissem W, Lange M (2003) Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana. Proc Natl Acad Sci USA 100: 6866–6871 [DOI] [PMC free article] [PubMed] [Google Scholar]
Li Z, Ratliff EA, Sharkey TD (2011) Effect of temperature on postillumination isoprene emission in oak and poplar. Plant Physiol 155: 1037–1046 [DOI] [PMC free article] [PubMed] [Google Scholar]
Li Z, Sharkey TD (2013a) Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illumination isoprene burst from leaves. Plant Cell Environ 36: 429–437 [DOI] [PubMed] [Google Scholar]
Li Z, Sharkey TD (2013b) Molecular and pathway controls on biogenic volatile organic compound emissions. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 119–151 [Google Scholar]
Lindert S, Zhu W, Liu YL, Pang R, Oldfield E, McCammon JA (2013) Farnesyl diphosphate synthase inhibitors from in silico screening. Chem Biol Drug Des 81: 742–748 [DOI] [PMC free article] [PubMed] [Google Scholar]
Liu YL, Lindert S, Zhu W, Wang K, McCammon JA, Oldfield E (2014) Taxodione and arenarone inhibit farnesyl diphosphate synthase by binding to the isopentenyl diphosphate site. Proc Natl Acad Sci USA 111: E2530–E2539 [DOI] [PMC free article] [PubMed] [Google Scholar]
Loreto F, Pinelli P, Brancaleoni E, Ciccioli P (2004) ¹³C labeling reveals chloroplastic and extrachloroplastic pools of dimethylallyl pyrophosphate and their contribution to isoprene formation. Plant Physiol 135: 1903–1907 [DOI] [PMC free article] [PubMed] [Google Scholar]
Loreto F, Schnitzler JP (2010) Abiotic stresses and induced BVOCs. Trends Plant Sci 15: 154–166 [DOI] [PubMed] [Google Scholar]
Loreto F, Velikova V (2001) Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol 127: 1781–1787 [PMC free article] [PubMed] [Google Scholar]
Mansouri H, Salari F (2014) Influence of mevinolin on chloroplast terpenoids in Cannabis sativa. Physiol Mol Biol Plants 20: 273–277 [DOI] [PMC free article] [PubMed] [Google Scholar]
Nakamura A, Shimada H, Masuda T, Ohta H, Takamiya K (2001) Two distinct isopentenyl diphosphate isomerases in cytosol and plastid are differentially induced by environmental stresses in tobacco. FEBS Lett 506: 61–64 [DOI] [PubMed] [Google Scholar]
Niinemets U. (2010) Mild versus severe stress and BVOCs: thresholds, priming and consequences. Trends Plant Sci 15: 145–153 [DOI] [PubMed] [Google Scholar]
Niinemets Ü, Tenhunen JD, Harley PC, Steinbrecher R (1999) A model of isoprene emission based on energetic requirements for isoprene synthesis and leaf photosynthetic properties for Liquidambar and Quercus. Plant Cell Environ 22: 1319–1335 [Google Scholar]
No JH, de Macedo Dossin F, Zhang Y, Liu YL, Zhu W, Feng X, Yoo JAY, Lee E, Wang K, Hui R. , et al. (2012) Lipophilic analogs of zoledronate and risedronate inhibit Plasmodium geranylgeranyl diphosphate synthase (GGPPS) and exhibit potent antimalarial activity. Proc Natl Acad Sci USA 109: 4058–4063 [DOI] [PMC free article] [PubMed] [Google Scholar]
Oberhauser V, Gaudin J, Fonné-Pfister R, Schär HP (1998) New target enzyme(s) for bisphosphonates: inhibition of geranylgeranyl diphosphate synthase. Pestic Biochem Physiol 60: 111–117 [Google Scholar]
Page JE, Hause G, Raschke M, Gao W, Schmidt J, Zenk MH, Kutchan TM (2004) Functional analysis of the final steps of the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway to isoprenoids in plants using virus-induced gene silencing. Plant Physiol 134: 1401–1413 [DOI] [PMC free article] [PubMed] [Google Scholar]
Peterson RB. (1983) Estimation of photorespiration based on the initial rate of postillumination CO₂ release: II. Effects of O₂, CO₂, and temperature. Plant Physiol 73: 983–988 [DOI] [PMC free article] [PubMed] [Google Scholar]
Piel J, Donath J, Bandemer K, Boland W (1998) Mevalonate-independent biosynthesis of terpenoid volatiles in plants: induced and constitutive emission of volatiles. Angew Chem Int Ed 37: 2478–2481 [DOI] [PubMed] [Google Scholar]
Possell M, Loreto F (2013) The role of volatile organic compounds in plant resistance to abiotic stresses: responses and mechanisms. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 209-235 [Google Scholar]
Rajabi Memari H, Pazouki L, Niinemets Ü (2013) The biochemistry and molecular biology of volatile messengers in trees. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 47-93 [Google Scholar]
Ramos-Valdivia AC, van der Heijden R, Verpoorte R (1997) Isopentenyl diphosphate isomerase: a core enzyme in isoprenoid biosynthesis. A review of its biochemistry and function. Nat Prod Rep 14: 591–603 [DOI] [PubMed] [Google Scholar]
Rasulov B, Bichele I, Hüve K, Vislap V, Niinemets Ü (2015) Acclimation of isoprene emission and photosynthesis to growth temperature in hybrid aspen: resolving structural and physiological controls. Plant Cell Environ 38: 751–766 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rasulov B, Bichele I, Laisk A, Niinemets Ü (2014) Competition between isoprene emission and pigment synthesis during leaf development in aspen. Plant Cell Environ 37: 724–741 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rasulov B, Copolovici L, Laisk A, Niinemets Ü (2009a) Postillumination isoprene emission: in vivo measurements of dimethylallyldiphosphate pool size and isoprene synthase kinetics in aspen leaves. Plant Physiol 149: 1609–1618 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rasulov B, Hüve K, Bichele I, Laisk A, Niinemets Ü (2010) Temperature response of isoprene emission in vivo reflects a combined effect of substrate limitations and isoprene synthase activity: a kinetic analysis. Plant Physiol 154: 1558–1570 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rasulov B, Hüve K, Laisk A, Niinemets Ü (2011) Induction of a longer term component of isoprene release in darkened aspen leaves: origin and regulation under different environmental conditions. Plant Physiol 156: 816–831 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rasulov B, Hüve K, Välbe M, Laisk A, Niinemets Ü (2009b) Evidence that light, carbon dioxide, and oxygen dependencies of leaf isoprene emission are driven by energy status in hybrid aspen. Plant Physiol 151: 448–460 [DOI] [PMC free article] [PubMed] [Google Scholar]
Roberts SC. (2007) Production and engineering of terpenoids in plant cell culture. Nat Chem Biol 3: 387–395 [DOI] [PubMed] [Google Scholar]
Rodríguez-Concepción M. (2006) Early steps in isoprenoid biosynthesis: multilevel regulation of the supply of common precursors in plant cells. Phytochem Rev 5: 1–15 [Google Scholar]
Rodríguez-Concepción M, Boronat A (2002) Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids: a metabolic milestone achieved through genomics. Plant Physiol 130: 1079–1089 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rodríguez-Concepción M, Forés O, Martinez-García JF, González V, Phillips MA, Ferrer A, Boronat A (2004) Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. Plant Cell 16: 144–156 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rohdich F, Zepeck F, Adam P, Hecht S, Kaiser J, Laupitz R, Gräwert T, Amslinger S, Eisenreich W, Bacher A. , et al. (2003) The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. Proc Natl Acad Sci USA 100: 1586–1591 [DOI] [PMC free article] [PubMed] [Google Scholar]
Rohmer M, Seemann M, Horbach S, Bringer-Meyer S, Sahm H (1996) Glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. J Am Chem Soc 118: 2564–2566 [Google Scholar]
Rosenkranz M, Schnitzler JP (2013) Genetic engineering of BVOC emissions from trees. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 95-118 [Google Scholar]
Russell RGG, Watts NB, Ebetino FH, Rogers MJ (2008) Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos Int 19: 733–759 [DOI] [PubMed] [Google Scholar]
Schwender J, Gemünden C, Lichtenthaler HK (2001) Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids. Planta 212: 416–423 [DOI] [PubMed] [Google Scholar]
Seemann M, Tse Sum Bui B, Wolff M, Miginiac-Maslow M, Rohmer M (2006) Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG. FEBS Lett 580: 1547–1552 [DOI] [PubMed] [Google Scholar]
Sharkey TD. (1988) Estimating the rate of photorespiration in leaves. Physiol Plant 73: 147–152 [Google Scholar]
Sharkey TD, Chen X, Yeh S (2001) Isoprene increases thermotolerance of fosmidomycin-fed leaves. Plant Physiol 125: 2001–2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
Sharkey TD, Gray DW, Pell HK, Breneman SR, Topper L (2013) Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family. Evolution 67: 1026–1040 [DOI] [PubMed] [Google Scholar]
Smith JM, Warrington NV, Vierling RJ, Kuhn ML, Anderson WF, Koppisch AT, Freel Meyers CL (2014) Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate. J Antibiot (Tokyo) 67: 77–83 [DOI] [PMC free article] [PubMed] [Google Scholar]
Szabo CM, Matsumura Y, Fukura S, Martin MB, Sanders JM, Sengupta S, Cieslak JA, Loftus TC, Lea CR, Lee HJ, et al. (2002) Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and diphosphates: a potential route to new bone antiresorption and antiparasitic agents. J Med Chem 45: 2185–2196 [DOI] [PubMed] [Google Scholar]
van Beek E, Pieterman E, Cohen L, Löwik C, Papapoulos S (1999) Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo. Biochem Biophys Res Commun 255: 491–494 [DOI] [PubMed] [Google Scholar]
Wasko BM. (2011) Novel bisphosphonates as inhibitors of isoprenoid biosynthesis. PhD. University of Iowa, Graduate College, Iowa City, Iowa [Google Scholar]
Weise SE, Li Z, Sutter AE, Corrion A, Banerjee A, Sharkey TD (2013) Measuring dimethylallyl diphosphate available for isoprene synthesis. Anal Biochem 435: 27–34 [DOI] [PubMed] [Google Scholar]
Wright LP, Rohwer JM, Ghirardo A, Hammerbacher A, Ortiz-Alcaide M, Raguschke B, Schnitzler JP, Gershenzon J, Phillips MA (2014) Deoxyxylulose 5-phosphate synthase controls flux through the methylerythritol 4-phosphate pathway in Arabidopsis. Plant Physiol 165: 1488–1504 [DOI] [PMC free article] [PubMed] [Google Scholar]
Xiao Y, Savchenko T, Baidoo EEK, Chehab WE, Hayden DM, Tolstikov V, Corwin JA, Kliebenstein DJ, Keasling JD, Dehesh K (2012) Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes. Cell 149: 1525–1535 [DOI] [PubMed] [Google Scholar]
Zeidler JG, Schwender J, Müller C, Wiesner J, Weidemeyer C, Beck E, Jomaa H, Lichtenthaler HK (1998) Inhibition of the non-mevalonate 1-deoxy-d-xylulose-5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Zeitschrift für Naturforschung, Section C. 53c: 980–986 [Google Scholar]
Zhou C, Li Z, Wiberley-Bradford AE, Weise SE, Sharkey TD (2013) Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase. Anal Biochem 440: 130–136 [DOI] [PubMed] [Google Scholar]

[bib1] Allen JF. (2003) Cyclic, pseudocyclic and noncyclic photophosphorylation: new links in the chain. Trends Plant Sci 8: 15–19 [DOI] [PubMed] [Google Scholar]

[bib2] Banerjee A, Wu Y, Banerjee R, Li Y, Yan H, Sharkey TD (2013) Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway. J Biol Chem 288: 16926–16936 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] Bergstrom JD, Bostedor RG, Masarachia PJ, Reszka AA, Rodan G (2000) Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch Biochem Biophys 373: 231–241 [DOI] [PubMed] [Google Scholar]

[bib4] Bick JA, Lange BM (2003) Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: unidirectional transport of intermediates across the chloroplast envelope membrane. Arch Biochem Biophys 415: 146–154 [DOI] [PubMed] [Google Scholar]

[bib5] Bojko M, Więckowski S (1999) Effect of pyrophosphate on the ferredoxin:NADP+ oxidoreductase activity in vitro. In G Garab, ed, Photosynthesis: mechanisms and effects. Proceedings of the XIth International Congress on Photosynthesis, Budapest, Hungary, August 17–22, Vol I. Kluwer, Dordrecht, pp 667–670 [Google Scholar]

[bib6] Brilli F, Ruuskanen TM, Schnitzhofer R, Müller M, Breitenlechner M, Bittner V, Wohlfahrt G, Loreto F, Hansel A (2011) Detection of plant volatiles after leaf wounding and darkening by proton transfer reaction “time-of-flight” mass spectrometry (PTR-TOF). PLoS ONE 6: e20419. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] Brüggemann N, Schnitzler JP (2002a) Diurnal variation of dimethylallyl diphosphate concentrations in oak (Quercus robur) leaves. Physiol Plant 115: 190–196 [DOI] [PubMed] [Google Scholar]

[bib8] Brüggemann N, Schnitzler JP (2002b) Relationship of isopentenyl diphosphate (IDP) isomerase activity to isoprene emission of oak leaves. Tree Physiol 22: 1011–1018 [DOI] [PubMed] [Google Scholar]

[bib9] Burke C, Klettke K, Croteau R (2004) Heteromeric geranyl diphosphate synthase from mint: construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs. Arch Biochem Biophys 422: 52–60 [DOI] [PubMed] [Google Scholar]

[bib10] Cromartie TH, Fisher KJ, Grossman JN (1999) The discovery of a novel site of action for herbicidal bisphosphonates. Pestic Biochem Physiol 63: 114–126 [Google Scholar]

[bib11] De-Eknamkul W, Potduang B (2003) Biosynthesis of β-sitosterol and stigmasterol in Croton sublyratus proceeds via a mixed origin of isoprene units. Phytochemistry 62: 389–398 [DOI] [PubMed] [Google Scholar]

[bib12] Eisenreich W, Rohdich F, Bacher A (2001) Deoxyxylulose phosphate pathway to terpenoids. Trends Plant Sci 6: 78–84 [DOI] [PubMed] [Google Scholar]

[bib13] Erixon KM, Dabalos CL, Leeper FJ (2008) Synthesis and biological evaluation of pyrophosphate mimics of thiamine pyrophosphate based on a triazole scaffold. Org Biomol Chem 6: 3561–3572 [DOI] [PubMed] [Google Scholar]

[bib14] Fall R, Karl T, Hansel A, Jordan A, Lindinger W (1999) Volatile organic compounds emitted after leaf wounding: on-line analysis by proton-transfer-reaction mass spectrometry. J Geophys Res 104: 15963–15974 [Google Scholar]

[bib15] Fall R, Karl T, Jordan A, Lindinger W (2001) Biogenic C5 VOCs: release from leaves after freeze-thaw wounding and occurrence in air at a high mountain observatory. Atmos Environ 35: 3905–3916 [Google Scholar]

[bib16] Forti G, Meyer EM (1969) Effect of pyrophosphate on photosynthetic electron transport reactions. Plant Physiol 44: 1511–1514 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib17] Foyer CH, Neukermans J, Queval G, Noctor G, Harbinson J (2012) Photosynthetic control of electron transport and the regulation of gene expression. J Exp Bot 63: 1637–1661 [DOI] [PubMed] [Google Scholar]

[bib18] Gershenzon J, Croteau RB (1993) Terpenoid biosynthesis: the basic pathway and formation of monoterpenes, sesquiterpenes, and diterpenes. In Moore TS, ed, Lipid metabolism in plants. CRC Press, Boca Raton - Ann Arbor - London -Tokyo, pp 339-388 [Google Scholar]

[bib19] Ghirardo A, Wright LP, Bi Z, Rosenkranz M, Pulido P, Rodríguez-Concepción M, Niinemets Ü, Brüggemann N, Gershenzon J, Schnitzler JP (2014) Metabolic flux analysis of plastidic isoprenoid biosynthesis in poplar leaves emitting and nonemitting isoprene. Plant Physiol 165: 37–51 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] Graus M, Schnitzler JP, Hansel A, Cojocariu C, Rennenberg H, Wisthaler A, Kreuzwieser J (2004) Transient release of oxygenated volatile organic compounds during light-dark transitions in grey poplar leaves. Plant Physiol 135: 1967–1975 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] Hemmerlin A, Hoeffler JF, Meyer O, Tritsch D, Kagan IA, Grosdemange-Billiard C, Rohmer M, Bach TJ (2003) Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells. J Biol Chem 278: 26666–26676 [DOI] [PubMed] [Google Scholar]

[bib22] Henkes S, Sonnewald U, Badur R, Flachmann R, Stitt M (2001) A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. Plant Cell 13: 535–551 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] Henneman L, van Cruchten AG, Kulik W, Waterham HR (2011) Inhibition of the isoprenoid biosynthesis pathway: detection of intermediates by UPLC-MS/MS. Biochim Biophys Acta 1811: 227–233 [DOI] [PubMed] [Google Scholar]

[bib24] Holloway PW, Popják G (1968) Isopentenyl pyrophosphate isomerase from liver. Biochem J 106: 835–840 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] Jahnke W, Rondeau JM, Cotesta S, Marzinzik A, Pellé X, Geiser M, Strauss A, Götte M, Bitsch F, Hemmig R, et al. (2010) Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery. Nat Chem Biol 6: 660–666 [DOI] [PubMed] [Google Scholar]

[bib26] Jomaa H, Wiesner J, Sanderbrand S, Altincicek B, Weidemeyer C, Hintz M, Türbachova I, Eberl M, Zeidler J, Lichtenthaler HK. , et al. (1999) Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285: 1573–1576 [DOI] [PubMed] [Google Scholar]

[bib27] Jux A, Gleixner G, Boland W (2001) Classification of terpenoids according to the methylerythritolphosphate or the mevalonate pathway with natural ¹²C/¹³C isotope ratios: dynamic allocation of resources in induced plants. Angew Chem Int Ed Engl 40: 2091–2094 [DOI] [PubMed] [Google Scholar]

[bib28] Kännaste A, Copolovici L, Niinemets Ü (2014) Gas chromatography mass-spectrometry method for determination of biogenic volatile organic compounds emitted by plants. In Rodríguez-Concepción M, ed, Plant isoprenoids: methods and protocols, Vol 1153 Humana Press, New York, pp 161-169 [DOI] [PubMed] [Google Scholar]

[bib29] Kloer DP, Welsch R, Beyer P, Schulz GE (2006) Structure and reaction geometry of geranylgeranyl diphosphate synthase from Sinapis alba. Biochemistry 45: 15197–15204 [DOI] [PubMed] [Google Scholar]

[bib30] Köksal M, Zimmer I, Schnitzler JP, Christianson DW (2010) Structure of isoprene synthase illuminates the chemical mechanism of teragram atmospheric carbon emission. J Mol Biol 402: 363–373 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] Kuzuyama T, Shimizu T, Takashashi S, Seto H (1998) Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis. Tetrahedron Lett 39: 7913–7916 [Google Scholar]

[bib32] Laisk A, Oja V, Rasulov B, Rämma H, Eichelmann H, Kasparova I, Pettai H, Padu E, Vapaavuori E (2002) A computer-operated routine of gas exchange and optical measurements to diagnose photosynthetic apparatus in leaves. Plant Cell Environ 25: 923–943 [Google Scholar]

[bib33] Lange BM, Ketchum REB, Croteau RB (2001) Isoprenoid biosynthesis: metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis. Plant Physiol 127: 305–314 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib34] Laule O, Fürholz A, Chang HS, Zhu T, Wang X, Heifetz PB, Gruissem W, Lange M (2003) Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana. Proc Natl Acad Sci USA 100: 6866–6871 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib35] Li Z, Ratliff EA, Sharkey TD (2011) Effect of temperature on postillumination isoprene emission in oak and poplar. Plant Physiol 155: 1037–1046 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib36] Li Z, Sharkey TD (2013a) Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illumination isoprene burst from leaves. Plant Cell Environ 36: 429–437 [DOI] [PubMed] [Google Scholar]

[bib37] Li Z, Sharkey TD (2013b) Molecular and pathway controls on biogenic volatile organic compound emissions. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 119–151 [Google Scholar]

[bib38] Lindert S, Zhu W, Liu YL, Pang R, Oldfield E, McCammon JA (2013) Farnesyl diphosphate synthase inhibitors from in silico screening. Chem Biol Drug Des 81: 742–748 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib39] Liu YL, Lindert S, Zhu W, Wang K, McCammon JA, Oldfield E (2014) Taxodione and arenarone inhibit farnesyl diphosphate synthase by binding to the isopentenyl diphosphate site. Proc Natl Acad Sci USA 111: E2530–E2539 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib40] Loreto F, Pinelli P, Brancaleoni E, Ciccioli P (2004) ¹³C labeling reveals chloroplastic and extrachloroplastic pools of dimethylallyl pyrophosphate and their contribution to isoprene formation. Plant Physiol 135: 1903–1907 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib41] Loreto F, Schnitzler JP (2010) Abiotic stresses and induced BVOCs. Trends Plant Sci 15: 154–166 [DOI] [PubMed] [Google Scholar]

[bib42] Loreto F, Velikova V (2001) Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiol 127: 1781–1787 [PMC free article] [PubMed] [Google Scholar]

[bib43] Mansouri H, Salari F (2014) Influence of mevinolin on chloroplast terpenoids in Cannabis sativa. Physiol Mol Biol Plants 20: 273–277 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib44] Nakamura A, Shimada H, Masuda T, Ohta H, Takamiya K (2001) Two distinct isopentenyl diphosphate isomerases in cytosol and plastid are differentially induced by environmental stresses in tobacco. FEBS Lett 506: 61–64 [DOI] [PubMed] [Google Scholar]

[bib45] Niinemets U. (2010) Mild versus severe stress and BVOCs: thresholds, priming and consequences. Trends Plant Sci 15: 145–153 [DOI] [PubMed] [Google Scholar]

[bib46] Niinemets Ü, Tenhunen JD, Harley PC, Steinbrecher R (1999) A model of isoprene emission based on energetic requirements for isoprene synthesis and leaf photosynthetic properties for Liquidambar and Quercus. Plant Cell Environ 22: 1319–1335 [Google Scholar]

[bib47] No JH, de Macedo Dossin F, Zhang Y, Liu YL, Zhu W, Feng X, Yoo JAY, Lee E, Wang K, Hui R. , et al. (2012) Lipophilic analogs of zoledronate and risedronate inhibit Plasmodium geranylgeranyl diphosphate synthase (GGPPS) and exhibit potent antimalarial activity. Proc Natl Acad Sci USA 109: 4058–4063 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib48] Oberhauser V, Gaudin J, Fonné-Pfister R, Schär HP (1998) New target enzyme(s) for bisphosphonates: inhibition of geranylgeranyl diphosphate synthase. Pestic Biochem Physiol 60: 111–117 [Google Scholar]

[bib49] Page JE, Hause G, Raschke M, Gao W, Schmidt J, Zenk MH, Kutchan TM (2004) Functional analysis of the final steps of the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway to isoprenoids in plants using virus-induced gene silencing. Plant Physiol 134: 1401–1413 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib50] Peterson RB. (1983) Estimation of photorespiration based on the initial rate of postillumination CO₂ release: II. Effects of O₂, CO₂, and temperature. Plant Physiol 73: 983–988 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib51] Piel J, Donath J, Bandemer K, Boland W (1998) Mevalonate-independent biosynthesis of terpenoid volatiles in plants: induced and constitutive emission of volatiles. Angew Chem Int Ed 37: 2478–2481 [DOI] [PubMed] [Google Scholar]

[bib52] Possell M, Loreto F (2013) The role of volatile organic compounds in plant resistance to abiotic stresses: responses and mechanisms. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 209-235 [Google Scholar]

[bib53] Rajabi Memari H, Pazouki L, Niinemets Ü (2013) The biochemistry and molecular biology of volatile messengers in trees. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 47-93 [Google Scholar]

[bib54] Ramos-Valdivia AC, van der Heijden R, Verpoorte R (1997) Isopentenyl diphosphate isomerase: a core enzyme in isoprenoid biosynthesis. A review of its biochemistry and function. Nat Prod Rep 14: 591–603 [DOI] [PubMed] [Google Scholar]

[bib55] Rasulov B, Bichele I, Hüve K, Vislap V, Niinemets Ü (2015) Acclimation of isoprene emission and photosynthesis to growth temperature in hybrid aspen: resolving structural and physiological controls. Plant Cell Environ 38: 751–766 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib56] Rasulov B, Bichele I, Laisk A, Niinemets Ü (2014) Competition between isoprene emission and pigment synthesis during leaf development in aspen. Plant Cell Environ 37: 724–741 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib57] Rasulov B, Copolovici L, Laisk A, Niinemets Ü (2009a) Postillumination isoprene emission: in vivo measurements of dimethylallyldiphosphate pool size and isoprene synthase kinetics in aspen leaves. Plant Physiol 149: 1609–1618 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib58] Rasulov B, Hüve K, Bichele I, Laisk A, Niinemets Ü (2010) Temperature response of isoprene emission in vivo reflects a combined effect of substrate limitations and isoprene synthase activity: a kinetic analysis. Plant Physiol 154: 1558–1570 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib59] Rasulov B, Hüve K, Laisk A, Niinemets Ü (2011) Induction of a longer term component of isoprene release in darkened aspen leaves: origin and regulation under different environmental conditions. Plant Physiol 156: 816–831 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib60] Rasulov B, Hüve K, Välbe M, Laisk A, Niinemets Ü (2009b) Evidence that light, carbon dioxide, and oxygen dependencies of leaf isoprene emission are driven by energy status in hybrid aspen. Plant Physiol 151: 448–460 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib61] Roberts SC. (2007) Production and engineering of terpenoids in plant cell culture. Nat Chem Biol 3: 387–395 [DOI] [PubMed] [Google Scholar]

[bib62] Rodríguez-Concepción M. (2006) Early steps in isoprenoid biosynthesis: multilevel regulation of the supply of common precursors in plant cells. Phytochem Rev 5: 1–15 [Google Scholar]

[bib63] Rodríguez-Concepción M, Boronat A (2002) Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids: a metabolic milestone achieved through genomics. Plant Physiol 130: 1079–1089 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib64] Rodríguez-Concepción M, Forés O, Martinez-García JF, González V, Phillips MA, Ferrer A, Boronat A (2004) Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. Plant Cell 16: 144–156 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib65] Rohdich F, Zepeck F, Adam P, Hecht S, Kaiser J, Laupitz R, Gräwert T, Amslinger S, Eisenreich W, Bacher A. , et al. (2003) The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. Proc Natl Acad Sci USA 100: 1586–1591 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib66] Rohmer M, Seemann M, Horbach S, Bringer-Meyer S, Sahm H (1996) Glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. J Am Chem Soc 118: 2564–2566 [Google Scholar]

[bib67] Rosenkranz M, Schnitzler JP (2013) Genetic engineering of BVOC emissions from trees. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 95-118 [Google Scholar]

[bib68] Russell RGG, Watts NB, Ebetino FH, Rogers MJ (2008) Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos Int 19: 733–759 [DOI] [PubMed] [Google Scholar]

[bib69] Schwender J, Gemünden C, Lichtenthaler HK (2001) Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids. Planta 212: 416–423 [DOI] [PubMed] [Google Scholar]

[bib70] Seemann M, Tse Sum Bui B, Wolff M, Miginiac-Maslow M, Rohmer M (2006) Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG. FEBS Lett 580: 1547–1552 [DOI] [PubMed] [Google Scholar]

[bib71] Sharkey TD. (1988) Estimating the rate of photorespiration in leaves. Physiol Plant 73: 147–152 [Google Scholar]

[bib72] Sharkey TD, Chen X, Yeh S (2001) Isoprene increases thermotolerance of fosmidomycin-fed leaves. Plant Physiol 125: 2001–2006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib73] Sharkey TD, Gray DW, Pell HK, Breneman SR, Topper L (2013) Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family. Evolution 67: 1026–1040 [DOI] [PubMed] [Google Scholar]

[bib74] Smith JM, Warrington NV, Vierling RJ, Kuhn ML, Anderson WF, Koppisch AT, Freel Meyers CL (2014) Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate. J Antibiot (Tokyo) 67: 77–83 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib75] Szabo CM, Matsumura Y, Fukura S, Martin MB, Sanders JM, Sengupta S, Cieslak JA, Loftus TC, Lea CR, Lee HJ, et al. (2002) Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and diphosphates: a potential route to new bone antiresorption and antiparasitic agents. J Med Chem 45: 2185–2196 [DOI] [PubMed] [Google Scholar]

[bib76] van Beek E, Pieterman E, Cohen L, Löwik C, Papapoulos S (1999) Nitrogen-containing bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo. Biochem Biophys Res Commun 255: 491–494 [DOI] [PubMed] [Google Scholar]

[bib77] Wasko BM. (2011) Novel bisphosphonates as inhibitors of isoprenoid biosynthesis. PhD. University of Iowa, Graduate College, Iowa City, Iowa [Google Scholar]

[bib78] Weise SE, Li Z, Sutter AE, Corrion A, Banerjee A, Sharkey TD (2013) Measuring dimethylallyl diphosphate available for isoprene synthesis. Anal Biochem 435: 27–34 [DOI] [PubMed] [Google Scholar]

[bib79] Wright LP, Rohwer JM, Ghirardo A, Hammerbacher A, Ortiz-Alcaide M, Raguschke B, Schnitzler JP, Gershenzon J, Phillips MA (2014) Deoxyxylulose 5-phosphate synthase controls flux through the methylerythritol 4-phosphate pathway in Arabidopsis. Plant Physiol 165: 1488–1504 [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib80] Xiao Y, Savchenko T, Baidoo EEK, Chehab WE, Hayden DM, Tolstikov V, Corwin JA, Kliebenstein DJ, Keasling JD, Dehesh K (2012) Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes. Cell 149: 1525–1535 [DOI] [PubMed] [Google Scholar]

[bib81] Zeidler JG, Schwender J, Müller C, Wiesner J, Weidemeyer C, Beck E, Jomaa H, Lichtenthaler HK (1998) Inhibition of the non-mevalonate 1-deoxy-d-xylulose-5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Zeitschrift für Naturforschung, Section C. 53c: 980–986 [Google Scholar]

[bib82] Zhou C, Li Z, Wiberley-Bradford AE, Weise SE, Sharkey TD (2013) Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase. Anal Biochem 440: 130–136 [DOI] [PubMed] [Google Scholar]

PERMALINK

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen1

Bahtijor Rasulov

Eero Talts

Astrid Kännaste

Ülo Niinemets

Abstract

RESULTS

Influence of Bisphosphonate Inhibitors on Isoprene Emission in Light and on Emission Dark Decay Kinetics

Figure 1.

Table I. Effects of alendronate and fosmidomycin treatments on isoprene emission rate, DMADP pool size, dark pool size, rate constant of isoprene synthase, and in vivo Km, and maximum activity (Vmax) of isoprene synthase in leaves of hybrid aspen.

Table II. Effects of zoledronate and fosmidomycin on average (±se) isoprene emission rate, DMADP pool size, dark pool size, rate constant of isoprene synthase reaction, and Km and Vmax of isoprene synthase in vivo in leaves of hybrid aspen.

Figure 2.

Influence of Bisphosphonate Inhibitors on Isoprene Synthase Kinetics

Figure 3.

Effects of Fosmidomycin on Isoprene Emission and Substrate Pool Size

Figure 4.

Figure 5.

Influences of Fosmidomycin on Bisphosphonate-Inhibited Leaves

Bisphosphonate Treatment following Fosmidomycin Application

Effects of Bisphosphonate Inhibitors and Fosmidomycin on Foliage Photosynthetic Characteristics

Figure 6.

Table III. Effects of alendronate and zoledronate inhibitors on the initial quantum yields for isoprene emission and net assimilation rates and on the ratio of quantum yields in hybrid aspen leaves.

The Relationships among Isoprene and Photosynthetic Characteristics through Bisphosphonate Treatments

Figure 7.

DISCUSSION

How Bisphosphonate Inhibition Affects Isoprene Emission: General Patterns

Figure 8.

Changes in the DXP/MEP Pathway Flux under Bisphosphonate Treatment: Feedback Inhibition and Pathway Elasticity

Figure 9.

Why Is the Large Dark Pool of Isoprenoid Substrates Not Converted to DMADP in Inhibited Leaves?

Figure 10.

Bisphosphonate Inhibition of End Reactions of the DXP/MEP Pathway at the Level of Isoprene Synthase

Interaction of Bisphosphonate Inhibitors with Fosmidomycin: Evidence of an Extra Precursor Source

Changes in IDP:DMADP Equilibrium as a Possible Further Determinant Affecting DMADP Pool Size

CONCLUSION

MATERIALS AND METHODS

Plant Material

Gas Exchange Measurements

Measurements of Chlorophyll Fluorescence and Initial Quantum Yields

Inhibitor Treatments

GC-MS Analyses

Estimation of Isoprene Precursor Pools from Postillumination Isoprene Emission Decay Data

Determination of in Vivo Isoprene Synthase Kinetic Characteristics

Quantitative Separation of Fluxes Due to Different Substrate Sources in Inhibited Leaves

Acknowledgments

Glossary

Footnotes

References

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen^¹

Table I. Effects of alendronate and fosmidomycin treatments on isoprene emission rate, DMADP pool size, dark pool size, rate constant of isoprene synthase, and in vivo K_m, and maximum activity (V_max) of isoprene synthase in leaves of hybrid aspen.

Table II. Effects of zoledronate and fosmidomycin on average (±se) isoprene emission rate, DMADP pool size, dark pool size, rate constant of isoprene synthase reaction, and K_m and V_max of isoprene synthase in vivo in leaves of hybrid aspen.