Article Figures & Data
Figures
- 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).
- 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).
- 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).
- 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).
- 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”).
- 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 (r 2 = 0.77, P < 0.001). The measurements were conducted at an incident quantum flux density of 650 μmol m−2 s−1.
- 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.
- 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.
- 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.
Tables
- 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
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 Km Vmax 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 - 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
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 Km Vmax 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 - 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 Isoprene Emission Net Assimilation 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
