Competition between isoprene emission and pigment synthesis during leaf development in aspen

Abstract

In growing leaves, lack of isoprene synthase (IspS) is considered responsible for delayed isoprene emission, but competition for dimethylallyl diphosphate (DMADP), the substrate for both isoprene synthesis and prenyltransferase reactions in photosynthetic pigment and phytohormone synthesis, can also play a role. We used a kinetic approach based on post-illumination isoprene decay and modelling DMADP consumption to estimate in vivo kinetic characteristics of IspS and prenyltransferase reactions, and to determine the share of DMADP use by different processes through leaf development in Populus tremula. Pigment synthesis rate was also estimated from pigment accumulation data and distribution of DMADP use from isoprene emission changes due to alendronate, a selective inhibitor of prenyltransferases. Development of photosynthetic activity and pigment synthesis occurred with the greatest rate in 1- to 5-day-old leaves when isoprene emission was absent. Isoprene emission commenced on days 5 and 6 and increased simultaneously with slowing down of pigment synthesis. In vivo Michaelis-Menten constant (Km ) values obtained were 265 nmol m(-2) (20 μm) for DMADP-consuming prenyltransferase reactions and 2560 nmol m(-2) (190 μm) for IspS. Thus, despite decelerating pigment synthesis reactions in maturing leaves, isoprene emission in young leaves was limited by both IspS activity and competition for DMADP by prenyltransferase reactions.

Keywords: competition within isoprenoid synthesis pathway; geranyl diphosphate synthase; isoprenoid synthesis; photosynthesis development; prenyltransferase reactions; terpenoid synthesis.

Figure 1

Age-dependent changes in (a) the size of main stem leaves, leaf dry mass per unit area (LMA), and (b) contents of total chlorophyll (Chl a+b) and total carotenoids (Car) in aspen (Populus tremula L.). In (b), the data were fitted by exponential relationships by minimizing the sum of the squares between predicted and measured values (Eq. 8; r² = 0.994 for chlorophyll and r² = 0.986 for carotenoids, P < 0.001 for both). The fitting coefficients (Eq. 8) obtained were for chlorophyll (a+b): a₁ = 601 μmol m⁻², a₂ = 0.443 d and a₃ = 0.125 d⁻¹ ; for carotenoids: a₁ = 185 μmol m⁻², a₂ = 0.775 d and a₃ = 0.130 d⁻¹. Data are means ± SE of at least four replicate experiments. Day 0 corresponds to the day of bud-burst (May 18). Data in the inset demonstrate variation in daytime minimum, maximum and mean temperature during the study period (derived from the data of the weather station of the Laboratory of Environmental Physics, University of Tartu, http://meteo.physic.ut.ee).

Figure 2

Dynamics of net CO₂ uptake, dark respiration, stomatal conductance to water vapor (a), quantum yields (QY) of Photosystem II (PSII) in dark- and light-adapted leaves and quantum yield of CO₂ uptake for an absorbed light (b), and the rate of photosynthetic electron transport and leaf absorptance (c) in aspen leaves. Data presentation as in Fig. 1.

Figure 3

Age-dependent changes in isoprene emission rate, apparent DMADP pool size and the apparent rate constant of isoprene synthase (a), and the apparent rate constant of isoprene synthase in relation to apparent DMADP pool size (b) in different-aged aspen leaves. Figure S1.1 demonstrates determinations of the apparent DMADP pool size and the rate constant of isoprene synthase (Supplemental Material S1). Data are means ± SE of at least four replicate measurements.

Figure 4

Simulation of isoprene emission rate (ν_Iso) vs. true (S_DAMAP, solid line) and apparent DMADP pool size (S_DMADP,app, dashed line) pool size relationships during light-dark transients (Supplemental Material S1, Fig. S1.1a for sample relationships) for a mature leaf (a) with a high capacity for isoprene emission and a low capacity for pigment synthesis and for a young leaf (b) with a low capacity for isoprene emission and a high capacity for pigment synthesis. After switching off the light, isoprene emission rate and DMADP pool size start to decrease until the preexisting pool of DMADP is exhausted. When isoprene emission rate is the only process consuming DMADP, integrating isoprene emission to a certain moment of time provides DMADP pool size that is remaining at that moment of time. However, when other processes compete for chloroplastic DMADP, this integration results in an apparent DMADP pool size (S_DMADP,app). In both panels, the relationships of ν_Iso vs. S_DAMAP were derived by Eq. 3 and 4 using constant K_m values for isoprene and pigments based on simultaneous fitting of all data. S_DMADP,app was thereafter derived by integrating the values of isoprene emission (Eq. 5), and the apparent K_m,iso values derived from ν_Iso vs. S_DMADP,app relationships (Hanes-Wolff plots). The pie diagrams show relative contributions of isoprene emission and pigment synthesis to overall consumption of DMADP pool size accumulated before the light-dark transient.

Figure 5

Comparison of the decay of DMADP pool size (S_DMADP for true pool, and S_DMADP,app for the apparent pool) and the contribution of isoprene emission rate to overall rate of DMADP consumption (f_Iso) following light-dark transients in hybrid aspen leaves of different age (Supplemental Material S1, Fig. S1.1a for corresponding measurements). f_Iso is defined as the ratio of isoprene emission, ν_Iso, to total rate of DMADP consumption (sum of ν_Iso and pigment synthesis, ν_Pig). For better visual comparison of patterns, DMADP pool sizes were normalized to DMADP pool size at the steady-state before the transient (S_DMADP,0). An iterative least squares approach was employed to fit simultaneously isoprene emission rate and DMADP pool size by Eq. 4 and 5 and estimate the Michaelis-Menten constants (K_m) for isoprene synthase (K_m,iso obtained was 2560 nmol m⁻²) and pigment synthesis (K_m,pig = 265 nmol m⁻²) for all leaves simultaneously, as well as to fit the capacities for isoprene synthase (V_m,iso) and pigment synthesis (V_m,pig) reactions and S_DMADP,0 for leaves of different age (Supplemental material S2, Fig. S2.1 for the overall fit of predicted and measured values). The pie graphs indicate relative contributions of isoprene emission and pigment synthesis to consumption of DMADP pool size accumulated before the light-dark transient.

Figure 6

Isoprene emission rate, DMADP pool size and the rate constant of isoprene synthase of young 6-day-old (a) and mature 20-day-old leaves (b) of aspen treated with 10 mM solution of alendronate for 0 (control) to 60 min. Alendronate specifically inhibits geranyl diphosphate (Lange et al. 2001; Burke et al. 2004) and farnesyl diphosphate synthase (Bergstrom et al. 2000; Burke et al. 2004) activities. Data are means + SE of at least four replicate measurements.

Figure 7

Effects of alendronate on the apparent characteristics of isoprene synthase, ν_m,iso,app and K_m,iso,app, in young 6-7-day-old (a) and mature 20-day-old leaves (b) of aspen treated for various times with alendronate. The isoprene synthase characteristics were derived from Hanes-Wolff plots as in Fig. S1.2 (Supplemental Material S1). If isoprene synthesis was the only process consuming DMADP as is expected in alendronate-inhibited leaves, the derived values reflected the true K_m and V_m of isoprene synthase. Data presentation and the number of replicates as in Fig. 6.

Figure 8

Dynamic changes in the rates of isoprene emission and synthesis of photosynthetic pigments (Chl+Car) (a) and the correlation between isoprene emission and pigment synthesis rate (b) through the development of aspen leaves. Both the rates of isoprene emission and pigment synthesis are expressed in nmol C m⁻² s⁻¹ (isoprene contains 5, phytyl residue of chlorophylls 20 and carotenoids 40 carbon atoms). The rates of pigment synthesis are calculated from the rate of pigment accumulation by Eq. 9 assuming a constant pigment synthesis rate for 12 h light period. The rates of isoprene emission were measured at a light intensity of 650 μmol m⁻² s⁻¹ and leaf temperature of 30 °C (data in the main panels). For quantitative comparison, isoprene emissions were also scaled to an average daytime temperature of 19.2 °C (inset in Fig. 1a for variations in temperature during the experiment) using the temperature response from Rasulov et al. (2010, inset in (b)). Data in (b) were fitted by a non-linear regression in the form y = aLn(x)+b.