Natural abundance carbon isotope composition of isoprene reflects incomplete coupling between isoprene synthesis and photosynthetic carbon flow

Abstract

Isoprene emission from leaves is dynamically coupled to photosynthesis through the use of primary and recent photosynthate in the chloroplast. However, natural abundance carbon isotope composition (delta(13)C) measurements in myrtle (Myrtus communis), buckthorn (Rhamnus alaternus), and velvet bean (Mucuna pruriens) showed that only 72% to 91% of the variations in the delta(13)C values of fixed carbon were reflected in the delta(13)C values of concurrently emitted isoprene. The results indicated that 9% to 28% carbon was contributed from alternative, slow turnover, carbon source(s). This contribution increased when photosynthesis was inhibited by CO(2)-free air. The observed variations in the delta(13)C of isoprene under ambient and CO(2)-free air were consistent with contributions to isoprene synthesis in the chloroplast from pyruvate associated with cytosolic Glc metabolism. Irrespective of alternative carbon source(s), isoprene was depleted in (13)C relative to mean photosynthetically fixed carbon by 4 per thousand to 11 per thousand. Variable (13)C discrimination, its increase by partially inhibiting isoprene synthesis with fosmidomicin, and the associated accumulation of pyruvate suggested that the main isotopic discrimination step was the deoxyxylulose-5-phosphate synthase reaction.

Figure 1

Changes in the isotopic composition of photosynthetically fixed carbon (from on-line gas exchange and isotopic measurements) and of isoprene emitted from a branch of myrtle-3, in response to a rapid change in the isotopic composition of the source CO₂ (at a time marked by the vertical lines). The numbers denote the discrimination in isoprene production relative to fixed carbon (Δ_c-isop in per mil). Photosynthesis was brought to steady state before the onset of the experiment and all conditions other than the δ¹³C of the source CO₂ were kept constant throughout the experiments.

Figure 2

Relationships between the isotopic composition of the photosynthetically fixed carbon (δ_fixed;3b from on-line gas exchange and isotopic measurements) and that of isoprene (δ_isop) emitted by leaves of myrtle-1 through -3 (a–c, ○), velvet bean (d), buckthorn (e), and reed (f). The vertical lines indicate the isotopic composition of leaf organic matter (δ_leaf; n between 4 and 10; se between 0.2 and 0.6). The horizontal lines denote the apparent discrimination (Δ_c-isop, calculated from the plot assuming that δ_leaf represents the mean δ_fixed during the growth period of the leaf). The slopes of myrtle, buckthorn, and velvet bean were significantly lower than 1 based on t test for ([slope − 1]/slope se), with n − 2 degrees of freedom. Slope se was 0.03 (n = 106), 0.02 (n = 83), 0.02 (n = 42), 0.04 (n = 33), 0.02 (n = 28), and 0.04 (n = 30) for plots a to f, respectively. The gray triangles and dashed line in plot a denote the influence of ABA on δ_fixed and δ_isop in a branch of myrtle-1.

Figure 3

Schematic representation of isoprene biosynthesis pathway and possible coupling to cytosolic Glc metabolism and IPP. Also noted are the sites of action of the inhibitor fosmidomycin (Fellermeier et al., 1999) and of the isotopic discrimination by DXS. DHAP, dihydroxyacetone phosphate; DMAPP, dimethylallyl pyrophosphate; TPP, thiamine pyrophosphate.

Figure 4

Effects of fosmidomycin inhibition on net assimilation (●) and isoprene emission rates (○) in a branch of myrtle-1. The vertical lines indicate the beginning of fosmidomycin feeding (5 μm at 12:30 and 10 μm at 16:20 pm). Leaf temperature was 26°C, light intensity was 250 μmol m⁻² s⁻¹, and c_i varied between 220 and 250 μL L⁻¹. Due to the decrease in net assimilation observed at the high fosmidomycin used here, we used only up to 5 μm fosmidomycin in the isotopic analysis experiments.