DOI: 10.5061/dryad.d7wm37q64
title: Data from: Hydroxymethylbutenyl diphosphate accumulation reveals MEP pathway regulation for high CO2-induced suppression of isoprene emission

description: Isoprene is emitted by some plants and is the most abundant biogenic
 hydrocarbon entering the atmosphere. Multiple studies have elucidated
 protective roles of isoprene against several environmental stresses,
 including high temperature, excessive ozone, and herbivory attack.
 However, isoprene emission adversely affects atmospheric chemistry by
 contributing to ozone production and aerosol formation. Thus,
 understanding the regulation of isoprene emission in response to varying
 environmental conditions, for example elevated CO2, is critical to
 comprehend how plants will respond to climate change. Isoprene emission
 decreases with increasing CO2 concentration; however, the underlying
 mechanism of this response is currently unknown. We demonstrated that
 high-CO2-mediated suppression of isoprene emission is independent of
 photosynthesis and light intensity, but it is reduced with increasing
 temperature. Furthermore, we measured methylerythritol 4-phosphate pathway
 metabolites in poplar leaves harvested at ambient and high CO2 to identify
 why isoprene emission is reduced under high CO2. We found that
 hydroxymethylbutenyl diphosphate (HMBDP) was increased and dimethylallyl
 diphosphate (DMADP) decreased at high CO2. This implies that high CO2
 impeded the conversion of HMBDP to DMADP, possibly through the inhibition
 of HMBDP reductase activity, resulting in reduced isoprene emission. We
 further demonstrated that although this phenomenon appears similar to
 ABA-dependent stomatal regulation, it is unrelated as abscisic acid
 treatment did not alter the effect of elevated CO2 on the suppression of
 isoprene emission. Thus, this study provides a comprehensive understanding
 of the regulation of the MEP pathway and isoprene emission in the face of
 increasing CO2.
description: a. Data for isoprene emission was collected using the Fast
 Isoprene Sensor (FIS)  b. Measurements of
 photosynthesis (<em>A</em>), stomatal conductance
 (<em>g<sub>sw</sub></em>), intercellular
 CO<sub>2</sub>
 (<em>C<sub>i</sub></em>) were conducted in a
 LI-COR 6800. c. Metabolite analysis was conducted on a
 Xevo TQ-XS mass spectrometer.
description: # Title of Dataset: Hydroxymethylbutenyl diphosphate accumulation reveals
 MEP pathway regulation for high CO2-induced suppression of isoprene
 emission Give a brief summary of dataset contents, contextualized in
 experimental procedures and results. This dataset contains- 1. Isoprene
 emission data from poplar leaves measured using the Fast Isoprene Sensor
 2. Assimilation rates or photosynthesis (A), intercellular CO2
 concentration (Ci), and stomatal conductance (gsw) measured using LI-COR
 6800 3. MEP pathway metabolite levels measured by LC-MS/MS Using these
 data, we showed that the decline in isoprene emission at high CO2 is due
 to accumulation of HMBDP which is an upstream precursor and reduction in
 DMADP, an immediate precursor of isoprene. We also showed that the CO2
 responsiveness of isoprene is independent of the ABA-dependent stomatal
 signaling pathway. Furthermore, high CO2 mediated suppression of isoprene
 emission is independent of varying light intensities but decreases at high
 temperature. ## Description of the data and file structure Raw data that
 were used to make the plots are provided as individual .csv files and each
 file name contains the figure number. \*Fig 1A. Isoprene emission measured
 at an interval of 1 min over 48 min as CO2 partial pressure is changed
 from 39 Pa to 78 Pa and back to 39 Pa \*Fig 1B. Isoprene emission measured
 in 14 poplar leaves at 39 Pa CO2 and 78 Pa CO2. Relative change in
 isoprene emission between 39 Pa and 78 Pa CO2 is calculated for each leaf.
 * Mean: Mean of isoprene emission at 39 Pa and 78 Pa CO2 * SD: standard
 deviation * p-value: calculated by Student's t-test (2 tailed,
 paired) <br> \*Fig 2A,B,C. Isoprene emission measured at 39 Pa and
 78 Pa CO2 under light intensities of 1000 umol m-2 s-1, 750 umol m-2 s-1,
 500 umol m-2 s-1, 250 umol m-2 s-1, and 100 umol m-2 s-1. * Absolute
 change: Difference in isoprene emission between 39 Pa and 78 Pa CO2 *
 Relative change: Difference in isoprene emission between 39 Pa and 78 Pa
 CO2 relative to isoprene emission at 39 Pa CO2, expressed in percent *
 Mean: Mean of absolute change and relative change in isoprene emission at
 39 Pa and 78 Pa CO2 * SD: standard deviation * p-value: calculated by
 Student's t-test (2 tailed, paired) between isoprene emission at 39
 Pa and 78 Pa CO2 \*Fig 2D,E,F. Isoprene emission measured at 39 Pa and 78
 Pa CO2 under temperatures 25degC, 30degC, and 35degC * Absolute change:
 Difference in isoprene emission between 39 Pa and 78 Pa CO2 * Relative
 change: Difference in isoprene emission between 39 Pa and 78 Pa CO2
 relative to isoprene emission at 39 Pa CO2, expressed in percent * Mean:
 Mean of absolute change and relative change in isoprene emission at 39 Pa
 and 78 Pa CO2 * SD: standard deviation * p-value: calculated by
 Student's t-test (2 tailed, paired) between isoprene emission at 39
 Pa and 78 Pa CO2 \*Fig 3B,D. Levels of MEP pathway metabolites DXP, MEP,
 CDP-ME, MEcDP, and HMBDP in 4 biological replicates R1, R2, R3, and R4
 collected at time points (i) at 39 Pa CO2 after isoprene emission has
 stabilized; T1 (ii) 3 min after changing CO2 to 78 Pa; T2 (iii) at 78 Pa
 CO2 after isoprene emission had stabilized; T3 (iv) 3 min after changing
 CO2 back to 39 Pa; T4 (v) at 39 Pa CO2 after isoprene emission has
 stabilized; T5. Each metabolite concentration is normalized to
 concentration of DXP. -Mean: Mean of 3-4 biological replicates at each
 time point * SD: standard deviation \*Fig 3C.DMADP measured by integrating
 isoprene emission after lights were turned off in 3 biological replicates
 R1, R2, and R3 at time points (i) at 39 Pa CO2 after isoprene emission has
 stabilized; T1 (ii) 3 min after changing CO2 to 78 Pa; T2 (iii) at 78 Pa
 CO2 after isoprene emission had stabilized; T3 (iv) 3 min after changing
 CO2 back to 39 Pa; T4 (v) at 39 Pa CO2 after isoprene emission has
 stabilized; T5. Each metabolite concentration is normalized to
 concentration of DXP. \*Fig 4. The following variables were measured: (i)
 Isoprene emission (ii) Assimilation rates or photosynthesis (A) (iii)
 intercellular CO2 concentration (Ci), and (iv) stomatal conductance (gsw)
 at an interval of 5 s for 54 min. Treatments used: (i) H2O at 39 Pa CO2
 (ii) H2O at 78 Pa CO2 (iii) H2O at 39 Pa CO2 (iv) ABA at 39 Pa CO2 (v) ABA
 at 78 Pa CO2 -H2O: water, ABA: abscisic acid \*Table 1. Isoprene emission
 and photosynthesis (A) measured at 25degC and 35degC at 39 Pa and 78 Pa
 CO2. Temperature coefficient (Q10) was measured for isoprene and
 photosynthesis at 39 Pa CO2 and 78 Pa CO2 using the formula:
 Q\_10=(R2/R1)^((10/(T2-T1))) \*Fig S1A,B. Photosynthesis (A) and stomatal
 conductance (gsw) measured in 10 biological replicates at 39 Pa CO2 and 78
 Pa CO2. * Relative change: Difference in photosynthesis (A) and stomatal
 conductance (gsw) between 39 Pa and 78 Pa CO2 relative to their values at
 39 Pa CO2, expressed in percent * Mean: Mean of relative change in
 photosynthesis (A) and stomatal conductance (gsw) at 39 Pa and 78 Pa CO2 *
 SD: standard deviation * p-value: calculated by Student's t-test (2
 tailed, paired) \*Fig S2A,B,C,D. Relative change in photosynthesis and
 carbon cost of isoprene emission (I/A) under (i) light intensities of 1000
 umol m-2 s-1, 750 umol m-2 s-1, 500 umol m-2 s-1, 250 umol m-2 s-1, and
 100 umol m-2 s-1 in 3 biological replicates rep 1, rep 2, and rep 3 (ii)
 temperatures 25degC, 30degC, and 35degC in 6 biological replicates rep 1,
 rep 2, rep 3, rep 4, rep5, and rep 6. -Mean: mean of biological replicates
 at each light intensity/temperature -SD: standard deviation \*Fig S4.
 Isoprene emission and stomatal conductance (gsw) under varying CO2. \*Fig
 S5. Levels of hydrogen peroxide (H2O2) measured in 5 biological replicates
 (R1, R2, R3, R4, R5) at 39 Pa CO2 and 78 Pa CO2. Rep1, rep2, and rep 3 are
 technical replicates. * Mean: Mean of technical replicates * SD: standard
 deviation * p-value: calculated by Student's t-test (2 tailed,
 unequal variance) \*Fig S6. Growth conditions (light and temperature) in
 the greenhouse for growing poplars. -Daily light integral (DLI) was
 calculated by multiplying each value of light intensity in umol m-2 s-1 by
 600 (since measurements were recorded every 600 s) then taking a sum of
 all the values over 24 h. -Day temperature was calculated by taking a mean
 of temperature readings from 6:13 am to 9:53 pm -Night temperature was
 calculated by taking a mean of temperature readings from 10:13 pm to 6:03
 am