DOI: 10.5061/dryad.51c59zwkh
title: Cryptic isoprene emission of soybeans under wounding and high temperature

description: Isoprene is the most abundant non-methane biogenic hydrocarbon emitted by
 some plants, mostly trees. It plays a critical role in atmospheric
 chemistry by contributing to ozone and aerosol formation. Isoprene also
 benefits plants, particularly under stress, through its signaling roles.
 Legume crops like soybean were thought to have evolutionarily lost
 isoprene synthase (ISPS) and are typically considered non-emitters. Here
 we report that damage to soybean leaves by wounding or burning triggered a
 burst of isoprene emission from undamaged parts of the leaves. In
 silico analysis identified intact ISPS genes in the soybean
 genome, with features similar to known ISPSs. Protein made from these gene
 sequences catalyzed isoprene production in the presence of dimethylallyl
 diphosphate. Isoprene emission in soybeans was linked to reduced
 photosynthesis rates and stomatal conductance. Metabolomic analysis showed
 that leaf damage caused a surge in glyceraldehyde 3-phosphate and pyruvate
 levels, leading to an increase of most of the methylerythritol 4-phosphate
 (MEP) pathway metabolites. Heat stress also led to significant isoprene
 emission from soybean leaves. We conclude that soybeans possess functional
 ISPSs and can make isoprene only under some conditions by regulating
 photosynthesis and MEP pathway.
description: # Cryptic isoprene emission of soybeans Dataset DOI:
 [10.5061/dryad.51c59zwkh](10.5061/dryad.51c59zwkh) ## Description of the
 data and file structure To understand the cryoptic isoprene emission
 nature of crop plant soybean.  ### Files and variables #### File:
 Raw\_data\_Mostofa\_et\_al.\_2025.xlsx **Description:** We measured
 changes in isoprene emission, photosynthesis and related metabolites, and
 functional validation of soybean isoprene synthase proteins.  #####
 Variables Figure 2: Expression and purification of recombinant soybean
 isoprene synthases (ISPSs) and in-vitro characterization of purified
 ISPSs. (a) Isoprene synthase (ISPS) genes from eucalyptus (Eg_ISPS) and
 soybean (TPS8 and TPS23) were cloned to an IPTG-inducible expression
 plasmid followed by expression in E. coli BL21, and purification using a
 Strep affinity column. (b) Diagram of Strep-tagged ISPS genes and
 chromatogram of ISPS detected by Fast protein liquid chromatography
 (FPLC). Strep affinity tag (green rectangle) and SUMO domain (gray
 rectangle) were fused to the N-terminus of ISPS genes (blue arrow)
 downstream to an IPTG-inducible promoter. Elution peaks are marked by
 black arrows. SDS-PAGE analysis shows specific band for each purified
 ISPS. (c, d) Isoprene emission capacity of EgISPS and TPS23 increases in a
 concentration and time dependent manner. (e) Purified EgISPS, TPS8, and
 TPS23 show isoprene emission capacity in presence of different
 concentration of dimethylallyl diphosphate (DMADP). (f) kcat and K1/2 of
 purified EgISPS, TPS8, and TPS23 based on their catalytic performance in
 conversion of DMADP to isoprene. S: total soluble proteins before Strep
 affinity purification, P: insoluble fraction, Elu: pooled and concentrated
 elution fractions. ISPS peaks are marked with blue arrows. Figure 3:
 Figure 3: Cryptic isoprene emission and physiological, biochemical, and
 gene expression changes in soybean leaves. (a) Soybean leaves transiently
 emit isoprene following wounding. (b) Isoprene burst is also evident in
 soybean plants after burning of the same, lateral, and distant leaflets.
 (c) Expression levels of GmTPS8 and GmTPS23 genes following wounding of
 soybean leaves. (d) Isoprene emission accompanies differential responses
 of CO2 assimilation (A) and stomatal conductance (gsw) upon wounding.
 Asterisk indicates significant differences in gene expression levels
 (P&lt;0.05; Student’s t-test). (e) A/Ci response curves illustrate rubisco
 activity, ribulose bisphosphate (RuBP) regeneration, and triose phosphate
 utilization (TPU) limitation in soybean leaves before, during, and after
 wounding. (f) Comparative responses of CO2 assimilation (A) in soybean
 leaves following wounding shows that photosynthesis was rubisco limited
 even at the lowest rates. Rd= respiration in the light, gm = mesophyll
 conductance, αG = the proportion of carbon from photorespiration lost when
 glycine exits the photorespiratory metabolism and αS = the carbon lost as
 serine exits the Calvin Benson cycle. (g) Photosynthetic efficiency of
 photosystem II (Phi II) in soybean leaves after wounding. (h) The level of
 hydrogen peroxide (H2O2) determined before and after wounding of soybean
 leaves (n=8). Asterisk indicates significant differences at P&lt;0.05
 according to Student’s t-test. SSR = sum of the squared residuals. Figure
 4: Wound-induced changes in MEP pathway and photosynthesis related
 metabolites. (a) Isoprene biosynthesis precursors originated from Calvin
 Benson cycle. Using pyruvate (PYR) and glyceraldehyde 3-phosphate (GAP),
 dimethylallyl diphosphate (DMADP) is synthesized from MEP pathway products
 by enzyme-mediated reactions and ultimately converted to isoprene by
 isoprene synthase (ISPS). (b) The levels of MEP pathway metabolites,
 including DXP, MEP, CDP-ME, MEcDP, and HMBDP were determined in soybean
 leaf samples collected before (S1), during (S2), and after (S3) wounding.
 (c) Calvin-Benson Cycle provides the precursors of PYR and GAP. The levels
 of metabolites, including 6-phosphogluconate (6PG), GAP/dihydroxyacetone
 phosphate (DHAP), fructose 6-phosphate (FBP)/glucose 6-phosphate (G6P),
 glucose 6-phosphate/fructose 6-phosphate (G6P/F6P), sedoheptulose
 7-phosphate (S7P), ribose 5-phosphate (R5P)/ ribulose 5-phosphate
 (Ru5P)/xylulose 5-phosphate (Xu5P), ribulose 1,5-bisphosphate (RuBP), and
 PYR were measured in soybean leaf samples before (S1), during (S2), and
 after (S3) wounding. DXP, 1-deoxyxylulose 5-phosphate; MEP,
 2-C-methyl-D-erythritol 4-phosphate; CDP-ME,
 4-diphosphocytidyl-2-C-methyl-D-erythritol; CDP-MEP,
 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; MEcDP,
 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBDP,
 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; DMADP, dimethylallyl
 diphosphate; IDP, isoprenyl diphosphate; DXS, 1-deoxyxylulose-5-phosphate
 synthase; DXR 1-deoxyxylulose-5-phosphate reductoisomerase; CMS,
 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase; CMK,
 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate kinase; MCS,
 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS,
 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, HDR,
 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; IDI, isopentenyl
 diphosphate isomerase. Statistically significant differences in MEP and
 CBC metabolites among S1, S2, and S3 were calculated by ANOVA and Tukey’s
 HSD and are indicated by lowercase letters. Figure 5: Isoprene emission in
 soybean leaves under different climate factors. (a) Effect of elevated CO2
 (HCO2) and high temperature (HT) on wound-induced isoprene emission from
 soybean leaves. (b) Carbon utilization for isoprene synthesis at ambient
 vs. high CO2 and temperature conditions in wounded leaves. Statistically
 significant differences in isoprene emission (a) and carbon utilization
 (b) among ambient, HT, and HCO2 were calculated by ANOVA and Tukey’s HSD
 and are indicated by lowercase letters. (c, d) Change in isoprene emission
 from soybean leaves during short-term heat stress (380C). (e, f, and g)
 Isoprene emission from soybean leaves at 25, 30, and 42 C after 2 h and
 20 h incubation in growth chambers. Statistically significant differences
 in isoprene emission with increase in temperature were calculated by ANOVA
 and Tukey’s HSD and are indicated by lowercase letters. (h) Mechanistic
 overview of cryptic isoprene emission and photosynthesis regulation upon
 wounding of soybean leaves. PYR, pyruvate; GAP, glyceraldehyde
 3-phosphate; G6PDH, glucose 6-phosphate dehydrogenase; 6-PG,
 6-phosphogluconate. Figure S9: MEP pathway and central carbon
 metabolism-related metabolites in soybean leaves before (S1), during (S2),
 and after (S3) burning. (a) The levels of MEP pathway metabolites,
 including 1-deoxyxylulose 5-phosphate (DXP), 2-C-methyl-D-erythritol
 4-phosphate (MEP), 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME),
 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-MEP),
 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP), and
 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) and (b) central
 carbon metabolism metabolites, including pyruvate (PYR), glyceraldehyde
 3-phosphate (GAP), 6-phosphogluconate (6PG), 3-hosphoglyceraldehyde (PGA),
 GAP/dihydroxyacetone phosphate (DHAP), fructose 6-bisphosphate
 (FBP)/glucose 6-phosphate (G6P), seduheptulose 7-phosphate (S7P), ribose
 5-phosphate (R5P)/ Ribulose 5-phosphate (Ru5P)/xylulose 5-phosphate
 (Xu5P), rubisco bisphosphate (RuBP), and pyruvate (PYR) are measured in
 soybean leaf samples collected S1, S2, and S3 upon burning of soybean
 leaves. Different alphabetical letters (a and b) indicate significant
 differences among the treatment conditions at p&lt;0.05 according to least
 significant difference (LSD). Statistically significant differences in MEP
 and CBC metabolites between S1, S2, and S3 were calculated by ANOVA and
 Tukey’s HSD and are indicated by lowercase letters. Figure S10: Hormonal
 profiles in soybean leaves before (S1), during (S2), and after (S3)
 wounding. Different alphabetical letters (a and b) indicate significant
 differences among the treatment conditions at p&lt;0.05 according to least
 significant difference (LSD). OPDA, 12-oxo-phytodienoic acid; JA, jasmonic
 acid; JA-Ile, jasmonic acid isoleucine; MeJA, methyl jasmonate; ABA,
 abscisic acid; SA, salicylic acid; SAG, salicylate glucoside.
 Statistically significant differences in hormone levels between S1, S2,
 and S3 were calculated by ANOVA and Tukey’s HSD and are indicated by
 lowercase letters. ## Code/software N/A ## Access information Other
 publicly accessible locations of the data: * N/A Data was derived from the
 following sources: * N/A