Cryptic isoprene emission of soybeans under wounding and high temperature

Mostofa, Mohammad Golam 1 ; Sahu, Abira 2 ; Xu, Yuan3; Basrai, Insiya3; Doron, Lior3; Lefrancois, Violet3; Sharkey, Thomas 3

Published May 14, 2025 on Dryad. https://doi.org/10.5061/dryad.51c59zwkh

Data files

May 14, 2025 version files 96.48 KB

Raw_data_Mostofa_et_al._2025.xlsx

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README.md

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Abstract

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.

Dataset DOI: 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<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<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<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<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.

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