Isoprene Acts as a Signaling Molecule in Gene Networks Important for Stress Responses and Plant Growth

Abstract

Isoprene synthase converts dimethylallyl diphosphate to isoprene and appears to be necessary and sufficient to allow plants to emit isoprene at significant rates. Isoprene can protect plants from abiotic stress but is not produced naturally by all plants; for example, Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) do not produce isoprene. It is typically present at very low concentrations, suggesting a role as a signaling molecule; however, its exact physiological role and mechanism of action are not fully understood. We transformed Arabidopsis with a Eucalyptus globulus isoprene synthase The regulatory mechanisms of photosynthesis and isoprene emission were similar to those of native emitters, indicating that regulation of isoprene emission is not specific to isoprene-emitting species. Leaf chlorophyll and carotenoid contents were enhanced by isoprene, which also had a marked positive effect on hypocotyl, cotyledon, leaf, and inflorescence growth in Arabidopsis. By contrast, leaf and stem growth was reduced in tobacco engineered to emit isoprene. Expression of genes belonging to signaling networks or associated with specific growth regulators (e.g. gibberellic acid that promotes growth and jasmonic acid that promotes defense) and genes that lead to stress tolerance was altered by isoprene emission. Isoprene likely executes its effects on growth and stress tolerance through direct regulation of gene expression. Enhancement of jasmonic acid-mediated defense signaling by isoprene may trigger a growth-defense tradeoff leading to variations in the growth response. Our data support a role for isoprene as a signaling molecule.

Figure 1.

Carbon assimilation and isoprene emission in Arabidopsis expressing ISPS. Photosynthesis (A) and isoprene emission (B) were measured simultaneously in Arabidopsis wild-type Col-0, empty vector controls (EV-A2, EV-B3, and EV-F1), and lines expressing ISPS (C3, B4, A1, F2, C1, C4, and B2). Measurements were made on plants 45 to 47 d old. All transgenic lines belonged to the T3 generation. Values in graphs represent means ± se (n = 5–7 plants per line). Arabidopsis lines expressing ISPS that are significantly different from Col-0 and empty vector lines EV-A2 and EV-B3 are marked with asterisks: **, P < 0.01 using one-way ANOVA followed by Fisher’s lsd test.

Figure 2.

Early seedling growth in Arabidopsis expressing ISPS. Comparison of hypocotyl length (A and B) and cotyledon size (C and D) in 7-d-old Arabidopsis wild-type Col-0, empty vector controls (EV-A2, EV-B3, and EV-F1), and lines expressing ISPS (C3, B4, A1, F2, C1, C4, and B2). All transgenic lines belonged to the T3 generation. Values in graphs represent means ± se. In B, n = 22 to 41 plants per line; in D, n = 12 to 26 plants per line. Arabidopsis lines expressing ISPS that are significantly different from Col-0 and empty vector lines EV-A2 and EV-B3 are marked with symbols: ⁺, P < 0.1; *, P < 0.05; and **, P < 0.01 using one-way ANOVA followed by Fisher’s lsd test. In D, C1 was only statistically different from Col-0, EV-B3, and EV-F1.

Figure 3.

Rosette and inflorescence growth in Arabidopsis expressing ISPS. Photographs comparing rosette size and projected leaf area of 21-d-old (A) and 50-d-old (B) T3 plants, and leaf dry weight (C), shoot (leaf + inflorescence) dry weight (D), number of days required for inflorescence initiation (E), and height of the primary (1°) inflorescence axis (F) in 50-d-old Arabidopsis wild-type Col-0, empty vector controls (EV-A2, EV-B3, and EV-F1), and lines expressing ISPS (C3, B4, A1, F2, C1, C4, and B2), are shown. All transgenic lines belonged to the T3 generation. Values in graphs represent means ± se. In C and D, n = 4 to 8 plants per line; in E and F, n = 3 to 6 plants per line. Arabidopsis lines expressing ISPS that are significantly different from Col-0 and empty vector lines EV-A2 and EV-B3 are marked with symbols: ⁺, P < 0.1; *, P < 0.05; and **, P < 0.01 using one-way ANOVA followed by Fisher’s lsd test; in E and F, symbols denote statistical comparisons relative to only EV-A2 and EV-B3.

Figure 4.

Rosette and inflorescence stem growth rates in Arabidopsis expressing ISPS. Projected leaf area over time (A), absolute growth rates calculated as the increase in projected leaf area per day (B), and increase in the height of the primary inflorescence axis (stem) per day (C) are shown. All transgenic lines belonged to the T3 generation. Values in graphs represent means ± se. In A and B, n = 4 to 6 plants per line. In C, n = 3 to 5 plants per line. Arabidopsis lines expressing ISPS (C3, B4, A1, F2, C1, C4, and B2) significantly different from Col-0 and empty vector lines EV-A2 and EV-B3 are marked with symbols. ⁺, P < 0.1; *, P < 0.05; and **, P < 0.01 using one-way ANOVA followed by Fisher’s lsd test. In C, symbols denote statistical comparisons relative to only EV-A2 and EV-B3.

Figure 5.

Photosynthesis and isoprene emission under varying environmental conditions in Arabidopsis expressing ISPS. The responses of photosynthesis (Photo) and isoprene emission to temperature (A), CO₂ (B), light (C), darkness (D), and N₂ break (E) in 39- to 42-d-old Arabidopsis empty vector control (EV-B3) and lines expressing ISPS (B2 and C4) are shown. In A, the wild type (WT) was used as the control. Data from B2 are shown in D and E. All plants were grown in growth chambers under a light intensity of 200 μmol m⁻² s⁻¹. Measurements were taken 30°C (B–E) and 500 μmol m⁻² s^-1 light intensity (A, B, and E). In D, plants were exposed to 500 μmol m⁻² s⁻¹ light before being exposed to sudden darkness. Values in A to C represent means ± se; values in D represent means (n = 5). In E, representative data from a single leaf are presented.

Figure 6.

MEP pathway metabolites in Arabidopsis and tobacco expressing ISPS. Metabolite levels in leaves harvested from 42-d-old Arabidopsis empty vector control (EV-B3) and lines expressing ISPS (B2 and C4; A), and 42-d-old NE and IE tobacco (B), are shown. Arabidopsis plants were grown in a growth chamber under a light intensity of 200 μmol m⁻² s⁻¹. Tobacco was grown in the greenhouse. CDP-ME, Diphosphocytidylyl methylerythritol; DXP, deoxyxylulose 5-phosphate; HMBDP, hydroxymethylbutenyl diphosphate. Lowercase letters indicate significant differences among the three Arabidopsis lines or between NE and IE at P < 0.05 using one-way ANOVA followed by Fisher’s lsd test (i.e. there were no differences). Values represent means ± se. For Arabidopsis, n ≥ 5 plants per line; for tobacco, n = 7. Because DMADP + IDP were measured by postillumination isoprene emission, there are no data for Arabidopsis EV-B3 and tobacco NE lines.

Figure 7.

Leaf growth in Arabidopsis expressing ISPS. A comparison of rosette size reflecting projected leaf area (A–C) and leaves after being separated from the rosettes reflecting apparent total leaf area (D–F) in 35-d-old Arabidopsis empty vector control (EV-B3) and lines expressing ISPS (B2 and C4) are shown. For A to F, all plants were grown in growth chambers under a light intensity of 200 μmol m⁻² s⁻¹. G shows a comparison of total leaf area of all lines used in this study. Plants used in G were 50 d old and grown at 120 μmol m⁻² s⁻¹. In G, Arabidopsis lines expressing ISPS that are significantly different from Col-0 and empty vector lines EV-A2 and EV-B3 are marked with asterisks: *, P < 0.05 and **, P < 0.01 using one-way ANOVA followed by Fisher’s lsd test.

Figure 8.

Comparison of photosynthetic pigment concentrations in Arabidopsis expressing ISPS. Concentrations of Chl a (A), Chl b (B), and total Chl (C), Chl a/b ratio, and carotenoid concentration (E) in leaves harvested from 49-d-old Arabidopsis wild-type Col-0, empty vector controls (EV-A2, EV-B3, and EV-F1), and lines expressing ISPS (A1, F2, C1, C4, and B2) are shown. All transgenic lines belonged to the T2 generation. Values represent means ± se (n = 4 or 5 plants per line). Arabidopsis lines expressing ISPS that are significantly different from Col-0 and empty vector lines EV-A2 and EV-B3 are marked with symbols: ⁺, P < 0.1 and *, P < 0.05 using one-way ANOVA followed by Fisher’s lsd test.

Figure 9.

Comparison of photosynthesis and isoprene emission under varying environmental conditions in NE and IE tobacco. Photosynthesis and isoprene emission measured simultaneously in 27-, 41-, and 54-d-old NE and IE tobacco (A), and responses of photosynthesis and isoprene emission to temperature (B), CO₂ (C), light (D), and darkness (E) in 43- to 47-d-old NE and IE tobacco, are shown. Measurements were taken at 30°C (A and C–E) and 800 μmol m⁻² s^-1 light intensity (A–C). In E, plants were under 800 μmol m⁻² s^-1 light before being exposed to sudden darkness. Values in A to D represent means ± se; values in E represent means (n = 5).

Figure 10.

Comparison of cotyledon and hypocotyl growth in NE and IE young tobacco seedlings. Cotyledon areas of NE and IE young tobacco seedlings grown in Suremix either in the greenhouse or in the growth chamber (under a light intensity of 200 μmol m⁻² s⁻¹) at 12 DAS (A), and representative photographs of cotyledons (B) and hypocotyls (C) of tobacco grown hydroponically in rock wool in 2-mL tubes in a chamber with a light intensity of 200 μmol m⁻² s⁻¹ at 10 DAS, are shown. Different lowercase letters indicate significant differences between NE and IE at P < 0.05 using one-way ANOVA followed by Fisher’s lsd test. Values in A represent means ± se (n ≥ 12 plants per line).

Figure 11.

Comparison of leaf and plant growth over time in NE and IE tobacco. Photographs depict the front and top views of tobacco plants taken at day 27 (A and B), day 41 (C and D), and day 54 (E and F) of plant growth. In G, the shape of leaf tips of the sixth mature leaf is compared.

Figure 12.

Proposed model for how isoprene signaling can affect GA-mediated growth regulation and JA-mediated defense responses. Transcriptomic data revealed that isoprene can alter genes required for both GA accumulation and JA synthesis (genes shown in green font), which can promote both GA-mediated growth and JA-mediated defense simultaneously. We speculate that the observed growth enhancement in Arabidopsis engineered to emit isoprene is likely a result of the up-regulation of PIF. However, interactions between GA and JA pathways occur through DELLA and JAZ proteins (Campos et al., 2016). JA synthesis leads to the degradation of JAZ proteins that release the inhibition of transcription factors to enhance defense-related processes (Campos et al., 2016). Antagonistic interactions between JAZ and DELLA proteins play a part in regulating the growth-defense tradeoff mediated by GA and JA (Campos et al., 2016). Therefore, one possible explanation for the observed variations in isoprene-mediated growth effects in different species is the likely effect of isoprene on the growth-defense tradeoff. Genes belonging to other signaling pathways whose expression was altered by isoprene were omitted from this diagram for the sake of simplicity. Dotted lines and genes written in green font denote isoprene-responsive gene expression revealed during this study. Asterisks denote genes differentially expressed in both Arabidopsis expressing ISPS and Arabidopsis fumigated by isoprene but not differentially expressed in tobacco. Up-regulation and down-regulation of gene expression are denoted by pointed and blunt-ended arrows, respectively (DFL2 and TEM1 expression was down-regulated in the presence of isoprene in Arabidopsis). Solid lines denote signaling pathways that are well established. BBD, Bifunctional nuclease in basal defense response; CBF, CRT/DRE-binding factor; COP1, Constitutively photomorphogenic1, a ubiquitin ligase; CRPK1, Cold-responsive protein kinase1; DELLA, PIF transcription factor repressors; DFL2, Dwarf in light2, a GH3-related (auxin response-related) protein; FT, Flowering locus T; HY5, Elongated hypocotyl5; JAZ, Jasmonate ZIM-domain repressors; JMT, Jasmonic acid carboxyl methyltransferase; LOX, Lipoxygenase; MARD1, Mediator of ABA-regulated dormancy1, a novel zinc finger protein; MYB59, MYB domain protein59; MYC2, a basic helix-loop-helix transcription factor and a master regulator of JA signaling; OPR3, Oxophytodienoate-reductase3; TEM1, Tempranillo1, a RAV transcription factor; TZF5,Tandem CCCH zinc finger protein5; 14-3-3, highly conserved acidic proteins of the 14-3-3 protein family.