EBSn, a robust synthetic reporter for monitoring ethylene responses in plants

Abstract

Ethylene is a gaseous plant hormone that controls a wide array of physiologically relevant processes, including plant responses to biotic and abiotic stress, and induces ripening in climacteric fruits. To monitor ethylene in plants, analytical methods, phenotypic assays, gene expression analysis, and transcriptional or translational reporters are typically employed. In the model plant Arabidopsis, two ethylene-sensitive synthetic transcriptional reporters have been described, 5xEBS:GUS and 10×2EBS-S10:GUS. These reporters harbor a different type, arrangement, and number of homotypic cis-elements in their promoters and thus may recruit the ethylene master regulator EIN3 in the context of alternative transcriptional complexes. Accordingly, the patterns of GUS activity in these transgenic lines differ and neither of them encompasses all plant tissues even in the presence of saturating levels of exogenous ethylene. Herein, we set out to develop and test a more sensitive version of the ethylene-inducible promoter that we refer to as EBSnew (abbreviated as EBSn). EBSn leverages a tandem of ten non-identical, natural copies of a novel, dual, everted, 11bp-long EIN3-binding site, 2EBS(-1). We show that in Arabidopsis, EBSn outperforms its predecessors in terms of its ethylene sensitivity, having the capacity to monitor endogenous levels of ethylene and displaying more ubiquitous expression in response to the exogenous hormone. We demonstrate that the EBSn promoter is also functional in tomato, opening new avenues to manipulating ethylene-regulated processes, such as ripening and senescence, in crops.

Figure 1.. Graphical representation of ethylene-inducible proximodistal promoters and transcriptional reporters utilized in this work.

A) Simplified representation for the size and number of EIN3 binding site (EBS) elements within different ethylene-inducible promoters experimentally tested in this study. Two of the EBS versions have previously been described, 5xEBS (Stepanova et al 2001) and 10×2EBS-S10 (Fernandez-Moreno et al. 2024), and two were generated and characterized in this work, EBSnew (EBSn) and mutatedEBSnew (mEBSn). B) Schematics of the transcriptional reporters harboring the EBS-containing promoters shown in panel A. For every reporter but (3), the GoldenBraid (GB) grammar for each assembled phytobrick is represented in purple at the flanks and between DNA parts marked with square brackets beneath the graphical representations. Reporters (1), (2), (4) and (5) harbors five phytobricks: [A1-A2] distal promoter (EBSn, 10×2EBS-S10 or mEBSn), [A3-B1] core promoter (mini35S/mini35S*), [B2] nuclear localization signal (3xSV40-NLS), [B3-B5] reporter CDS (3xYPet or intronless--GUS), and [C1] terminator (term35S) (Supplementary Table S1A). Reporter (6) lacks a subcellular localization signal (no 3xSV40-NLS) and harbors a [B2-B5] GUS-1xYPet double reporter assembled from a [B2] intronless-stopless--GUS CDS and a [B3-B5] (Gly-Gly-Gly-Gly-Ser)2 linker-1xYPet phythobricks (Supplementary Table S1A). Finally, reporter (3) represents the original 5xEBS reporter (Stepanova 2001) which harbors a distal promoter with five EBS elements in tandem spaced by a BamH/BgIII cloning scare, a core promoter (mini35S*), an intronless GUS CDS reporter, and a terminator (term35S). Core promoters mini35S (54bp long) and mini35S* (60bp long) have 49bp in common, with variable 5bp flanks (Supplementary Fig. S1C).

Figure 2.. The EBSn:3xYPet reporter is induced by ethylene and ACC in three-day-old dark-grown Arabidopsis seedlings.

A) Whole-plant fluorescence images of wild-type (WT) and transgenic (EBSn:3xYPet, lines A and C) seedlings germinated in AT media in the presence of hydrocarbon-free air (AIR) or 10 ppm ethylene (ETH). B) Close-up apical hook and root tip images of seedlings from panel A. AIR and ETH images are shown, along with ENHANCED AIR images that were digitally enhanced (0.1% contrast enhancement, CE, for apical hooks and 2–20 min-max brightness/contrast, B/C, for root tips in ImageJ v1.52, Fiji 2015) to show nuclear localization of 3xYPet in apical hooks and a more diffuse 3xYPet signal in root tips (arrowheads) not seen in WT plants. C), D) Confocal microscopy images displaying optical cross-sections (C) and Z-stacks (D) of propidium iodide-stained (pink) root tips of EBSn:3xYPet seedlings (lines A and C) germinated in AT media or AT media supplemented with 10 μM ACC.

Figure 3.. Robust fluorescence of EBSn-driven reporters in three-day-old dark-grown Arabidopsis seedlings is ethylene-mediated.

Seeds were germinated in AT plates in the presence of hydrocarbon-free air (AIR) or 10 ppm ethylene (ETH). A) The loss of functional EIN2 in ein2–5 EBSn:3xYPet (lines A and C) abolishes the ethylene-dependent triple response and YPet activity in 10 ppm ethylene, and the patterns of YPet fluorescence in EBSn:GUS-1xYPet dual reporter lines 1 (weak) and 4 (strong) are comparable to that of the EBSn:3xYPet lines A and C. B) Confocal images of the EBSn activity in the apical hooks of three-day-old dark-grown EBSn:3xYPet and EBSn:GUS-1xYPet seedlings in Arabidopsis. Seeds were germinated in AT plates in the presence of hydrocarbon-free air (AIR) or 10 ppm ethylene (ETH). Fluorescence and bright-field images are displayed.

Figure 4.. EBSn is more uniformly activated by ethylene in Arabidopsis seedlings than are other synthetic ethylene-regulated EBS promoters.

Three-day-old seedlings germinated in AT plates in the dark for three days in the presence of 200 ppm 1-MCP (ethylene receptor inhibitor), hydrocarbon-free air (AIR), or 10 ppm ethylene (ETH) were stained for GUS overnight and photographed. A) Expression of EBSn:GUS in representative moderate (4.8) and strong (2.6) lines. B), C), D) A side-by-side comparison of GUS expression in EBSn:GUS (lines 4.8 and 2.6), 5xEBS:GUS, 10×2EBS-S10:GUS, mEBSn:GUS, EBSn:GUS-1xYPet, ein2 EBSn:GUS (2.6), and WT Col-0 in 1-MCP (panel A), air (panel B) or ethylene (panel C). Scale bars represent 1mm.

Figure 5.. The EBSn promoter is active in soil-grown Arabidopsis plants.

A) The side-by-side comparison of GUS expression patterns of EBSn-driven reporters and other synthetic ethylene reporters in adult Arabidopsis plants. First column: 21-day-old rosettes (scale bar 5mm). Second column: inflorescences of 45-day-old plants (scale bar 1 mm). Third column: close-ups of mature flowers (scale bar 0.5mm). Fourth column: close-ups of young siliques (scale bar 1 mm). B) Developmental regulation of the EBSn:GUS reporter (lines 4.8 and 2.6) in rosette leaves. GUS staining of rosettes of 18-, 24-, and 31-day-old plants of the T5 generation was compared. The staining patterns are consistent with that observed in earlier generations of EBSn:GUS plants. Scale bars represent 10mm. No exogenous ethylene or ACC was provided. All samples were stained for GUS overnight.

Figure 6.. The EBSn:GUS reporter is expressed in tomato and inducible by exogenous ACC.

A) The EBSn promoter is active in three-day-old etiolated T2 tomato seedlings and strongly inducible by 1 μM ACC (equivalent to 0.1 μM ACC in Arabidopsis based on ACC/control relative root length). Two independent EBSn:GUS transgenic lines (0 and 13) germinated for three days in the dark in magenta boxes containing either plain AT or AT supplemented with ACC is displayed. GUS staining was performed overnight. B) EBSn:GUS is active in leaves and roots of three-week-old T2 tomato plants (lines 13 and 14) grown under long day conditions in soil. Seeds were germinated in AT media supplemented with 100μg/mL kanamycin (except for WT germinated in plain AT media) in the dark for three days and transferred to continuous light for an additional week. After a total of 10 days, seedlings were transferred to soil and grown at 22 °C in long day conditions (16-h light:8-h darkness) for four more days. GUS staining was performed overnight. C,D) The EBSn activity is strongly induced in the vasculature and rachis of detached three-week-old leaves of T2 tomato plants (lines 29 and 37) upon 24-hour exposure to 50 μM ACC. Leaves were detached, and the abaxial side of the leaf was imbibed for 24 hours in plain 0.6% (w/v) bactoagar or bactoagar supplemented with 1 μM ACC. Images of GUS staining (panel C) or 3xYPet fluorescence (panel D) are displayed. WT = Solanum lycopersicum M82.

Figure 7.. The EBSn activity in tomato fruits is enhanced upon ripening.

A) YPet fluorescence in T2 EBSn:3xYPet tomato lines 29 and 37 is weak in green fruits and strongly induced in red fruits. B) GUS staining in T2 EBSn:GUS tomato lines 13 and 14 is weak in green fruits and prominently induced in red fruits. Fruit slices were fixed and stained for GUS for one hour. C) Potential future application of the EBSn synthetic promoter. By expressing a negative regulator of ethylene signaling under the control of EBSn, it should be possible to counteract the effects of ripening-induced ethylene and avoid over-ripening. In developing green fruits, the ethylene production is low, the EIN3 transcription factor is destabilized, and the expression of a negative regulator such as EBF1/2 (or, alternatively, of an antisense version of a positive regulator such as EIN2) is off, and fruits develop normally. In maturing fruits, ethylene production and signaling are triggered at the onset of fruit ripening, thus stabilizing EIN3/EILs and activating the expression of the negative regulator (or the antisense version of the positive regulator), inhibiting further ethylene signaling, and preventing fruit over-ripening. As a result, transgenic fruits should develop normally until the boost in ethylene production at the onset of ripening, at which point ethylene would self-inhibit further ethylene signaling, delaying fruit decay. WT = Solanum lycopersicum M82.