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StrigoQuant: A genetically encoded biosensor for quantifying strigolactone activity and specificity

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Science Advances  04 Nov 2016:
Vol. 2, no. 11, e1601266
DOI: 10.1126/sciadv.1601266
  • Fig. 1 StrigoQuant design, characterization, and analysis of receptor complex requirements.

    (A) General structure and configuration of the canonical SLs, strigol and orobanchol. Note the stereocenters at the BC-junction (carbons 3a and 8b) and in the D-ring (carbon 2′). (B) Scheme of the SL perception machinery in Arabidopsis. In the presence of SLs, receptor complex formation takes place, with SLs binding to the receptor protein AtD14, with recruitment of SMXLs to the MAX2 and SKP1/CUL1/F-box E2 ubiquitin ligase complex (SCFMAX2). SMXLs are ubiquitinated (U) and consequently degraded by the 26S proteasome. (C) StrigoQuant construct expressing a renilla luciferase (REN; green) connected via a 2A peptide to the sensor module (SM), AtSMXL6 (SMXL6), fused to a firefly luciferase (FF; yellow), under the control of a constitutive 35S promoter. The 2A peptide in the synthetic construct leads to stoichiometric coexpression of REN (normalization element) and SM (SMXL6-FF). Upon the addition of SLs, SMXL6-FF becomes ubiquitinated and degraded, whereas REN expression remains constant, leading to a decrease in the FF/REN ratio.

  • Fig. 2 StrigoQuant biosensor for the study of the SL perception machinery.

    (A) StrigoQuant biosensor for the study of the SL perception machinery Characterization of the StrigoQuant sensor construct in protoplasts isolated from WT [Columbia (col-0), ecotype] and max2 (col) mutant Arabidopsis backgrounds upon addition of increasing concentrations of rac-GR24 (left). Protoplasts were isolated from the seedlings of each genotype and transformed with StrigoQuant. Twenty-four hours after transformation, protoplasts were supplemented with increasing concentrations of a rac-GR24 serial dilution for 2 hours before luciferase activity determination. On the right, the schematic principle of the experiment is shown, where the functionality of the F-box protein MAX2 for SL-mediated SMXL6 degradation was analyzed. (B) Characterization of the StrigoQuant sensor construct in protoplasts isolated from WT and mutant Arabidopsis backgrounds for potential SL receptors upon addition of increasing concentrations of rac-GR24 (left). Protoplasts were isolated from Atd14 (col), dlk2 (col), WT [Landsberg erecta (ler), ecotype], and kai2 (ler) seedlings and transformed with StrigoQuant. Twenty-four hours after transformation, protoplasts were supplemented with increasing concentrations of a rac-GR24 serial dilution for 2 hours before luciferase activity determination. On the right, the schematic principle of the experiment is shown, where the functionality of AtD14, DLK2, and KAI2 in mediating SMXL6 degradation was tested. (C) Characterization of the effect of overexpression of OsD14 in Atd14 (col) protoplasts. Protoplasts isolated from Atd14 (col) seedlings were transformed either only with StrigoQuant and a stuffer plasmid (pGEN16; with equal amounts of 15 μg each) or with StrigoQuant and a plasmid harboring OsD14 (with equal amounts of 15 μg each) and induced with rac-GR24 for 2 hours before luminescence determination. On the right, a schematic principle of the experiment is shown, where OsD14 was overexpressed in protoplasts lacking AtD14 to rescue functional SMXL6 degradation. Results for each panel are averaged FF/REN ratios, normalized to the sample without addition of any inducer substrate for each genotype. The data shown correspond to one representative experiment of four replicated experiments for (A) and (B) and three experiments for (C). Error bars represent SEM from the individual experimental data shown. n = 6. Statistical significance between the tested concentrations within a genotype is indicated with lowercase letters above each bar, where “a” significantly differs from “b,” “b” from “c,” and so on. One-way analysis of variance (ANOVA), P < 0.01.

  • Fig. 3 Specificity and sensitivity of the sensor to strigol- and orobanchol-like SLs.

    WT Arabidopsis protoplasts transformed with StrigoQuant were induced with racemic mixtures of strigol-like (rac-strigol and rac-5DS) and orobanchol-like (rac-orobanchol and rac-4DO) SLs for 2 hours before luminescence activity determination. Results are averaged FF/REN ratios, normalized to the sample without addition of inducer for each substrate tested. The data shown correspond to one representative experiment of three replicated experiments. Error bars represent SEM from the individual experimental data shown. n = 6. Statistical significance between the tested concentrations for each substrate is indicated with lowercase letters above each bar, where “a” significantly differs from “b,” “b” from “c,” and so on. One-way ANOVA, P < 0.01.

  • Fig. 4 Stereoselectivity analysis of SL species.

    (A) Chemical structures of enantiomers of selected SLs. The differential stereochemistry at the carbon 2′ in the D-ring is highlighted (gray boxes). (B) WT protoplasts were transformed with StrigoQuant and induced with the individual enantiomers GR24 and ent-GR24, 5DS and ent-5DS, and 4DO and ent-4DO for 2 hours before luminescence activity determination. Dose-response curves for substrates with the 2′R (+) D-ring configuration are shown with dashed lines, whereas those for 2′S (−)–configured substrates are in solid black. The data shown correspond to one representative experiment of three replicated experiments. Results are averaged FF/REN ratios, normalized to the sample without addition of inducer for each substrate tested. Error bars represent SEM from the individual experimental data shown. n = 6.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/11/e1601266/DC1

    fig. S1. CtrlQuant activity upon incubation with racemic SLs in WT and mutant Arabidopsis protoplasts.

    fig. S2. SL-dependent degradation of the SMXL6-FF sensor component is mediated by the 26S proteasome.

    fig. S3. Specificity and sensitivity of StrigoQuant to 5DS.

    fig. S4. Stereoselectivity analysis of SL species with CtrlQuant.

    table S1. Amino acid sequences of the components of the StrigoQuant and CtrlQuant sensors.

    table S2. Oligonucleotides used for the cloning of the sensor constructs.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. CtrlQuant activity upon incubation with racemic SLs in WT and mutant Arabidopsis protoplasts.
    • fig. S2. SL-dependent degradation of the SMXL6-FF sensor component is mediated by the 26S proteasome.
    • fig. S3. Specificity and sensitivity of StrigoQuant to 5DS.
    • fig. S4. Stereoselectivity analysis of SL species with CtrlQuant.
    • table S1. Amino acid sequences of the components of the StrigoQuant and CtrlQuant sensors.
    • table S2. Oligonucleotides used for the cloning of the sensor constructs.

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