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Fig. 1 | Plant Methods

Fig. 1

From: A foliar pigment-based bioassay for interrogating chloroplast signalling revealed that carotenoid isomerisation regulates chlorophyll abundance

Fig. 1

Optimisation of a pigment-based signalling bioassay in Arabidopsis detached rosettes. A Pathway for carotenoid biosynthesis and catabolism into an apocarotenoid signal (ACS) or phytohormone such as strigolactone (SL) and abscisic acid (ABA). Norflurazon (NFZ) inhibits PDS activity and aryl-C3N hydroxamic acid (D15) impairs CCD activity. Green arrows and red lines represent positive and negative regulation, respectively. Blue lines denote a pathway towards the generation of a carotenoid cleavage product specified in the grey box. Mutants used in this study include; ζ-carotene isomerase (z-iso) and carotenoid chloroplast regulatory 2 (ccr2), lutein deficient 2 (lut2), nonphotochemical quenching 1 (npq1), nonphotochemical quenching 2 (npq2), abscisic acid deficient 4 (aba4), more axillary branching 3 (max3). Abbreviations: GERANYLGERANYL PYROPHOSPHATE (GGPP), PHYTOENE SYNTHASE (PSY), PHYTOENE DESATURASE (PDS), ζ-CAROTENE DESATURASE (ZDS), ZETA-CAROTENE ISOMERASE (Z-ISO), CAROTENOID ISOMERASE (CRTISO), LYCOPENE EPSILON CYCLASE (LCY), and LYCOPENE BETA CYCLASE (bLCY), ZEAXANTHIN EPOXIDASE (ZE), VIOLAXANTHIN DEEPOXIDASE (VDE), NEOAXANTHIN SYNTHASE (NXS), CAROTENOID CLEAVAGE DIOXYGENASE (CCD). B Visual display of the whole rosette bioassay showing the rosettes incubating on kim wipes saturated with NFZ contained with a petri dish. The numbered rosette shows the leaf position by chronological age (1 to 15; oldest to youngest). Three week old Arabidopsis rosettes were treated with different NFZ concentrations (0–100 μM) for 24 h (DF) and various time points over a 24 h period (GI). Arabidopsis trays were kept in dark for 4–5 h before transferring Arabidopsis plants to NFZ under continuous light (130–150 µmol m−2 s−1, cool fluorescent lamps) at 22 °C. Mature (leaf 1–2; old) and recently emerged (leaf 9–11, young) leaves were collected after treatments and pigment levels quantified. Absolute concentrations of Phytofluene (C), Phytoene (D), Total carotenoids (E), and Total chlorophylls (F) in response to the different concentration of NFZ. Absolute concentrations of Phytoene (G), Total carotenoids (H), and Total chlorophylls (I) in response to the different NFZ incubation times over a 24 h period. Plots represent the mean values with standard error of means (n = 3–4; C) from a representative dataset of at least two independent experimental repetitions. Letter codes in the plots indicate the level of statistical variation (p < 0.05) in carotenoid content within and across the test groups determined by One-Way ANOVA adopting Holm-Sidak post-hoc multiple comparisons

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