Abscisic acid and other plant hormones: Methods to visualize distribution and signaling

Abstract

The exploration of plant behavior on a cellular scale in a minimal invasive manner is key to understanding plant adaptations to their environment. Plant hormones regulate multiple aspects of growth and development and mediate environmental responses to ensure a successful life cycle. To monitor the dynamics of plant hormone actions in intact tissue, we need qualitative and quantitative tools with high temporal and spatial resolution. Here, we describe a set of biological instruments (reporters) for the analysis of the distribution and signaling of various plant hormones. Furthermore, we provide examples of their utility for gaining novel insights into plant hormone action with a deeper focus on the drought hormone abscisic acid.

Keywords: abscisic acid; fluorescent reporter; in vivo visualization; plant hormone.

Figure 1

Schematic presentation of the FRET-based ABA reporters ABACUS1-2µ (A) and ABAleon2.1 (B). As FRET donor and acceptor pair, ABACUS1-2µ uses the enhanced dimerization CFP- and YFP-variants edCerulean and edCitrine. ABAleon2.1 encompasses mTurquoise and Venus circularly permutated at amino acid 173 (cpVenus173) as FRET-pair. The N- and C-terminal orientation of the respective fluorescent protein is interchanged in both reporters. The sensory modules consist of a full length ABA receptor protein (PYL1 with the H87P mutation in ABACUS1-2µ and PYR1 in ABAleon2.1) fused to a fragment of the type 2C protein phosphatase (PP2C) ABI1. ABACUS1-2µ harbors 49 amino acids of the ABI1 phosphatase domain (amino acids H279-D327), while ABAleon2.1 contains the complete phosphatase domain (amino acids S125-N434), but with a D413L mutation that abolishes phosphatase activity. The orientation between PYR1/PYL1 and the ABI1 fragment is interchanged in both reporters. PYR1/PYL1 and ABI1 are linked through a 52 amino acid spring linker in ABACUS1-2 µ, and through a flexible 29 amino acid glycine-serine-(GS)-linker in ABAleon2.1. ABACUS1-2µ was generated using the gateway-cloning strategy, therefore the sensory module is linked to the fluorescent protein pair via 13 amino acid attB1 and attB2 sites. ABAleon2.1 was generated using classical cloning and linked to the fluorescent proteins through GP and PG linkers encoded in the Apal and XmaI restriction sites. Informations about both reporters are derived from [82, 83].

Figure 2

ABAleon2.1 plants exhibit a growth phenotype and a reduced sensitivity to ABA. A: Twenty-five-day-old plants grown in parallel in a growth chamber at a relative humidity of ~40%. B: Four-day-old seedlings were transferred to 0.5 MS agar plates supplemented with 0 µM ABA (top row) or 30 µM ABA (bottom row) and grown for 5 additional days. C and D: Quantification of growth parameters (C) fresh weight and (D) root growth of seedlings presented in (B) 5 days after transfer to media supplemented with 0 µM ABA (blue bars) or 30 µM ABA (red bars). Data represent means ±SEM of n = 5 experiments with seven seedlings per experiment. Plants were grown and analyzed as described in [83].

Figure 3

Distribution of (A) [ABA] and (B) ABA signaling. Five-day-old seedlings of (A) ABAleon2.1 line 10 and (B) pRAB18-GFP were imaged before (left panel) or 2 hours (ABAleon2.1) or 4 hours (pRAB18-GFP) after application of 50 µM ABA (right panel). Shown are manually assembled emission ratio (ABAleon2.1 – reports [ABA]) or background subtracted fluorescence emission (pRAB18-GFP – reports ABA signaling) images calibrated to the respective calibration bar. Blue color indicates high [ABA] or signaling and red indicates low [ABA] or signaling. Background colors are according to the respective calibration bars [values are ~1 in (A) and 0 in (B)]. Plants were grown, imaged, and analyzed as described in [83], except that imaging of pRAB18-GFP was conducted using a 71012pH sensitive GFP filter set (CHROMA Technology Corp.).

Figure 4

ABA signaling in response to salt and osmotic stress. Fluorescence emission of the expression-based ABA signaling reporter pRAB18-GFP in (A and B) the hypocotyl, (C and D) the root maturation zone, and (E and F) the early maturation zone, elongation zone, and division zone in response to 6 hours control, 10 µM ABA, 100mM NaCl (salt stress), and 300 µM sorbitol (osmotic stress) treatments. Images were calibrated according to the adjacent calibration bar. B, D, and F: Background subtracted fluorescence emission values measured from two color-coded regions depicted in the right images of (A, C, and E) and normalized to the control. Data represent means ± SEM of n = 8 seedlings. The indicated color-coded regions are representative for regions analyzed in all acquired images with identical positions on the Y-axis.