Abstract
Abscisic acid (ABA) is a plant hormone that regulates plant growth and development and mediates abiotic stress responses. Direct cellular monitoring of dynamic ABA concentration changes in response to environmental cues is essential for understanding ABA action. We have developed ABAleons: ABA-specific optogenetic reporters that instantaneously convert the phytohormone-triggered interaction of ABA receptors with PP2C-type phosphatases to send a fluorescence resonance energy transfer (FRET) signal in response to ABA. We report the design, engineering and use of ABAleons with ABA affinities in the range of 100-600 nM to map ABA concentration changes in plant tissues with spatial and temporal resolution. High ABAleon expression can partially repress Arabidopsis ABA responses. ABAleons report ABA concentration differences in distinct cell types, ABA concentration increases in response to low humidity and NaCl in guard cells and to NaCl and osmotic stress in roots and ABA transport from the hypocotyl to the shoot and root. DOI: http://dx.doi.org/10.7554/eLife.01739.001.
Highlights
Plant hormones control plant growth and development
Based on structural analyses of PYRABACTIN RESISTANCE 1 (PYR1) (Nishimura et al, 2009) and the PYL1-Abscisic acid (ABA)-ABI1 complex (Miyazono et al, 2009) and a fluorescence resonance energy transfer (FRET) cassette consisting of the fluorescent proteins mTurquoise (Goedhart et al, 2010) and Venus circularly permutated at amino acid 173, FRET-based reporters for ABA, named ABAleons, were designed (Figure 1A)
Full length PYR1 and ABI1 truncated at amino acid S125 (ΔNABI1) were fused via a flexible ASGGSGGTS(GGGGS)4-linker (Arai et al, 2004; Nagai et al, 2004) and inserted into the mTurquoisecpVenus173 FRET cassette using short two amino acid GP- and PG-linkers (Piljić et al, 2011) resulting in the FRET reporter ABAleon1.1 (Figure 1A)
Summary
Plant hormones control plant growth and development. Knowledge of the locations of hormone synthesis and transport, and the resulting hormone gradients and distributions in plants is important for understanding how plants respond to their environment via hormone signaling and cross talk of hormone and other small molecule signaling pathways (Hetherington and Woodward, 2003; Israelsson et al, 2006; Nemhauser et al, 2006; Muday et al, 2012). Reporter genes developed for ABA-induced gene expression (pRD29A/B, pRAB18 and pAtHB6) are used (Lång and Palva, 1992; Yamaguchi-Shinozaki and Shinozaki, 1993; Ishitani et al, 1997; Christmann et al, 2005; Kim et al, 2011; Duan et al, 2013). Despite their potential, such promoter-linked reporters respond indirectly and slowly to their respective plant hormone. To unequivocally investigate dynamic models of hormone distribution and dissect the complex functions and interconnection of these signaling molecules, direct plant hormone reporters that act instantaneously and reversibly are essential
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