Self-reporting Arabidopsis expressing pH and [Ca2+] indicators unveil ion dynamics in the cytoplasm and in the apoplast under abiotic stress

Abstract

For noninvasive in vivo measurements of intra- and extracellular ion concentrations, we produced transgenic Arabidopsis expressing pH and calcium indicators in the cytoplasm and in the apoplast. Ratiometric pH-sensitive derivatives of the green fluorescent protein (At-pHluorins) were used as pH indicators. For measurements of calcium ([Ca(2+)]), luminescent aequorin variants were expressed in fusion with pHluorins. An Arabidopsis chitinase signal sequence was used to deliver the indicator complex to the apoplast. Responses of pH and [Ca(2+)] in the apoplast and in the cytoplasm were studied under salt and "drought" (mannitol) stress. Results are discussed in the frame of ion flux, regulation, and signaling. They suggest that osmotic stress and salt stress are differently sensed, compiled, and processed in plant cells.

Figure 1.

Schematic structures of the gene cassettes. A, Fusion construct from pCM2. B, Apoplast targeting construct: insertion of chitinase signal sequences (66 bp) upstream of GFP5 giving pchitGFP5:AQ. C, Fragment exchange (Cla-Xho) to yield pH sensitivity and (XhoI-PstI) for insertion of low-affinity AQ (LAAQ), leading to pchitratioGFP:LAAQ. D, Same as C but insertion of ecliptic pHluorin fragment giving pchiteclipGFP:LAAQ. E and F, Replacement of Cla-Xho fragment for introducing pH sensitivity and introduction of point mutation for increased solubility. This produced cytoplasmic expression of ratioGFP (E) and ecliptic green fluorescent protein (GFP; F), i.e. psmratioGFP:AQ and psmeclipGFP:AQ, respectively.

Figure 2.

A, Excitation spectra of ratiometric GFP at different pHs (emission wavelength = 508 nm). Curves were normalized by F(428ex; 508em). B, Corresponding calibration curve for fluorescence excitation ratio R(395ex/475ex; 508em); dissociation constant, i.e. midpoint of the fitted curve = pK_d = 6.9 ± 0.03; optimal dynamic range for pH measurements is between 5.6 and 8.0.

Figure 3.

A, Emission spectra of ecliptic GFP at different pHs (excitation wavelength = 400 nm). Curves were normalized by F(400ex; 490em). B, Corresponding calibration curve for fluorescence emission ratio R(400ex; 490em/510em); dissociation constant, i.e. midpoint of the fitted curve = pK_d = 7.25 ± 0.02; optimal dynamic range for pH measurements is between 6.5 and 8.0.

Figure 4.

Development of basal AQ luminescence during in vivo reconstitution of Arabidopsis roots with different coelenterazine (CTZ) derivatives. Squares, Reconstitution with native CTZ; circles, reconstitution with cp-CTZ. Addition of CTZ at t = 0.6 h to 10 μm final concentration. Note: Absolute luminescence (×10,000) is given here, which is luminescence of each integration interval divided by total luminescence produced by the specimen. Relative luminescence (×10,000) is given in all other figures, which is luminescence of each integration interval divided by luminescence still remaining in the specimen. A, Reconstitution of AQ expressed in the cytoplasm. B, Reconstitution of AQ targeted to the apoplast.

Figure 5.

Luminescence response to cold from Arabidopsis expressing AQ in the cytoplasm (A and B) and in the apoplast (C and D). A and C, Relative luminescence; B and D, corresponding temperature measured in parallel.

Figure 6.

In vivo spectra of ratioGFP expressed in Arabidopsis roots (black curve, cytoplasmic expression; gray curve, apoplastic expression). Curves were normalized by F(428ex; 530em).

Figure 7.

pH changes in the apoplast (gray curve) and the cytoplasm (black curve) of Arabidopsis roots in response to changes in external pH. External pH was adjusted with 10 mm HEPES (pH 7.6) and 10 mm MES (pH 6.0) in standard medium (KCl, CaCl₂, and MgCl₂: 1 mm each).

Figure 8.

A, Cytoplasmic and apoplastic [Ca²⁺] in response to “drought” stress (200 mm mannitol versus water) measured by AQ luminescence from Arabidopsis roots. B, Cytoplasmic and apoplastic [Ca²⁺] in response to NaCl stress (100 mm NaCl versus water). C and D, Close-ups of A, and B, respectively.

Figure 9.

Calcium responses upon drought and NaCl stress (200 mm mannitol versus 100 mm NaCl versus water) in Arabidopsis roots.

Figure 10.

A, Cytoplasmic and apoplastic pH responses upon drought stress (water versus 200 mm mannitol) in Arabidopsis roots measured by fluorescence ratiometry. B, Cytoplasmic and apoplastic pH responses upon NaCl stress (water versus 100 mm NaCl) in Arabidopsis roots.