Inhibitor-induced oxidation of the nucleus and cytosol in Arabidopsis thaliana: implications for organelle to nucleus retrograde signalling

Abstract

Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction-oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1, WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.

Keywords: alternative oxidase; antimycin A; lincomycin; norflurazon; nuclear-encoded mitochondrial proteins; photosynthesis-associated nuclear genes.

Figure 1.

Typical examples of ro-GFP2 fluorescence images of the root and cotyledons of A. thaliana seedlings in the absence of inhibitors. Root tip (a), leaf epidermal cells (b) and stomata guard cell (c). In each case, the reduced form (left panels) was determined following incubation with 5 mM DTT, and the oxidized form was determined following incubation with 2 mM hydrogen peroxide (middle panels). The overlaid images of reduced and oxidised forms (right panels) are shown for completeness. Wide arrows indicate nuclei and thin arrows indicate cytosol. Scale bar, 25 µm.

Figure 2.

The 405/488 nm fluorescence ratios (a), the degree of oxidation (b) and the glutathione redox potential (c) measured in the cytosol (dark bar) and in the nuclei (grey bar) of the stomatal cells of seedlings grown for 5 days either in the absence (CTR) or the presence of LINC, NF, LINC + NF or ANT. Data are the mean ± s.d. Asterisks indicate the statistical significance of p-value: *p ≤ 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

The 405/488 nm fluorescence ratios (a), the degree of oxidation (b) and the glutathione redox potential (c) measured in the cytosol (dark bar) and in the nuclei (grey bar) of the epidermal cells of the cotyledons of seedlings grown for 5 days either in the absence (CTR) or presence of LINC, NF, LINC + NF or ANT. Data are the mean ± s.d. For statistical significance of p-values, see figure 2.

Figure 4.

The 405/488 nm fluorescence ratios (a), the degree of oxidation (b) and the glutathione redox potential (c) measured in the cytosol (dark bar) and in the nuclei (grey bar) of the root tips of seedlings grown for 5 days either in the absence (CTR) or the presence of LINC, NF, LINC + NF or ANT. Data are the mean ± s.d. For statistical significance of p-values, see figure 2.

Figure 5.

The relative abundance of transcripts in 5-day-old seedlings grown for 5 days either in the absence or presence of inhibitors of chloroplast (a) or mitochondrial (b) functions. In (a), seedlings were either grown in the absence of inhibitors (black bars) or in the presence of LINC (grey bars), NF (white bar with black border) or LINC + NF (white bars with grey border), In (b), seedlings were either grown in the absence of inhibitors (black bars) or in the presence of ANT (grey bars), SHAM (white bar with black border), or SHAM plus ANT (white bar with grey border). Data are expressed relative to UBIQUITIN 10. Data are the mean ± s.d. For statistical significance of p-values, see figure 2. (a) LHCA1, LIGHT-HARVESTING CHLOROPHYLL A BINDING PROTEN 1; LHCB1, LIGHT-HARVESTING CHLOROPHYLL A BINDING B1; AOXA1, ALTERNATIVE OXIDASE A1; (b) WHIRLY1 (WHY1, WHIRLY1; WHY3, WHIRLY3; LEA5, LATE EMBRIOGENESIS 5; RRTF1, REDOX-REGULATED TRANSCRIPTION FACTOR 1.