Dual role of the plastid terminal oxidase in tomato

Abstract

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase whose absence in tomato (Solanum lycopersicum) results in the ghost (gh) phenotype characterized by variegated leaves (with green and bleached sectors) and by carotenoid-deficient ripe fruit. We show that PTOX deficiency leads to photobleaching in cotyledons exposed to high light primarily as a consequence of reduced ability to synthesize carotenoids in the gh mutant, which is consistent with the known role of PTOX as a phytoene desaturase cofactor. In contrast, when entirely green adult leaves from gh were produced and submitted to photobleaching high light conditions, no evidence for a deficiency in carotenoid biosynthesis was obtained. Rather, consistent evidence indicates that the absence of PTOX renders the tomato leaf photosynthetic apparatus more sensitive to light via a disturbance of the plastoquinone redox status. Although gh fruit are normally bleached (most likely as a consequence of a deficiency in carotenoid biosynthesis at an early developmental stage), green adult fruit could be obtained and submitted to photobleaching high light conditions. Again, our data suggest a role of PTOX in the regulation of photosynthetic electron transport in adult green fruit, rather than a role principally devoted to carotenoid biosynthesis. In contrast, ripening fruit are primarily dependent on PTOX and on plastid integrity for carotenoid desaturation. In summary, our data show a dual role for PTOX. Its activity is necessary for efficient carotenoid desaturation in some organs at some developmental stages, but not all, suggesting the existence of a PTOX-independent pathway for plastoquinol reoxidation in association with phytoene desaturase. As a second role, PTOX is implicated in a chlororespiratory mechanism in green tissues.

Figure 1.

Pigment content of cotyledons from SM and gh tomato lines. Seedlings were grown in darkness for 4 and 6 d after seed imbibition to obtain white (A) and pale yellow (B) cotyledons (T0 time point), respectively. Seedlings were then transferred to either low light (20 μmol m⁻² s⁻¹ PAR) or to a higher light intensity (200 μmol m⁻² s⁻¹ PAR). Samples were analyzed after 24 h of light for white cotyledons and after 6 h of light for yellow cotyledons. Eight cotyledons were pooled for a given measurement. Each measurement was repeated three times with a different pool of cotyledons. ses of the mean values were below 12% (data not shown). Whether changes are statistically significant is discussed in the “Results” section. PAR, Photosynthetically active radiation.

Figure 2.

Bleaching of SM and gh adult leaves. A, Carotenoid content in SM (1) and different types of gh leaves (2–4) as shown in the inserted photograph: 2, green gh; 3, variegated; 4, mainly white. For details, see “Materials and Methods.” Statistically significant differences are indicated compared to SM leaves: *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, Chlorophyll content in the four leaf types. C, Phytoene content in the four leaf types. D, HPLC elution profiles of pigments (A₂₈₇). The top trace shows an extract from sectors bleaching after 24 h on green gh leaves submitted to high light conditions (1,000 μmol m⁻² s⁻¹), as shown in the inset photograph. The bottom trace shows an extract from mainly white gh leaves, as shown in A. Thick arrows point to the two major phytoene isomers that are quantified in C; the thin arrow points to a minor phytoene isomer.

Figure 3.

PTOX protein levels in SM leaves. A, Immunodetection of PTOX and PSI-D (a PSI protein used as a control for equal gel loading). Plants were grown under low light (60 μmol m⁻² s⁻¹) and at T0 detached leaves were incubated during a 16-h time course under low (60 μmol m⁻² s⁻¹) or high (1,000 μmol m⁻² s⁻¹) light. Leaf protein samples were separated by gel electrophoresis and immunodetected as described in “Materials and Methods.” B, Densitometric scanning values of the PTOX band in A after normalization using the PSI-D values as a standard.

Figure 4.

Photoinhibition of SM and gh fully green leaves. The maximal quantum yield of PSII is expressed as the F_v/F_m ratio. SM and gh leaves were incubated at 24°C or 15°C for 6 h under either low (60 μmol m⁻² s⁻¹) or high light (1,000 μmol m⁻² s⁻¹) and then kept for 30 min in the dark before chlorophyll fluorescence measurement. Mean values of six measurements (i.e. six different leaves) are shown.

Figure 5.

Damage to PSII and oxidative stress in SM and gh fully green leaves. A, Lipid peroxidation in SM (black columns) and gh (white columns) leaves. Plants were exposed to high light stress (1,000 μmol m⁻² s⁻¹) for 6 h. Lipid peroxidation was estimated using the MDA method. Data are mean values of a minimum of five experiments. Statistically significant differences are indicated compared to T0: *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, Thermoluminescence (TL) measurements in SM (top) and gh (bottom) leaf discs from control leaves (low light) or leaves treated for 6 h under high light (1,000 μmol m⁻² s⁻¹) or leaves treated under the same high light conditions but allowed to recover for 24 h under the light conditions used for the control leaves. Samples were then treated as described in “Materials and Methods,” cooled to 2°C, and flash illuminated to induce charge separation. The samples were then heated to induce charge recombination of PSII radical pairs (2°C–60°C; the B band represents the thermoinduced light emission from the recombination of S₂Q_B⁻ radical pairs after one light flash) and chemiluminescent reactions (70°C–180°C) representing the chemical stress status (HTL2 band). n.s., Not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to control). TL measurements were performed under N₂ atmosphere to avoid autooxidation at high temperatures. There was no influence on the B band by N₂ flushing compared to air (data not shown).

Figure 6.

Fast fluorescence kinetics (OJIP) for fully green leaves from gh and SM plants pulse illuminated after different times of dark adaptation. The O level at 0.01 ms represents a fluorescence value close to F₀. The fluorescence rise from the O to the J level (2 ms) indicates the reduction of Q_A. The fluorescence rise from the J to the I level (30 ms) indicates the reduction of Q_B and partially of the PQ pool. The rise from the I to the P level (200–300 ms) indicates the electron flow through PSI (PQ pool fully reduced at P level).

Figure 7.

Induction kinetics for the chlorophyll fluorescence parameters ΦPSII (A), 1 − qP (B), and qN (C). SM and green gh leaves were dark adapted 30 min and then illuminated with actinic light of 200 μmol m⁻² s⁻¹ for 10 min. Mean values (n = 6) were used.

Figure 8.

Pigment content of mature and ripe SM and gh fruits. The colored carotenoid (A), chlorophyll (B), and phytoene (C) contents were analyzed in mature green (adult size) fruit. The phytoene (D) and colored carotenoid (E) contents were analyzed in ripe fruit (10 d after the breaker stage). Samples were taken from SM green and red fruit (histogram columns indicated by 1), gh green and their derived orangey fruit (2), and gh bleached and their derived yellow fruit (3). Statistically significant differences are indicated compared to SM fruit: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 9.

Photoinhibition of SM and gh green fruit. The maximal quantum yield of PSII is expressed as the F_v/F_m ratio. Fruit pericarp of SM and gh were incubated at 24°C or 15°C for 6 h under either low (60 μmol m⁻² s⁻¹) or high light (1,000 μmol m⁻² s⁻¹) and then kept 30 min in the dark before chlorophyll fluorescence measurement. Mean values are shown (n = 3).