Chlororespiration and grana hyperstacking: how an Arabidopsis double mutant can survive despite defects in starch biosynthesis and daily carbon export from chloroplasts

Abstract

An Arabidopsis (Arabidopsis thaliana) double mutant impaired in starch biosynthesis and the triose phosphate/phosphate translocator (adg1-1/tpt-1) is characterized by a diminished utilization of photoassimilates and the concomitant consumption of reducing power and energy produced in the photosynthetic light reaction. In order to guarantee survival, the double mutant responds to this metabolic challenge with growth retardation, an 80% decline in photosynthetic electron transport, diminished chlorophyll contents, an enhanced reduction state of plastoquinone in the dark (up to 50%), a perturbation of the redox poise in leaves (increased NADPH/NADP ratios and decreased ascorbate/dehydroascorbate ratios), hyperstacking of grana thylakoids, and an increased number of plastoglobules. Enhanced oxygen consumption and applications of inhibitors of alternative mitochondrial and chloroplast oxidases (AOX and PTOX) suggest that chlororespiration as well as mitochondrial respiration are involved in the enhanced plastoquinone reduction state in the dark. Transcript amounts of PTOX and AOX were diminished and nucleus-encoded components related to plastidic NADH reductase (NDH1) were increased in adg1-1/tpt-1 compared with the wild type. Cytochrome b559, proposed to be involved in the reoxidation of photosystem II, was not regulated at the transcriptional level. The hyperstacking of grana thylakoids mimics adaptation to low light, and increased plastoglobule numbers suggest a response to enhanced oxidative stress. Altered chloroplast organization combined with perturbations in the redox poise suggests that adg1-1/tpt-1 could be a tool for the in vivo study of retrograde signaling mechanisms controlling the coordinated expression of nucleus- and plastome-encoded photosynthetic genes.

Figure 1.

Typical traces of modulated Chl a fluorescence determined with adg1-1/tpt-1 or wild-type plants after 20 min of dark adaptation (A and B) or in actinic light (C and D). The arrows indicate the time points when FR illumination or actinic light (AL) was turned on or off. The spikes in the kinetics during illumination indicate the applications of saturated light pulses. The dashed lines represent F_o and F_m determined in the dark-adapted state following the application of a saturated light pulse (duration, 1 s).

Figure 2.

Effects of oxygen deprivation and FR illumination on traces of modulated Chl a fluorescence yield of dark-adapted leaves of adg1-1/tpt-1 (A) or wild-type (B) plants. Rosette leaves were fumigated in a Perspex chamber with either air or, in order to create an oxygen-free atmosphere, N₂ gas. Where indicated, FR was either turned on or off. In order to detect small changes in the dark fluorescence yield (F_o) in leaves of wild-type plants in response to N₂ or FR treatment, the signal had to be amplified 4-fold compared with adg1-1/tpt-1 (B, inset). F_m indicates the maximum Chl a fluorescence yield following the application of a saturated light pulse (duration, 1 s).

Figure 3.

Impact of inhibitor treatment on modulated Chl a fluorescence parameters in leaves of the wild type, the adg1-1/tpt-1 double mutant, and the tpt-1 and adg1-1 single mutants. F_v/F_m of dark-adapted plants (A), ΦPSII after 1 min of illumination with actinic light at a PFD of 120 μmol m⁻² s⁻¹ (B), and F_v/F_m during FR illumination at 1 min after actinic light was turned off (C) were determined in a time course after inhibitor application. Excised leaves of the wild type or adg1-1/tpt-1 were plunged into solutions containing 1 mm OG and 2.5 mm SHAM either individually or in combination. Chl a fluorescence parameters were determined at 0 h (white bars), 3.5 h (light gray bars), and 6 h (dark gray bars) after inhibitor treatment. As a control, leaves were incubated in 1% methanol. The leaf samples were kept in low light (PFD of 5 μmol m⁻² s⁻¹) during incubation. In a second experiment (D), F_v/F_m was determined after treatment of leaves of the wild type, both single mutants, and the double mutant with increasing concentrations of OG, SHAM, or antimycin A for 3 h. All data represent means ± se of three replicates.

Figure 4.

A, Time course of O₂ gas exchange of wild-type and adg1-1/tpt-1 plants. Excised rosette leaves were kept in the dark for at least 30 min and then illuminated with PFD of 80 μmol m⁻² s⁻¹ (L1), 392 μmol m⁻² s⁻¹ (L2), or 780 μmol m⁻² s⁻¹ (L3) interrupted by dark periods (D) as indicated by arrows. B to D, Combined determinations of O₂ gas exchange (B) and photosynthetic ETR (C) in response to increasing PFDs in wild-type and adg1-1/tpt-1 plants as well as light dependencies of the ratios of ETR and O₂ evolution (ETR/O₂ ratio) in the wild type (circles) and adg1-1/tpt-1 (squares; D). The data represent two independent sets of experiments. The leaf area inside the oxygen electrode chamber varied between 4 and 5 cm². The chamber temperature was kept constant at 25°C.

Figure 5.

Chloroplast ultrastructure analyses of wild-type, tpt-1, adg1-1, and adg1-1/tpt-1 plants. A, The images taken by an transmission electron microscope represent typical examples of chloroplasts from the individual lines either as overviews or as close-ups of the same chloroplasts. Bars = 1 μm and 0.5 μm for the overviews and close-ups, respectively. The cross-section areas of grana thylakoids (containing more than two stacks) were determined for eight to 20 chloroplast images per line and grouped into size classes. B, The relative grana size class distribution was compared between the lines. C, The number of plastoglobules was counted in 20 chloroplast cross sections per line and grouped into plastoglobule number classes per chloroplast, and the relative distribution of plastoglobule numbers per chloroplast was compared between the individual lines.

Figure 6.

Schematic overview of the response of Arabidopsis to a block of daily carbon export combined with a lack of starch in adg1-1/tpt-1 plants (B, D, F, and G) compared with wild-type plants (A, C, E, and G). The impairment of primary metabolism, as indicated in A and B, results in smaller chloroplasts, grana hyperstacking, and an increased number of plastoglobules (C and D). The enhanced reduction state of Q_A in the dark combined with increased NADPH/NADP ratios (G) in the light and dark point at an increased flux through chlororespiration in adg1-1/tpt-1 (E and F; modified from Rumeau et al., 2007). The gray arrows indicate the path of electrons in the light, starting from water splitting to NADPH generation, whereas the black arrows mark the probable path of electrons from NAD(P)H or reduced metabolites to molecular oxygen via NDH, PQ, and PTOX. Fd, Ferredoxin; FNR, ferredoxin NADPH reductase; PC, plastocyanin. The enhanced requirement to detoxify ROS is reflected in a large decrease in the Asc/DAsc ratio in adg1-1/tpt-1 compared with the wild type (G). Note that the thickness of the arrows in the metabolic diagrams reflects the relative velocities through individual pathways.