Figure 5. Altered electron transfer between the organelles in rcd1.

(A) Leaf discs were pre-treated with 1 μM MV or MV plus 2 mM SHAM for 1 hr in the darkness. Then light was turned on (80 µmol m⁻² s⁻¹) and chlorophyll fluorescence under light (Fs) was recorded by Imaging PAM. Application of the two chemicals together caused Fs rise in rcd1, but not Col-0, suggesting increase in the reduction state of the chloroplast ETC in rcd1. For analysis of photochemical quenching see Figure 5—figure supplement 1. (B) Malate levels are significantly decreased in Col-0 but not in rcd1 after MV treatment in light. Malate level was measured in extracts from Col-0 and rcd1 seedlings that were pre-treated overnight with 50 μM MV or water control and collected either dark-adapted or after exposure to 4 hr of light. Mean ±SE are shown. Asterisks indicate values significantly different from those in the similarly treated wild type, ***P value < 0.001, **P value < 0.01, Student’s t-test). For statistics, see Figure 5—source data 1. (C) NADPH-MDH activity is increased in rcd1. To measure the activity of chloroplastic NADPH-MDH, plants were grown at 100–120 µmol m⁻² s⁻¹ at an 8 hr day photoperiod, leaves were collected in the middle of the day and freeze-dried. The extracts were prepared in the buffer supplemented with 250 μM thiol-reducing agent DTT, and initial activity was measured (top left). The samples were then incubated for 2 hr in the presence of additional 150 mM DTT, and total activity was measured (top right). The activation state of NADPH-MDH (bottom) is presented as the ratio of the initial and the total activity. Mean ±SE are shown. Asterisks indicate values significantly different from the wild type, **P value < 0.01, *P value < 0.05, Student’s t-test. For statistics, see Figure 5—source data 1.

Figure 5—figure supplement 1. Alterations in chloroplast electron transfer induced by MV and SHAM.

During the first 20 min of light exposure, MV-pre-treated Col-0 and rcd1 experienced transient decrease in PSII photochemical quenching (qP). Within the next hour, photosynthesis recovered in rcd1 to the level observed in the non-treated control, while only very mild recovery was observed in Col-0. In rcd1, the recovery was significantly inhibited by co-application of SHAM together with MV. Leaf discs were pre-treated with MV and SHAM for 1 hr in the darkness. SHAM stock solution was prepared in DMSO, thus pure DMSO was added in the SHAM-minus controls. To calculate qP, Fs was recorded as in Figure 5A; saturating pulses were introduced every 10 min to measure Fm’. Data are presented as mean ±SD, for source data and statistics, see Figure 5—source data 1.

Figure 5—figure supplement 2. Distribution of malate in subcellular compartments of Col-0 and rcd1.

Distribution of malate was assessed by non-aqueous fractionation metabolomics as described in Figure 1—figure supplement 5C. Mean values ± SE are presented. For source data and statistics, see Figure 5—source data 1.