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. 2018 Oct 12;7:e38194. doi: 10.7554/eLife.38194

Figure 5. Inhibition of NADPH-MDH and ribulose-5-phosphate kinase in leaves upon light-dark transitions, and non-soluble sugar contents in WT and 2cysprxAB during a 24 hr day-night cycle.

(A) MDH activity during a light-dark transition: WT and 2cysprxAB plants were exposed to 650 µmol quanta m−2 s−1 for 30 min and then darkened at t = 0 s. Proteins were rapidly extracted prior to darkening and after different time points in the dark as indicated. Initial (upper figure) and total MDH activity (lower figure) after full activation with 20 mM DTT were determined. Inactivation of MDH was slightly delayed in 2cysprxAB than in WT. This is shown for the time point 10 s. Data are means ± SD from n ≥ 5 determinations. (B) Ribulose-5-phosphate kinase (PRK) activity during a light-dark transition plotted as ratio of initial to total activity. WT and 2cysprxAB plants were illuminated with 650 µmol quanta m−2s−1 for 30 min and darkened as above. Initial and total PRK activities were determined and are represented as ratio. Initial inactivation was insignificantly delayed in 2cysprxAB, but while PRK in WT continued to be inactivated until 5 min, the PRK activity essentially remained unaltered and became significantly different at t = 300 s (n = 6). Complemented lines behaved like WT (supplements). (C) Changes in insoluble carbohydrates during a 24 hr day-night cycle. Leaves of WT and 2cysprxAB plants were harvested at 10 o’clock (1 hr after start of light phase), 15, 18, 20 (1 hr after end of light phase), 0 and 8 (1 hr before end of dark phase). Insoluble acid hydrolysable carbohydrates were quantified in the washed sediment of leaf homogenates using the Anthrone reagent following 1 hr boiling in sulfuric acid. Data are means ± SD of n = 3 extracts of different plants with three technical replicates each in Anthrone quantification. Statistics was calculated using one-sided ANOVA followed by Tukey’s HSD, while * = p ≤ 0.05.

Figure 5—source data 1. Altered MDH and PRK activities in 2cysprxAB mutants after light-dark transitions.
elife-38194-fig5-data1.docx (21.8KB, docx)
DOI: 10.7554/eLife.38194.016

Figure 5.

Figure 5—figure supplement 1. Genotyping and protein detection in 2cysprxAB and two independent 2-CysPrxA-complemented lines (C1 and C2).

Figure 5—figure supplement 1.

(A) PCR-based genotyping: Used primers are found in Supplementary file 1. The target sequence confirming T-DNA insertion in 2cysprxA had a length of 479 bp (At2-CysPrxgenR + GK RBfor), the PCR product from the WT gene: 1160 bp (At2-CysPrxgenF + At2 CysPrxgenR), and the complementation with p2cysprxA:2CysPrxA (cds) gave 801 bp (At2CPCDS-NcoIrev + At2 CPCDS-XhoIfor). (B) 2-CysPrx detection by Western Blot for plant extracts. Loading control for large RuBisCO LS subunit and Ponceau red stain.

Figure 5—figure supplement 2. PRK activity in the complemented lines C1 and C2 in comparison to WT and 2cysprxAB.

Figure 5—figure supplement 2.

PRK activity was determined 300 s after transfer of light-exposed plants to darkness. PRK was efficiently inhibited in WT and both complemented lines C1 and C2 after darkening, but was still highly active in the 2cysprxAB-line. Data are means ± SD of n = 6 for WT and 2cysprxAB and n = 3 for C1 and C2. Statistics was calculated using one-sided ANOVA followed by Tukey’s HSD.

Figure 5—figure supplement 3. Simulation of redox change of FBPase upon darkening.

Figure 5—figure supplement 3.

(A) The model assumes reduction of ferredoxin (Fd) by the photosynthetic electron transport chain in the light during the first 200 s. This process also reduces Fd-dependent thioredoxin reductase. Then Fd and FTR are set to full oxidation. Following this simulation of light switch off, the oxidized fractions of Trx and FBPase increase at the expense of reduced fraction. (B) Pathway of regulatory electron flow at the start of the experiment. (C) Pathway of electrons simulating darkness after 200 s. The parameters and equations employed in this model are found in Appendix 2.