Antisense suppression of the small chloroplast protein CP12 in tobacco alters carbon partitioning and severely restricts growth

Abstract

The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants.

Figure 1.

Amount of CP12 protein quantified in control and antisense tobacco plants. Values are expressed as percentage of control. Error bars represent se; n = 3.

Figure 2.

Phenotypic changes observed in antisense CP12 tobacco lines. A, Plant growth 6 weeks from sowing in control and antisense tobacco lines (left to right: control, AS1, AS43, AS2/1, and AS6). B and C, Altered leaf morphology in control (B) and AS2/1 (C) tobacco. D, Loss of apical dominance. E and F, Altered leaf morphology at maturity (E) and at the cotyledon stage (F; control [left] and AS2/1 [right]). G, Transverse sections of leaves from control (left) and AS2/1 (right), viewed at the same magnification. Bars = 200 μm.

Figure 3.

PRK and GAPDH aggregation and enzyme activities in antisense CP12 plants. A and B, Immunoblot detection of PRK (A) and GAPDH (B) proteins separated using BN-PAGE. Stromal proteins prepared from darkened leaves were incubated either in the absence (−) or presence (+) of 2.5 mm NAD⁺ for 30 min prior to separation. Arrows indicate the locations of PRK and GAPDH protein complexes as identified previously (Howard et al., 2011): i, PRK/GAPDH/CP12; ii, PRK homodimer; iii, A8B8 GAPDH; iv, A4B4 GAPDH; v, A2B2 GAPDH; vi, A4 GAPDH. C and D, PRK (C) and GAPDH (D) activities in leaves from control (black bars), AS2/1 (white bars), and AS6 (gray bars) sampled during darkness, following illumination or following full activation with DTT. Error bars represent se; n = 4. FW, Fresh weight; WT, wild type.

Figure 4.

Photosynthetic carbon assimilation rates in antisense CP12 plants. A, Carbon assimilation rates at different light intensities. B, Carbon assimilation rates at different CO₂ concentrations. Controls (black squares) and antisense CP12 lines AS43 (white squares) and AS2/1 (white diamonds) are represented. PPFD, Photosynthetic photon flux density.

Figure 5.

Glc-6-P dehydrogenase, NADP-MDH, and pyridine nucleotides in antisense CP12 plants. A, DTT-sensitive (plastidial) G6PDH activity (g⁻¹ fresh weight [FW]), expressed as a percentage of total extractable G6PDH activity from leaves sampled in the light or dark: control (black bars), AS2/1 (white bars), and AS6 (gray bars). B, NADP-MDH activity in the dark, following illumination or following full activation with DTT: control (black bars), AS1 (white bars; light activation not determined), AS2/1 (gray bars), and AS6 (dark gray bars). C, Immunoblot detection of MDH following separation of stromal proteins prepared from darkened leaves using BN-PAGE. D, Pyridine nucleotide content of illuminated leaves: control (black bars), AS1 (white bars), AS32 (gray bars), AS2/1 (dark gray bars), and AS6 (light gray bars). Error bars represent se; n = 3 to 6 replicates. Asterisks indicate significant differences determined by ANOVA followed by Tukey’s test (P < 0.05).

Figure 6.

Metabolism of [U-¹⁴C]Glc in illuminated leaves. Control (black bars) and antisense CP12 lines AS2/1 (white bars) and AS6 (gray bars) are represented. A, Partitioning between respiration, soluble, and insoluble fractions. B, Partitioning within the insoluble fraction. C, Partitioning within the soluble fraction. Error bars represent se; n = 3. Asterisks indicate significant differences determined by ANOVA followed by Tukey’s test (P < 0.05).

Figure 7.

Metabolite composition of illuminated leaves of antisense CP12 lines. Antisense lines AS1 (black bars), AS43 (white bars), AS2/1 (gray bars), and AS6 (dark gray bars) are represented. All values are expressed as percentage of control values. A, Soluble carbohydrate content. B, Tricarboxylic acid cycle intermediates. C, Shikimate pathway metabolites and branched amino acids. D, Metabolites related to polyamine metabolism. Error bars represent se; n = 4 replicates. Asterisks indicate significant differences determined by ANOVA followed by Tukey’s test (P < 0.05).