Two cyclic electron flows around photosystem I differentially participate in C4 photosynthesis

Abstract

C4 plants assimilate CO2 more efficiently than C3 plants because of their C4 cycle that concentrates CO2. However, the C4 cycle requires additional ATP molecules, which may be supplied by cyclic electron flow (CEF) around photosystem I. One CEF route, which depends on a chloroplast NADH dehydrogenase-like (NDH) complex, is suggested to be crucial for C4 plants despite the low activity in C3 plants. The other route depends on proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic phenotype 1 (PGRL1) and is considered a major CEF route to generate the proton gradient across the thylakoid membrane in C3 plants. However, its contribution to C4 photosynthesis is still unclear. In this study, we investigated the contribution of the two CEF routes to the NADP-malic enzyme subtype of C4 photosynthesis in Flaveria bidentis. We observed that suppressing the NDH-dependent route drastically delayed growth and decreased the CO2 assimilation rate to approximately 30% of the wild-type rate. On the other hand, suppressing the PGR5/PGRL1-dependent route did not affect plant growth and resulted in a CO2 assimilation rate that was approximately 80% of the wild-type rate. Our data indicate that the NDH-dependent CEF substantially contributes to the NADP-malic enzyme subtype of C4 photosynthesis and that the PGR5/PGRL1-dependent route cannot complement the NDH-dependent route in F. bidentis. These findings support the fact that during C4 evolution, photosynthetic electron flow may have been optimized to provide the energy required for C4 photosynthesis.

Figure 1

Knockdown of PGR5, PGRL1, and NdhO in F. bidentis. A, Expression of PGR5A, PGR5B, PGR5C, and PGRL1 in PGR5-RNAi lines, PGRL1 in PGRL1-RNAi lines, and NdhO in NdhO-RNAi lines relative to the corresponding expression in the WT controls. Vertical bars indicate the Sd (n = 4–5). Asterisks indicate significant differences (Student t-test, P < 0.05) between WT and PGR5-RNAi, PGRL1-RNAi, or NdhO-RNAi lines. B, Immunoblot analysis of the membrane proteins extracted from the leaves of the WT, VC, PGR5-RNAi, PGRL1-RNAi, and NdhO-RNAi lines. Lanes were loaded with 20 μg protein to detect PGR5 and 10 μg to detect PGRL1, NdhH, Rieske, PsaD, and PsbO. The dilution series for the WT plants is indicated. C, Relative content of membrane proteins involved in cyclic or LEF. The amount of membrane proteins was quantified by chemiluminescence signal intensities of immunoblot analysis, and the signal intensity of the WT plants was set to 1. Vertical bars indicate the Sd (n = 3). Asterisks indicate significant differences (Student t-test, P < 0.05) between VC and PGR5-RNAi, PGRL1-RNAi, or NdhO-RNAi lines.

Figure 2

Electron transfer to PQ in ruptured chloroplasts (20 μg chlorophyll ml⁻¹) following the addition of 250 μM NADPH and 5 μM ferredoxin (fd). The electron transfer was based on the chlorophyll fluorescence under weak light (0.25 μmol photons m⁻² s⁻¹). Data are presented as the average of three measurements. Black line, no antimycin A; gray line, in the presence of 5 μM antimycin A.

Figure 3

Photosynthetic activity assessed based on growth under medium light conditions (250 μmol photons m⁻² s⁻¹) in WT, VC, PGR5-RNAi, PGRL1-RNAi, and NdhO-RNAi lines. A, Observable phenotypes of 45-day-old plants. The bar indicates 5 cm. B, Leaf area per plant of 45-day-old plants. C, Days to flowering. Vertical bars indicate the Sd (n = 5). Asterisks indicate significant differences (Student t-test, P < 0.05) between WT and PGR5-RNAi, PGRL1-RNAi, or NdhO-RNAi lines.

Figure 4

Light intensity dependence of photosynthetic activity and electron transfer activity in WT, VC, PGR5-RNAi, PGRL1-RNAi, and NdhO-RNAi lines. Response curve of the net CO₂ assimilation rate per leaf area (A), rETR (B), NPQ (C), and the ECSt parameter (D) to light intensity. A–C, Chlorophyll fluorescence parameters were measured along with the CO₂ assimilation rate under 400 ppm ambient CO₂. D, The ECSt parameter was estimated by measuring the rapid decline of the ECS after the cessation of actinic light. Black closed circles, WT; black open circles, VC; light blue squares, PGR5-RNAi #3; blue diamonds, PGRL1-RNAi #4; red triangles, NdhO-RNAi #1; pink inverted triangles, NdhO-RNAi #18. Vertical bars indicate the Sd (A–C, n = 3–6; D, n = 4–6). Light blue, blue, red, or pink asterisks indicate significant differences (Student t-test, P < 0.05) between WT and PGR5-RNAi #3, PGRL1-RNAi #4, NdhO-RNAi #1 or NdhO-RNAi #18, respectively.

Figure 5

Effects on CO₂ concentration mechanism in PGR5-RNAi, PGRL1-RNAi, and NdhO-RNAi lines. Response curve of the net CO₂ assimilation rate per leaf area to intercellular CO₂ concentration under 1,500 μmol m⁻² s⁻¹ illumination (A) and location of chloroplasts in the cell (B). A, Black closed circles, WT; black open circles, VC; light blue squares, PGR5-RNAi #3; blue diamonds, PGRL1-RNAi #4; red triangles, NdhO-RNAi #1; pink inverted triangles, NdhO-RNAi #18. Vertical bars indicate the Sd (n = 3–7). Light blue, blue, red, or pink asterisks indicate significant differences (Student t-test, P < 0.05) between WT and PGR5-RNAi #3, PGRL1-RNAi #4, NdhO-RNAi #1 or NdhO-RNAi #18, respectively. B, Cross section of WT and RNAi plant leaves stained with toluidine blue. Arrows indicate chloroplasts in BSC. Bars indicate 20 μm.