Differential use of two cyclic electron flows around photosystem I for driving CO2-concentration mechanism in C4 photosynthesis

Abstract

Whereas linear electron flow (LEF) in photosynthesis produces both ATP and NADPH, the cyclic electron flow (CEF) around photosystem I has been shown to produce only ATP. Two alternative routes have been shown for CEF; NAD(P)H dehydrogenase (NDH)- and ferredoxin:plastoquinone oxidoreductase (FQR)-dependent flows, but their physiological relevance has not been elucidated in detail. Meanwhile, because C(4) photosynthesis requires more ATP than does C(3) photosynthesis to concentrate CO(2), it has not been clear how the extra ATP is produced. In this study, to elucidate whether CEF contributes to the additional ATP needed in C(4) photosynthesis, we estimated the amounts of PGR5, which participates in FQR-dependent flow, and NDH-H, a subunit of NDH, in four C(4) species. Although the expression profiles of PGR5 did not correlate well with the additional ATP requirement, NDH was greatly expressed in mesophyll cells in the NAD-malic enzyme (ME) species, and in bundle-sheath cells in NADP-ME species, where there is a strong need for ATP in the respective cells. Our results indicate that CEF via NDH plays a central role in driving the CO(2)-concentrating mechanism in C(4) photosynthesis.

Fig. 1.

Estimation of cross-contamination. (a) Immunoblot analysis of the mesophyll-specific phosphoenolpyruvate carboxylase (PEPC) and (b) bundle-sheath-specific Rubisco (RBCL). Soluble proteins were extracted from mesophyll preparations (MC) and bundle-sheath preparations (BC). For the 1/1 samples, 4 μg of proteins were loaded per lane. As judged from a dilution series of immunoblot analysis (1/1, 1/2, and 1/4), the amounts of PEPC in all MC were ≈4-fold more than those in the corresponding BC, suggesting that all MC contained <20% contamination. Likewise, all BC contained <20% contamination based on the results of immunoblot analysis of Rubisco. (c) Immunoblot analysis of extrinsic 33-kDa proteins (PsbO) in the PSII. Equal amounts of proteins (10 μg) extracted from MC and BC were loaded per lane. The PsbO proteins in the PS II were greatly decreased in the BC of NADP-ME species, as in previous reports (16, 17, 22).

Fig. 2.

Relative amounts of CEF-related enzymes and transcripts of mesophyll cells to bundle-sheath cells. (a) Immunoblot analysis of NDH-H, PGR5, and cytochrome f. (b-d) Densitometric analysis of immunoblots using polyclonal antibodies against NDH-H (b), PGR5 (c), and cytochrome f (d) proteins. Each value represents the relative intensity of mesophyll preparations to bundle-sheath preparations. Data are means ± SD (n = 3). (e) Relative pgr5 gene transcripts of mesophyll preparations to bundle-sheath preparations. The amounts of pgr5 gene transcripts were estimated by qPCR as described in Materials and Methods, normalized to the corresponding actin mRNA levels. Data are the means ± SD (n = 5).

Fig. 3.

In vivo measurement of NDH activity. After the actinic light illumination (ACT1; 85 μmol photons m^-2·s^-1), the transient increases in chlorophyll fluorescence were measured under dim light (Act2; 3 μmol m^-2·s^-1), recorded, and are shown in the enlarged boxes. This transient increase represents the reduction of the plastoquinone pool by the reducing equivalents accumulated during actinic illumination, confirmed by the experimental result that the transient increase disappeared when illuminated by far-red light after actinic illumination.

Fig. 4.

Sequence comparison of higher plant pgr5 genes. The deduced amino acid sequences from Arabidopsis thaliana (AAK53038), Amaranthus hybridus (AB218879), P. oleracea (AB218880), Z. mays (AB218881), P. grandiflora (AB218882), and Synechocystis sp. PCC6803 (S77542) were compared by using the CLUSTALW analysis algorithm and visually refined by using the BioEdit sequence-alignment editor (34). Identical residues are shaded in black, and similar residues are in gray. Dashes are gaps introduced to maximize the alignment.

Fig. 5.

Estimation of NDH and PGR5 in C₃ and C₄ plants. Ten micrograms of total cell (TC) or thylakoid membrane (TM) fraction proteins were subjected to SDS/PAGE and immunodetected by antibodies against NDH-H or PGR5. Proteins were extracted from wild-type tobacco (Nt), ndhB-disrupted tobacco (ΔB), Amaranthus hybridus (Ah), P. oleracea (Po), Z. mays (Zm) and P. grandiflora (Pg).

Fig. 6.

Schematic model of ATP:NADPH production in the electron-transport chain and consumption in CO₂ concentration and fixation. The energy requirements in each cell type are based on calculations by Edwards and Walker (16). II, I, N, and F represent PS II, PS I, NDH, and FQR, respectively. Values in parentheses represent the production ratio of ATP:NADPH in each cell type. This model implies that the NDH-dependent pathway meets the need for the extra ATP in C₄ photosynthesis. In reality, CO₂ is transported from mesophyll to bundle sheath in the form of C₄-carbonic acids, whereas ATP and NADPH are transported in the form of triosephosphate and malate.