SummaryChloroplastic NAD(P)H dehydrogenase (NDH) plays a role in cyclic electron flow around photosystem I to produce ATP, especially in adaptation to environmental changes. Although the NDH complex contains 11 subunits that are homologous to NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3), recent genetic and biological studies have indicated that NDH also comprises unique subunits. We describe here an in silico approach based on co-expression analysis and phylogenetic profiling that was used to identify 65 genes as potential candidates for NDH subunits. Characterization of 21 Arabidopsis T-DNA insertion mutants among these ndh gene candidates indicated that three novel ndf (NDH-dependent cyclic electron flow) mutants (ndf1, ndf2 and ndf4) had impaired NDH activity as determined by measurement of chlorophyll fluorescence. The amount of NdhH subunit was greatly decreased in these mutants, suggesting that the loss of NDH activity was caused by a defect in accumulation of the NDH complex. In addition, NDF1, NDF2 and NDF4 proteins co-migrated with the NdhH subunit, as shown by blue native electrophoresis. These results strongly suggest that NDF proteins are novel subunits of the NDH complex. Further analysis revealed that the NDF1 and NDF2 proteins were unstable in the mutants lacking hydrophobic subunits of the NDH complex, but were stable in mutants lacking the hydrophilic subunits, suggesting that NDF1 and NDF2 interact with a hydrophobic sub-complex. NDF4 protein was predicted to possess a redox-active iron-sulfur cluster domain that may be involved in the electron transfer.
Arabidopsis has three PsbQ-like (PQL) proteins in addition to the PsbQ subunit of the oxygen-evolving complex of PSII. Recent bioinformatic and proteomic studies suggested that the two PQL proteins, PQL1 (At1g14150) and PQL2 (At3g01440), might function in the chloroplast NAD(P)H dehydrogenase (NDH) complex; however, their molecular function has not been characterized. In this study, we examined the function of the chloroplast NDH in the Arabidopsis pql1 and pql2 mutants. Post-illumination increases in Chl fluorescence, which are caused by an NDH-dependent cyclic electron flow, were absent in both mutants, indicating that PQL1 and PQL2 are required for NDH activity. In the thylakoid membranes of wild-type plants, PQL1 and PQL2 were tightly associated with the NDH-PSI supercomplex and protected from protease treatments, while unassembled PQLs were not stably accumulated in mutants lacking known NDH subunits. Subunit stability of the NDH complex was affected differently in the thylakoid membranes of the pql1 and pql2 mutants. These data indicate that PQL1 and PQL2 are novel NDH subunits and differ in their functional roles and in their binding sites in the NDH complex. Furthermore, functional analysis on PQL3 (At2g01918) using the pql3 mutant suggests that PQL3 is also required for NDH activity. Proteins homologous to each PQL protein are found in various plant species, but not in cyanobacteria, algae, mosses or ferns. These results suggest that seed plants that have NDH activity in chloroplasts specifically developed three PQL proteins for the function of the chloroplast NDH complex.
C photosynthesis exhibits efficient CO assimilation in ambient air by concentrating CO around ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) through a metabolic pathway called the C cycle. It has been suggested that cyclic electron flow (CEF) around PSI mediated by chloroplast NADH dehydrogenase-like complex (NDH), an alternative pathway of photosynthetic electron transport (PET), plays a crucial role in C photosynthesis, although the contribution of NDH-mediated CEF is small in C photosynthesis. Here, we generated NDH-suppressed transformants of a C plant, Flaveria bidentis, and showed that the NDH-suppressed plants grow poorly, especially under low-light conditions. CO assimilation rates were consistently decreased in the NDH-suppressed plants under low and medium light intensities. Measurements of non-photochemical quenching (NPQ) of Chl fluorescence, the oxidation state of the reaction center of PSI (P700) and the electrochromic shift (ECS) of pigment absorbance indicated that proton translocation across the thylakoid membrane is impaired in the NDH-suppressed plants. Since proton translocation across the thylakoid membrane induces ATP production, these results suggest that NDH-mediated CEF plays a role in the supply of ATP which is required for C photosynthesis. Such a role is more crucial when the light that is available for photosynthesis is limited and the energy production by PET becomes rate-determining for C photosynthesis. Our results demonstrate that the physiological contribution of NDH-mediated CEF is greater in C photosynthesis than in C photosynthesis, suggesting that the mechanism of PET in C photosynthesis has changed from that in C photosynthesis accompanying the changes in the mechanism of CO assimilation.
By concentrating CO2, C4 photosynthesis can suppress photorespiration and achieve high photosynthetic efficiency, especially under conditions of high light, high temperature, and drought. To concentrate CO2, extra ATP is required, which would also require a change in photosynthetic electron transport in C4 photosynthesis from that in C3 photosynthesis. Several analyses have shown that the accumulation of the components of cyclic electron flow (CEF) around photosystem I, which generates the proton gradient across thylakoid membranes (ΔpH) and functions in ATP production without producing NADPH, is increased in various NAD-malic enzyme and NADP-malic enzyme C4 plants, suggesting that CEF may be enhanced to satisfy the increased need for ATP in C4 photosynthesis. However, in C4 plants, the accumulation patterns of the components of two partially redundant pathways of CEF, NAD(P)H dehydrogenase-like complex and PROTON GRADIENT REGULATION5-PGR5-like1 complex, are not identical, suggesting that these pathways may play different roles in C4 photosynthesis. Accompanying the increase in the amount of NDH, the expression of some genes which encode proteins involved in the assembly of NDH is also increased at the mRNA level in various C4 plants, suggesting that this increase is needed to increase the accumulation of NDH. To better understand the relation between CEF and C4 photosynthesis, a reverse genetic approach to generate C4 transformants with respect to CEF will be necessary.
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