Physiological links among alternative electron transport pathways that reduce and oxidize plastoquinone in Arabidopsis

In addition to linear electron transport from water to NADP⁺, alternative electron transport pathways are believed to regulate photosynthesis. In the two routes of photosystem I (PSI) cyclic electron transport, electrons are recycled from the stromal reducing pool to plastoquinone (PQ), generating additional ΔpH (proton gradient across thylakoid membranes). Plastid terminal oxidase (PTOX) accepts electrons from PQ and transfers them to oxygen to produce water. Although both electron transport pathways share the PQ pool, it is unclear whether they interact in vivo. To investigate the physiological link between PSI cyclic electron transport‐dependent PQ reduction and PTOX‐dependent PQ oxidation, we characterized mutants defective in both functions. Impairment of PSI cyclic electron transport suppressed leaf variegation in the Arabidopsis immutans (im) mutant, which is defective in PTOX. The im variegation was more effectively suppressed in the pgr5 mutant, which is defective in the main pathway of PSI cyclic electron transport, than in the crr2‐2 mutant, which is defective in the minor pathway. In contrast to this chloroplast development phenotype, the im defect alleviated the growth phenotype of the crr2‐2 pgr5 double mutant. This was accompanied by partial suppression of stromal over‐reduction and restricted linear electron transport. We discuss the function of the alternative electron transport pathways in both chloroplast development and photosynthesis in mature leaves.     

Chloroplast Structure and Function Is Altered in the NCS2 Maize Mitochondrial Mutant

The nonchromosomal stripe 2 (NCS2) mutant of maize (Zea mays L.) has a DNA rearrangement in the mitochondrial genome that segregates with the abnormal growth phenotype. Yet, the NCS2 characteristic phenotype includes striped sectors of pale-green tissue on the leaves. This suggests a chloroplast abnormality. To characterize the chloroplasts present in the mutant sectors, we examined the chloroplast structure by electron microscopy, chloroplast function by radiolabeled carbon dioxide fixation and fluorescence induction kinetics, and thylakoid protein composition by polyacrylamide gel electrophoresis. The data from these analyses suggest abnormal or prematurely arrested chloroplast development. Deleterious effects of the NCS2 mutant mitochondria upon the cells of the leaf include structural and functional alterations in the both the bundle sheath and mesophyll chloroplasts.     

Analysis of Leaf Sectors in the NCS6 Mitochondrial Mutant of Maize

The nonchromosomal stripe (NCS6) mutation of maize is a partial deletion of the mitochondrial cytochrome oxidase subunit 2 (Cox2) gene. The Cox2 deletion and a narrow yellow striping phenotype are inherited together in a maternal fashion. The striped plants are heteroplasmic for mutant and normal Cox2 genes. Only the mutant Cox2 gene is detected within the yellow stripes, whereas both normal and mutant forms of the gene are present in the green sectors of the NCS6 plants. In the green leaves of nonstriped relatives, only the normal Cox2 gene is found. Both the structure and functioning of the chloroplasts in the yellow leaf sectors of NCS6 plants are altered. The pleiotropic effects of the NCS6 mutation suggest that mitochondrial function is required for the development of photosynthetically competent chloroplasts.     

Differential expression of alternative oxidase genes in maize mitochondrial mutants

We have examined the expression of three alternative oxidase (aox) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.     

Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex

Eleven genes (ndhA-ndhK) encoding proteins homologous to the subunits of bacterial and mitochondrial NADH dehydrogenase (complex I) were found in the plastid genome of most land plants. These genes encode subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex involved in photosystem I (PSI) cyclic electron transport and chlororespiration. Although the chloroplast NDH is believed to be closely and functionally related to the cyanobacterial NDH-1L complex, extensive proteomic, genetic and bioinformatic studies have discovered many novel subunits that are specific to higher plants. On the basis of extensive mutant characterization, the chloroplast NDH complex is divided into four parts, the A, B, membrane and lumen subcomplexes, of which subunits in the B and lumen subcomplexes are specific to higher plants. These results suggest that the structure of NDH has been drastically altered during the evolution of land plants. Furthermore, chloroplast NDH interacts with multiple copies of PSI to form the unique NDH-PSI supercomplex. Two minor light-harvesting-complex I (LHCI) proteins, Lhca5 and Lhca6, are required for the specific interaction between NDH and PSI. The evolution of chloroplast NDH in land plants may be required for development of the function of NDH to alleviate oxidative stress in chloroplasts. In this review, we summarize recent progress on the subunit composition and structure of the chloroplast NDH complex, as well as the information on some factors involved in its assembly. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green‐ and white‐sectored leaves. Cells in the green sectors contain normal‐appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.     

Dual roles of photosynthetic electron transport in photosystem I biogenesis: light induction of mRNAs and chromatic regulation at post-mRNA level

Light regulation of photosystem I (PSI) biogenesis was studied in a unicellular green alga, Chlamydomonas reinhardtii. When Chlamydomonas cells were transferred from darkness to the light, mRNAs for both nuclear- and chloroplast-encoded PSI subunits were induced in concert. This light induction was inhibited by photosynthetic electron transport (PET) inhibitors, 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6 isopropyl-p-benzoquinone, but not by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone. This indicated that PET plays a pivotal role in the light induction of PSI subunit mRNAs, but that photophosphorylation is not necessary. When we irradiated the Chlamydomonas cells with PSI-light (695 nm) or PSII-light (644 nm), which makes the plastoquinone pool oxidative and reductive, respectively, PSII-light caused the accumulation of PSI proteins more abundantly than did PSI-light. However, there was no difference for the PSI subunit mRNA levels between these light sources. From these results, we conclude that PET plays dual roles in the regulation of PSI biogenesis in Chlamydomonas: when cells are illuminated, PET first induces the PSI subunit mRNAs irrespective of the redox state of the intersystem electron carriers, and then their redox state fine-tunes PSI biogenesis at translational and/or post-translational steps to fulfil the chromatic adaptation.     

Specific substitutions of light‐harvesting complex I proteins associated with photosystem I are required for supercomplex formation with chloroplast NADH dehydrogenase‐like complex

In Arabidopsis, the chloroplast NADH‐dehydrogenase‐like (NDH) complex is sandwiched between two copies of photosystem I (PSI) supercomplex, consisting of a PSI core and four light‐harvesting complex I (LHCI) proteins (PSI‐LHCI) to form the NDH–PSI supercomplex. Two minor LHCI proteins, Lhca5 and Lhca6, contribute to the interaction of each PSI–LHCI copy with the NDH complex. Here, large‐pore blue‐native gel electrophoresis revealed that, in addition to this complex, there were at least two types of higher‐order association of more LHCI copies with the NDH complex. In single‐particle images, this higher‐order association of PSI–LHCI preferentially occurs at the left side of the NDH complex when viewed from the stromal side, placing subcomplex A at the top (Yadav et al., Biochim. Biophys. Acta ‐ Bioenerg., 1858, 2017, 12). The association was impaired in the lhca6 mutant but not in the lhca5 mutant, suggesting that the left copy of PSI–LHCI was linked to the NDH complex via Lhca6. From an analysis of subunit compositions of the NDH–PSI supercomplex in lhca5 and lhca6 mutants, we propose that Lhca6 substitutes for Lhca2 in the left copy of PSI–LHCI, whereas Lhca5 substitutes for Lhca4 in the right copy. In the lhca2 mutant, Lhca3 was specifically stabilized in the NDH–PSI supercomplex through heterodimer formation with Lhca6. In the left copy of PSI–LHCI, subcomplex B, Lhca6 and NdhD likely formed the core of the supercomplex interaction. In contrast, a larger protein complex, including at least subcomplexes B and L and NdhB, was needed to form the contact site with Lhca5 in the right copy of PSI–LHCI.     

The NdhV subunit is required to stabilize the chloroplast NADH dehydrogenase‐like complex in Arabidopsis

The chloroplast NADH dehydrogenase‐like (NDH) complex is involved in cyclic electron transport around photosystem I (PSI) and chlororespiration. Although the NDH complex was discovered more than 20 years ago, its low abundance and fragile nature render it recalcitrant to analysis, and it is thought that some of its subunits remain to be identified. Here, we identified the NDH subunit NdhV that readily disassociates from the NDH complex in the presence of detergent, salt and alkaline solutions. The Arabidopsis ndhv mutant is partially defective in the accumulation of NDH subcomplex A (SubA) and SubE, resulting in impaired NDH activity. NdhV was mainly detected in the wild‐type thylakoid membrane, and its accumulation in thylakoids strictly depended on the presence of the NDH complex. Quantitative immunoblot analysis revealed that NdhV and NdhN occur at close to equimolar concentrations. Furthermore, several NDH subunits were co‐immunopurified with NdhV using a combination of chemical crosslinking and an affinity chromatography assay. These data indicate that NdhV is an intrinsic subunit of NDH. We found that NdhV did not directly affect NDH activity, but that NDH SubA and SubE were more rapidly degraded in ndhv than in the wild type under high‐light treatment. We propose that NdhV is an NDH subunit that stabilizes this complex, especially under high‐light conditions.     

Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b6f complex in maize

Nuclear genes essential for the biogenesis of the chloroplast cytochrome b ₆ f complex were identified by mutations that cause the specific loss of the complex. We describe four transposon-induced maize mutants that lack cytochrome b ₆ f proteins but contain normal levels of other photosynthetic complexes. The four mutations define two nuclear genes. To identify the step at which each mutation blocks protein accumulation, mRNAs encoding each subunit were examined by Northern hybridization analysis and the rates of subunit synthesis were examined in pulse-labeling experiments. In each mutant the mRNAs encoding the known subunits of the complex were normal in size and abundance and the major subunits were synthesized at normal rates. Thus, these mutations block the biogenesis of the cytochrome b ₆ f complex at a post-translational step. The two nuclear genes identified by these mutations may encode previously unknown subunits, be involved in prosthetic group synthesis or attachment, or facilitate assembly of the complex. These mutations were also used to provide evidence for the authenticity of a proposed fifth subunit of the complex and to demonstrate a role for the cytochrome b ₆ f complex in protecting photosystem 11 from light-induced degradation.     

Contribution of NDH-dependent cyclic electron transport around photosystem I to the generation of proton motive force in the weak mutant allele of pgr5

In angiosperms, cyclic electron transport (CET) around photosystem I (PSI) consists of two pathways, depending on PGR5/PGRL1 proteins and the chloroplast NDH complex. In single mutants defective in chloroplast NDH, photosynthetic electron transport is only slightly affected at low light intensity, but in double mutants impaired in both CET pathways photosynthesis and plant growth are severely affected. The question is whether this strong mutant phenotype observed in double mutants can be simply explained by the additive effect of defects in both CET pathways. In this study, we used the weak mutant allele of pgr5-2 for the background of double mutants to avoid possible problems caused by the secondary effects due to the strong mutant phenotype. In two double mutants, crr2-2 pgr5-2 and ndhs-1 pgr5-2, the plant growth was unaffected and linear electron transport was only slightly affected. However, NPQ induction was more severely impaired in the double mutants than in the pgr5-2 single mutant. A similar trend was observed in the size of the proton motive force. Despite the slight reduction in photosystem II parameters, PSI parameters were severely affected in the pgr5-2 single mutant, the phenotype that was further enhanced by adding the NDH defects. Despite the lack of ?pH-dependent regulation at the cytochrome b₆f complex (donor-side regulation of PSI), the plastoquinone pool was more reduced in the double mutants than in the pgr5-2 single mutants. This phenotype suggests that both PGR5/PGRL1- and NDH-dependent CET contribute to supply sufficient acceptors from PSI by balancing the ATP/NADPH production ratio.     

Thylakoid Transit Peptide Is Related to the Expression and Localization of NdhB Subunits in Soybean

The chloroplast NAD(P)H dehydrogenase (NDH) complex, as one of the most important photosynthesis protein complexes in thylakoid membrane, is involved in photosystem I (PSI) cyclic electron transport (CEF). Under abiotic environmental stress, the photosynthetic apparatus is susceptible to the damage caused by the strong light illumination. However, the enhancement of NDHdependent CEF could facilitate the alleviation of the damage to the photosynthetic apparatus. The NdhB subunit encoded by chloroplast genome is one of most important subunits of NDH complex and consists of 510 amino acids. Here, according to cloning ndhB from Melrose (cultivated soybean), ACC547 (wild salt-tolerant soybean), S113-6 and S111-9 (hybrid descendant), based on the comparison and analysis of the sequences of NdhB subunits, we found that there is a novel thylakoid transit peptide of NdhB subunit in S111-9. In addition, crosslink immunoprecipitation, immunogold labeling and co-expression of GFP fusion protein indicated that the novel thylakoid transit peptide is favorable to the expression and localization of NdhB subunit in chloroplast. Therefore, we suggest that this novel thylakoid transit peptide plays the same role as chaperonin and contributes to facilitating the expression and localization of NdhB subunit.     

A partially assembled complex I in NAD4-deficient mitochondria of maize

The proton-translocating NADH:ubiquinone oxidoreductase (respiratory complex I) consists of at least 32 subunits in higher plants, nine of which are mitochondrially encoded (NAD 1–7, NAD4L, NAD9). Complex I (CI) has been analyzed from a mitochondrial mutant of maize, NCS2, that carries a deletion for the 3′ end of the nad4 gene. Mitochondria from highly defective, near-homoplasmic mutant plants have only trace amounts of the normal complex I. Instead, a reduced amount of a smaller complex, which also exhibits NADH dehydrogenase activity, is detected on ‘blue-native’ polyacrylamide gels. Subunits of 76 kDa, 40 kDa and 55 kDa, as well as NAD7 and NAD9, have been identified in the subcomplex by their cross-reactivity with heterologous antisera. The corresponding subunits in Neurospora are localized in a ‘peripheral arm’ of CI, which is known to assemble independently of a ‘membrane arm’. The maize NCS2 CI subcomplex is loosely bound to the membrane and is missing several subunits that could be membrane components. Thus, the mutant CI subcomplex may consist of a peripheral arm. A reduction in the steady-state levels of NAD7 and NAD9 in NCS2 mitochondria occurs despite normal rates of biosynthesis and there is a concomitant decrease of the nuclear encoded 76 kDa subunit. The reduction in CI-associated NADH dehydrogenase activity in the nad4 -deficient NCS2 mutant mitochondria is not associated with a compensatory increase in the activities or amounts of the putative ‘exogenous’ NAD(P)H dehydrogenases that are found in plant mitochondria.