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.     

Identification of a novel protein, CRR7, required for the stabilization of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

An Arabidopsis thaliana mutant, crr7 (chlororespiratory reduction), was isolated using chlorophyll fluorescence imaging to detect reduced activity in NAD(P)H dehydrogenase (NDH). The chloroplast NDH complex is considered to have originated from cyanobacteria in which the NDH complex is involved in respiration, photosystem I (PSI) cyclic electron transport and CO2 uptake. In higher plants the NDH complex functions in PSI cyclic electron transport within the chloroplast. Despite exhaustive biochemical approaches, the entire subunit composition of the NDH complex is unclear in both cyanobacteria and chloroplasts. In crr7 accumulation of the NDH complex was specifically impaired. In vivo analysis of electron transport supported the specific loss of the NDH complex in crr7. CRR7 (At5g39210) encodes a protein of 156 amino acids, including a putative plastid target signal, and does not contain any known motifs. In contrast to CRR2 and CRR4, involved in the expression of chloroplast ndh genes, CRR7 is conserved in cyanobacterial genomes. Although CRR7 did not contain any transmembrane domains, it localized to the membrane fraction of the chloroplast. CRR7 was unstable in the crr2-2 mutant background, in which the expression of ndhB was impaired. These results strongly suggest that CRR7 is a novel subunit of the chloroplast NDH complex.     

Dihydrodipicolinate reductase-like protein, CRR1, is essential for chloroplast NAD(P)H dehydrogenase in Arabidopsis

Chloroplast NAD(P)H dehydrogenase (NDH) is a homolog of the bacterial NADH dehydrogenase NDH-1 and is involved in cyclic electron transport around photosystem I. In higher plants, 14 subunits of the NDH complex have been identified. The subunit that contains the electron donor-binding site or an electron donor to NDH has not been determined. Arabidopsis crr1 (chlororespiratory reduction 1) mutants were isolated by chlorophyll fluorescence imaging on the basis of their lack of NDH activity. CRR1 is homologous to dihydrodipicolinate reductase (DHPR), which functions in a lysine biosynthesis pathway. However, the dihydrodipicolinate-binding motif was not conserved in CRR1, and the crr1 defect was specific to accumulation of the NDH complex, implying that CRR1 is not involved in lysine biosynthesis in Arabidopsis. Similarly to other nuclear-encoded genes for NDH subunits, CRR1 was expressed only in photosynthetic tissue. CRR1 contained a NAD(P)H-binding motif and was a candidate electron donor-binding subunit of the NDH complex. However, CRR1 was detected in the stroma but not in the thylakoid membranes, where the NDH complex is localized. Furthermore, CRR1 was stable in crr2-2 lacking the NDH complex. These results suggest that CRR1 is involved in biogenesis or stabilization of the NDH complex, possibly via the reduction of an unknown substrate.     

Composition and physiological function of the chloroplast NADH dehydrogenase‐like complex in Marchantia polymorpha

The chloroplast NADH dehydrogenase‐like (NDH) complex mediates cyclic electron transport and chloro‐respiration and consists of five sub‐omplexes, which in angiosperms further associate with photosystem I (PSI) to form a super‐complex. In Marchantia polymorpha, 11 plastid‐encoded subunits and all the nuclear‐encoded subunits of the A, B, membrane and ferredoxin‐binding sub‐complexes are conserved. However, it is unlikely that the genome of this liverwort encodes Lhca5 and Lhca6, both of which mediate NDH–PSI super‐complex formation. It is also unlikely that the subunits of the lumen sub‐complex, PnsL1–L4, are encoded by the genome. Consistent with this in silico prediction, the results of blue‐native gel electrophoresis showed that NDH subunits were detected in a protein complex with lower molecular mass in Marchantia than the NDH–PSI super‐complex in Arabidopsis. Using the plastid transformation technique, we knocked out the ndhB gene in Marchantia. Although the wild‐type genome copies were completely segregated out, the ΔndhB lines grew like the wild‐type photoautotrophically. A post‐illumination transient increase in chlorophyll fluorescence, which reflects NDH activity in vivo in angiosperms, was absent in the thalli of the ΔndhB lines. In ruptured chloroplasts, antimycin A‐insensitive, and ferredoxin‐dependent plastoquinone reduction was impaired, suggesting that chloroplast NDH mediates similar electron transport in Marchantia and Arabidopsis, despite its possible difference in structure. As in angiosperms, linear electron transport was not strongly affected in the ΔndhB lines. However, the plastoquinone pool was slightly more reduced at low light intensity, suggesting that chloroplast NDH functions in redox balancing of the inter system, especially under low light conditions.     

A cellulose synthase‐like protein is required for osmotic stress tolerance in Arabidopsis

Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root‐bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6‐1, which defines a locus essential for osmotic stress tolerance. sos6‐1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase‐like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6‐1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress.     

Chlororespiration is involved in the adaptation of Brassica plants to heat and high light intensity

Two species of Brassica were used to study their acclimation to heat and high illumination during the first stages of development. One, Brassica fruticulosa, is a wild species from south-east Spain and is adapted to both heat and high light intensity in its natural habitat, while the other, Brassica oleracea, is an agricultural species that is widely cultivated throughout the world. Growing Brassica plants under high irradiance and moderate heat was seen to affect the growth parameters and the functioning of the photosynthetic apparatus. The photosystem II (PSII) quantum yields and the capacity of photosynthetic electron transport, which were lower in B. fruticulosa than in B. oleracea, decreased in B. oleracea plants when grown under stress conditions, indicating inhibition of PSII. However, in B. fruticulosa, the values of these parameters were similar to the values of control plants. Photosystem I (PSI) activity was higher in B. fruticulosa than in B. oleracea, and in both species this activity increased in plants exposed to heat and high illumination. Immunoblot analysis of thylakoid membranes using specific antibodies raised against the NDH-K subunit of the thylakoidal NADH dehydrogenase complex (NADH DH) and against plastid terminal oxidase (PTOX) revealed a higher amount of both proteins in B. fruticulosa than in B. oleracea. In addition, PTOX activity in plastoquinone oxidation, and NADH DH activity in thylakoid membranes were higher in the wild species (B. fruticulosa) than in the agricultural species (B. oleracea). The results indicate that tolerance to high illumination and heat of the photosynthetic activity was higher in the wild species than in the agricultural species, suggesting that plant adaptation to these stresses in natural conditions favours subsequent acclimation, and that the chlororespiration process is involved in adaptation to heat and high illumination in Brassica.     

The ANGULATA7 gene encodes a DnaJ‐like zinc finger‐domain protein involved in chloroplast function and leaf development in Arabidopsis

The characterization of mutants with altered leaf shape and pigmentation has previously allowed the identification of nuclear genes that encode plastid‐localized proteins that perform essential functions in leaf growth and development. A large‐scale screen previously allowed us to isolate ethyl methanesulfonate‐induced mutants with small rosettes and pale green leaves with prominent marginal teeth, which were assigned to a phenotypic class that we dubbed Angulata. The molecular characterization of the 12 genes assigned to this phenotypic class should help us to advance our understanding of the still poorly understood relationship between chloroplast biogenesis and leaf morphogenesis. In this article, we report the phenotypic and molecular characterization of the angulata7‐1 (anu7‐1) mutant of Arabidopsis thaliana, which we found to be a hypomorphic allele of the EMB2737 gene, which was previously known only for its embryonic‐lethal mutations. ANU7 encodes a plant‐specific protein that contains a domain similar to the central cysteine‐rich domain of DnaJ proteins. The observed genetic interaction of anu7‐1 with a loss‐of‐function allele of GENOMES UNCOUPLED1 suggests that the anu7‐1 mutation triggers a retrograde signal that leads to changes in the expression of many genes that normally function in the chloroplasts. Many such genes are expressed at higher levels in anu7‐1 rosettes, with a significant overrepresentation of those required for the expression of plastid genome genes. Like in other mutants with altered expression of plastid‐encoded genes, we found that anu7‐1 exhibits defects in the arrangement of thylakoidal membranes, which appear locally unappressed.     

The Na-translocating NADH:quinone oxidoreductase (Na-NQR) from Vibrio cholerae enhances insertion of FeS in overproduced NqrF subunit

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae is a membrane-bound, respiratory Na⁺ pump. Its NqrF subunit contains one FAD and a [2Fe–2S] cluster and catalyzes the initial oxidation of NADH. A soluble variant of NqrF lacking its hydrophobic, N-terminal helix (NqrF′) was produced in V. cholerae wild type and nqr deletion strain. Under identical conditions of growth and induction, the yield of NqrF′ increased by 30% in the presence of the Na⁺-NQR. FAD-containing NqrF′ species with or without the FeS cluster were observed, indicating that assembly of the FeS center, but not insertion of the flavin cofactor, was limited during overproduction in V. cholerae. A comparison of these distinct NqrF′ species with regard to specific NADH dehydrogenase activity, pH dependence of activity and thermal inactivation showed that NqrF′ lacking the [2Fe–2S] cluster was less stable, partially unfolded, and therefore prone to proteolytic degradation in V. cholerae. We conclude that the overall yield of NqrF′ critically depends on the amount of fully assembled, FeS-containing NqrF′ in the V. cholerae host cells. The Na⁺-NQR is proposed to increase the stability of NqrF′ by stimulating the maturation of FeS centers.     

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae enhances insertion of FeS in overproduced NqrF subunit

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae is a membrane-bound, respiratory Na⁺ pump. Its NqrF subunit contains one FAD and a [2Fe–2S] cluster and catalyzes the initial oxidation of NADH. A soluble variant of NqrF lacking its hydrophobic, N-terminal helix (NqrF′) was produced in V. cholerae wild type and nqr deletion strain. Under identical conditions of growth and induction, the yield of NqrF′ increased by 30% in the presence of the Na⁺-NQR. FAD-containing NqrF′ species with or without the FeS cluster were observed, indicating that assembly of the FeS center, but not insertion of the flavin cofactor, was limited during overproduction in V. cholerae. A comparison of these distinct NqrF′ species with regard to specific NADH dehydrogenase activity, pH dependence of activity and thermal inactivation showed that NqrF′ lacking the [2Fe–2S] cluster was less stable, partially unfolded, and therefore prone to proteolytic degradation in V. cholerae. We conclude that the overall yield of NqrF′ critically depends on the amount of fully assembled, FeS-containing NqrF′ in the V. cholerae host cells. The Na⁺-NQR is proposed to increase the stability of NqrF′ by stimulating the maturation of FeS centers.     

crinkled leaves 8--a mutation in the large subunit of ribonucleotide reductase--leads to defects in leaf development and chloroplast division in Arabidopsis thaliana

The crinkled leaves8 (cls8) mutant of Arabidopsis thaliana displays a developmental phenotype of abnormal leaf and flower morphology, reduced root growth and bleached leaf sections. Map-based cloning identified the mutation as being within the gene encoding the large subunit of ribonucleotide reductase (RNR1), the enzyme that catalyses the rate-limiting step in the production of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Levels of dTTP and dATP were significantly reduced in cls8. Two further mutant cls8 alleles and cls8::RNAi plants show similar or more severe phenotypes. The cls8-1 mutant has fewer copies of the chloroplast genome, and fewer, larger chloroplasts than wild-type plants. The ultrastructure of the chloroplast, however, appears normal in cls8-1 leaves. We present evidence that, under conditions of limited dNTP supply, the inhibition of chloroplast DNA replication may be the primary factor in inducing aberrant growth.     

茄科作物青枯菌NADH脱氢酶F亚基在细胞运动和致病性中的功能研究

[目的]劳尔氏菌（Ralstonia solanacearum）在茄科作物上引起严重的细菌性青枯病,本研究旨在发掘青枯劳尔氏菌与致病相关的基因。[方法]利用Tn5转座子构建随机插入突变体,分析生物膜形成、细胞运动和致病性;对有表型变化的突变体,运用TAIL-PCR方法鉴定Tn5插入位点,确定所突变的基因。[结果]以模式菌株GMI000为出发菌,总共获得了400个突变体,其中2个突变体不能形成生物膜,在软琼脂平板上的运动能力下降;接种感病番茄植物,这2个突变体都不能引起萎焉症状。TAIL-PCR结果显示,2个突变体的Tn5插入位点都在NADH脱氢酶F亚基（nuoF）中,距离翻译起始位点分别为103-bp和225-bp。ripAY基因启动子推动的nuoF基因互补载体,完全恢复了2个突变体的表型。[结论]NADH脱氢酶复合物是微生物呼吸电子传递链中的第一步催化酶。我们的结果表明,NADH脱氢酶复合物对R.solanacearum生物膜形成、细胞运动和致病性也有重要作用。     

The NADH dehydrogenase subunit 7 gene is interrupted by four group II introns in the wheat mitochondrial genome

Two loci FRI (FRIGIDA) and KRY (KRYOPHILA) have previously been identified as having major influences on the flowering time of the late-flowering, vernalization-responsive Arabidopsis ecotype, Stockholm. We report here on the mapping and subsequent analysis of these two loci. FRI was mapped to the top of chromosome 4 between markers w122 and m506, using restriction fragment length polymorphism (RFLP) analysis. Due to lack of segregation in of the late-flowering phenotype under the environmental conditions used, KRY could only be localized, by subtractive genotyping, to chromosome 5 or part of chromosome 3. The map position of FRI indicates that it is not allelic to any of the late-flowering loci identified by mutagenesis of the early-flowering ecotype Landsberg erecta. The late-flowering phenotype conferred by the Stockholm allele of FRI is modified (towards earlier flowering) by Landsberg erecta alleles at an unknown number of loci, perhaps accounting for the absence of fri mutations among mutant lines recovered in Landsberg erecta.     

Supercomplex formation with photosystem I is required for the stabilization of the chloroplast NADH dehydrogenase-like complex in Arabidopsis

In higher plants, the chloroplast NADH dehydrogenase-like complex (NDH) interacts with photosystem I (PSI) to form the NDH-PSI supercomplex via two minor light-harvesting complex I (LHCI) proteins, Lhca5 and Lhca6. Previously, we showed that in lhca5 and lhca6, NDH still associates with PSI to form smaller versions of the NDH-PSI supercomplex, although their molecular masses are far smaller than that of the full-size NDH-PSI supercomplex. In this study, we show that the NDH complex is present in the monomeric form in Arabidopsis (Arabidopsis thaliana) lhca5 lhca6, implying that NDH interacts with multiple copies of PSI. NDH subunit levels were slightly reduced in immature leaves and more drastically (approximately 50%) in mature leaves of the lhca5 lhca6 double mutant compared with the wild type. Chlorophyll fluorescence analyses detected NDH activity of lhca5 lhca6, suggesting that the supercomplex formation is not essential for NDH activity. However, the severe phenotypes of the lhca5 lhca6 proton gradient regulation5 triple mutant in both plant growth rate and photosynthesis suggest that the function of NDH was impaired in this mutant in vivo. Accumulation of NDH subunits was drastically reduced in lhca5 lhca6 when the light intensity was shifted from 50 to 500 μmol photons m(-2) s(-1). Furthermore, the half-life of NDH subunits, especially that of NDH18, was shorter in monomeric NDH than in the NDH-PSI supercomplex under the high-light conditions. We propose that NDH-PSI supercomplex formation stabilizes NDH and that the process is especially required under stress conditions.