ndhB Gene Research Articles

Comparative analyses of complete chloroplast genomes reveal interspecific difference and intraspecific variation of Tripterygium genus.

The genus Tripterygium was of great medicinal value and attracted much attention on the taxonomic study using morphological and molecular methods. In this study, we assembled 12 chloroplast genomes of Tripterygium to reveal interspecific difference and intraspecific variation. The sequence length (156,692-157,061 bp) and structure of Tripterygium were conserved. Comparative analyses presented abundant variable regions for further study. Meanwhile, we determined the ndhB gene under positive selection through adaptive evolution analysis. And the phylogenetic analyses based on 15 chloroplast genomes supported the monophyly of Tripterygium hypoglaucum and the potential sister relationship between Tripterygium wilfordii and Tripterygium regelii. Molecular dating analysis indicated that the divergence time within Tripterygium was approximately 5.99 Ma (95% HPD = 3.11-8.68 Ma). The results in our study provided new insights into the taxonomy, evolution process, and phylogenetic construction of Tripterygium using complete plastid genomes.

Comparative chloroplast genome analysis of four Polygonatum species insights into DNA barcoding, evolution, and phylogeny

The Polygonatum genus represents a perennial herb with the Liliaceae family, boasting substantial economic and medicinal significance. The majority of Polygonatum plants exhibit notable similarity while lacking distinctive identifying characteristics, thus resulting in the proliferation of adulterated medicinal materials within the market. Within this study, we conducted an in-depth analysis of the complete chloroplast (cp) genomes of four Polygonatum plants and compared them with four closely akin species. The primary objectives were to unveil structural variations, species divergence, and the phylogenetic interrelations among taxa. The cp genomes of the four Polygonatum species were typified by a conventional quadripartite structure, incorporating a large single copy region (LSC), a small single copy region (SSC), and a pair of inverted repeat regions. In total, we annotated a range of 131 to 133 genes, encompassing 84 to 86 protein-coding genes, 38 transfer RNA (tRNA) genes, 8 ribosomal RNA (rRNA) genes, and 0 to 2 pseudogenes (ycf1, infA). Our comparative analyses unequivocally revealed a remarkable consistency in gene order and GC content within the Polygonatum genus. Furthermore, we predicted a potential 59 to 64 RNA editing sites distributed across 22 protein-coding genes, with the ndhB gene exhibiting the most prominent propensity for RNA editing sites, boasting a tally of 15 sites. Notably, six regions of substantial potential variability were ascertained, characterized by elevated Pi values. Noteworthy, molecular markers for species identification, population genetic scrutiny, and phylogenetic investigations within the genus were identified in the form of the psaJ-rpl33 and trnS + trnT-psaD barcodes. The resultant phylogenetic tree unequivocally depicted the formation of a monophyletic clade comprising species within the evolutionary framework of Liliaceae, demonstrating closer evolutionary affinities with Maianthemum, Dracaeneae, and Asparageae. This comprehensive compendium of findings collectively contributes to the advancement of molecular species identification, elucidation of phylogenetic interrelationships, and the establishment of DNA barcodes tailored to the Polygonatum species.

OTP970 Is Required for RNA Editing of Chloroplast ndhB Transcripts in Arabidopsis thaliana.

RNA editing is essential for compensating for defects or mutations in haploid organelle genomes and is regulated by numerous trans-factors. Pentatricopeptide repeat (PPR) proteins are the prime factors that are involved in RNA editing; however, many have not yet been identified. Here, we screened the plastid-targeted PLS-DYW subfamily of PPR proteins belonging to Arabidopsis thaliana and identified ORGANELLE TRANSCRIPT PROCESSING 970 (OTP970) as a key player in RNA editing in plastids. A loss-of-function otp970 mutant was impaired in RNA editing of ndhB transcripts at site 149 (ndhB-C149). RNA-immunoprecipitation analysis indicated that OTP970 was associated with the ndhB-C149 site. The complementation of the otp970 mutant with OTP970 lacking the DYW domain (OTP970∆DYW) failed to restore the RNA editing of ndhB-C149. ndhB gene encodes the B subunit of the NADH dehydrogenase-like (NDH) complex; however, neither NDH activity and stability nor NDH-PSI supercomplex formation were affected in otp970 mutant compared to the wild type, indicating that alteration in amino acid sequence is not necessary for NdhB function. Together, these results suggest that OTP970 is involved in the RNA editing of ndhB-C149 and that the DYW domain is essential for its function.

Complete chloroplast genome of Stephania tetrandra (Menispermaceae) from Zhejiang Province: insights into molecular structures, comparative genome analysis, mutational hotspots and phylogenetic relationships

BackgroundThe Stephania tetrandra S. Moore (S. tetrandra) is a medicinal plant belonging to the family Menispermaceae that has high medicinal value and is well worth doing further exploration. The wild resources of S. tetrandra were widely distributed in tropical and subtropical regions of China, generating potential genetic diversity and unique population structures. The geographical origin of S. tetrandra is an important factor influencing its quality and price in the market. In addition, the species relationship within Stephania genus still remains uncertain due to high morphological similarity and low support values of molecular analysis approach. The complete chloroplast (cp) genome data has become a promising strategy to determine geographical origin and understand species evolution for closely related plant species. Herein, we sequenced the complete cp genome of S. tetrandra from Zhejiang Province and conducted a comparative analysis within Stephania plants to reveal the structural variations, informative markers and phylogenetic relationship of Stephania species.ResultsThe cp genome of S. tetrandra voucher ZJ was 157,725 bp, consisting of a large single copy region (89,468 bp), a small single copy region (19,685 bp) and a pair of inverted repeat regions (24,286 bp each). A total of 134 genes were identified in the cp genome of S. tetrandra, including 87 protein-coding genes, 8 rRNA genes, 37 tRNA genes and 2 pseudogene copies (ycf1 and rps19). The gene order and GC content were highly consistent in the Stephania species according to the comparative analysis results, with the highest RSCU value in arginine (1.79) and lowest RSCU value in serine of S. tetrandra, respectively. A total of 90 SSRs have been identified in the cp genome of S. tetrandra, where repeats that consisting of A or T bases were much higher than that of G or C bases. In addition, 92 potential RNA editing sites were identified in 25 protein-coding genes, with the most predicted RNA editing sites in ndhB gene. The variations on length and expansion extent to the junction of ycf1 gene were observed between S. tetrandra vouchers from different regions, indicating potential markers for further geographical origin discrimination. Moreover, the values of transition to transversion ratio (Ts/Tv) in the Stephania species were significantly higher than 1 using Pericampylus glaucus as reference. Comparative analysis of the Stephania cp genomes revealed 5 highly variable regions, including 3 intergenic regions (trnH-psbA, trnD-trnY, trnP) and two protein coding genes (rps16 and ndhA). The identified mutational hotspots of Stephania plants exhibited multiple SNP sites and Gaps, as well as different Ka/Ks ratio values. In addition, five pairs of specific primers targeting the divergence regions were accordingly designed, which could be utilized as potential molecular markers for species identification, population genetic and phylogenetic analysis in Stephania species. Phylogenetic tree analysis based on the conserved chloroplast protein coding genes indicated a sister relationship between S. tetrandra and the monophyletic group of S. japonica and S. kwangsiensis with high support values, suggesting a close genetic relationship within Stephania plants. However, two S. tetrandra vouches from different regions failed to cluster into one clade, confirming the occurrences of genetic diversities and requiring further investigation for geographical tracing strategy.ConclusionsOverall, we provided comprehensive and detailed information on the complete chloroplast genome and identified nucleotide diversity hotspots of Stephania species. The obtained genetic resource of S. tetrandra from Zhejiang Province would facilitate future studies in DNA barcode, species discrimination, the intraspecific and interspecific variability and the phylogenetic relationships of Stephania plants.

Light/heat effects on RNA editing in chloroplast NADH-plastoquinone oxidoreductase subunit 2 (ndhB) gene of Calotropis (Calotropis procera)

BackgroundRNA editing is common in terrestrial plants, especially in mitochondria and chloroplast. In the photosynthesis process, NAD dehydrogenase plays a very important role. Subunit 2 of NADH-dehydrogenase is one of the major subunits in NAD dehydrogenase complex. Using desert plant Calotropis (Calotropis procera), this study focuses on the RNA editing activity of ndhB based on light time. ResultsNdhB (NADH-dehydrogenase subunit 2) gene accession no. MK144329 was isolated from Calotropis procera genomic data (PRJNA292713). Additionally, using RNA-seq data, the cDNA of the ndhB gene of C. procera was isolated at three daylight periods, i.e., dawn (accession no. MK165161), at midday (accession no. MK165160), and pre-dusk (accession no. MK165159). Seven RNA editing sites have been found in several different positions (nucleotide no. C467, C586, C611, C737, C746, C830, and C1481) within the ndhB coding region. The rate of these alterations was deferentially edited across the three daylight periods. RNA editing rate of ndhB gene was highest at dawn, (87.5, 79.6, 78.5, 76, 68.6, 39.3, and 96.9%, respectively), less in midday (74.8, 54.1, 62.6, 47.4, 45.5, 47.4, and 93.4%, respectively), and less at pre-dusk (67, 52.6, 56.9, 40.1, 40.7, 33.2, and 90%, respectively), also all these sites were validated by qRT-PCR. ConclusionThe differential editing of chloroplast ndhB gene across light periods may be led to a somehow relations between the RNA editing and control of photosynthesis.

Plastome Sequences of Lygodium japonicum and Marsilea crenata Reveal the Genome Organization Transformation from Basal Ferns to Core Leptosporangiates

Previous studies have shown that core leptosporangiates, the most species-rich group of extant ferns (monilophytes), have a distinct plastid genome (plastome) organization pattern from basal fern lineages. However, the details of genome structure transformation from ancestral ferns to core leptosporangiates remain unclear because of limited plastome data available. Here, we have determined the complete chloroplast genome sequences of Lygodium japonicum (Lygodiaceae), a member of schizaeoid ferns (Schizaeales), and Marsilea crenata (Marsileaceae), a representative of heterosporous ferns (Salviniales). The two species represent the sister and the basal lineages of core leptosporangiates, respectively, for which the plastome sequences are currently unavailable. Comparative genomic analysis of all sequenced fern plastomes reveals that the gene order of L. japonicum plastome occupies an intermediate position between that of basal ferns and core leptosporangiates. The two exons of the fern ndhB gene have a unique pattern of intragenic copy number variances. Specifically, the substitution rate heterogeneity between the two exons is congruent with their copy number changes, confirming the constraint role that inverted repeats may play on the substitution rate of chloroplast gene sequences.

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.

Templated synthesis of monodisperse mesoporous maghemite/silica microspheres for magnetic separation of genomic DNA

A novel method is described for the preparation of superparamagnetic mesoporous maghemite (γ-Fe 2O 3)/silica (SiO 2) composite microspheres to allow rapid magnetic separation of DNA from biological samples. With magnetite (Fe 3O 4) and silica nanoparticles as starting materials, such microspheres were synthesized by the following two consecutive steps: (1) formation of monodispersed organic/inorganic hybrid microspheres through urea–formaldedyde (UF) polymerization and (2) removal of the organic template and phase transformation of Fe 3O 4 to γ-Fe 2O 3 by calcination at elevated temperatures. The as-synthesized particles obtained by heating at temperature 300 °C feature spherical shape and uniform particle size ( d particle=1.72 μm), high saturation magnetization ( M s=17.22 emu/g), superparamagnetism ( M r/ M s=0.023), high surface area ( S BET=240 m 2/g), and mesoporosity ( d pore=6.62 nm). The composite microsphere consists of interlocked amorphous SiO 2 nanoparticles, in which cubic γ-Fe 2O 3 nanocrystals are homogeneously dispersed and thermally stable against γ- to α-phase transformation at temperatures up to 600 °C. With the exposed iron oxide nanoparticles coated with a thin layer of silica shell, the magnetic microspheres were used as a solid-phase adsorbent for rapid extraction of genomic DNA from plant samples. The results show that the DNA templates isolated from pea and green pepper displayed single bands with molecular weights greater than 8 kb and A 260/ A 280 values of 1.60–1.72. The PCR amplification of a fragment encoding the endogenous chloroplast ndhB gene confirmed that the DNA templates obtained were inhibitor-free and amenable to sensitive amplification-based DNA technologies.

Novel Nuclear-encoded Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex

The NAD(P)H dehydrogenase (NDH) complex functions in photosystem I cyclic electron transfer in higher plant chloroplasts and is crucial for plant responses to environmental stress. Chloroplast NDH complex is a close relative to cyanobacterial NDH-1L complex, and all fifteen subunits so far identified in NDH-1L have homologs in the chloroplast NDH complex. Here we report on the identification of two nuclear-encoded proteins NDH48 and NDH45 in higher plant chloroplasts and show their intimate association with the NDH complex. These two membrane proteins are shown to interact with each other and with the NDH complex enriched in stroma thylakoids. Moreover, the deficiency of either the NDH45 protein or the NDH48 protein in respective mutant plants leads to severe defects in both the accumulation and the function of the NDH complex. The NDH48 and NDH45 proteins are not components of the hydrophilic connecting domain of the NDH complex but are strongly attached to the hydrophobic membrane domain. We conclude that NDH48 and NDH45 are novel nuclear-encoded subunits of the chloroplast NDH complex and crucial both for the stable structure and function of the NDH complex.

The molecular mechanism of menadione resistance in the Synechocystis sp. PCC 6803 cyanobacterium

The effect of mutations in the genes encoding dehydrogenases and oxidases on the resistance of the Synechocystis sp. PCC 6803 cyanobacterium to menadione, an oxidative stress inducer, was studied. An enhanced sensitivity to menadione was observed in the mutants carrying inserts in the drgA gene encoding the NAD(P)H:quinone oxidoreductase (NQR) and in the ndhB gene encoding the subunit of NDH-1 complex. The menadione resistance in the mutants lacking oxidases (Ox), succinate dehydrogenase (SDH), and NDH-2 dehydrogenase do not differ from those in wild-type cells. An additional mutation in the drgA gene increased the sensitivity to menadione in the NDH-2 and Ox mutants. The double mutant that lacks both SDH and NQR was not viable. The expression of the drgA gene decreased during cell incubation in the dark but increased in the presence of glucose both in the dark and in light. Under photoautotrophic growth conditions, the dehydrogenase activity of the cells mainly depends on the NQR and NDH-1 functions. The re-reduction rate of the photosystem I reaction center (P700+) increased in wild-type and NDH-1 mutants after its oxidation with white light in the presence of DCMU after addition of menadione, and it decreased in the NQR mutant. The reduction of P700+ was accelerated in the presence of menadiol in all the strains studied. These results suggest that NQR provides defense of cyanobacterium cells from the toxic effect of menadione via its two-electron reduction to menadiol. An increased sensitivity of the NDH-1 mutant to menadione may result from the inhibition of respiration and the cyclic electron transport in photosystem I.

Ferredoxin-quinone reductase benefits cyclic electron flow around photosystem 1 in tobacco leaves upon exposure to chilling stress under low irradiance

The function of chloroplast ferredoxin quinone reductase (FQR)-dependent flow was examined by comparing a wild type tobacco and a tobacco transformant (ΔndhB) in which the ndhB gene had been disrupted with their antimycin A (AA)-fed leaves upon exposure to chilling temperature (4 °C) under low irradiance (100 µmol m−2 s−1 photon flux density). During the chilling stress, the maximum photochemical efficiency of photosystem (PS) 2 (Fv/Fm) decreased markedly in both the controls and AA-fed leaves, and P700+ was also lower in AA-fed leaves than in the controls, implying that FQR-dependent cyclic electron flow around PS1 functioned to protect the photosynthetic apparatus from chilling stress under low irradiance. Under such stress, non-photochemical quenching (NPQ), particularly the fast relaxing NPQ component (qf) and the de-epoxidized ratio of the xanthophyll cycle pigments, (A+Z)/(V+A+Z), formed the difference between AA-fed leaves and controls. The lower NPQ in AA-fed leaves might be related to an inefficient proton gradient across thylakoid membranes (ΔpH) because of inhibiting an FQR-dependent cyclic electron flow around PS1 at chilling temperature under low irradiance.

The Function of Chloroplastic NAD(P)H Dehydrogenase in Tobacco during Chilling Stress under Low Irradiance

The function of chloroplastic NAD(P)H dehydrogenase (NDH) was examined by comparing a tobacco transformant (DeltandhB) in which the ndhB gene had been disrupted with its wild type, upon exposure to chilling temperature (4 degrees C) under low irradiance (100 micro mol m(-2) s(-1) PFD). During the chilling stress, the maximum photochemical efficiency of PSII (F(v)/F(m)) decreased markedly in both the wild type and DeltandhB. However, both F(v)/F(m) and P700(+), as well as the PSII-driven electron transport rate (ETR), in DeltandhB were lower than that in the wild type, implying that NDH-dependent cyclic electron flow around PSI functioned to protect the photosynthetic apparatus from chilling stress under low irradiance. Under the stress, non-photochemical quenching (NPQ), particularly the fast relaxing NPQ component (qf) and the de-epoxidized ratio of the xanthophyll cycle pigments, (A+Z)/(V+A+Z), were distinguishable in DeltandhB from those in the wild type. The lower NPQ in DeltandhB might be related to an inefficient proton gradient across thylakoid membranes (DeltapH) because of lacking an NDH-dependent cyclic electron flow around PSI at chilling temperature under low irradiance.

Post-illumination Reduction of the Plastoquinone Pool in Chloroplast Transformants in which Chloroplastic NAD(P)H Dehydrogenase was Inactivated

We reported previously that an ndhB gene disruptant, delta ndhB, had the same phenotype as wild-type tobacco plants under normal growth conditions. Two other groups have reported conflicting phenotypes with each other for ndhCKJ operon disruptants. Here, we generated two transformants in which the ndhCKJ operon was disrupted, and found that new transformants had the same phenotype as delta ndhB. After illumination with visible light, all ndh disruptants had higher levels of steady-state fluorescence than wild-type controls when measured under weak light, suggesting that reduction of the plastoquinone pool in ndh disruptants was greater than that in wild-type controls. The weak light itself could not reduce the plastoquinone much, so the reduction in the plastoquinone in the mutant was due to electron donation from stromal reductants generated during illumination with the strong light. These results supported the hypothesis that NAD(P)H dehydrogenase prevents overreduction in chloroplasts and suggested that chlororespiratory oxidase did not function under low light or in the dark.

Lack of conservation of editing sites in mRNAs that encode subunits of the NAD(P)H dehydrogenase complex in plastids and mitochondria of Arabidopsis thaliana.

RNA editing in the plastids and mitochondria of higher plants involves C to U conversion of specific nucleotides in the mRNA. This leads to the synthesis of proteins that are different from those predicted by the DNA sequence. Editing appears to have arisen at about the same time in both plastids and mitochondria, suggesting a common evolutionary origin. The problem we address here is whether or not there has been co-evolution of the editing systems in the two organelles. Our test system was editing of the Arabidopsis thaliana mRNAs for ndhB and nad2, and for ndhD and nad4, which encode homologous subunits of the plastid and mitochondrial NAD(P)H dehydrogenases, respectively. The editing sites in the Arabidopsis nad2 and nad4 mRNAs have previously been determined and we report here 19 editing sites in eight mRNAs in Arabidopsis plastids. Out of these, eight sites are localized in the ndhB mRNA. In its mitochondrial counterpart, nad2, 31 editing sites are present, none of which are shared with the ndhB gene. The Arabidopsis ndhD mRNA is edited at four positions, only one of which is shared by its mitochondrial homologue, nad4, which contains 32 editing sites. These findings suggest that, although editing in the two organelles may have derived from a single system, there is no significant conservation of editing sites in cognate mRNAs in plastids and mitochondria.

Targeted inactivation of the plastid ndhB gene in tobacco results in an enhanced sensitivity of photosynthesis to moderate stomatal closure.

The ndh genes encoding for the subunits of NAD(P)H dehydrogenase complex represent the largest family of plastid genes without a clearly defined function. Tobacco (Nicotiana tabacum) plastid transformants were produced in which the ndhB gene was inactivated by replacing it with a mutant version possessing translational stops in the coding region. Western-blot analysis indicated that no functional NAD(P)H dehydrogenase complex can be assembled in the plastid transformants. Chlorophyll fluorescence measurements showed that dark reduction of the plastoquinone pool by stromal reductants was impaired in ndhB-inactivated plants. Both the phenotype and photosynthetic performance of the plastid transformants was completely normal under favorable conditions. However, an enhanced growth retardation of ndhB-inactivated plants was revealed under humidity stress conditions causing a moderate decline in photosynthesis via stomatal closure. This distinctive phenotype was mimicked under normal humidity by spraying plants with abscisic acid. Measurements of CO(2) fixation demonstrated an enhanced decline in photosynthesis in the mutant plants under humidity stress, which could be restored to wild-type levels by elevating the external CO(2) concentration. These results suggest that the plastid NAD(P)H:plastoquinone oxidoreductase in tobacco performs a significant physiological role by facilitating photosynthesis at moderate CO(2) limitation.

Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around photosystem I.

To evaluate the physiological significance of cyclic electron flow around photosystem (PS) I, we used a reverse genetic approach to focus on 11 chloroplast genes that encode homologs of mitochondrial complex I subunits (ndhA-K). Since their discovery, the exact function of the respiratory components in plant chloroplasts has been a matter of discussion. We disrupted one of these genes (ndhB) in tobacco by chloroplast transformation. Analysis of the transient increase in chlorophyll fluorescence after actinic light illumination and the redox kinetics of P700 (reaction center chlorophylls of PS I) suggest that the cyclic electron flow around PS I is impaired in the ndhB-deficient transformants. Transformants grew normally in a greenhouse, suggesting that the cyclic electron flow around PS I mediated by ndh gene products is dispensable in tobacco under mild environmental conditions.

Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803

The expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803 was continuously monitored as bioluminescence by an automated monitoring system, using the bacterial luciferase genes (luxAB) of Vibrio harveyi as a reporter of promoter activity. A dnaK-reporting bioluminescent Synechocystis strain was constructed by fusing a promoterless segment of the luxAB gene set downstream of the promoter region of the Synechocystis dnaK gene and introduction of this gene fusion into a BglII site downstream of the ndhB gene in the Synechocystis chromosome. Bioluminescence from this strain was continuously monitored and oscillated with a period of about 22 h for at least 5 days in continuous light. The phase of the rhythm was reset by the timing of the 12-h dark period administered prior to the continuous light. The period of the rhythm was temperature compensated between 25 and 35 degrees C. Thus, the bioluminescence rhythm satisfied the three criteria of circadian rhythms. Furthermore, the abundance of dnaK mRNA also oscillated with a period of about 1 day for at least 2 days in continuous light conditions, indicating circadian control of dnaK gene expression in Synechocystis sp. strain PCC 6803.

Functional loss of all ndh genes in an otherwise relatively unaltered plastid genome of the holoparasitic flowering plant Cuscuta reflexa

We have cloned and sequenced an area of about 9.0 kb of the plastid DNA (ptDNA) from the holoparasitic flowering plant Cuscuta reflexa to investigate the evolutionary response of plastid genes to a reduced selective pressure. The region contains genes for the 16S rRNA, a subunit of a plastid NAD(P)H dehydrogenase (ndhB), three transfer RNAs (trnA, trnI, trnV) as well as the gene coding for the ribosomal protein S7 (rps7). While the other genes are strongly conserved in C. reflexa, the ndhB gene is a pseudogene due to many frameshift mutations. In addition we used heterologous gene probes to identify the other ndh genes encoded by the plastid genome in higher plants. No hybridization signals could be obtained, suggesting that these genes are either lost or strongly altered in the ptDNA of C. reflexa. Together with evidence of deleted genes in the ptDNA of C. reflexa, the plastid genome can be grouped into four classes reflecting a different evolutionary rate in each case. The phylogenetic position of Cuscuta and the significance of ndh genes in the plastid genome of higher plants are discussed.

Identification and Characterization of the ictA/ndhL Gene Product Essential to Inorganic Carbon Transport of Synechocystis PCC6803

The ictA gene, renamed ndhL in this paper, essential to inorganic carbon transport of Synechocystis PCC6803, was expressed in Eschericia coli as a fusion protein with glutathione S-transferase. An antibody was raised against this fusion protein. Western analysis of the thylakoid membrane of wild-type (WT) Synechocystis revealed that a protein with an apparent molecular mass of 6.7 kilodaltons cross-reacted with this antibody. No immunoreactive protein was present in the thylakoid membranes of the Synechocystis mutants, RKb and M9, which have defects in the ictA/ndhL gene, or in the cytoplasmic membranes of the WT and mutant cells. Thus, the protein reacted with the antibody is the ictA gene product (IctA) and is localized in the thylakoid membrane of WT cells. IctA was absent in the thylakoid membranes of the M55 mutant, in which the ndhB gene is inactivated, and was poorly immunostained in the membranes of the mutants (M-ndhC and M-ndhK) constructed by inactivating the ndhC and ndhK genes of WT Synechocystis, respectively. The carbon dioxide uptake activity was nearly zero in M-ndhK and was about 40% of the activity of WT cells in M-ndhC. The RKb, M-ndhC, and M-ndhK mutants were unable to grow or grew very slowly under photoheterotrophic conditions. These results indicated that NADH dehydrogenase is essential to inorganic carbon transport and photoheterotrophic growth of Synechocystis and that IctA is one of the subunits of this enzyme.

Identification of editing positions in the ndhB transcript from maize chloroplasts reveals sequence similarities between editing sites of chloroplasts and plant mitochondria.

A comparison of the nucleotide sequences from genomic DNA and cDNA of the ndhB gene from maize chloroplasts shows that the ndhB transcript is edited by C-to-U transitions at six positions which appear to exist as editing sites also in the chloroplast ndhB genes from rice and tobacco but not from liverwort. In order to identify possible sequence determinants necessary for editing, the sequences surrounding the newly identified ndhB and previously identified ndhA editing sites were compared with each other and with editing sites observed in plant mitochondrial transcripts. Among the chloroplast editing sites two closely positioned ndhB sites show similarity by sharing a common octanucleotide. The existence of the identical octanucleotide in the ndhJ gene whose transcript is not edited at the respective position, shows, however, that this octanucleotide is not sufficient to elicit the editing process. On the other hand, several of the chloroplast editing sites show sequence similarities with certain sets of consensus sequences reported earlier for editing sites of plant mitochondria. This supports the view that the editing processes of both plant organelles share common components and/or mechanistic steps and that the consensus sequences are part of the determinants necessary for editing.