Mutagenesis of the genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)H-plastoquinone-oxidoreductase in tobacco by polyethylene glycol-mediated plastome transformation

Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the metabolic function of the enzyme is unknown. The enzyme consists of at least eleven subunits (A-K), which are all encoded on the plastid chromosome. We have mutagenized ndhC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertion, of a cassette which carried a spectinomycin resistance gene as a marker. The transformation was carried out by the polyethylene glycol-mediated plastid transformation method. Southern analysis revealed that even after repeated regeneration cycles each of the four different types of transformants had retained 1–5% of wild-type gene copies. This suggests that complete deletion of ndh genes is not compatible with viability. The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark after illumination with actinic light for 5 min; in the wild-type this dark-rise reflects a transient reduction of the plastoquinone pool by reduction equivalents generated in the stroma; and (ii) transformants with defects in the ndhC-K-J operon accumulate starch, indicating inefficient oxidation of glucose via glycolysis and the oxidative pentose phosphate pathway. Both observations support the theory of chlororespiration, which postulates that the NDH complex acts as a valve to remove excess reduction equivalents in the chloroplast. Received: 15 December 1997 / Accepted: 15 January 1998     

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.     

Deletion of the tobacco plastid psbA gene triggers an upregulation of the thylakoid-associated NAD(P)H dehydrogenase complex and the plastid terminal oxidase (PTOX)

We have constructed a tobacco psbA gene deletion mutant that is devoid of photosystem II (PSII) complex. Analysis of thylakoid membranes revealed comparable amounts, on a chlorophyll basis, of photosystem I (PSI), the cytochrome b6f complex and the PSII light-harvesting complex (LHCII) antenna proteins in wild-type (WT) and Δ psbA leaves. Lack of PSII in the mutant, however, resulted in over 10-fold higher relative amounts of the thylakoid-associated plastid terminal oxidase (PTOX) and the NAD(P)H dehydrogenase (NDH) complex. Increased amounts of Ndh polypeptides were accompanied with a more than fourfold enhancement of NDH activity in the mutant thylakoids, as revealed by in-gel NADH dehydrogenase measurements. NADH also had a specific stimulating effect on P700⁺ re-reduction in the Δ psbA thylakoids. Altogether, our results suggest that enhancement of electron flow via the NDH complex and possibly other alternative electron transport routes partly compensates for the loss of PSII function in the Δ psbA mutant. As mRNA levels were comparable in WT and Δ psbA plants, upregulation of the alternative electron transport pathways (NDH complex and PTOX) occurs apparently by translational or post-translational mechanisms.     

Anaerobiosis Induced State Transition: A Non Photochemical Reduction of PQ Pool Mediated by NDH in Arabidopsis thaliana

Background

Non photochemical reduction of PQ pool and mobilization of LHCII between PSII and PSI are found to be linked under abiotic stress conditions. The interaction of non photochemical reduction of PQ pool and state transitions associated physiological changes are critically important under anaerobic condition in higher plants.

Methodology/Findings

The present study focused on the effect of anaerobiosis on non-photochemical reduction of PQ pool which trigger state II transition in Arabidopsis thaliana. Upon exposure to dark-anaerobic condition the shape of the OJIP transient rise is completely altered where as in aerobic treated leaves the rise is unaltered. Rise in F _o and F _J was due to the loss of oxidized PQ pool as the PQ pool becomes more reduced. The increase in F_o′ was due to the non photochemical reduction of PQ pool which activated STN7 kinase and induced LHCII phosphorylation under anaerobic condition. Further, it was observed that the phosphorylated LHCII is migrated and associated with PSI supercomplex increasing its absorption cross-section. Furthermore, evidences from crr2-2 (NDH mutant) and pgr5 mutants (deficient in non NDH pathway of cyclic electron transport) have indicated that NDH is responsible for non photochemical reduction of the PQ pool. We propose that dark anaerobic condition accelerates production of reducing equivalents (such as NADPH by various metabolic pathways) which reduce PQ pool and is mediated by NDH leading to state II transition.

Conclusions/Significance

Anaerobic condition triggers non photochemical reduction of PQ pool mediated by NDH complex. The reduced PQ pool activates STN7 kinase leading to state II transition in A. thaliana.     

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.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Induction and Transmission of a Merodiploid Condition near the Terminal Area of the Chromosome of BACILLUS SUBTILIS

A small fraction (about 0.5%) of the transformants for a particular marker of B. subtilis (ilvA4; most probably a deletion) were found to be relatively unstable merodiploids. They possess a redundancy of the metB–ilvA chromosome segment. When their DNA is used as donor in transformation a merodiploid condition for the whole of this segment is created in all ilvA4⁺ transformants. For several of the duplicated loci both copies often are of recipient strain origin. Markers originally belonging to different copies of the diploidized region can be contransferred in PBS1-mediated transduction. The data are well in agreement with the hypothesis that the merodiploids carry a tandem duplication. An alternative hypothesis which does not call for integration of the exogenote within the recipient chromosome was also considered. Models are proposed for interpreting the segregation of the merodiploids, the transmission of the diploid state and its generation during transformation of the ilvA4 marker by wild-type DNA.     

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.     

Instability of Plastid DNA in the Nuclear Genome

Functional gene transfer from the plastid (chloroplast) and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo) readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.     

Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation

We describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aad A-based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atp B promoter region. Efficient recombination between atp B repeats deletes the intervening foreign genes and 1984 bp of plastid DNA (co-ordinates 57 424–59 317) containing the rbc L gene. Only five foreign bases are present in Δ rbc L plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbc L-related sequences in Δ rbc L plants indicated an extra-plastidic origin. Mutant Δ rbc L plants were heterotrophic, pale-green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aad A-based transformation with the wild-type rbc L gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aad A genes. Rescue of the Δ rbc L mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbc L deletion mutant isolated here is readily transformed with the efficient aad A marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods.     

Deletions in the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA₁₂-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.     

Extensive homologous recombination between introduced and native regulatory plastid DNA elements in transplastomic plants

Homologous recombination within plastids directs plastid genome transformation for foreign gene expression and study of plastid gene function. Though transgenes are generally efficiently targeted to their desired insertion site, unintended homologous recombination events have been observed during plastid transformation. To understand the nature and abundance of these recombination events, we analyzed transplastomic tobacco lines derived from three different plastid transformation vectors utilizing two different loci for foreign gene insertion. Two unintended recombinant plastid DNA species were formed from each regulatory plastid DNA element included in the transformation vector. Some of these recombinant DNA species accumulated to as much as 10–60% of the amount of the desired integrated transgenic sequence in T0 plants. Some of the recombinant DNA species undergo further, “secondary” recombination events, resulting in an even greater number of recombinant plastid DNA species. The abundance of novel recombinant DNA species was higher in T0 plants than in T1 progeny, indicating that the ancillary recombination events described here may have the greatest impact during selection and regeneration of transformants. A line of transplastomic tobacco was identified containing an antibiotic resistance gene unlinked from the intended transgene insertion as a result of an unintended recombination event, indicating that the homologous recombination events described here may hinder efficient recovery of plastid transformants containing the desired transgene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An Arabidopsis Mutant with High Cyclic Electron Flow around Photosystem I (hcef) Involving the NADPH Dehydrogenase Complex

Cyclic electron flow (CEFI) has been proposed to balance the chloroplast energy budget, but the pathway, mechanism, and physiological role remain unclear. We isolated a new class of mutant in Arabidopsis thaliana, hcef for high CEF1, which shows constitutively elevated CEF1. The first of these, hcef1, was mapped to chloroplast fructose-1,6-bisphosphatase. Crossing hcef1 with pgr5, which is deficient in the antimycin A–sensitive pathway for plastoquinone reduction, resulted in a double mutant that maintained the high CEF1 phenotype, implying that the PGR5-dependent pathway is not involved. By contrast, crossing hcef1 with crr2-2, deficient in thylakoid NADPH dehydrogenase (NDH) complex, results in a double mutant that is highly light sensitive and lacks elevated CEF1, suggesting that NDH plays a direct role in catalyzing or regulating CEF1. Additionally, the NdhI component of the NDH complex was highly expressed in hcef1, whereas other photosynthetic complexes, as well as PGR5, decreased. We propose that (1) NDH is specifically upregulated in hcef1, allowing for increased CEF1; (2) the hcef1 mutation imposes an elevated ATP demand that may trigger CEF1; and (3) alternative mechanisms for augmenting ATP cannot compensate for the loss of CEF1 through NDH.     

A novel nucleus-encoded chloroplast protein, PIFI, is involved in NAD(P)H dehydrogenase complex-mediated chlororespiratory electron transport in Arabidopsis

A transient rise in chlorophyll fluorescence after turning off actinic light reflects nonphotochemical reduction of the plastoquinone (PQ) pool. This process is dependent on the activity of the chloroplast NAD(P)H dehydrogenase (NDH) complex, which mediates electron flow from stromal reductants to the PQ pool. In this study, we characterized an Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant pifi (for postillumination chlorophyll fluorescence increase), which possesses an intact NDH complex, but lacks the NDH-dependent chlorophyll fluorescence increase after turning off actinic light. The nuclear gene PIFI (At3g15840) containing the T-DNA insertion encodes a chloroplast-targeted protein localized in the stroma and is annotated as a protein of unknown function. The pifi mutant exhibited a lower capacity for nonphotochemical quenching, but similar CO(2) assimilation rates, photosystem II (PSII) quantum efficiencies (PhiPSII), and reduction levels of the primary electron acceptor of PSII (1 - qL) as compared with the wild type. The pifi mutant grows normally under optimal conditions, but exhibits greater sensitivity to photoinhibition and long-term mild heat stress than wild-type plants, which is consistent with lower capacity of nonphotochemical quenching. We conclude that PIFI is a novel component essential for NDH-mediated nonphotochemical reduction of the PQ pool in chlororespiratory electron transport.     

Chloramphenicol acetyltransferase as selectable marker for plastid transformation

Chloroplast transformation remains a demanding technique and is still restricted to relatively few plant species. The limited availability of selectable marker genes and the lack of selection markers that would be universally applicable to all plant species represent some of the most serious technical problems involved in extending the species range of plastid transformation. Here we report the development of the chloramphenicol acetyltransferase gene cat as a new selectable marker for plastid transformation. We show that, by selecting for chloramphenicol resistance, tobacco chloroplast transformants are readily obtained. Transplastomic lines quickly reach the homoplasmic state (typically in one additional regeneration round), accumulate the chloramphenicol acetyltransferase enzyme to high levels and transmit their plastid transgenes maternally into the next generation. No spontaneous antibiotic resistance mutants appear upon chloramphenicol selection. Several lines of evidence support the assumption that plant mitochondria are also sensitive to chloramphenicol suggesting that the chloramphenicol acetyltransferase may be a good candidate selectable marker for plant mitochondrial transformation.     

Stable transformation of petunia plastids

Plastid transformation results in stably expressed foreign genes, which for most Angiosperms are largely excluded from sperm cells, thereby greatly reducing the risk of foreign gene spread through pollen. Prior to this work, fertile plastid transformants were restricted to tobacco, tomato and Lesquerella . Application of plastid engineering in the important floriculture industry requires the development of stable plastid transformation in a major ornamental plant species such as Petunia hybrida. Here we describe the successful isolation of fertile and stable plastid transformants in a commercial cultivar of P. hybrida (var. Pink Wave). Plastid targeting regions from tobacco were used to integrate aad A and gusA between the acc D and rbc L genes of P. hybrida plastid DNA following particle bombardment of leaves. For three spectinomycin and streptomycin resistant lines, DNA blot analysis confirmed transgene integration into plastid DNA and homoplasmy. Maternal inheritance and homoplasmy resulted in 100 transmission of spectinomycin resistance to progeny after selfing. Plastid transformants expressed the gusA gene uniformly within leaves and to comparable levels in all three lines. Insertion of trait genes in place of gusA coding sequences enables immediate applications of our plastid transformation vector. Establishment of plastid transformation in P. hybrida facilitates a safe and reliable use of this important ornamental plant for research and plant biotechnology.These two authors contributed equally to this work.     

Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient

The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the distinctive tip-high Ca²⁺ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea.