The Arabidopsis SR45 splicing factor bridges the splicing machinery and the exon-exon junction complex.

The Arabidopsis splicing factor serine/arginine-rich 45 (SR45) contributes to several biological processes. The sr45-1 loss-of-function mutant exhibits delayed root development, late flowering, unusual numbers of floral organs, shorter siliques with decreased seed sets, narrower leaves and petals, and altered metal distribution. SR45 bears a unique RNA recognition motif (RRM) flanked by one serine/arginine-rich (RS) domain on both sides. Here, we studied the function of each SR45 domains by examining their involvement in: (i) the spatial distribution of SR45; (ii) the establishment of a protein-protein interaction network including spliceosomal and exon-exon junction complex (EJC) components; and (iii) the RNA binding specificity. We report that the endogenous SR45 promoter is active during vegetative and reproductive growth, and that the SR45 protein localizes in the nucleus. We demonstrate that the C-terminal arginine/serine-rich domain is a determinant of nuclear localization. We show that the SR45 RRM domain specifically binds purine-rich RNA motifs via three residues (H101, H141, and Y143), and is also involved in protein-protein interactions. We further show that SR45 bridges both mRNA splicing and surveillance machineries as a partner of EJC core components and peripheral factors, which requires phosphoresidues probably phosphorylated by kinases from both the CLK and SRPK families. Our findings provide insights into the contribution of each SR45 domain to both spliceosome and EJC assemblies.

Altered metal distribution in the sr45-1 Arabidopsis mutant causes developmental defects.

The plant serine/arginine-rich (SR) splicing factor SR45 plays important roles in several biological processes, such as splicing, DNA methylation, innate immunity, glucose regulation, and abscisic acid signaling. A homozygous Arabidopsis sr45-1 null mutant is viable, but exhibits diverse phenotypic alterations, including delayed root development, late flowering, shorter siliques with fewer seeds, narrower leaves and petals, and unusual numbers of floral organs. Here, we report that the sr45-1 mutant presents an unexpected constitutive iron deficiency phenotype characterized by altered metal distribution in the plant. RNA-Sequencing highlighted severe perturbations in metal homeostasis, the phenylpropanoid pathway, oxidative stress responses, and reproductive development. Ionomic quantification and histochemical staining revealed strong iron accumulation in the sr45-1 root tissues accompanied by iron starvation in aerial parts. Mis-splicing of several key iron homeostasis genes, including BTS, bHLH104, PYE, FRD3, and ZIF1, was observed in sr45-1 roots. We showed that some sr45-1 developmental abnormalities can be complemented by exogenous iron supply. Our findings provide new insight into the molecular mechanisms governing the phenotypes of the sr45-1 mutant.

Correction: Co-Evolution of Mitochondrial tRNA Import and Codon Usage Determines Translational Efficiency in the Green Alga Chlamydomonas.

[This corrects the article DOI: 10.1371/journal.pgen.1002946.].

Long-term acclimation to cadmium exposure reveals extensive phenotypic plasticity in Chlamydomonas.

Increasing industrial and anthropogenic activities are producing and releasing more and more pollutants in the environment. Among them, toxic metals are one of the major threats for human health and natural ecosystems. Because photosynthetic organisms play a critical role in primary productivity and pollution management, investigating their response to metal toxicity is of major interest. Here, the green microalga Chlamydomonas (Chlamydomonas reinhardtii) was subjected to short (3 d) or chronic (6 months) exposure to 50 µM cadmium (Cd), and the recovery from chronic exposure was also examined. An extensive phenotypic characterization and transcriptomic analysis showed that the impact of Cd on biomass production of short-term (ST) exposed cells was almost entirely abolished by long-term (LT) acclimation. The underlying mechanisms were initiated at ST and further amplified after LT exposure resulting in a reversible equilibrium allowing biomass production similar to control condition. This included modification of cell wall-related gene expression and biofilm-like structure formation, dynamics of metal ion uptake and homeostasis, photosynthesis efficiency recovery and Cd acclimation through metal homeostasis adjustment. The contribution of the identified coordination of phosphorus and iron homeostasis (partly) mediated by the main phosphorus homeostasis regulator, Phosphate Starvation Response 1, and a basic Helix-Loop-Helix transcription factor (Cre05.g241636) was further investigated. The study reveals the highly dynamic physiological plasticity enabling algal cell growth in an extreme environment.

ZRT-IRT-Like PROTEIN 6 expression perturbs local ion homeostasis in flowers and leads to anther indehiscence and male sterility.

Metallic micronutrients are essential throughout the plant life cycle. Maintaining metal homeostasis in plant tissues requires a highly complex and finely tuned network controlling metal uptake, transport, distribution and storage. Zinc and cadmium hyperaccumulation, such as observed in the model plant Arabidopsis halleri, represents an extreme evolution of this network. Here, non-ectopic overexpression of the A. halleri ZIP6 (AhZIP6) gene, encoding a zinc and cadmium influx transporter, in Arabidopsis thaliana enabled examining the importance of zinc for flower development and reproduction. We show that AhZIP6 expression in flowers leads to male sterility resulting from anther indehiscence in a dose-dependent manner. The sterility phenotype is associated to delayed tapetum degradation and endothecium collapse, as well as increased magnesium and potassium accumulation and higher expression of the MHX gene in stamens. It is rescued by the co-expression of the zinc efflux transporter AhHMA4, linking the sterility phenotype to zinc homeostasis. Altogether, our results confirm that AhZIP6 is able to transport zinc in planta and highlight the importance of fine-tuning zinc homeostasis in reproductive organs. The study illustrates how the characterization of metal hyperaccumulation mechanisms can reveal key nodes and processes in the metal homeostasis network.

Intertwined metal homeostasis, oxidative and biotic stress responses in the Arabidopsis frd3 mutant.

FRD3 (FERRIC REDUCTASE DEFECTIVE 3) plays a major role in iron (Fe) and zinc (Zn) homeostasis in Arabidopsis. It transports citrate, which enables metal distribution in the plant. An frd3 mutant is dwarf and chlorotic and displays a constitutive Fe-deficiency response and strongly altered metal distribution in tissues. Here, we have examined the interaction between Fe and Zn homeostasis in an frd3 mutant exposed to varying Zn supply. Detailed phenotyping using transcriptomic, ionomic, histochemical and spectroscopic approaches revealed the full complexity of the frd3 mutant phenotype, which resulted from altered transition metal homeostasis, manganese toxicity, and oxidative and biotic stress responses. The cell wall played a key role in these processes, as a site for Fe and hydrogen peroxide accumulation, and displayed modified structure in the mutant. Finally, we showed that Zn excess interfered with these mechanisms and partially restored root growth of the mutant, without reverting the Fe-deficiency response. In conclusion, the frd3 mutant molecular phenotype is more complex than previously described and illustrates how the response to metal imbalance depends on multiple signaling pathways.

Transcriptional regulation of ZIP genes is independent of local zinc status in Brachypodium shoots upon zinc deficiency and resupply.

The biological processes underlying zinc homeostasis are targets for genetic improvement of crops to counter human malnutrition. Detailed phenotyping, ionomic, RNA-Seq analyses and flux measurements with 67 Zn isotope revealed whole-plant molecular events underlying zinc homeostasis upon varying zinc supply and during zinc resupply to starved Brachypodium distachyon (Brachypodium) plants. Although both zinc deficiency and excess hindered Brachypodium growth, accumulation of biomass and micronutrients into roots and shoots differed depending on zinc supply. The zinc resupply dynamics involved 1,893 zinc-responsive genes. Multiple zinc-regulated transporter and iron-regulated transporter (IRT)-like protein (ZIP) transporter genes and dozens of other genes were rapidly and transiently down-regulated in early stages of zinc resupply, suggesting a transient zinc shock, sensed locally in roots. Notably, genes with identical regulation were observed in shoots without zinc accumulation, pointing to root-to-shoot signals mediating whole-plant responses to zinc resupply. Molecular events uncovered in the grass model Brachypodium are useful for the improvement of staple monocots.

The two copies of the zinc and cadmium ZIP6 transporter of Arabidopsis halleri have distinct effects on cadmium tolerance.

Plants have the ability to colonize highly diverse environments. The zinc and cadmium hyperaccumulator Arabidopsis halleri has adapted to establish populations on soils covering an extreme range of metal availabilities. The A. halleri ZIP6 gene presents several hallmarks of hyperaccumulation candidate genes: it is constitutively highly expressed in roots and shoots and is associated with a zinc accumulation quantitative trait locus. Here, we show that AhZIP6 is duplicated in the A. halleri genome. The two copies are expressed mainly in the vasculature in both A. halleri and Arabidopsis thaliana, indicative of conserved cis regulation, and acquired partial organ specialization. Yeast complementation assays determined that AhZIP6 is a zinc and cadmium transporter. AhZIP6 silencing in A. halleri or expression in A. thaliana alters cadmium tolerance, but has no impact on zinc and cadmium accumulation. AhZIP6-silenced plants display reduced cadmium uptake upon short-term exposure, adding AhZIP6 to the limited number of Cd transporters supported by in planta evidence. Altogether, our data suggest that AhZIP6 is key to fine-tune metal homeostasis in specific cell types. This study additionally highlights the distinct fates of duplicated genes in A. halleri.

Homology modeling and in vivo functional characterization of the zinc permeation pathway in a heavy metal P-type ATPase.

The P1B ATPase heavy metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements, enabling metal transport across the membrane. The detailed mechanism of metal permeation by HMA4 through the membrane remains elusive. Here, homology modeling of the HMA4 transmembrane region was conducted based on the crystal structure of a ZntA bacterial homolog. The analysis highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. Although the zinc pathway displayed overall conservation among the two proteins, significant differences were observed, especially in the entrance area with altered electronegativity and the presence of a ionic interaction/hydrogen bond network. The analysis also newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation. These observations could be instrumental to design low cadmium-accumulating crops, hence decreasing human cadmium exposure.

Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors.

Although the involvement of Ser/Arg-rich (SR) proteins in RNA metabolism is well documented, their role in vertebrate development remains elusive. We, therefore, elected to take advantage of the zebrafish model organism to study the SR genes' functions using the splicing morpholino (sMO) microinjection and the programmable site-specific nucleases. Consistent with previous research, we revealed discrepancies between the mutant and morphant phenotypes and we show that these inconsistencies may result from a large number of unsuspected inadvertent morpholino RNA targets. While microinjection of MOs directed against srsf5a (sMOsrsf5a) led to developmental defects, the corresponding homozygous mutants did not display any phenotypic traits. Furthermore, microinjection of sMOsrsf5a into srsf5a-/- led to the previously observed morphant phenotype. Similar findings were observed for other SR genes. sMOsrsf5a alternative target genes were identified using deep mRNA sequencing. We uncovered that only 11 consecutive bases complementary to sMOsrsf5a are sufficient for binding and subsequent blocking of splice sites. In addition, we observed that sMOsrsf5a secondary targets can be reduced by increasing embryos growth temperature after microinjection. Our data contribute to the debate about MO specificity, efficacy and the number of unknown targeted sequences.

Adaptation to high zinc depends on distinct mechanisms in metallicolous populations of Arabidopsis halleri.

Zinc (Zn) hyperaccumulation and hypertolerance are highly variable traits in Arabidopsis halleri. Metallicolous populations have evolved from nearby nonmetallicolous populations in multiple independent adaptation events. To determine whether these events resulted in similar or divergent adaptive strategies to high soil Zn concentrations, we compared two A. halleri metallicolous populations from distant genetic units in Europe (Poland (PL22) and Italy (I16)). The ionomic (Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)) and transcriptomic (RNA sequencing (RNA-Seq)) responses to growth at 5 and 150 µM Zn were analyzed in root and shoot tissues to examine the contribution of the geographic origin and treatment to variation among populations. These analyses were enabled by the generation of a reference A. halleri transcriptome assembly. The genetic unit accounted for the largest variation in the gene expression profile, whereas the two populations had contrasting Zn accumulation phenotypes and shared little common response to the Zn treatment. The PL22 population displayed an iron deficiency response at high Zn in roots and shoots, which may account for higher Zn accumulation. By contrast, I16, originating from a highly Zn-contaminated soil, strongly responded to control conditions. Our data suggest that distinct mechanisms support adaptation to high Zn in soils among A. halleri metallicolous populations.

di-Cysteine motifs in the C-terminus of plant HMA4 proteins confer nanomolar affinity for zinc and are essential for HMA4 function in vivo.

The PIB ATPase heavy metal ATPase 4 (HMA4) has a central role in the zinc homeostasis network of Arabidopsis thaliana. This membrane protein loads metal from the pericycle cells into the xylem in roots, thereby allowing root to shoot metal translocation. Moreover, HMA4 is key for zinc hyperaccumulation as well as zinc and cadmium hypertolerance in the pseudometallophyte Arabidopsis halleri. The plant-specific cytosolic C-terminal extension of HMA4 is rich in putative metal-binding residues and has substantially diverged between A. thaliana and A. halleri. To clarify the function of the domain in both species, protein variants with truncated C-terminal extension, as well as with mutated di-Cys motifs and/or a His-stretch, were functionally characterized. We show that di-Cys motifs, but not the His-stretch, contribute to high affinity zinc binding and function in planta. We suggest that the HMA4 C-terminal extension is at least partly responsible for protein targeting to the plasma membrane. Finally, we reveal that the C-terminal extensions of both A. thaliana and A. halleri HMA4 proteins share similar function, despite marginally different zinc-binding capacity.

Dynamic Distribution and Interaction of the Arabidopsis SRSF1 Subfamily Splicing Factors.

Ser/Arg-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis (Arabidopsis thaliana) RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two pathways, either dependent or independent on the XPO1 receptor. Here, we examined the expression profiles and shuttling dynamics of the Arabidopsis SRSF1 subfamily (SR30, SR34, SR34a, and SR34b) under control of their endogenous promoter in Arabidopsis and in transient expression assay. Due to its rapid nucleocytoplasmic shuttling and high expression level in transient assay, we analyzed the multiple determinants that regulate the localization and shuttling dynamics of SR34. By site-directed mutagenesis of SR34 RNA-binding sequences and Arg/Ser-rich (RS) domain, we further show that functional RRM1 or RRM2 are dispensable for the exclusive protein nuclear localization and speckle-like distribution. However, mutations of both RRMs induced aggregation of the protein whereas mutation in the RS domain decreased the stability of the protein and suppressed its nuclear accumulation. Furthermore, the RNA-binding motif mutants are defective for their export through the XPO1 (CRM1/Exportin-1) receptor pathway, but retain nucleocytoplasmic mobility. We performed a yeast two hybrid screen with SR34 as bait and discovered SR45 as a new interactor. SR45 is an unusual SR splicing factor bearing two RS domains. These interactions were confirmed in planta by FLIM-FRET and BiFC and the roles of SR34 domains in protein-protein interactions were further studied. Altogether, our report extends our understanding of shuttling dynamics of Arabidopsis SR splicing factors.

Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.

Hard selective sweep and ectopic gene conversion in a gene cluster affording environmental adaptation.

Among the rare colonizers of heavy-metal rich toxic soils, Arabidopsis halleri is a compelling model extremophile, physiologically distinct from its sister species A. lyrata, and A. thaliana. Naturally selected metal hypertolerance and extraordinarily high leaf metal accumulation in A. halleri both require Heavy Metal ATPase4 (HMA4) encoding a PIB-type ATPase that pumps Zn(2+) and Cd(2+) out of specific cell types. Strongly enhanced HMA4 expression results from a combination of gene copy number expansion and cis-regulatory modifications, when compared to A. thaliana. These findings were based on a single accession of A. halleri. Few studies have addressed nucleotide sequence polymorphism at loci known to govern adaptations. We thus sequenced 13 DNA segments across the HMA4 genomic region of multiple A. halleri individuals from diverse habitats. Compared to control loci flanking the three tandem HMA4 gene copies, a gradual depletion of nucleotide sequence diversity and an excess of low-frequency polymorphisms are hallmarks of positive selection in HMA4 promoter regions, culminating at HMA4-3. The accompanying hard selective sweep is segmentally eclipsed as a consequence of recurrent ectopic gene conversion among HMA4 protein-coding sequences, resulting in their concerted evolution. Thus, HMA4 coding sequences exhibit a network-like genealogy and locally enhanced nucleotide sequence diversity within each copy, accompanied by lowered sequence divergence between paralogs in any given individual. Quantitative PCR corroborated that, across A. halleri, three genomic HMA4 copies generate overall 20- to 130-fold higher transcript levels than in A. thaliana. Together, our observations constitute an unexpectedly complex profile of polymorphism resulting from natural selection for increased gene product dosage. We propose that these findings are paradigmatic of a category of multi-copy genes from a broad range of organisms. Our results emphasize that enhanced gene product dosage, in addition to neo- and sub-functionalization, can account for the genomic maintenance of gene duplicates underlying environmental adaptation.

Zinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives.

In Arabidopsis thaliana, FRD3 (FERRIC CHELATE REDUCTASE DEFECTIVE 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of ß-glucuronidase and green fluorescent protein reporter constructs to compare the expression profile and transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes displayed a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determined transcript variants that were differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc-excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation.

Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri.

In Arabidopsis halleri, the HMA4 gene has an essential function in Zn/Cd hypertolerance and hyperaccumulation by mediating root-to-shoot translocation of metals. Constitutive high expression of AhHMA4 results from a tandem triplication and cis-activation of the promoter of all three copies. The three AhHMA4 copies possess divergent promoter sequences, but highly conserved coding sequences, and display identical expression profiles in the root and shoot vascular system. Here, an AhHMA4::GFP fusion was expressed under the control of each of the three A. halleri HMA4 promoters in a hma2hma4 double mutant of A. thaliana to individually examine the function of each AhHMA4 copy. The protein showed non-polar localization at the plasma membrane of the root pericycle cells of both A. thaliana and A. halleri. The expression of each AhHMA4::GFP copy complemented the severe Zn-deficiency phenotype of the hma2hma4 mutant by restoring root-to-shoot translocation of Zn. However, each copy had a different impact on metal homeostasis in the A. thaliana genetic background: AhHMA4 copies 2 and 3 were more highly expressed and provided higher Zn tolerance in roots and accumulation in shoots than copy 1, and AhHMA4 copy 3 also increased Cd tolerance in roots. These data suggest a certain extent of functional differentiation among the three A. halleri HMA4 copies, stemming from differences in expression levels rather than in expression profile. HMA4 is a key node of the Zn homeostasis network and small changes in expression level can have a major impact on Zn allocation to root or shoot tissues.

Co-evolution of mitochondrial tRNA import and codon usage determines translational efficiency in the green alga Chlamydomonas.

Mitochondria from diverse phyla, including protozoa, fungi, higher plants, and humans, import tRNAs from the cytosol in order to ensure proper mitochondrial translation. Despite the broad occurrence of this process, our understanding of tRNA import mechanisms is fragmentary, and crucial questions about their regulation remain unanswered. In the unicellular green alga Chlamydomonas, a precise correlation was found between the mitochondrial codon usage and the nature and amount of imported tRNAs. This led to the hypothesis that tRNA import might be a dynamic process able to adapt to the mitochondrial genome content. By manipulating the Chlamydomonas mitochondrial genome, we introduced point mutations in order to modify its codon usage. We find that the codon usage modification results in reduced levels of mitochondrial translation as well as in subsequent decreased levels and activities of respiratory complexes. These effects are linked to the consequential limitations of the pool of tRNAs in mitochondria. This indicates that tRNA mitochondrial import cannot be rapidly regulated in response to a novel genetic context and thus does not appear to be a dynamic process. It rather suggests that the steady-state levels of imported tRNAs in mitochondria result from a co-evolutive adaptation between the tRNA import mechanism and the requirements of the mitochondrial translation machinery.

Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4.

Little is known about the types of mutations underlying the evolution of species-specific traits. The metal hyperaccumulator Arabidopsis halleri has the rare ability to colonize heavy-metal-polluted soils, and, as an extremophile sister species of Arabidopsis thaliana, it is a powerful model for research on adaptation. A. halleri naturally accumulates and tolerates leaf concentrations as high as 2.2% zinc and 0.28% cadmium in dry biomass. On the basis of transcriptomics studies, metal hyperaccumulation in A. halleri has been associated with more than 30 candidate genes that are expressed at higher levels in A. halleri than in A. thaliana. Some of these genes have been genetically mapped to broad chromosomal segments of between 4 and 24 cM co-segregating with Zn and Cd hypertolerance. However, the in planta loss-of-function approaches required to demonstrate the contribution of a given candidate gene to metal hyperaccumulation or hypertolerance have not been pursued to date. Using RNA interference to downregulate HMA4 (HEAVY METAL ATPASE 4) expression, we show here that Zn hyperaccumulation and full hypertolerance to Cd and Zn in A. halleri depend on the metal pump HMA4. Contrary to a postulated global trans regulatory factor governing high expression of numerous metal hyperaccumulation genes, we demonstrate that enhanced expression of HMA4 in A. halleri is attributable to a combination of modified cis-regulatory sequences and copy number expansion, in comparison to A. thaliana. Transfer of an A. halleri HMA4 gene to A. thaliana recapitulates Zn partitioning into xylem vessels and the constitutive transcriptional upregulation of Zn deficiency response genes characteristic of Zn hyperaccumulators. Our results demonstrate the importance of cis-regulatory mutations and gene copy number expansion in the evolution of a complex naturally selected extreme trait. The elucidation of a natural strategy for metal hyperaccumulation enables the rational design of technologies for the clean-up of metal-contaminated soils and for bio-fortification.

Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas.

Defects in complex I (NADH:ubiquinone oxidoreductase (EC 1.6.5.3)) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate because the mitochondrial genome harbors large numbers of polymorphic base changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in the heteroplasmic state, which may hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (i) respiratory complex-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (ii) transformation of the mitochondrial genome is feasible, and (iii) Chlamydomonas complex I is similar to that of humans. To illustrate this proposal, we introduced a Leu157Pro substitution into the Chlamydomonas ND4 subunit of complex I in two recipient strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas mitochondrial genome is possible. This substitution did not lead to any respiratory enzyme defects when present in the heteroplasmic state in a patient with chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent assembly of whole complex I (950 kDa) and the NADH dehydrogenase activity of the peripheral arm of the complex is mildly affected. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect binding of ubiquinone to the membrane domain. The in vitro defects correlate with a decrease in dark respiration and growth rate in vivo.