Overexpression of mtDNA-associated AtWhy2 compromises mitochondrial function.

BACKGROUND: StWhy1, a member of the plant-specific Whirly single-stranded DNA-binding protein family, was first characterized as a transcription factor involved in the activation of the nuclear PR-10a gene following defense-related stress in potato. In Arabidopsis thaliana, Whirlies have recently been shown to be primarily localized in organelles. Two representatives of the family, AtWhy1 and AtWhy3 are imported into plastids while AtWhy2 localizes to mitochondria. Their function in organelles is currently unknown. RESULTS: To understand the role of mitochondrial Whirlies in higher plants, we produced A. thaliana lines with altered expression of the atwhy2 gene. Organellar DNA immunoprecipitation experiments demonstrated that AtWhy2 binds to mitochondrial DNA. Overexpression of atwhy2 in plants perturbs mitochondrial function by causing a diminution in transcript levels and mtDNA content which translates into a low activity level of respiratory chain complexes containing mtDNA-encoded subunits. This lowered activity of mitochondria yielded plants that were reduced in size and had distorted leaves that exhibited accelerated senescence. Overexpression of atwhy2 also led to early accumulation of senescence marker transcripts in mature leaves. Inactivation of the atwhy2 gene did not affect plant development and had no detectable effect on mitochondrial morphology, activity of respiratory chain complexes, transcription or the amount of mtDNA present. This lack of phenotype upon abrogation of atwhy2 expression suggests the presence of functional homologues of the Whirlies or the activation of compensating mechanisms in mitochondria. CONCLUSION: AtWhy2 is associated with mtDNA and its overexpression results in the production of dysfunctional mitochondria. This report constitutes the first evidence of a function for the Whirlies in organelles. We propose that they could play a role in the regulation of the gene expression machinery of organelles.

Histochemical staining and quantification of plant mitochondrial respiratory chain complexes using blue-native polyacrylamide gel electrophoresis.

Our knowledge of the respiratory chain and associated defects depends on the study of the multisubunit protein complexes in the inner mitochondrial membrane. Functional analysis of the plant mitochondrial respiratory chain has been successfully achieved by a combination of blue-native polyacrylamide gel electrophoresis (BN-PAGE) for separation of the protein complexes, and in-gel histochemical staining of the enzyme activities. We have optimized this powerful technique by determining linear ranges of amount of protein and enzyme activity for each respiratory complex. Time courses of the in-gel enzyme activities were also performed to determine optimal reaction times. Using the in-gel activity staining method we have previously shown decreased activity of complex V (F(1)F(0)-ATPase) in male-sterile sunflowers (Sabar et al., 2003). Here we have identified unique supercomplexes comprising complex IV (cytochrome c oxidase) in sunflower mitochondria. This method therefore represents a reliable tool for the diagnosis of respiratory dysfunction. In addition, the wider application of BN-PAGE in combination with enzyme activity staining is discussed.

Comparison of changes in fruit gene expression in tomato introgression lines provides evidence of genome-wide transcriptional changes and reveals links to mapped QTLs and described traits.

Total soluble solids content is a key determinant of tomato fruit quality for processing. Several tomato lines carrying defined introgressions from S. pennellii in a S. lycopersicum background produce fruit with elevated Brix, a refractive index measure of soluble solids. The genetic basis for this trait can be determined by fine-mapping each QTL to a single gene, but this is time-consuming and technically demanding. As an alternative, high-throughput analytical technologies can be used to provide useful information that helps characterize molecular changes in the introgression lines. This paper presents a study of transcriptomic changes in six introgression lines with increased fruit Brix. Each line also showed altered patterns of fruit carbohydrate accumulation. Transcriptomic changes in fruit at 20 d after anthesis (DAA) were assessed using a 12 000-element EST microarray and significant changes analysed by SAM (significance analysis of microarrays). Each non-overlapping introgression resulted in a unique set of transcriptomic changes with 78% of significant changes being unique to a single line. Principal components analysis allowed a clear separation of the six lines, but also revealed evidence of common changes; lines with quantitatively similar increases in Brix clustered together. A detailed examination of genes encoding enzymes of primary carbon metabolism demonstrated that few of the known introgressed alleles were altered in expression at the 20 DAA time point. However, the expression of other metabolic genes did change. Particularly striking was the co-ordinated up-regulation of enzymes of sucrose mobilization and respiration that occurred only in the two lines with the highest Brix increase. These common downstream changes suggest a similar mechanism is responsible for large Brix increases.

Extensive phenotypic variation in early flowering mutants of Arabidopsis.

Flowering time, the major regulatory transition of plant sequential development, is modulated by multiple endogenous and environmental factors. By phenotypic profiling of 80 early flowering mutants of Arabidopsis, we examine how mutational reduction of floral repression is associated with changes in phenotypic plasticity and stability. Flowering time measurements in mutants reveal deviations from the linear relationship between the number of leaves and number of days to bolting described for natural accessions and late flowering mutants. The deviations correspond to relative early bolting and relative late bolting phenotypes. Only a minority of mutants presents no detectable phenotypic variation. Mutants are characterized by a broad release of morphological pleiotropy under short days, with leaf characters being most variable. They also exhibit changes in phenotypic plasticity across environments for florigenic-related responses, including the reaction to light and dark, photoperiodic behavior, and Suc sensitivity. Morphological pleiotropy and plasticity modifications are differentially distributed among mutants, resulting in a large diversity of multiple phenotypic changes. The pleiotropic effects observed may indicate that floral repression defects are linked to global developmental perturbations. This first, to our knowledge, extensive characterization of phenotypic variation in early flowering mutants correlates with the reports that most factors recruited in floral repression at the molecular genetic level correspond to ubiquitous regulators. We discuss the importance of functional ubiquity for floral repression with respect to robustness and flexibility of network biological systems.

ORFB is a subunit of F1F(O)-ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower.

ORFB is the product of a gene that is conserved in plant mitochondrial genomes, and which, on the basis of sequence motif and structural similarity, is predicted to be the homologue of yeast and mammalian ATP8, part of the F(O) component of the F1F(O)-ATP synthase. We have shown that, in sunflower, orfB transcripts are edited, increasing the similarity of the predicted protein to ATP8 proteins from non-plant species. Blue-native polyacrylamide gel electrophoresis and peptide sequencing confirm that ORFB localizes to the ATP synthase complex. The predicted amino-terminal 19 amino acids of ORFB are identical to those in the chimeric mitochondrial ORF522 protein, which is associated with cytoplasmic male sterility (CMS) in sunflower. Assays comparing respiratory complexes from a male-sterile line expressing ORF522 with those from a male-fertile line show a specific decrease in ATP hydrolysis by the ATP synthase. These observations allow us to propose a mechanism underlying CMS that is associated with the expression of chimeric open reading frames containing part of the orfB gene.