Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas.
Larosa, Véronique ; Coosemans, Nadine ; Motte, Patrick et al
in Plant Journal : for Cell & Molecular Biology (2012)
Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate ... [more ▼]
Defects in complex I (NADH:ubiquinone oxidoreductase) 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 could hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (1) respiratory-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (2) transformation of the mitochondrial genome is feasible, (3) Chlamydomonas complex I is close to that of humans. To illustrate that, we have introduced a Leu157Pro substitution in the Chlamydomonas ND4 subunit of complex I of two different 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 defect when it is present in the heteroplasmic state in a patient presenting chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent the assembly of the 950 kDa whole complex I which conserves nearly all the NADH dehydrogenase activity of the peripheral arm. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect ubiquinone fixation to the membrane domain. The in vitro defects are correlated in vivo with a decrease in dark respiration and growth rate. [less ▲]Detailed reference viewed: 31 (6 ULg)
Cytokinin promotes flowering of Arabidopsis via transcriptional activation of the FT paralogue TSF
D'Aloia, Maria ; Bonhomme, Delphine ; Bouché, Frédéric et al
in Plant Journal : for Cell & Molecular Biology (2011), 65
Cytokinins are involved in many aspects of plant growth and development and physiological evidence also indicates that they have a role in floral transition. In order to integrate these phytohormones into ... [more ▼]
Cytokinins are involved in many aspects of plant growth and development and physiological evidence also indicates that they have a role in floral transition. In order to integrate these phytohormones into the current knowledge of genetically defined molecular pathways to flowering, we performed exogenous treatments of adult wild-type and mutant Arabidopsis plants and analysed the expression of candidate genes. We used a hydroponic system that enables synchronous growth and flowering of Arabidopsis and allows precise application of chemicals to the roots for defined periods of time. We show that application of N6-benzylaminopurine (BAP) promotes flowering of plants grown in non-inductive short days. The response to cytokinin treatment does not require FLOWERING LOCUS T (FT) but activates its paralogue TWIN SISTER OF FT (TSF), as well as FD, which encodes a partner protein of TSF, and the downstream gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1). Treatment of selected mutants confirmed that TSF and SOC1 are necessary for the flowering response to BAP while the activation cascade might partially act independently of FD. These experiments provide a mechanistic basis for the role of cytokinins in flowering and demonstrate that the redundant genes FT and TSF are differently regulated by distinct floral inducing signals. [less ▲]Detailed reference viewed: 29 (10 ULg)
Sucrose non-fermenting kinase 1 (SnRK1) coordinates metabolic and hormonal signals during pea cotyledon growth and differentiation.
; ; et al
in Plant Journal : for Cell & Molecular Biology (2010), 61(2), 324-38
Seed development passes through developmental phases such as cell division, differentiation and maturation: each have specific metabolic demands. The ubiquitous sucrose non-fermenting-like kinase (SnRK1 ... [more ▼]
Seed development passes through developmental phases such as cell division, differentiation and maturation: each have specific metabolic demands. The ubiquitous sucrose non-fermenting-like kinase (SnRK1) coordinates and adjusts physiological and metabolic demands with growth. In protoplast assays sucrose deprivation and hormone supplementation, such as with auxin and abscisic acid (ABA), stimulate SnRK1-promoter activity. This indicates regulation by nutrients: hormonal crosstalk under conditions of nutrient demand and cell proliferation. SnRK1-repressed pea (Pisum sativum) embryos show lower cytokinin levels and deregulation of cotyledonary establishment and growth, together with downregulated gene expression related to cell proliferation, meristem maintenance and differentiation, leaf formation, and polarity. This suggests that at early stages of seed development SnRK1 regulates coordinated cotyledon emergence and growth via cytokinin-mediated auxin transport and/or distribution. Decreased ABA levels and reduced gene expression, involved in ABA-mediated seed maturation and response to sugars, indicate that SnRK1 is required for ABA synthesis and/or signal transduction at an early stage. Metabolic profiling of SnRK1-repressed embryos revealed lower levels of most organic and amino acids. In contrast, levels of sugars and glycolytic intermediates were higher or unchanged, indicating decreased carbon partitioning into subsequent pathways such as the tricarbonic acid cycle and amino acid biosynthesis. It is hypothesized that SnRK1 mediates the responses to sugar signals required for early cotyledon establishment and patterning. As a result, later maturation and storage activity are strongly impaired. Changes observed in SnRK1-repressed pea seeds provide a framework for how SnRK1 communicates nutrient and hormonal signals from auxins, cytokinins and ABA to control metabolism and development. [less ▲]Detailed reference viewed: 20 (7 ULg)
Genetical metabolomics of flavonoid biosynthesis in Populus: a case study.
; ; et al
in Plant Journal : for Cell & Molecular Biology (2006), 47(2), 224-37
Genetical metabolomics [metabolite profiling combined with quantitative trait locus (QTL) analysis] has been proposed as a new tool to identify loci that control metabolite abundances. This concept was ... [more ▼]
Genetical metabolomics [metabolite profiling combined with quantitative trait locus (QTL) analysis] has been proposed as a new tool to identify loci that control metabolite abundances. This concept was evaluated in a case study with the model tree Populus. Using HPLC, the peak abundances were analyzed of 15 closely related flavonoids present in apical tissues of two full-sib poplar families, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24, and correlation and QTL analysis were used to detect flux control points in flavonoid biosynthesis. Four robust metabolite quantitative trait loci (mQTL), associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of these mQTL, and the corresponding candidate genes were mapped. The data indicate that the combination of metabolite profiling with QTL analysis is a valuable tool to identify control points in a complex metabolic pathway of closely related compounds. [less ▲]Detailed reference viewed: 4 (0 ULg)
Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system.
; Vigeolas, Hélène ; et al
in Plant Journal : for Cell & Molecular Biology (2002), 30(2), 221-35
Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important ... [more ▼]
Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol-3-phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration. [less ▲]Detailed reference viewed: 7 (0 ULg)
FIL2, an extracellular Leucine-Rich Repeat protein, is specifically expressed in Antirrhinum flowers.
; Motte, Patrick ; et al
in Plant Journal : for Cell & Molecular Biology (1994), 5(4), 459-67
The expression of the Antirrhinum gene FIL2 is affected in mutants of the homeotic transcription factor DEFICIENS. Northern and Western blot analyses showed that FIL2 in wild-type Antirrhinum flowers is ... [more ▼]
The expression of the Antirrhinum gene FIL2 is affected in mutants of the homeotic transcription factor DEFICIENS. Northern and Western blot analyses showed that FIL2 in wild-type Antirrhinum flowers is expressed weakly in the petals and more abundantly in the reproductive organs; the gene is active in the filaments and anthers of stamens, and in the stigma and transmitting tissue of the carpels. The FIL2 protein is glycosylated with high mannose type glycan chains and is located in the middle lamella of the extracellular matrix. The amino acid sequence contains 10 tandem repeats, the composition of which is similar to the Leucine-Rich Repeat (LRR) motif found in mammals, Drosophila and yeast. The possibility that FIL2 might be a component of a cellular signalling mechanism, involving LRR-mediated protein-protein interactions is discussed. [less ▲]Detailed reference viewed: 8 (0 ULg)