| 1. |
- Engqvist, Martin, 1983, et al.
(author)
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Two D-2-hydroxy-acid dehydrogenases in arabidopsis thaliana with catalytic capacities to participate in the last reactions of the methylglyoxal and β-oxidation pathways
- 2009
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:37, s. 25026-25037
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Journal article (peer-reviewed)abstract
- The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae D-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of D- and L-lactate, D-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for D-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for L-lactate and glycolate, respectively, and a Km value for D-lactate of ∼160 μM. Knock-out mutants showed impaired growth in the presence of D-lactate or methylglyoxal. Collectively, the data indicated that the protein is a D-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific D-2-hydroxyglutarate (D-2HG) conversion in the presence of an organic cofactor with a Km value of ∼580 μM. Thus, the enzyme was characterized as a D-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total D-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in β-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of D-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.
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| 2. |
- Gigolashvili, T., et al.
(author)
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HAG2/MYB76 and HAG3/MYB29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in Arabidopsis thaliana
- 2008
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In: New Phytologist. - : Wiley. - 1469-8137 .- 0028-646X. ; 177:3, s. 627-642
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Journal article (peer-reviewed)abstract
- In a previous transactivation screen, two Arabidopsis thaliana R2R3-MYB transcription factors, HAG2/MYB76 and HAG3/MYB29, along with the already characterized HAG1/MYB28, were identified as putative regulators of aliphatic glucosinolate biosynthesis. • Molecular and biochemical characterization of HAG2/MYB76 and HAG3/MYB29 functions was performed using transformants with increased or repressed transcript levels. Real-time PCR assays, cotransformation assays and measurements of glucosinolate contents were used to assess the impact of both MYB factors on the steady-state level of glucosinolate biosynthetic genes and accumulation of aliphatic glucosinolates. • Both HAG2/MYB76 and HAG3/MYB29 were shown to be positive regulators of aliphatic glucosinolate biosynthesis. Expression of promoter-β- glucuronidase (GUS) fusions indicated GUS activities in both vegetative and generative organs, with distinct characteristics for each MYB factor. HAG1/MYB28, HAG2/MYB76 and HAG3/MYB29 reciprocally transactivated each other in the control of aliphatic glucosinolate biosynthesis and downregulated the expression of genes involved in the control of indolic glucosinolate biosynthesis, pointing to a reciprocal negative regulation of these two pathways. • All three HAG transcription factors exert a coordinated control on aliphatic glucosinolate biosynthesis.
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| 3. |
- Maier, A., et al.
(author)
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Transgenic introduction of a glycolate oxidative cycle into A. thaliana chloroplasts leads to growth improvement
- 2012
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In: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 3:FEB
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Journal article (peer-reviewed)abstract
- The photorespiratory pathway helps illuminated C3-plants under conditions of limited CO2availability by effectively exporting reducing equivalents in form of glycolate out of the chloroplast and regenerating glycerate-3-P as substrate for RubisCO. On the other hand, this pathway is considered as probably futile because previously assimilated CO2 is released in mitochondria. Consequently, a lot of effort has been made to reduce this CO2 loss either by reducing fluxes via engineering RubisCO or circumventing mitochondrial CO2 release by the introduction of new enzyme activities. Here we present an approach following the latter route, introducing a complete glycolate catabolic cycle in chloroplasts ofArabidopsis thalianacomprising glycolate oxidase (GO), malate synthase (MS), and catalase (CAT). Results from plants bearing both GO and MS activities have already been reported. This previous work showed that the H2O2produced by GO had strongly negative effects. These effects can be prevented by introducing a plastidial catalase activity, as reported here. Transgenic lines bearing all three transgenic enzyme activities were identified and some with higher CAT activity showed higher dry weight, higher photosynthetic rates, and changes in glycine/serine ratio compared to the wild type. This indicates that the fine-tuning of transgenic enzyme activities in the chloroplasts seems crucial and strongly suggests that the approach is valid and that it is possible to improve the growth of A. thaliana by introducing a synthetic glycolate oxidative cycle into chloroplasts.
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| 4. |
- Schneider, A., et al.
(author)
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The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis
- 2016
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In: Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 28:4, s. 892-910
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Journal article (peer-reviewed)abstract
- In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn2+ and Ca2+ homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn2+ and Ca2+ ions were differently sequestered in pam71, with Ca2+ enriched in pam71 thylakoids relative to the wild type. The changes in Ca2+ homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn2+, but not Ca2+. Furthermore, PAM71 suppressed the Mn2+-sensitive phenotype of the yeast mutant Delta pmr1. Therefore, PAM71 presumably functions in Mn2+ uptake into thylakoids to ensure optimal PSII performance.
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| 5. |
- Wienstroer, J., et al.
(author)
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D-Lactate dehydrogenase as a marker gene allows positive selection of transgenic plants
- 2012
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In: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 586:1, s. 36-40
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Journal article (peer-reviewed)abstract
- d-Lactate negatively affects Arabidopsis thaliana seedling development in a concentration-dependent manner. At media d-lactate concentrations greater than 5-10 mM the development of wild-type plants is arrested shortly after germination whereas plants overexpressing the endogenous d-lactate dehydrogenase (d-LDH) detoxify d-lactate to pyruvate and survive. When the transgenic plants are further transferred to normal growth conditions they develop indistinguishably from the wild type. Thus, d-LDH was successfully established as a new marker in A. thaliana allowing selecting transgenic plants shortly after germination. The selection on d-lactate containing media adds a new optional marker system, which is especially useful if the simultaneous selection of multiple constructs is desired.
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