GATAs are evolutionarily conserved zinc-finger transcription factors from eukaryotes. In plants, ... more GATAs are evolutionarily conserved zinc-finger transcription factors from eukaryotes. In plants, GATAs can be subdivided into four classes, A – D, based on their DNA-binding domain, and into further subclasses based on additional protein motifs. B-GATAs with a so-called LLM-domain can already be found in algae, but bryophytes and angiosperms also contain B-GATAs with a HAN-domain. The angiosperm B-GATA family is expanded and can be subdivided into family members with either a HAN- or an LLM-domain. The B-GATA family in the liverwort Marchantia polymorpha and the moss Physcomitrium patens is restricted to one and four family members, respectively, and all family members contain a HAN- as well as an LLM-domain. Here, we characterize mutants of the single B-GATA from Marchantia polymorpha. We reveal that this mutant has defects in thallus growth and in gemma formation. Transcriptomic studies uncover that the B-GATA mutant displays a constitutive high-light stress response, a phenotype ...
All land plants encode two isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominan... more All land plants encode two isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis, the effects of PPO2 deficiency have not been investigated in detail. We identified twoppo2T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, significant changes in PPO activity were measured in etiolated and root tissues. However,ppo1ppo2double mutants are embryo-lethal. To shed light on possible functional differences between the two isoforms, PPO2 was overexpressed in theppo1background. Although theppo1phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of its subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid membranes. A mitochondria...
The Chloroplast: From Molecular Biology to Biotechnology, 1999
Plastids harbor a multi-subunit RNA polymerase (RNAP) highly similar to eubacterial RNA polymeras... more Plastids harbor a multi-subunit RNA polymerase (RNAP) highly similar to eubacterial RNA polymerase, encoded by the plastid rpoA, rpoB and rpoC genes. The rpo genes have been found in all plant species so far investigated, with the exception of the parasite Epifagus virginiana. The eubacterial-like plastid RNA polymerase has been designated PEP (= plastid encoded polymerase). Several lines of evidence implicate the existence of a second RNA polymerase in the plastids of higher plants which is encoded in the nucleus (NEP = nuclear encoded polymerase. The plastid enzyme, NEP, functions in a coordinated manner, which can be described as „division of labour“, together with the plastid encoded PEP [4, 10, 14, 17].
All land plants encode 2 isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantl... more All land plants encode 2 isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis (Arabidopsis thaliana), the effects of PPO2 deficiency have not been investigated in detail. We identified 2 ppo2 T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, substantial changes in PPO activity were measured in etiolated and root tissues. However, ppo1 ppo2 double mutants were embryo-lethal. To shed light on possible functional differences between the 2 isoforms, PPO2 was overexpressed in the ppo1 background. Although the ppo1 phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid me...
The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme p... more The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme protochlorophyllide oxidoreductase (POR). The interruption of Chl synthesis during darkness requires suppression of the synthesis of 5-aminolevulinic acid (ALA), the first precursor molecule specific for Chl synthesis. The inactivation of glutamyl-tRNA reductase (GluTR), the first enzyme in tetrapyrrole biosynthesis, accomplished the decreased ALA synthesis by the membrane-bound protein FLUORESCENT (FLU) and prevents overaccumulation of protochlorophyllide (Pchlide) in the dark. We set out to elucidate the molecular mechanism of FLU-mediated inhibition of ALA synthesis, and explored the role of each of the three structural domains of mature FLU, the transmembrane, coiled-coil and tetratricopeptide repeat (TPR) domains, in this process. Efforts to rescue the FLU knockout mutant with truncated FLU peptides revealed that, on its own, the TPR domain is insufficient to inactivate GluTR, although tight binding of the TPR domain to GluTR was detected. A truncated FLU peptide consisting of transmembrane and TPR domains also failed to inactivate GluTR in the dark. Similarly, suppression of ALA synthesis could not be achieved by combining the coiled-coil and TPR domains. Interaction studies revealed that binding of GluTR and POR to FLU is essential for inhibiting ALA synthesis. These results imply that all three FLU domains are required for the repression of ALA synthesis, in order to avoid the overaccumulation of Pchlide in the dark. Only complete FLU ensures the formation of a membrane-bound ternary complex consisting at least of FLU, GluTR and POR to repress ALA synthesis.
The synthesis of 5-aminolevulinic acid (ALA) determines adequate amounts of metabolites for the t... more The synthesis of 5-aminolevulinic acid (ALA) determines adequate amounts of metabolites for the tetrapyrrole biosynthetic pathway. Glutamyl-tRNA reductase (GluTR) catalyzes the rate-limiting step of ALA synthesis and was previously considered to be exclusively localized in the chloroplast stroma of light-exposed plants. To assess the intraplastidic localization of GluTR, we developed a fast separation protocol of soluble and membrane-bound proteins and reassessed the subplastidal allocation of GluTR in stroma and membrane fractions of Arabidopsis plants grown under different light regimes as well as during de-etiolation and dark incubations. Under the examined conditions, the amount of stroma-localized GluTR correlated with the ALA synthesis rate. The transfer to dark repression of ALA synthesis resulted in a loss of soluble GluTR. Arabidopsis mutants lacking one of the GluTR-interacting factors FLUORESCENT (FLU), the GluTR-binding protein (GBP) or ClpC, a chaperone of the Clp protease system, were applied to examine the amount of GluTR and its distribution to the stroma or membrane in darkness and light. Taking into consideration the different compartmental allocation of GluTR, its stability and ALA synthesis rates, the post-translational impact of these regulatory factors on GluTR activity and plastidic sublocalization is discussed.
Chlorophyll accumulation and chloroplast development are regulated at multiple levels during plan... more Chlorophyll accumulation and chloroplast development are regulated at multiple levels during plant development. The paralogous LLM-domain B-GATA transcription factors GNC and GNL contribute to chlorophyll biosynthesis and chloroplast formation in light-grown Arabidopsis thaliana seedlings. Whereas there is already ample knowledge about the transcriptional regulation of GNC and GNL, the identity of their downstream targets is largely unclear. Here, we identified genes controlling greening directly downstream of the GATAs by integrating data from RNA-sequencing and microarray data sets. We found that genes encoding subunits of the Mg-chelatase complex and 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR) likely function directly downstream of the GATAs and that DVR expression is limiting in the pale-green gnc gnl mutants. The GATAs also regulate the nucleus-encoded SIGMA (SIG) factor genes, which control transcription in the chloroplast and suppress the greening defects of sig mutants. Furthermore, GNC and GNL act, at the gene expression level, in an additive manner with the GOLDEN2-LIKE1 (GLK1) and GLK2 transcription factor genes, which are also important for proper chlorophyll accumulation. We thus reveal that chlorophyll biosynthesis genes are directly controlled by LLM-domain B-GATAs and demonstrate that these transcription factors play an indirect role in the control of greening through regulating SIGMA factor genes.
Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5-aminolev... more Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5-aminolevulinic acid (ALA) synthesis and interacts with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme of tetrapyrrole biosynthesis. In this study, we investigated FLU's regulatory function in light-exposed FLU-overexpressing (FLUOE) Arabidopsis lines and under fluctuating light intensities in wild-type (WT) and flu seedlings. FLUOE lines suppress ALA synthesis in the light, resulting in reduced chlorophyll content, but more strongly in low and high light than in medium growth light. This situation indicates that FLU's impact on chlorophyll biosynthesis depends on light intensity. FLU overexpressors contain strongly increased amounts of mainly membrane-associated GluTR. These findings correlate with FLU-dependent localization of GluTR to plastidic membranes and concomitant inhibition, such that only the soluble GluTR fraction is active. The overaccumulation of membrane-associated GluTR indicates that FLU binding enhances GluTR stability. Interestingly, under fluctuating light, the leaves of flu mutants contain less chlorophyll compared with WT and become necrotic. We propose that FLU is basically required for fine-tuned ALA synthesis. FLU not only mediates dark repression of ALA synthesis, but functions also to control balanced ALA synthesis under variable light intensities to ensure the adequate supply of chlorophyll.
The author names were shown incorrectly in the initial online publication. They are correct in th... more The author names were shown incorrectly in the initial online publication. They are correct in this erratum. The original article has been corrected.
In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyr... more In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyrin IX at the final step of heme biosynthesis. FERROCHELATASE1 (FC1) is continuously, but weakly expressed in roots and leaves, while FC2 is dominantly active in leaves. As a continuation of previous studies on the physiological consequences of FC2 inactivation in tobacco, we aimed to assign FC1 function in plant organs. While reduced FC2 expression leads to protoporphyrin IX accumulation in leaves, FC1 downregulation and overproduction caused reduced and elevated FC activity in root tissue, respectively, but were not associated with changes in macroscopic phenotype, plant development or leaf pigmentation. In contrast to the lower heme content resulting from a deficiency of the dominant FC2 expression in leaves, a reduction of FC1 in roots and leaves does not significantly disturb heme accumulation. The FC1 overexpression was used for an additional approach to reexamine FC activity in mitochondria. Transgenic FC1 protein was immunologically shown to be present in mitochondria. Although matching only a small portion of total cellular FC activity, the mitochondrial FC activity in a FC1 overexpressor line increased 5-fold in comparison with wild-type mitochondria. Thus, it is suggested that FC1 contributes to mitochondrial heme synthesis.
5-Aminolevulinic acid (ALA) is the first committed substrate of tetrapyrrole biosynthesis and is ... more 5-Aminolevulinic acid (ALA) is the first committed substrate of tetrapyrrole biosynthesis and is formed from glutamyl-tRNA by two enzymatic steps. Glutamyl-tRNA reductase (GluTR) as the first enzyme of ALA synthesis is encoded by HEMA genes and tightly regulated at the transcriptional and posttranslational level. Here we show that the Clp protease substrate adaptor ClpS1 and the ClpC1 chaperone as well as the GluTR-binding protein (GBP) interact with the N-terminus of GluTR. Loss-of function mutants of ClpR2 and ClpC1 proteins show increased GluTR stability, whereas absence of GBP results in decreased GluTR stability. Thus the Clp protease system and GBP contribute to GluTR accumulation levels, and thereby of the rate-limiting ALA synthesis. These findings are supported with Arabidopsis hema1 mutants expressing a truncated GluTR lacking the 29 N-terminal amino acid residues of the mature protein. Accumulation of this truncated GluTR is higher in dark periods resulting in increased p...
Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the p... more Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signalling). A screen designed to identify components of retrograde signalling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression (PGE) and tetrapyrrole biosynthesis (TPB) in Arabidopsis seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signalling in the chloroplast is unclear. Here we show that GUN1 operates also in adult plants, contributing to operational control of chloroplasts. The gun1 mutation g...
Tetrapyrroles are essential cofactors and pigments. Photosynthetic organisms share the use of the... more Tetrapyrroles are essential cofactors and pigments. Photosynthetic organisms share the use of the highest diversity of tetrapyrrole end products. This report intends to call readers' attention to the recent achievements in research on tetrapyrrole biosynthesis and the current hot topics in tetrapyrrole biosynthesis, including posttranslational control mechanisms and intracellular signaling between the nucleus and the two organelles, plastids and mitochondria. Moreover, the functions of tetrapyrroles in the photosynthetic organisms and valuable information about the current analytical techniques to determine steady state levels of tetrapyrrole intermediates from cyanobacteria, green algae and plant will be surveyed.
International journal of molecular sciences, Jan 31, 2012
Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living... more Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interesting...
In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA po... more In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identi®ed six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, ®rst described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.
GATAs are evolutionarily conserved zinc-finger transcription factors from eukaryotes. In plants, ... more GATAs are evolutionarily conserved zinc-finger transcription factors from eukaryotes. In plants, GATAs can be subdivided into four classes, A – D, based on their DNA-binding domain, and into further subclasses based on additional protein motifs. B-GATAs with a so-called LLM-domain can already be found in algae, but bryophytes and angiosperms also contain B-GATAs with a HAN-domain. The angiosperm B-GATA family is expanded and can be subdivided into family members with either a HAN- or an LLM-domain. The B-GATA family in the liverwort Marchantia polymorpha and the moss Physcomitrium patens is restricted to one and four family members, respectively, and all family members contain a HAN- as well as an LLM-domain. Here, we characterize mutants of the single B-GATA from Marchantia polymorpha. We reveal that this mutant has defects in thallus growth and in gemma formation. Transcriptomic studies uncover that the B-GATA mutant displays a constitutive high-light stress response, a phenotype ...
All land plants encode two isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominan... more All land plants encode two isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis, the effects of PPO2 deficiency have not been investigated in detail. We identified twoppo2T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, significant changes in PPO activity were measured in etiolated and root tissues. However,ppo1ppo2double mutants are embryo-lethal. To shed light on possible functional differences between the two isoforms, PPO2 was overexpressed in theppo1background. Although theppo1phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of its subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid membranes. A mitochondria...
The Chloroplast: From Molecular Biology to Biotechnology, 1999
Plastids harbor a multi-subunit RNA polymerase (RNAP) highly similar to eubacterial RNA polymeras... more Plastids harbor a multi-subunit RNA polymerase (RNAP) highly similar to eubacterial RNA polymerase, encoded by the plastid rpoA, rpoB and rpoC genes. The rpo genes have been found in all plant species so far investigated, with the exception of the parasite Epifagus virginiana. The eubacterial-like plastid RNA polymerase has been designated PEP (= plastid encoded polymerase). Several lines of evidence implicate the existence of a second RNA polymerase in the plastids of higher plants which is encoded in the nucleus (NEP = nuclear encoded polymerase. The plastid enzyme, NEP, functions in a coordinated manner, which can be described as „division of labour“, together with the plastid encoded PEP [4, 10, 14, 17].
All land plants encode 2 isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantl... more All land plants encode 2 isoforms of protoporphyrinogen oxidase (PPO). While PPO1 is predominantly expressed in green tissues and its loss is seedling-lethal in Arabidopsis (Arabidopsis thaliana), the effects of PPO2 deficiency have not been investigated in detail. We identified 2 ppo2 T-DNA insertion mutants from publicly available collections, one of which (ppo2-2) is a knock-out mutant. While the loss of PPO2 did not result in any obvious phenotype, substantial changes in PPO activity were measured in etiolated and root tissues. However, ppo1 ppo2 double mutants were embryo-lethal. To shed light on possible functional differences between the 2 isoforms, PPO2 was overexpressed in the ppo1 background. Although the ppo1 phenotype was partially complemented, even strong overexpression of PPO2 was unable to fully compensate for the loss of PPO1. Analysis of subcellular localization revealed that PPO2 is found exclusively in chloroplast envelopes, while PPO1 accumulates in thylakoid me...
The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme p... more The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme protochlorophyllide oxidoreductase (POR). The interruption of Chl synthesis during darkness requires suppression of the synthesis of 5-aminolevulinic acid (ALA), the first precursor molecule specific for Chl synthesis. The inactivation of glutamyl-tRNA reductase (GluTR), the first enzyme in tetrapyrrole biosynthesis, accomplished the decreased ALA synthesis by the membrane-bound protein FLUORESCENT (FLU) and prevents overaccumulation of protochlorophyllide (Pchlide) in the dark. We set out to elucidate the molecular mechanism of FLU-mediated inhibition of ALA synthesis, and explored the role of each of the three structural domains of mature FLU, the transmembrane, coiled-coil and tetratricopeptide repeat (TPR) domains, in this process. Efforts to rescue the FLU knockout mutant with truncated FLU peptides revealed that, on its own, the TPR domain is insufficient to inactivate GluTR, although tight binding of the TPR domain to GluTR was detected. A truncated FLU peptide consisting of transmembrane and TPR domains also failed to inactivate GluTR in the dark. Similarly, suppression of ALA synthesis could not be achieved by combining the coiled-coil and TPR domains. Interaction studies revealed that binding of GluTR and POR to FLU is essential for inhibiting ALA synthesis. These results imply that all three FLU domains are required for the repression of ALA synthesis, in order to avoid the overaccumulation of Pchlide in the dark. Only complete FLU ensures the formation of a membrane-bound ternary complex consisting at least of FLU, GluTR and POR to repress ALA synthesis.
The synthesis of 5-aminolevulinic acid (ALA) determines adequate amounts of metabolites for the t... more The synthesis of 5-aminolevulinic acid (ALA) determines adequate amounts of metabolites for the tetrapyrrole biosynthetic pathway. Glutamyl-tRNA reductase (GluTR) catalyzes the rate-limiting step of ALA synthesis and was previously considered to be exclusively localized in the chloroplast stroma of light-exposed plants. To assess the intraplastidic localization of GluTR, we developed a fast separation protocol of soluble and membrane-bound proteins and reassessed the subplastidal allocation of GluTR in stroma and membrane fractions of Arabidopsis plants grown under different light regimes as well as during de-etiolation and dark incubations. Under the examined conditions, the amount of stroma-localized GluTR correlated with the ALA synthesis rate. The transfer to dark repression of ALA synthesis resulted in a loss of soluble GluTR. Arabidopsis mutants lacking one of the GluTR-interacting factors FLUORESCENT (FLU), the GluTR-binding protein (GBP) or ClpC, a chaperone of the Clp protease system, were applied to examine the amount of GluTR and its distribution to the stroma or membrane in darkness and light. Taking into consideration the different compartmental allocation of GluTR, its stability and ALA synthesis rates, the post-translational impact of these regulatory factors on GluTR activity and plastidic sublocalization is discussed.
Chlorophyll accumulation and chloroplast development are regulated at multiple levels during plan... more Chlorophyll accumulation and chloroplast development are regulated at multiple levels during plant development. The paralogous LLM-domain B-GATA transcription factors GNC and GNL contribute to chlorophyll biosynthesis and chloroplast formation in light-grown Arabidopsis thaliana seedlings. Whereas there is already ample knowledge about the transcriptional regulation of GNC and GNL, the identity of their downstream targets is largely unclear. Here, we identified genes controlling greening directly downstream of the GATAs by integrating data from RNA-sequencing and microarray data sets. We found that genes encoding subunits of the Mg-chelatase complex and 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR) likely function directly downstream of the GATAs and that DVR expression is limiting in the pale-green gnc gnl mutants. The GATAs also regulate the nucleus-encoded SIGMA (SIG) factor genes, which control transcription in the chloroplast and suppress the greening defects of sig mutants. Furthermore, GNC and GNL act, at the gene expression level, in an additive manner with the GOLDEN2-LIKE1 (GLK1) and GLK2 transcription factor genes, which are also important for proper chlorophyll accumulation. We thus reveal that chlorophyll biosynthesis genes are directly controlled by LLM-domain B-GATAs and demonstrate that these transcription factors play an indirect role in the control of greening through regulating SIGMA factor genes.
Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5-aminolev... more Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5-aminolevulinic acid (ALA) synthesis and interacts with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme of tetrapyrrole biosynthesis. In this study, we investigated FLU's regulatory function in light-exposed FLU-overexpressing (FLUOE) Arabidopsis lines and under fluctuating light intensities in wild-type (WT) and flu seedlings. FLUOE lines suppress ALA synthesis in the light, resulting in reduced chlorophyll content, but more strongly in low and high light than in medium growth light. This situation indicates that FLU's impact on chlorophyll biosynthesis depends on light intensity. FLU overexpressors contain strongly increased amounts of mainly membrane-associated GluTR. These findings correlate with FLU-dependent localization of GluTR to plastidic membranes and concomitant inhibition, such that only the soluble GluTR fraction is active. The overaccumulation of membrane-associated GluTR indicates that FLU binding enhances GluTR stability. Interestingly, under fluctuating light, the leaves of flu mutants contain less chlorophyll compared with WT and become necrotic. We propose that FLU is basically required for fine-tuned ALA synthesis. FLU not only mediates dark repression of ALA synthesis, but functions also to control balanced ALA synthesis under variable light intensities to ensure the adequate supply of chlorophyll.
The author names were shown incorrectly in the initial online publication. They are correct in th... more The author names were shown incorrectly in the initial online publication. They are correct in this erratum. The original article has been corrected.
In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyr... more In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyrin IX at the final step of heme biosynthesis. FERROCHELATASE1 (FC1) is continuously, but weakly expressed in roots and leaves, while FC2 is dominantly active in leaves. As a continuation of previous studies on the physiological consequences of FC2 inactivation in tobacco, we aimed to assign FC1 function in plant organs. While reduced FC2 expression leads to protoporphyrin IX accumulation in leaves, FC1 downregulation and overproduction caused reduced and elevated FC activity in root tissue, respectively, but were not associated with changes in macroscopic phenotype, plant development or leaf pigmentation. In contrast to the lower heme content resulting from a deficiency of the dominant FC2 expression in leaves, a reduction of FC1 in roots and leaves does not significantly disturb heme accumulation. The FC1 overexpression was used for an additional approach to reexamine FC activity in mitochondria. Transgenic FC1 protein was immunologically shown to be present in mitochondria. Although matching only a small portion of total cellular FC activity, the mitochondrial FC activity in a FC1 overexpressor line increased 5-fold in comparison with wild-type mitochondria. Thus, it is suggested that FC1 contributes to mitochondrial heme synthesis.
5-Aminolevulinic acid (ALA) is the first committed substrate of tetrapyrrole biosynthesis and is ... more 5-Aminolevulinic acid (ALA) is the first committed substrate of tetrapyrrole biosynthesis and is formed from glutamyl-tRNA by two enzymatic steps. Glutamyl-tRNA reductase (GluTR) as the first enzyme of ALA synthesis is encoded by HEMA genes and tightly regulated at the transcriptional and posttranslational level. Here we show that the Clp protease substrate adaptor ClpS1 and the ClpC1 chaperone as well as the GluTR-binding protein (GBP) interact with the N-terminus of GluTR. Loss-of function mutants of ClpR2 and ClpC1 proteins show increased GluTR stability, whereas absence of GBP results in decreased GluTR stability. Thus the Clp protease system and GBP contribute to GluTR accumulation levels, and thereby of the rate-limiting ALA synthesis. These findings are supported with Arabidopsis hema1 mutants expressing a truncated GluTR lacking the 29 N-terminal amino acid residues of the mature protein. Accumulation of this truncated GluTR is higher in dark periods resulting in increased p...
Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the p... more Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signalling). A screen designed to identify components of retrograde signalling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression (PGE) and tetrapyrrole biosynthesis (TPB) in Arabidopsis seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signalling in the chloroplast is unclear. Here we show that GUN1 operates also in adult plants, contributing to operational control of chloroplasts. The gun1 mutation g...
Tetrapyrroles are essential cofactors and pigments. Photosynthetic organisms share the use of the... more Tetrapyrroles are essential cofactors and pigments. Photosynthetic organisms share the use of the highest diversity of tetrapyrrole end products. This report intends to call readers' attention to the recent achievements in research on tetrapyrrole biosynthesis and the current hot topics in tetrapyrrole biosynthesis, including posttranslational control mechanisms and intracellular signaling between the nucleus and the two organelles, plastids and mitochondria. Moreover, the functions of tetrapyrroles in the photosynthetic organisms and valuable information about the current analytical techniques to determine steady state levels of tetrapyrrole intermediates from cyanobacteria, green algae and plant will be surveyed.
International journal of molecular sciences, Jan 31, 2012
Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living... more Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interesting...
In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA po... more In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identi®ed six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, ®rst described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.
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Papers by Boris Hedtke