Dietmar Funck

Universität Konstanz, Constance, Baden-Württemberg, Germany

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Publications (15)82.82 Total impact

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    ABSTRACT: Δ(1) -pyrroline-5-carboxylate (P5C) reductase (P5CR) catalyses the final step of proline synthesis in plants. In Arabidopsis thaliana, protein levels are correlated neither to the corresponding mRNA copy numbers, nor to intracellular proline concentrations. The occurrence of post-translational regulatory mechanisms has therefore been hypothesized, but never assessed. The purification of A. thaliana P5CR was achieved through either a six-step protocol from cultured cells, or heterologous expression of AtP5CR in Escherichia coli. The protein was characterized with respect to structural, kinetic, and biochemical properties. P5CR was able to use either NADPH or NADH as the electron donor, with contrasting affinities and maximum reaction rates. The presence of equimolar concentrations of NADP(+) completely suppressed the NADH-dependent activity, whereas the NADPH-dependent reaction was mildly affected. Proline inhibited only the NADH-dependent reaction. At physiological values, increasing concentrations of salt progressively inhibited the NADH-dependent activity, but were stimulatory of the NADPH-dependent reaction. The biochemical properties of A. thaliana P5CR suggest a complex regulation of enzyme activity by the redox status of the pyridine nucleotide pools, and the concentrations of proline and chloride in the cytosol. Data support a to date underestimated role of P5CR in controlling stress-induced proline accumulation.
    New Phytologist 01/2014; · 6.74 Impact Factor
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    ABSTRACT: BACKGROUND: Gamete and embryo development are crucial for successful reproduction and seed set in plants, which is often the determining factor for crop yield. Proline accumulation was largely viewed as a specific reaction to overcome stress conditions, while recent studies suggested important functions of proline metabolism also in reproductive development. Both the level of free proline and proline metabolism were proposed to influence the transition to flowering, as well as pollen and embryo development. RESULTS: In this study, we performed a detailed analysis of the contribution of individual proline biosynthetic enzymes to vegetative development and reproductive success in Arabidopsis. In contrast to previous reports, we found that pyrroline-5-carboxylate (P5C) synthetase 2 (P5CS2) is not essential for sexual reproduction although p5cs2 mutant plants were retarded in vegetative development and displayed reduced fertility under long-day conditions. Single mutant plants devoid of P5CS1 did not show any developmental defects. Simultaneous absence of both P5CS isoforms resulted in pollen sterility, while fertile egg cells could still be produced. Expression of P5C reductase (P5CR) was indispensable for embryo development but surprisingly not needed for pollen or egg cell fertility. The latter observation could be explained by an extreme stability of P5CR activity, which had a half-life time of greater than 3 weeks in vitro. Expression of P5CR-GFP under the control of the endogenous P5CR promoter was able to restore growth of homozygous p5cr mutant embryos. The analysis of P5CR-GFP-fluorescence in planta supported an exclusively cytoplasmatic localisation of P5CR. CONCLUSIONS: Our results demonstrate that potential alternative pathways for proline synthesis or inter-generation transfer of proline are not sufficient to overcome a defect in proline biosynthesis from glutamate during pollen development. Proline biosynthesis through P5CS2 and P5CR is limiting for vegetative and reproductive development in Arabidopsis, whereas disruption of P5CS1 alone does not affect development of non-stressed plants.
    BMC Plant Biology 10/2012; 12(1):191. · 4.35 Impact Factor
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    ABSTRACT: Mechanisms for sensing and regulating metabolic processes at the cellular level are critical for the general physiology and development of living organisms. In higher plants, sugar signaling is crucial for adequate regulation of carbon and energy metabolism and affects virtually every aspect of development. Although many genes are regulated by sugar levels, little is known on how sugar levels are measured by plants. Several components of the sugar signaling network have been unraveled and demonstrated to have extensive overlap with hormone signaling networks. Here we describe the reduced sugar response1-1 (rsr1-1) mutant as a new early flowering mutant that displays decreased sensitivity to abscisic acid. Both hexokinase1 (HXK1)-dependent and glucose phosphorylation-independent signaling is reduced in rsr1-1. Map-based identification of the affected locus demonstrated that rsr1-1 carries a premature stop codon in the gene for a CstF64-like putative RNA processing factor, ESP1, which is involved in mRNA 3'-end formation. The identification of RSR1/ESP1 as a nuclear protein with a potential threonine phosphorylation site may explain the impact of protein phosphorylation cascades on sugar-dependent signal transduction. Additionally, RSR1/ESP1 may be a crucial factor in linking sugar signaling to the control of flowering time.
    Frontiers in Plant Science 01/2012; 3:80. · 3.60 Impact Factor
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    ABSTRACT: Proline fulfils diverse functions in plants. As amino acid it is a structural component of proteins, but it also plays a role as compatible solute under environmental stress conditions. Proline metabolism involves several subcellular compartments and contributes to the redox balance of the cell. Proline synthesis has been associated with tissues undergoing rapid cell divisions, such as shoot apical meristems, and appears to be involved in floral transition and embryo development. High levels of proline can be found in pollen and seeds, where it serves as compatible solute, protecting cellular structures during dehydration. The proline concentrations of cells, tissues and plant organs are regulated by the interplay of biosynthesis, degradation and intra- as well as intercellular transport processes. Among the proline transport proteins characterized so far, both general amino acid permeases and selective compatible solute transporters were identified, reflecting the versatile role of proline under stress and non-stress situations. The review summarizes our current knowledge on proline metabolism and transport in view of plant development, discussing regulatory aspects such as the influence of metabolites and hormones. Additional information from animals, fungi and bacteria is included, showing similarities and differences to proline metabolism and transport in plants.
    Amino Acids 03/2010; 39(4):949-62. · 3.91 Impact Factor
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    Dietmar Funck, Sonja Eckard, Gudrun Müller
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    ABSTRACT: Proline (Pro) accumulation is a widespread response of prokaryotic and eukaryotic cells subjected to osmotic stress or dehydration. When the cells are released from stress, Pro is degraded to glutamate by Pro-dehydrogenase (ProDH) and Pyrroline-5-carboxylate dehydrogenase (P5CDH), which are both mitochondrial enzymes in eukaryotes. While P5CDH is a single copy gene in Arabidopsis, two ProDH genes have been identified in the genome. Until now, only ProDH1 (At3g30775) had been functionally characterised. We demonstrate vasculature specific expression of the Arabidopsis ProDH2 gene (At5g38710) as well as enzymatic activity and mitochondrial localisation of the encoded protein. Expression levels of ProDH2 are generally low, but increased in senescent leaves and in the abscission zone of floral organs. While sucrose represses ProDH2 expression, Pro and NaCl were identified as inducers. Endogenous ProDH2 expression was not able to overcome Pro sensitivity of ProDH1 mutants, but overexpression of a GFP-tagged form of ProDH2 enabled the utilisation of Pro as single nitrogen source for growth. Amongst two intronic insertion mutants, one was identified as a null allele, whereas the other still produced native ProDH2 transcripts. Arabidopsis possesses two functional ProDHs, which have non-redundant, although partially overlapping physiological functions. The two ProDH isoforms differ with respect to spatial, developmental and environmental regulation of expression. While ProDH1 appears to be the dominant isoform under most conditions and in most tissues, ProDH2 was specifically upregulated during salt stress, when ProDH1 was repressed. The characterisation of ProDH2 as a functional gene requires a careful re-analysis of mutants with a deletion of ProDH1, which were so far considered to be devoid of ProDH activity. We hypothesise that ProDH2 plays an important role in Pro homeostasis in the vasculature, especially under stress conditions that promote Pro accumulation.
    BMC Plant Biology 01/2010; 10:70. · 4.35 Impact Factor
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    ABSTRACT: Like many other plant species, Arabidopsis uses arginine (Arg) as a storage and transport form of nitrogen, and proline (Pro) as a compatible solute in the defence against abiotic stresses causing water deprivation. Arg catabolism produces ornithine (Orn) inside mitochondria, which was discussed controversially as a precursor for Pro biosynthesis, alternative to glutamate (Glu). We show here that ornithine-delta-aminotransferase (deltaOAT, At5g46180), the enzyme converting Orn to pyrroline-5-carboxylate (P5C), is localised in mitochondria and is essential for Arg catabolism. Wildtype plants could readily catabolise supplied Arg and Orn and were able to use these amino acids as the only nitrogen source. Deletion mutants of deltaOAT, however, accumulated urea cycle intermediates when fed with Arg or Orn and were not able to utilize nitrogen provided as Arg or Orn. Utilisation of urea and stress induced Pro accumulation were not affected in T-DNA insertion mutants with a complete loss of deltaOAT expression. Our findings indicate that deltaOAT feeds P5C exclusively into the catabolic branch of Pro metabolism, which yields Glu as an end product. Conversion of Orn to Glu is an essential route for recovery of nitrogen stored or transported as Arg. Pro biosynthesis occurs predominantly or exclusively via the Glu pathway in Arabidopsis and does not depend on Glu produced by Arg and Orn catabolism.
    BMC Plant Biology 02/2008; 8:40. · 4.35 Impact Factor
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    ABSTRACT: Fragilariopsis cylindrus (Grunow) Willi Krieger, a bipolar psychrophilic and highly abundant diatom, experiences strong shifts in salinity during the formation of sea ice. We investigated the effects of osmotic stress due to an increased salt concentration from 34 to 70 practical salinity units (PSU) in conjunction with a temperature decrease from 0˚C to -4˚C on the anabolic and catabolic pathways of proline metabolism. Gene expression levels of Δ­­­1-pyrroline-5-carboxylate synthase (P5CS) strongly decreased by a factor of 17.3 in the 70 PSU ⁄ 0˚­­­C treatment, whereas copy numbers of ornithine d-aminotransferase (­­­δ-OAT) increased 7.6-fold. Transcript levels of D1-pyrroline-5-carboxylate reductase (P5CR) and proline dehydrogenase (ProDH) were also slightly up-regulated by 2.5 and 2.88, respectively. This contrasts with findings in higher plants where a reverse regulation of P5CS and ­­­δ-OAT was observed and indicates that under elevated external salinities, the ornithine route is preferred to the glutamate pathway in F. cylindrus. Photosynthetic quantum yield at PSII instantly dropped from 0.61 to 0.24 after the upshift in salinity, which confirmed a detrimental effect of elevated salt concentrations on the photosynthetic machinery. Reduced photosynthetic energy capture might explain the preference for the ornithine route over the more energyconsuming proline route. Salt proved to be the dominating stressor, while an additional temperature decrease appeared to have an ameliorating effect.
    Journal of Phycology 01/2007; · 2.24 Impact Factor
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    ABSTRACT: Sudden exposure of plants to high light (HL) leads to metabolic and physiological disruption of the photosynthetic cells. Changes in ROS content, adjustment of photosynthetic processes and the antioxidant pools and, ultimately, gene induction are essential components for a successful acclimation to the new light conditions. The influence of salicylic acid (SA) on plant growth, short-term acclimation to HL, and on the redox homeostasis of Arabidopsis thaliana leaves was assessed here. The dwarf phenotype displayed by mutants with high SA content (cpr1-1, cpr5-1, cpr6-1, and dnd1-1) was less pronounced when these plants were grown in HL, suggesting that the inhibitory effect of SA on growth was partly overcome at higher light intensities. Moreover, higher SA content affected energy conversion processes in low light, but did not impair short-term acclimation to HL. On the other hand, mutants with low foliar SA content (NahG and sid2-2) were impaired in acclimation to transient exposure to HL and thus predisposed to oxidative stress. Low and high SA levels were strictly correlated to a lower and higher foliar H(2)O(2) content, respectively. Furthermore high SA was also associated with higher GSH contents, suggesting a tight correlation between SA, H(2)O(2) and GSH contents in plants. These observations implied an essential role of SA in the acclimation processes and in regulating the redox homeostasis of the cell. Implications for the role of SA in pathogen defence signalling are also discussed.
    Journal of Experimental Botany 02/2006; 57(8):1795-807. · 5.79 Impact Factor
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    ABSTRACT: In response to stress, plants accumulate Pro, requiring degradation after release from adverse conditions. Delta1-Pyrroline-5-carboxylate dehydrogenase (P5CDH), the second enzyme for Pro degradation, is encoded by a single gene expressed ubiquitously. To study the physiological function of P5CDH, T-DNA insertion mutants in AtP5CDH were isolated and characterized. Although Pro degradation was undetectable in p5cdh mutants, neither increased Pro levels nor an altered growth phenotype were observed under normal conditions. Thus AtP5CDH is essential for Pro degradation but not required for vegetative plant growth. External Pro application caused programmed cell death, with callose deposition, reactive oxygen species production, and DNA laddering, involving a salicylic acid signal transduction pathway. p5cdh mutants were hypersensitive toward Pro and other molecules producing P5C, such as Arg and Orn. Pro levels were the same in the wild type and mutants, but P5C was detectable only in p5cdh mutants, indicating that P5C accumulation may be the cause for Pro hypersensitivity. Accordingly, overexpression of AtP5CDH resulted in decreased sensitivity to externally supplied Pro. Thus, Pro and P5C/Glu semialdehyde may serve as a link between stress responses and cell death.
    The Plant Cell 01/2005; 16(12):3413-25. · 9.25 Impact Factor
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    ABSTRACT: The mutant regulator of APX2 1-1 (rax1-1) was identified in Arabidopsis thaliana that constitutively expressed normally photooxidative stress-inducible ASCORBATE PEROXIDASE2 (APX2) and had >/=50% lowered foliar glutathione levels. Mapping revealed that rax1-1 is an allele of gamma-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic gamma-glutamylcysteine synthetase, the controlling step of glutathione biosynthesis. By comparison of rax1-1 with the GSH1 mutant cadmium hypersensitive 2, the expression of 32 stress-responsive genes was shown to be responsive to changed glutathione metabolism. Under photo-oxidative stress conditions, the expression of a wider set of defense-related genes was altered in the mutants. In wild-type plants, glutathione metabolism may play a key role in determining the degree of expression of defense genes controlled by several signaling pathways both before and during stress. This control may reflect the physiological state of the plant at the time of the onset of an environmental challenge and suggests that changes in glutathione metabolism may be one means of integrating the function of several signaling pathways.
    The Plant Cell 10/2004; 16(9):2448-62. · 9.25 Impact Factor
  • The Plant Cell 01/2004; 9.26(16). · 9.25 Impact Factor
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    ABSTRACT: Delta1-pyrroline-5-carboxylate (P5C), an intermediate in biosynthesis and degradation of proline (Pro), is assumed to play a role in cell death in plants and animals. Toxicity of external Pro and P5C supply to Arabidopsis suggested that P5C dehydrogenase (P5CDH; EC 1.2.1.12) plays a crucial role in this process by degrading the toxic Pro catabolism intermediate P5C. Also in a Deltaput2 yeast mutant, lacking P5CDH, Pro led to growth inhibition and formation of reactive oxygen species (ROS). Complementation of the Deltaput2 mutant allowed identification of the Arabidopsis P5CDH gene. AtP5CDH is a single-copy gene and the encoded protein was localized to the mitochondria. High homology of AtP5CDH to LuFIS1, an mRNA up-regulated during susceptible pathogen attack in flax, suggested a role for P5CDH in inhibition of hypersensitive reactions. An Arabidopsis mutant (cpr5) displaying a constitutive pathogen response was found to be hypersensitive to external Pro. In agreement with a role in prevention of cell death, AtP5CDH was expressed at a basal level in all tissues analysed. The highest expression was found in flowers that are known to contain the highest Pro levels under normal conditions. External supply of Pro induced AtP5CDH expression, but much more slowly than Pro dehydrogenase (AtProDH) expression. Uncoupled induction of the AtProDH and AtP5CDH genes further supports the hypothesis that P5C levels have to be tightly controlled. These results indicate that, in addition to the well-studied functions of Pro, for example in osmoregulation, the Pro metabolism intermediate P5C also serves as a regulator of cellular stress responses.
    The Plant Journal 09/2001; 27(4):345-56. · 6.58 Impact Factor
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    ABSTRACT: SummaryΔ1-pyrroline-5-carboxylate (P5C), an intermediate in biosynthesis and degradation of proline (Pro), is assumed to play a role in cell death in plants and animals. Toxicity of external Pro and P5C supply to Arabidopsis suggested that P5C dehydrogenase (P5CDH; EC 1.2.1.12) plays a crucial role in this process by degrading the toxic Pro catabolism intermediate P5C. Also in a Δput2 yeast mutant, lacking P5CDH, Pro led to growth inhibition and formation of reactive oxygen species (ROS). Complementation of the Δput2 mutant allowed identification of the Arabidopsis P5CDH gene. AtP5CDH is a single-copy gene and the encoded protein was localized to the mitochondria. High homology of AtP5CDH to LuFIS1, an mRNA up-regulated during susceptible pathogen attack in flax, suggested a role for P5CDH in inhibition of hypersensitive reactions. An Arabidopsis mutant (cpr5) displaying a constitutive pathogen response was found to be hypersensitive to external Pro. In agreement with a role in prevention of cell death, AtP5CDH was expressed at a basal level in all tissues analysed. The highest expression was found in flowers that are known to contain the highest Pro levels under normal conditions. External supply of Pro induced AtP5CDH expression, but much more slowly than Pro dehydrogenase (AtProDH) expression. Uncoupled induction of the AtProDH and AtP5CDH genes further supports the hypothesis that P5C levels have to be tightly controlled. These results indicate that, in addition to the well-studied functions of Pro, for example in osmoregulation, the Pro metabolism intermediate P5C also serves as a regulator of cellular stress responses.
    The Plant Journal 12/2000; 27(4):345 - 356. · 6.58 Impact Factor
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    ABSTRACT: In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutant rsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS and P5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Delta(1)-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.
    Plant physiology 07/2000; 123(2):779-89. · 6.56 Impact Factor
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    ABSTRACT: In higher plants, sugars possess multiple functions: transport and storage of carbon and energy as well as signal molecules. A variety of sugar transporters have been cloned that show differential expression between source and sink tissues. Expression of these transporters is highly regulated, according to the local metabolic status and the demands of long distance transport. Very little knowledge is available on mechanisms underlying the regulation of sugar transporter expression in plants. Studies in E. coli, yeast and mammals have unravelled complex regulatory pathways with crosstalk between sugar transport and metabolism. Recent studies in plants provide increasing evidence for the existence of similar regulatory mechanisms. In many cases, connections have been found between C and N-metabolism, implicating a tight network of signal transduction and metabolism. Some aspects of this network are presented in this review, emphasising sugar transport and sugar signal transduction.
    Australian journal of plant physiology 01/2000; 27:583-594.