Dietmar Funck

Evidence for a Direct Link between Glutathione Biosynthesis and Stress Defense Gene Expression in Arabidopsis

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 γ-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic γ-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.

Proline metabolism and transport in plant development

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.

Ornithine-δ-aminotransferase is essential for Arginine Catabolism but not for Proline Biosynthesis

BackgroundLike 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).ResultsWe show here that ornithine-δ-aminotransferase (δOAT, 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 δOAT, 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 δOAT expression.ConclusionOur findings indicate that δOAT 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.

Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis

BackgroundProline (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.ResultsWe 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.ConclusionsArabidopsis 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.

Physiological implications of arginine metabolism in plants

Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions.

Requirement of proline synthesis during Arabidopsis reproductive development

BackgroundGamete 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.ResultsIn 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.ConclusionsOur 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.

Regulation of proline metabolism under salt stress in the psychrophilic diatom Fragilariopsis cylindrus (Bacillariophyceae)

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 δ‐aminotransferase (δ‐OAT) increased 7.6‐fold. Transcript levels of Δ1‐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.

The regulation of assimilate allocation and transport

In higher plants, sugars possess multiplefunctions: transport and storage of carbon and energy as well as signalmolecules. A variety of sugar transporters have been cloned that showdifferential expression between source and sink tissues. Expression of thesetransporters is highly regulated, according to the local metabolic status andthe demands of long distance transport. Very little knowledge is available onmechanisms underlying the regulation of sugar transporter expression inplants. Studies in E. coli, yeast and mammals haveunravelled complex regulatory pathways with crosstalk between sugar transportand metabolism. Recent studies in plants provide increasing evidence for theexistence of similar regulatory mechanisms. In many cases, connections havebeen found between C-and N-metabolism, implicating a tight network of signaltransduction and metabolism. Some aspects of this network are presented inthis review, emphasising sugar transport and sugar signaltransduction.

Δ1-pyrroline-5-carboxylate reductase from Arabidopsis thaliana: stimulation or inhibition by chloride ions and feedback regulation by proline depend on whether NADPH or NADH acts as co-substrate

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.

Small One-Helix Proteins Are Essential for Photosynthesis in Arabidopsis

The extended superfamily of chlorophyll a/b binding proteins comprises the Light-Harvesting Complex Proteins (LHCs), the Early Light-Induced Proteins (ELIPs) and the Photosystem II Subunit S (PSBS). The proteins of the ELIP family were proposed to function in photoprotection or assembly of thylakoid pigment-protein complexes and are further divided into subgroups with one to three transmembrane helices. Two small One-Helix Proteins (OHPs) are expressed constitutively in green plant tissues and their levels increase in response to light stress. In this study, we show that OHP1 and OHP2 are highly conserved in photosynthetic eukaryotes, but have probably evolved independently and have distinct functions in Arabidopsis. Mutations in OHP1 or OHP2 caused severe growth deficits, reduced pigmentation and disturbed thylakoid architecture. Surprisingly, the expression of OHP2 was severely reduced in ohp1 T-DNA insertion mutants and vice versa. In both ohp1 and ohp2 mutants, the levels of numerous photosystem components were strongly reduced and photosynthetic electron transport was almost undetectable. Accordingly, ohp1 and ohp2 mutants were dependent on external organic carbon sources for growth and did not produce seeds. Interestingly, the induction of ELIP1 expression and Cu/Zn superoxide dismutase activity in low light conditions indicated that ohp1 mutants constantly suffer from photo-oxidative stress. Based on these data, we propose that OHP1 and OHP2 play an essential role in the assembly or stabilization of photosynthetic pigment-protein complexes, especially photosystem reaction centers, in the thylakoid membrane.