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author:("funk, dietary")
1.  Requirement of proline synthesis during Arabidopsis reproductive development 
BMC Plant Biology  2012;12:191.
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.
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.
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.
PMCID: PMC3493334  PMID: 23062072
Proline metabolism; Gamete and embryo development; Enzyme stability; Subcellular localisation
2.  The Arabidopsis CstF64-Like RSR1/ESP1 Protein Participates in Glucose Signaling and Flowering Time Control 
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.
PMCID: PMC3355569  PMID: 22629280
Arabidopsis; 3-O-methylglucose; patatin; mRNA processing; protein phosphorylation; signal transduction; proline-rich protein
3.  Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis 
BMC Plant Biology  2010;10:70.
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.
PMCID: PMC3095344  PMID: 20403182
4.  Ornithine-δ-aminotransferase is essential for Arginine Catabolism but not for Proline Biosynthesis 
BMC Plant Biology  2008;8:40.
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-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.
Our 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.
PMCID: PMC2377265  PMID: 18419821

Results 1-4 (4)