Pyrimidine degradation influences germination seedling growth and production of Arabidopsis seeds

PYD1 (dihydropyrimidine dehydogenase) initiates the degradation of pyrimidine nucleobases and is located in plastids. In this study, a physiological analysis of PYD1 employing T-DNA knockout mutants and overexpressors was carried out. PYD1 knockout mutants were restricted in degradation of exogenously provided uracil and accumulated high uracil levels in plant organs throughout development, especially in dry seeds. Moreover, PYD1 knockout mutants showed delayed germination which was accompanied by low invertase activity and decreased monosaccharide levels. Abscisic acid (ABA) is an important regulator of seed germination, and ABA-responsive genes were deregulated in PYD1 knockout mutants. Together with an observed increased PYD1 expression in wild-type seedlings upon ABA treatment, an interference of PYD1 with ABA signalling can be assumed. Constitutive PYD1 overexpression mutants showed increased growth and higher seed number compared with wild-type and knockout mutant plants. During senescence PYD1 expression increased to allow uracil catabolism. From this it is concluded that early in development and during seed production PYD1 is needed to balance pyrimidine catabolism versus salvage

In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Background: Adenine nucleotide/phosphate carriers (APCs) from mammals and yeast are commonly known to adapt the mitochondrial adenine nucleotide pool in accordance to cellular demands. They catalyze adenine nucleotide - particularly ATP-Mg - and phosphate exchange and their activity is regulated by calcium. Our current knowledge about corresponding proteins from plants is comparably limited. Recently, the three putative APCs from Arabidopsis thaliana were shown to restore the specific growth phenotype of APC yeast loss-of-function mutants and to interact with calcium via their N-terminal EF-hand motifs in vitro. In this study, we performed biochemical characterization of all three APC isoforms from A. thaliana to gain further insights into their functional properties. Results: Recombinant plant APCs were functionally reconstituted into liposomes and their biochemical characteristics were determined by transport measurements using radiolabeled substrates. All three plant APCs were capable of ATP, ADP and phosphate exchange, however, high preference for ATP-Mg, as shown for orthologous carriers, was not detectable. By contrast, the obtained data suggest that in the liposomal system the plant APCs rather favor ATP-Ca as substrate. Moreover, investigation of a representative mutant APC protein revealed that the observed calcium effects on ATP transport did not primarily/essentially involve Ca2+-binding to the EF-hand motifs in the N-terminal domain of the carrier. Conclusion: Biochemical characteristics suggest that plant APCs can mediate net transport of adenine nucleotides and hence, like their pendants from animals and yeast, might be involved in the alteration of the mitochondrial adenine nucleotide pool. Although, ATP-Ca was identified as an apparent import substrate of plant APCs in vitro it is arguable whether ATP-Ca formation and thus the corresponding transport can take place in vivo

Neuroendocrine–immune disequilibrium and endometriosis: an interdisciplinary approach

Endometriosis, a chronic disease characterized by endometrial tissue located outside the uterine cavity, affects one fourth of young women and is associated with chronic pelvic pain and infertility. However, an in-depth understanding of the pathophysiology and effective treatment strategies of endometriosis is still largely elusive. Inadequate immune and neuroendocrine responses are significantly involved in the pathophysiology of endometriosis, and key findings are summarized in the present review. We discuss here the role of different immune mechanisms particularly adhesion molecules, protein–glycan interactions, and pro-angiogenic mediators in the development and progression of the disease. Finally, we introduce the concept of endometrial dissemination as result of a neuroendocrine-immune disequilibrium in response to high levels of perceived stress caused by cardinal clinical symptoms of endometriosis

Pyrimidine salvage: Physiological functions and interaction with chloroplast viogenesis

International audienceThe synthesis of pyrimidine nucleotides, an essential process in every organism, is accomplished by de novo synthesis or by salvaging pyrimdines from e.g. nucleic acid turnover. Here, we identify two Arabidopsis (Arabidopsis thaliana) uridine/cytidine kinases, UCK1 and UCK2, which are located in the cytosol and are responsible for the majority of pyrimidine salvage activity in vivo. In addition, the chloroplast has an active uracil salvage pathway. Uracil phosphoribosyltransferase (UPP) catalyzes the initial step in this pathway and is required for the establishment of photosynthesis, as revealed by analysis of upp mutants. The upp knockout mutants are unable to grow photoautotrophically, and knockdown mutants exhibit a variegated phenotype, with leaves that have chlorotic pale areas. Moreover, the upp mutants did not show altered expression of chloroplast-encoded genes, but transcript accumulation of the LIGHT HARVESTING COMPLEX B nuclear genes LHCB1.2 and LHCB2.3 was markedly reduced. An active UPP homolog from Escherichia colt failed to complement the upp mutant phenotype when targeted to the chloroplast, suggesting that the catalytic function of UPP is not the important factor for the chloroplast phenotype. Indeed, the expression of catalytically inactive Arabidopsis UPP, generated by introduction of point mutations, did complement the upp chloroplast phenotype. These results suggest that UPP has a vital function in chloroplast biogenesis unrelated to its catalytic activity and driven by a moonlighting function

Nucleobase and nucleoside transport and integration into plant metabolism

Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides i.e. nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level

Apoplastic nucleoside accumulation in Arabidopsis leads to reduced photosynthetic performance and increased susceptibility against Botrytis cinerea

ABSTRACT Interactions between plant and pathogen often occur in the extracellular space and especially nucleotides like ATP and NAD have been identified as key players in this scenario. Arabidopsis mutants accumulating nucleosides in the extracellular space were generated and studied with respect to susceptibility against Botrytis cinerea infection and general plant fitness determined as photosynthetic performance. The mutants used are deficient in the main nucleoside uptake system ENT3 and the extracellular nucleoside hydrolase NSH3. When grown on soil but not in hydroponic culture, these plants markedly accumulate adenosine and uridine in leaves. This nucleoside accumulation was accpmpanied by reduced photosystem II efficiency and altered expression of photosynthesis related genes. Moreover, a higher susceptibility towards Botrytis cinerea infection and a reduced induction of pathogen related genes PR1 and WRKY33 was observed. All these effects did not occur in hydroponically grown plants substantiating a contribution of extracellular nucleosides to these effects. Whether reduced general plant fitness, altered pathogen response capability or more direct interactions with the pathogen are responsible for these observations is discussed

Additional file 2: Figure S2. of In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Time dependent ADP transport via AtAPC1-3. Transport of 50 ÎźM [Îą32P]-ADP into Pi (A, C, E) and into ADP (B, D, F) loaded proteoliposomes with reconstituted AtAPC1 (A, B), AtAPC2 (C, D) and AtAPC3 (E, F). Non-loaded liposomes (non-filled rhombs; negative control) showed only marginal accumulation of radioactivity when compared to proteoliposomes loaded with Pi or ADP (black rhombs). Data represent mean values of three independent replicates, standard errors are given. (PDF 82 kb

Additional file 4: Figure S4. of In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Heterologous expression and ATP transport analysis of N- terminally truncated AtAPC2. (A) SDS-PAGE of 5μg and (B) Western-blot and immunodetection of 0.5 μg of the inclusion bodies fraction from E. coli cells expressing the N-terminally truncated (lanes 1). To enable detection of the molecular mass reduction due to loss of the N-terminal extension the full-length protein was included in this analysis (lanes 2). The Western-blot was immuno-decorated with a monoclonal anti poly His IgG (Sigma, Taufkirchen, Germany). M, prestained molecular weight marker (Thermo Fisher Scientific). (C) Time dependent import of 50 μM [α32P]-ATP via N- terminally truncated AtAPC2 into ATP loaded (black rhombs), Pi loaded (gray circles) and non-loaded (non-filled rhombs) liposomes. (PDF 156 kb

Additional file 6: Figure S6. of In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects

Effects of rising MgCl2 concentrations on [45Ca] transport via the N- terminally truncated AtAPC2. Transport of 20 ÎźM [45Ca] into Pi loaded (dark gray bars) and non- loaded (light gray bars) proteoliposomes was allowed for 10 min (given as nmol mg protein-1 h- 1). The transport medium was supplemented with 100 ÎźM non-labeled ATP and the indicated MgCl2 concentrations. Data represent mean values of three independent replicates. Standard errors are indicated. (PDF 102 kb