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

Cytosolic CTP Production Limits the Establishment of Photosynthesis in Arabidopsis

CTP synthases (CTPS) comprise a protein family of the five members CTPS1-CTPS5 in Arabidopsis, all located in the cytosol. Specifically, downregulation of CTPS2 by amiRNA technology results in plants with defects in chlorophyll accumulation and photosynthetic performance early in development. CTP and its deoxy form dCTP are present at low levels in developing seedlings. Thus, under conditions of fast proliferation, the synthesis of CTP (dCTP) can become a limiting factor for RNA and DNA synthesis. The higher sensitivity of ami-CTPS2 lines toward the DNA-Gyrase inhibitor ciprofloxacin, together with reduced plastid DNA copy number and 16S and 23S chloroplast ribosomal RNA support this view. High expression and proposed beneficial biochemical features render CTPS2 the most important isoform for early seedling development. In addition, CTPS2 was identified as an essential enzyme in embryo development before, as knock-out mutants were embryo lethal. In line with this, ami-CTPS2 lines also exhibited reduced seed numbers per plant

Acclimation in plants – the Green Hub consortium

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to ‘smart breeding’ methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast‐related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Peer Reviewe

Limitation of Cytokinin Export to the Shoots by Nucleoside Transporter ENT3 and Its Linkage with Root Elongation in Arabidopsis

The trans-membrane carrier AtENT3 is known to transport externally supplied cytokinin ribosides and thus promote uptake by cells. However, its role in distributing either exogenous or endogenous cytokinins within the intact plant has not hitherto been reported. To test this, we used ent3-1 mutant Arabidopsis seedlings in which the gene is not expressed due to a T-DNA insertion, and examined the effect on the concentration and distribution of either endogenous cytokinins or exogenous trans-zeatin riboside applied to the roots. In the mutant, accumulation of endogenous cytokinins in the roots was reduced and capacity to deliver externally supplied trans-zeatin riboside to the shoots was increased suggesting involvement of equilibrative nucleoside (ENT) transporter in the control of cytokinin distribution in the plants. Roots of ent3-1 were longer in the mutant in association with their lower cytokinin concentration. We concluded that the ENT3 transporter participates in partitioning endogenous cytokinins between the apoplast and the symplast by facilitating their uptake by root cells thereby limiting cytokinin export to the shoots through the xylem. Dilution of the mineral nutrient solution lowered endogenous cytokinin concentration in the roots of both wild type (WT) and ent3-1 plants accompanied by promotion of root elongation. Nevertheless, cytokinin content was lower, while roots were longer in the ent3-1 mutant than in the WT under either normal or deficient mineral nutrition suggesting a significant role of ENT3 transporter in the control of cytokinin level in the roots and the rate of their elongation

Embryo-Specific Reduction of ADP-Glc Pyrophosphorylase Leads to an Inhibition of Starch Synthesis and a Delay in Oil Accumulation in Developing Seeds of Oilseed Rape

In oil-storing Brassica napus (rape) seeds, starch deposition occurs only transiently in the early stages of development, and starch is absent from mature seeds. This work investigates the influence of a reduction of ADP-Glc pyrophosphorylase (AGPase) on storage metabolism in these seeds. To manipulate the activity of AGPase in a seed-specific manner, a cDNA encoding the small subunit of AGPase was expressed in the sense or antisense orientation under the control of an embryo-specific thioesterase promoter. Lines were selected showing an embryo-specific decrease in AGPase due to antisense and cosuppression at different stages of development. At early developmental stages (25 days after flowering), a 50% decrease in AGPase activity was accompanied by similar decreases in starch content and the rate of starch synthesis measured by injecting (14)C-Suc into seeds in planta. In parallel to inhibition of starch synthesis, the level of ADP-Glc decreased, whereas Glc 1-phosphate levels increased, providing biochemical evidence that inhibition of starch synthesis was due to repression of AGPase. At 25 days after flowering, repression of starch synthesis also led to a decrease in the rate of (14)C-Suc degradation and its further metabolism via other metabolic pathways. This was not accompanied by an increase in the levels of soluble sugars, indicating that Suc import was inhibited in parallel. Flux through glycolysis, the activities of hexokinase, and inorganic pyrophosphate-dependent phosphofructokinase, and the adenylate energy state (ATP to ADP ratio) of the transgenic seeds decreased, indicating inhibition of glycolysis and respiration compared to wild type. This was accompanied by a marked decrease in the rate of storage lipid (triacylglycerol) synthesis and in the fatty acid content of seeds. In mature seeds, glycolytic enzyme activities, metabolite levels, and ATP levels remained unchanged, and the fatty acid content was only marginally lower compared to wild type, indicating that the influence of AGPase on carbon metabolism and oil accumulation was largely compensated for in the later stages of seed development. Results indicate that AGPase exerts high control over starch synthesis at early stages of seed development where it is involved in establishing the sink activity of the embryo and the onset of oil accumulation

Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase

13 páginas, 6 figuras.Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth.This work was supported by funding from the Spanish Ministry of Science, Innovation and Universities (BFU2016-80570-R and RTI2018-098084-B-100; AEI/FEDER, UE) to S.R.-M., DFG (Mo 1032/4-1 and CRC175-B08) to T.M., DFG-IRTG 1830 to T.M. and L.B., and Bayer AG Crop Science (Grants for Targets 2017-02-005) to S.R.-M. and T.M. We thank the staff from ALBA (Barcelona, Spain) and ESRF (Grenoble, France) synchrotron facilities for help during crystallographic data collection; R. Campos-Olivas and C.M. Santiveri for support with SEC-MALS analyses; S. Niopek-Witz and N. Navaseelan for support with ATC expression and kinetic analysis; L. Ohler, A. John, and A. Grande-García for support during protein purification, mutant generation, and crystallization trials; and M. Moreno-Morcillo for support with crystal structure analysis.Peer reviewe

Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants

We have isolated an Arabinopsis thaliana cDNA encoding a highly hydrophobic membrane protein of 589 amino acids which contains 12 potential transmembrane helices and shows a high degree of similarity (43.5% identity, 66.2% similarity) to the ATP/ADP translocase of the Gram-negative bacterium Rickettsia prowazekii, an obligate intracellular parasite responsible for the epidemic typhus. This rickettsial translocator resides in the cytoplasmic membrane and allows the bacterium to exploit the host cytoplasmic ATP pool. We hypothesize that the A. thaliana homolog of the R. prowazekii ATP/ADP translocase is the functional eukaryotic equivalent and resides in the plastid inner envelope membrane where it functions as an ATP importer

Characterization of filament‐forming CTP synthases from Arabidopsis thaliana

Summary Cytidine triphosphate (CTP) is essential for DNA, RNA and phospholipid biosynthesis. De novo synthesis is catalyzed by CTP synthases (CTPS). Arabidopsis encodes five CTPS isoforms that unanimously share conserved motifs found across kingdoms, suggesting all five are functional enzymes. Whereas CTPS1–4 are expressed throughout Arabidopsis tissues, CTPS5 reveals exclusive expression in developing embryos. CTPS activity and substrates affinities were determined for a representative plant enzyme on purified recombinant CTPS3 protein. As demonstrated in model organisms such as yeast, fruit fly and mammals, CTPS show the capacity to assemble into large filaments called cytoophidia. Transient expression of N‐ and C‐terminal YFP‐CTPS fusion proteins in Nicotiana benthamiana allowed to monitor such filament formation. Interestingly, CTPS1 and 2 always appeared as soluble proteins, whereas filaments were observed for CTPS3, 4 and 5 independent of the YFP‐tag location. However, when similar constructs were expressed in Saccharomyces cerevisiae, no filaments were observed, pointing to a requirement for organism‐specific factors in vivo. Indications for filament assembly were also obtained in vitro when recombinant CTPS3 protein was incubated in the presence of CTP. T‐DNA‐insertion mutants in four CTPS loci revealed no apparent phenotypical alteration. In contrast, CTPS2 T‐DNA‐insertion mutants did not produce homozygous progenies. An initial characterization of the CTPS protein family members from Arabidopsis is presented. We provide evidence for their involvement in nucleotide de novo synthesis and show that only three of the five CTPS isoforms were able to form filamentous structures in the transient tobacco expression system. This represents a striking difference from previous observations in prokaryotes, yeast, Drosophila and mammalian cells. This finding will be highly valuable to further understand the role of filament formation to regulate CTPS activity. Significance Statement An initial characterization of the CTP synthase protein family members from Arabidopsis is presented. We provide evidence for their involvement in nucleotide de novo synthesis and show that only three of the five CTPS isoforms were able to form filamentous structures in the transient tobacco expression system. This represents a striking difference from previous observations in prokaryotes, yeast, drosophila and mammalian cells. This finding will be highly valuable to further understand the role of filament formation to regulate CTP Synthase activity

Nucleotide Imbalance, Provoked by Downregulation of Aspartate Transcarbamoylase Impairs Cold Acclimation in Arabidopsis

Aspartate transcarbamoylase (ATC) catalyzes the first committed step in pyrimidine de novo synthesis. As shown before, mutants with 80% reduced transcript and protein levels exhibit reduced levels of pyrimidine metabolites and thus nucleotide limitation and imbalance. Consequently, reduced photosynthetic capacity and growth, accompanied by massive transcriptional changes, were observed. Here, we show that nucleotide de novo synthesis was upregulated during cold acclimation of Arabidopsis thaliana (ecotype Columbia, Col-0) plants, but ATC knockdown mutants failed to acclimate to this condition as they did not accumulate neutral sugars and anthocyanins. A global transcriptome analysis revealed that most of the transcriptional changes observed in Col-0 plants upon cold exposure were also evident in ATC knockdown plants. However, several responses observed in cold-treated Col-0 plants could already be detected in knockdown plants when grown under standard conditions, suggesting that these mutants exhibited typical cold responses without prior cold stimulation. We believe that nucleotide signaling is involved in “cold-like priming” and “cold acclimation” in general. The observed transcript levels of genes involved in central carbon metabolism and respiration were an exception to these findings. These were upregulated in the cold but downregulated in warm-grown ATC mutants