Gene expression changes in phosphorus deficient potato (Solanum tuberosum L.) leaves and the potential for diagnostic gene expression markers

Background

There are compelling economic and environmental reasons to reduce our reliance on inorganic phosphate (Pi) fertilisers. Better management of Pi fertiliser applications is one option to improve the efficiency of Pi fertiliser use, whilst maintaining crop yields. Application rates of Pi fertilisers are traditionally determined from analyses of soil or plant tissues. Alternatively, diagnostic genes with altered expression under Pi limiting conditions that suggest a physiological requirement for Pi fertilisation, could be used to manage Pifertiliser applications, and might be more precise than indirect measurements of soil or tissue samples.

Results

We grew potato (Solanum tuberosum L.) plants hydroponically, under glasshouse conditions, to control their nutrient status accurately. Samples of total leaf RNA taken periodically after Pi was removed from the nutrient solution were labelled and hybridised to potato oligonucleotide arrays. A total of 1,659 genes were significantly differentially expressed following Pi withdrawal. These included genes that encode proteins involved in lipid, protein, and carbohydrate metabolism, characteristic of Pi deficient leaves and included potential novel roles for genes encoding patatin like proteins in potatoes. The array data were analysed using a support vector machine algorithm to identify groups of genes that could predict the Pi status of the crop. These groups of diagnostic genes were tested using field grown potatoes that had either been fertilised or unfertilised. A group of 200 genes could correctly predict the Pi status of field grown potatoes.

Conclusions

This paper provides a proof-of-concept demonstration for using microarrays and class prediction tools to predict the Pi status of a field grown potato crop. There is potential to develop this technology for other biotic and abiotic stresses in field grown crops. Ultimately, a better understanding of crop stresses may improve our management of the crop, improving the sustainability of agriculture.     

Oligopeptidase B from Trypanosoma evansi. A parasite peptidase that inactivates atrial natriuretic factor in the bloodstream of infected hosts

Serine oligopeptidases of trypanosomatids are emerging as important virulence factors and therapeutic targets in trypanosome infections. We report here the isolation and characterization of oligopeptidase B (OpdB) and its corresponding gene from Trypanosoma evansi, a pathogen of significant veterinary importance. The T. evansi opdB gene was present as a single copy per haploid genome containing an open reading frame of 2148 bp encoding a protein of 80.664 kDa. Purified OpdB hydrolyzed substrates with basic residues in P1 (k(cat)/K(m) for carbobenzyloxy-L-arginyl-L-arginyl-7-amido-4-methylcoumarin, 337 s(-1) x microm(-1)) and exhibited potent arginyl carboxypeptidase activity (k(cat)/K(m) for Val-Lys-Arg Arg-OH, 231 s(-1) x mM(-1)). While not secreted, T. evansi released OpdB into the plasma of infected hosts where it retained catalytic activity. Plasma OpdB levels correlated with blood parasitemia. In vitro, OpdB cleaved the peptide hormone atrial natriuretic factor (ANF) at four sites: Arg3 Arg4, Arg4 Ser5, Arg11 Ile12, and Arg27 Tyr28, thereby abrogating smooth muscle relaxant and prohypotensive properties of ANF. Circulating plasma ANF levels in T. evansi-infected rats were depressed from 130 to 8 pg x ml(-1), and plasma ANF levels inversely correlated with plasma OpdB activity. The in vitro half-life of ANF in rat plasma was reduced 300-fold in plasma from T. evansi-infected rodents, which contains high levels of OpdB activity. Addition of OpdB inhibitors to cell-free plasma from infected rodents significantly abrogated this ANF hydrolysis. Furthermore the in vivo ANF half-life was reduced 5-fold in T. evansi-infected rats. Thus, we propose a role for OpdB in peptide hormone dysregulation in trypanosomiasis, specifically in generating the depressed plasma levels of ANF in mammals infected with T. evansi.     

Identification of the reactive cysteine residues in oligopeptidase B from Trypanosoma brucei

Oligopeptidase B (OpdB) from Trypanosoma brucei is a candidate therapeutic target in African trypanosomiasis. OpdB is an atypical serine peptidase, since activity is inhibited by thiol-blocking reagents and enhanced by reducing agents. We have identified C256 as the reactive cysteine residue that mediates OpdB inhibition by N-ethylmaleimide and iodoacetic acid. Modeling studies suggest that C256 adducts occlude the P(1) substrate-binding site, preventing substrate binding. We further demonstrate that C559 and C597 are responsible for the thiol-enhancement of OpdB activity. These studies may facilitate the development of specific OpdB inhibitors with therapeutic potential, by exploiting these unique properties of this enzyme.     

Evidence that Ubiquinone Is a Required Intermediate for Rhodoquinone Biosynthesis in Rhodospirillum rubrum

Rhodoquinone (RQ) is an important cofactor used in the anaerobic energy metabolism of Rhodospirillum rubrum. RQ is structurally similar to ubiquinone (coenzyme Q or Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is also found in several eukaryotic species that utilize a fumarate reductase pathway for anaerobic respiration, an important example being the parasitic helminths. RQ is not found in humans or other mammals, and therefore inhibition of its biosynthesis may provide a parasite-specific drug target. In this report, we describe several in vivo feeding experiments with R. rubrum used for the identification of RQ biosynthetic intermediates. Cultures of R. rubrum were grown in the presence of synthetic analogs of ubiquinone and the known Q biosynthetic precursors demethylubiquinone, demethoxyubiquinone, and demethyldemethoxyubiquinone, and assays were monitored for the formation of RQ₃. Data from time course experiments and S-adenosyl-l-methionine-dependent O-methyltransferase inhibition studies are discussed. Based on the results presented, we have demonstrated that Q is a required intermediate for the biosynthesis of RQ in R. rubrum.Rhodospirillum rubrum is a well-characterized and metabolically diverse member of the family of purple nonsulfur bacteria (29, 61). R. rubrum is typically found in aquatic environments and can adapt to a variety of growth conditions by using photosynthesis, respiration, or fermentation pathways (28, 70). In the light, R. rubrum exhibits photoheterotrophic growth using organic substrates or photoautotrophic growth using CO₂ and H₂ (15, 70). In the dark, R. rubrum can utilize either aerobic respiration (70, 73) or anaerobic respiration with a fumarate reduction pathway or with nonfermentable substrates in the presence of oxidants such as dimethyl sulfoxide (DMSO) or trimethylamine oxide (15, 58, 73). R. rubrum can also grow anaerobically in the dark by fermentation of sugars in the presence of bicarbonate (58). The focus of this work was the biosynthesis of quinones used by R. rubrum for aerobic and anaerobic respiration.Rhodoquinone (RQ; compound 1 in Fig. ​Fig.1)1) is an aminoquinone structurally similar to ubiquinone (coenzyme Q or Q [compound 2]) (44); however, the two differ considerably in redox potential (that of RQ is −63 mV, and that of Q is +100 mV) (2). Both RQ and Q have a fully substituted benzoquinone ring and a polyisoprenoid side chain that varies in length (depending on the species; see Fig. ​Fig.11 for examples). The only difference between the structures is that RQ has an amino substituent (NH₂) instead of a methoxy substituent (OCH₃) on the quinone ring. While Q is a ubiquitous lipid component involved in aerobic respiratory electron transport (9, 36, 60), RQ functions in anaerobic respiration in R. rubrum (19) and in several other phototrophic purple bacteria (21, 22, 41) and is also present in a few aerobic chemotrophic bacteria, including Brachymonas denitrificans and Zoogloea ramigera (23). In these varied species of bacteria, RQ has been proposed to function in fumarate reduction to maintain NAD⁺/NADH redox balance, either during photosynthetic anaerobic metabolism (12, 15-18, 64) or in chemotrophic metabolism when the availability of oxygen as a terminal oxidant is limiting (23). Another recent finding is that RQH₂ is capable of inducing Q-cycle bypass reactions in the cytochrome bc₁ complex in Saccharomyces cerevisiae, resulting in superoxide formation (7). If RQ/RQH₂ coexists in the cytoplasmic membrane with Q/QH₂ in R. rubrum, it might serve as both a substrate for and an inhibitor of the bc₁ complex (47).Open in a separate windowFIG. 1.Proposed pathways for RQ biosynthesis. The number of isoprene units (n) varies by species (in S. cerevisiae, n = 6; in E. coli, n = 8; in C. elegans, n = 9; in helminth parasites, n = 9 or 10; in R. rubrum, n = 10; in humans, n = 10). RQ is not found in S. cerevisiae, E. coli, or humans. Known Coq (from S. cerevisiae) and Ubi (from E. coli) gene products required for the biosynthesis of ubiquinone (Q, compound 2) are labeled. A polyisoprenyl diphosphate (compound 5) is assembled from dimethylallyl disphosphate (compound 3) and isopentyl diphosphate (compound 4). Coupling of compound 5 with p-hydroxybenzoic acid (compound 6) yields 3-polyprenyl-4-hydroxybenzoic acid (compound 7). The next three steps differ between S. cerevisiae and E. coli. However, they merge at the common intermediate (compound 8), which is oxidized to demethyldemethoxyubiquinone (DDMQ_n, compound 9). RQ (compound 1) has been proposed to arise from compound 9, demethoxyubiquinone (DMQ_n; compound 10), demethylubiquinone (DMeQ_n; compound 11), or compound 2 (by pathway A, B, C, or D). Results presented in this work support pathway D as the favored route for RQ biosynthesis in R. rubrum.RQ is also found in the mitochondrial membrane of eukaryotic species capable of fumarate reduction, such as the flagellate Euglena gracilis (25, 53), the free-living nematode Caenorhabditis elegans (62), and the parasitic helminths (65, 66, 68, 72). Similar to R. rubrum, these species can adapt their metabolism to both aerobic and anaerobic conditions throughout their life cycle. For example, most adult parasitic species (e.g., Ascaris suum, Fasciola hepatica, and Haemonchus contortus) rely heavily on fumarate reduction for their energy generation while inside a host organism, where the oxygen tension is very low (30, 65, 72). Under these conditions, the biosynthesis of RQ is upregulated; however, during free-living stages of their life cycle, the helminth parasites use primarily aerobic respiration, which requires Q (30, 65, 72). The anaerobic energy metabolism of the helminthes has been reviewed (63, 67). Humans and other mammalian hosts use Q for aerobic energy metabolism but do not produce or require RQ; therefore, selective inhibition of RQ biosynthesis may lead to highly specific antihelminthic drugs that do not have a toxic effect on the host (35, 48).R. rubrum is an excellent facultative model system for the study of RQ biosynthesis. The complete genome of R. rubrum has recently been sequenced by the Department of Energy Joint Genome Institute, finished by the Los Alamos Finishing Group, and further validated by optical mapping (57). The 16S rRNA sequence of R. rubrum is highly homologous to cognate eukaryotic mitochondrial sequences (46). Due to the similarities in structure, the biosynthetic pathways of RQ and Q have been proposed to diverge from a common precursor (67). Proposed pathways for RQ biosynthesis (A to D), in conjunction with the known steps in Q biosynthesis, are outlined in Fig. ​Fig.11 (31, 34, 60). Parson and Rudney previously showed that when R. rubrum was grown anaerobically in the light in the presence of [U-¹⁴C]p-hydroxybenzoate, ¹⁴C was incorporated into both Q₁₀ and RQ₁₀ (50). In their growth experiments, the specific activity of Q₁₀ was measured at its maximal value 15 h after inoculation and then began to decrease. However, the specific activity of RQ₁₀ continued to increase for 40 h before declining. These results suggested that Q₁₀ was a biosynthetic precursor of RQ₁₀, although this was not directly demonstrated using radiolabeled Q₁₀; hence, the possibility remained that the labeled RQ₁₀ was derived from another radiolabeled lipid species. We have done this feeding experiment with a synthetic analog of Q where n = 3 (Q₃) and monitored for the production of RQ₃. The synthesis and use of farnesylated quinone and aromatic intermediates for characterization of the Q biosynthetic pathway in S. cerevisiae and Escherichia coli has been well documented (4, 5, 38, 52, 59). The other proposed precursors of RQ shown in Fig. ​Fig.11 were also fed to R. rubrum, and the lipid extracts from these assays were analyzed for the presence of RQ₃, i.e., demethyldemethoxyubiquinone-3 (DDMQ₃; compound 9), demethoxyubiquinone-3 (DMQ₃; compound 10), and demethylubiquinone-3 (DMeQ₃; compound 11).In S. cerevisiae and E. coli, the last O-methylation step in Q biosynthesis is catalyzed by the S-adenosyl-l-methionine (SAM)-dependent methyltransferases Coq3 and UbiG, respectively (26, 52); this final methylation step converts DMeQ to Q. Using the NCBI Basic Local Alignment Search Tool, an O-methyltransferase (GeneID no. 3834724 Rru_A0742) that had 41% and 59% sequence identity with Coq3 and UbiG, respectively, was identified in R. rubrum. S-Adenosyl-l-homocysteine (SAH) is a well-known inhibitor of SAM-dependent methyltransferases (13, 24). Because SAH is the transmethylation by-product of SAM-dependent methyltransferases, it is not readily taken up by cells and must be generated in vivo (24). SAH can be produced in vivo from S-adenosine and l-homocysteine thiolactone by endogenous SAH hydrolase (SAHH) (37, 71). A search of the R. rubrum genome also confirmed the presence of a gene encoding SAHH (GeneID no. 3836896 Rru_A3444). It was proposed that if DMeQ is the immediate precursor of RQ, then SAH inhibition of the methyltransferase required for Q biosynthesis should have little effect on RQ production. Conversely, if Q is required for RQ synthesis, then inhibition of Q biosynthesis should have a significant effect on RQ production. Assays were designed to quantify the levels of RQ₃ produced from DMeQ₃ and Q₃ in R. rubrum cultures at various concentrations of SAH.     

Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF)

In this study we have shown that N376 to D mutation in the conserved NPxxY motif within the carboxy terminal tail domain (CT) of the 5-HT2A receptor alters the binding preference of GST-fusion protein constructs of the CT domain from ARF1 to an alternative isoform, ARF6. These findings were corroborated by experiments investigating co-immunoprecipitation of the wild type (WT) and N376D mutant of the 5-HT2A receptor with ARF1 or 6 or dominant negative ARF1/6 constructs co-expressed in COS7 cells. In functional assays of 5-HT-induced phospholipase D (PLD) activation responses of the WT receptor were inhibited by a dominant negative mutant of ARF1 but not ARF6, whereas responses of the N376D mutant were strongly inhibited by negative mutant ARF6. No equivalent effect of the ARF mutants was seen on phospholipase C activation. In experiments assaying 5-HT-induced increases in [35S]GTPgammaS binding to ARF 1/6 immunoprecipitates as a measure of ARF activation, increased ARF6 activation was seen only with the mutant receptor. When cellular PLD responses of other NPxxY- or a DPxxY-containing GPCRs were measured in the presence of dominant negative ARF1/6 constructs, the majority, but not all, fitted the pattern exemplified by the 5-HT2A receptor and its N376D mutant. These data suggest that the presence of the N or a D in this highly conserved motif is an important, but not exclusive, determinant of which ARF isoform interacts with the GPCR.     

The involvement of replication in single stranded oligonucleotide-mediated gene repair

Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the ‘annealing-integration’ model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a λ-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased.     

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.