Effect of phytase supplementation on tissue inositol phosphate levels in broiler chickens

Phytate, myo-inositol hexakisphosphate, serves as an essential phosphorus store in plant seeds, but has antinutritive properties in the animals that consume it. Phytases, enzymes that degrade phytate, are added to the diets of monogastric animals. Many studies have correlated the addition of phytase with improved animal performance, with a subset of these seeking to correlate animal performance with phytase-mediated release of inositol or phosphate. This study aimed to develop methods to measure inositol phosphates in poultry tissues and to determine the effect of phytase supplementation on poultry tissues, as the effect of dietary phytase on tissue inositol and inositol phosphates and phytase mediated release of inositol and phosphate had not been studied. The study comprised wheat/soy-based diets containing one of three levels of phytase and one level of d30‰ inositol equivalent to the inositol released from total dietary phytate hydrolysis (0, 500 and 6000 FTU/kg of modified E. coli 6-phytase and 2 g/kg inositol). Diets were provided for 21 days and on day 21, digesta were collected from the gizzard and ileum, and tissues were harvested from brain, liver, kidney, breast and leg muscle, and intestinal segments. Myo-inositol and inositol phosphates were measured in diet, digesta and tissues. Gizzard and ileal content inositol increased and total inositol phosphates reduced progressively by phytase supplementation. The predominant higher inositol phosphates detected in tissues, D- and/or L-Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5 different from those generated by phytate degradation by E. coli 6-phytase or endogenous feed phytase, suggesting tissue inositol phosphates are not the result of direct absorption. Kidney inositol phosphates were reduced progressively with increasing phytase supplementation. These data suggest that tissue inositol phosphate concentrations can be influenced by dietary phytase inclusion rates, and that such effects are tissue specific, though the consequences of this for animal physiology and performance are yet to be elucidated

Accentuating the positive and eliminating the negative: Efficacy of TiO2 as digestibility index marker for poultry nutrition studies

Inert digestibility index markers such as titanium dioxide are universally accepted to provide simple measurement of digestive tract retention and relative digestibility in poultry feeding trials. Their use underpins industry practice: specifically dosing regimens for adjunct enzymes added to animal feed. Among these, phytases, enzymes that degrade dietary phytate, inositol hexakisphosphate, represent a billion-dollar sector in an industry that raises ca. 70 billion chickens/annum. Unbeknown to the feed enzyme sector, is the growth in cell biology of use of titanium dioxide for enrichment of inositol phosphates from extracts of cells and tissues. The adoption of titanium dioxide in cell biology arises from its affinity under acid conditions for phosphates, suggesting that in feeding trial contexts that target phytate degradation this marker may not be as inert as assumed. We show that feed grade titanium dioxide enriches a mixed population of higher and lower inositol phosphates from acid solutions. Additionally, we compared the extractable inositol phosphates in gizzard and ileal digesta of 21day old male Ross 308 broilers fed three phytase doses (0, 500 and 6000 FTU/kg feed) and one inositol dose (2g/kg feed). This experiment was performed with or without titanium dioxide added as a digestibility index marker at a level of 0.5%, with all diets fed for 21 days. Analysis yielded no significant difference in effect of phytase inclusion in the presence or absence of titanium dioxide. Thus, despite the utility of titanium dioxide for recovery of inositol phosphates from biological samples, it seems that its use as an inert marker in digestibility trials is justified—as its inclusion in mash diets does not interfere with the recovery of inositol phosphates from digesta samples

An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels

Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called 'high-energy' phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology

LC-ICP-MS analysis of inositol phosphate isomers in soil offers improved sensitivity and fine‐scale mapping of inositol phosphate distribution

1. Organic forms of phosphorus (P) prevail in soils and their quantification is vital to better understand global biogeochemical cycles. P speciation in soil is commonly assessed by 31P NMR spectroscopy of sodium hydroxide-EDTA (NaOH-EDTA) extracts. 3. A liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) method that employs NaOH-EDTA is described. 3. Comparison with 31P NMR shows that LC-ICP-MS is up to three orders of magnitude more sensitive. It allows measurement in samples as small as 1 mg. We reveal variation of inositol phosphate distribution in Swedish boreal forest soil and identify myo- and scyllo-inositol hexakisphosphates and other isomers including scyllo-inositol pentakisphosphate. 4. Speciation of the major inositol phosphates was not altered by long-term nitrogen fertilization

The PRT6 N‐degron pathway restricts VERNALIZATION 2 to endogenous hypoxic niches to modulate plant development

VERNALIZATION2 (VRN2), an angiosperm‐specific subunit of the polycomb repressive complex 2 (PRC2), is an oxygen (O2) regulated target of the PCO branch of the PRT6 N‐degron pathway of ubiquitin‐mediated proteolysis. How this post‐translational regulation coordinates VRN2 activity remains to be fully established. Here we use Arabidopsis thaliana ecotypes, mutants and transgenic lines to determine how control of VRN2 stability contributes to its functions during plant development. VRN2 localises to endogenous hypoxic regions in aerial and root tissues. In the shoot apex, VRN2 differentially modulates flowering time dependent on photoperiod, whilst its presence in lateral root primordia and the root apical meristem negatively regulates root system architecture. Ectopic accumulation of VRN2 does not enhance its effects on flowering, but does potentiate its repressive effects on root growth. In late‐flowering vernalization‐dependent ecotypes, VRN2 is only active outside meristems when its proteolysis is inhibited in response to cold exposure, since its function requires concomitant cold‐triggered increases in other PRC2 subunits and co‐factors. We conclude that the O2‐sensitive N‐degron of VRN2 has a dual function, confining VRN2 to meristems and primordia, where it has specific developmental roles, whilst also permitting broad accumulation outside of meristems in response to environmental cues, leading to other functions

Diversification in the inositol tris/tetrakisphosphate kinase (ITPK) family: crystal structure and enzymology of the outlier AtITPK4

Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Å resolution that, along with a description of the enantiospecificity of the enzyme, affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a KM for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologues in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology

Phytase dose-dependent response of kidney inositol phosphate levels in poultry

Phytases, enzymes that degrade phytate present in feedstuffs, are widely added to the diets of monogastric animals. Many studies have correlated phytase addition with improved animal productivity and a subset of these have sought to correlate animal performance with phytase-mediated generation of inositol phosphates in different parts of the gastro-intestinal tract or with release of inositol or of phosphate, the absorbable products of phytate degradation. Remarkably, the effect of dietary phytase on tissue inositol phosphates has not been studied. The objective of this study was to determine effect of phytase supplementation on liver and kidney myo-inositol and myo-inositol phosphates in broiler chickens. For this, methods were developed to measure inositol phosphates in chicken tissues. The study comprised wheat/soy-based diets containing one of three levels of phytase (0, 500 and 6,000 FTU/kg of modified E. coli 6-phytase). Diets were provided to broilers for 21 D and on day 21 digesta were collected from the gizzard and ileum. Liver and kidney tissue were harvested. Myo-inositol and inositol phosphates were measured in diet, digesta, liver and kidney. Gizzard and ileal content inositol was increased progressively, and total inositol phosphates reduced progressively, by phytase supplementation. The predominant higher inositol phosphates detected in tissues, D-and/or L-Ins(3,4,5,6)P(4) and Ins(1,3,4,5,6)P(5), differed from those (D-and/or L-Ins(1,2,3,4)P(4), D-and/or L-Ins(1,2,5,6)P(4), Ins(1,2,3,4,6)P(5), D-and/or L-Ins(1,2,3,4,5)P(5) and D-and/or L-Ins(1,2,4,5,6)P(5)) generated from phytate (InsP(6)) degradation by E. coli 6-phytase or endogenous feed phytase, suggesting tissue inositol phosphates are not the result of direct absorption. Kidney inositol phosphates were reduced progressively by phytase supplementation. These data suggest that tissue inositol phosphate concentrations can be influenced by dietary phytase inclusion rate and that such effects are tissue specific, though the consequences for physiology of such changes have yet to be elucidated

Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

Phylogenetic analysis, homology modelling and biochemical methods have been employed to characterize a phytase from a Gram-negative soil bacterium. Acinetobacter sp. AC1-2 phytase belongs to clade 2 of the histidine (acid) phytases, to the Multiple Inositol Polyphosphate Phosphatase (MINPP) subclass. The enzyme was extraordinarily stable in solution both at room temperature and 4 °C, retaining near 100% activity over 755 days. It showed a broad pH activity profile from 2-8.5 with maxima at 3, 4.5-5 and 6. The enzyme showed Michaelis-Menten kinetics and substrate inhibition (Vmax, Km, and Ki, 228 U/mg, 0.65 mM and 2.23 mM, respectively). Homology modelling using the crystal structure of a homologous MINPP from a human gut commensal bacterium indicated the presence of a potentially stabilising polypeptide loop (a U-loop) straddling the active site. By employ of the enantiospecificity of Arabidopsis inositol tris/tetrakisphosphate kinase 1 for inositol pentakisphosphates, we show AC1-2 MINPP to possess D6-phytase activity, which allowed modelling of active site specificity pockets for InsP6 substrate. While phytase gene transcription was unaltered in rich media, it was repressed in minimal media with phytic acid and orthophosphate as phosphate sources. The results of this study reveal AC1-2 MINPP to possess desirable attributes relevant to biotechnological use

Oxygen-dependent proteolysis regulates the stability of angiosperm polycomb repressive complex 2 subunit VERNALIZATION 2

The polycomb repressive complex 2 (PRC2) regulates epigenetic gene repression in eukaryotes. Mechanisms controlling its developmental specificity and signal-responsiveness are poorly understood. Here, we identify an oxygen-sensitive N-terminal (N-) degron in the plant PRC2 subunit VERNALIZATION(VRN) 2, a homolog of animal Su(z)12, that promotes its degradation via the N-end rule pathway. We provide evidence that this N-degron arose early during angiosperm evolution via gene duplication and N-terminal truncation, facilitating expansion of PRC2 function in flowering plants. We show that proteolysis via the N-end rule pathway prevents ectopic VRN2 accumulation, and that hypoxia and long-term cold exposure lead to increased VRN2 abundance, which we propose may be due to inhibition of VRN2 turnover via its N-degron. Furthermore, we identify an overlap in the transcriptional responses to hypoxia and prolonged cold, and show that VRN2 promotes tolerance to hypoxia. Our work reveals a mechanism for post-translational regulation of VRN2 stability that could potentially link environmental inputs to the epigenetic control of plant development