Molecular characterization and localization of Plasmodium falciparum choline kinase

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K_m values for choline and ATP are found to be 145 ± 20 μM and 2.5 ± 0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the α and β content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.     

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.     

The Accumulation of Glycine Betaine Is Dependent on Choline Monooxygenase (OsCMO), Not on Phosphoethanolamine N-Methyltransferase (OsPEAMT1), in Rice (Oryza sativa L. ssp. japonica)

Glycine betaine (GB) is an important osmoprotectant, which improves plant tolerance to various abiotic stresses. In higher plants, GB is synthesized through two-step oxidations of choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Choline, the precursor of GB, is synthesized by phosphoethanolamine N-methyltransferase (PEAMT). Rice is known as a typical non-GB-accumulated species. However, the underlying mechanism related to GB accumulation remains elusive. Here, we determined whether the endogenous accumulation of choline is sufficient to GB biosynthesis in rice and whether the rice CMO protein has the function of oxidizing choline to generate betaine aldehyde. The results showed that overexpression of the rice PEAMT1 gene (OsPEAMT1) resulted in increased levels of choline, while GB content remained unchanged in the transgenic rice plants overexpressing OsPEAMT1. However, the intracellular GB level and the tolerance to salt stress of the transgenic lines overexpressing OsCMO were significantly enhanced. Immunoblotting analysis demonstrated that abundant functional OsCMO proteins with correct size were detected in OsCMO-overexpressing transgenic rice plants, but rarely accumulated in the wild type. Collectively, these results implicated that the endogenous accumulation level of choline is not the major factor leading to non-GB accumulation in rice. Instead, the defective expression of OsCMO resulted in non-GB accumulation.     

Inoculation of Rhizobium (VR-1 and VA-1) induces an increasing growth and metal accumulation potential in Vigna radiata and Vigna angularis L. growing under fly-ash

Fly-ash-tolerant Rhizobium strains were isolated from plants grown in fly-ash-contaminated soil, axenically under laboratory conditions. Saplings of both plants were raised in N₂-free Jenson medium and inoculated with 2.6 × 10⁸ cell ml⁻¹ and 5.2 × 10⁸ cell ml⁻¹ of culture after 10 d of growth. Plants were transferred into 100% fly-ash under natural condition. Rhizobium-inoculated plants grown on 100% fly-ash showed marked increase in relation to root-shoot length, biomass yield, photosynthetic pigment, protein content and nodulation frequency compared to uninoculated plant grown in control (100% fly-ash). Inoculation of fly-ash-tolerant Rhizobium increased the accumulation of Fe, Zn, Cu Cd and Cr in different tissues vis-à-vis enhanced translocation of metals to the aboveground part of plant. Although inoculation of fly-ash-tolerant Rhizobium strains (VR-1 and VA-1) enhanced the translocation of more Fe to shoot parts, nevertheless, the amount of Rhizobium inoculants supplied to the plant was found to be very important since it has a positive role in increasing plant growth through increased N₂ supply via nitrogenase activity. Results suggest that an integrated approach employing biotechnological means and inoculation of plants with host-specific fly-ash-tolerant Rhizobium strain may prove a stimulus to a fly-ash management programme.     

Effect of low light intensity on growth and accumulation of secondary metabolites in roots of Glycyrrhiza uralensis Fisch

Effects of low light intensity on growth and accumulation of secondary metabolites of a medicinal plant Glycyrrhiza uralensis Fisch. were investigated. Hydroponic-cultivated one year-old rhizome seedlings were grown under three low irradiances, 200, 100, and 50 μmol m⁻² s⁻¹ for 135 days. Control plants were cultured under natural light conditions. Low light intensity stress decreased leaf thickness, photosynthesis and biomass, but increased leaf area and chlorophyll concentrations. Low light intensity also significantly increased accumulation of glycyrrhizic acid and liquiritin in the root, while the maximum values of both secondary metabolites were obtained under an irradiance of 100 μmol m⁻² s⁻¹. Concentrations of both secondary metabolites were negatively correlated with root biomass. The results suggested that G. uralensis could endure an environment with low light intensity and suitable light control might increase the secondary metabolite contents within agroforestry systems.     

Ectomycorrhizal Pisolithus albus inoculation of Acacia spirorbis and Eucalyptus globulus grown in ultramafic topsoil enhances plant growth and mineral nutrition while limits metal uptake

Ectomycorrhizal fungi (ECM) isolates of Pisolithus albus (Cooke and Massee) from nickel-rich ultramafic topsoils in New Caledonia were inoculated onto Acacia spirorbis Labill. (an endemic Fabaceae) and Eucalyptus globulus Labill. (used as a Myrtaceae plant host model). The aim of the study was to analyze the growth of symbiotic ECM plants growing on the ultramafic substrate that is characterized by high and toxic metal concentrations i.e. Co, Cr, Fe, Mn and Ni, deficient concentrations of plant essential nutrients such as N, P, K, and that presents an unbalanced Ca/Mg ratio (1/19). ECM inoculation was successful with a plant level of root mycorrhization up to 6.7%. ECM symbiosis enhanced plant growth as indicated by significant increases in shoot and root biomass. Presence of ECM enhanced uptake of major elements that are deficient in ultramafic substrates; in particular P, K and Ca. On the contrary, the ECM symbioses strongly reduced transfer to plants of element in excess in soils; in particular all metals. ECM-inoculated plants released metal complexing molecules as free thiols and oxalic acid mostly at lower concentrations than in controls. Data showed that ECM symbiosis helped plant growth by supplying uptake of deficient elements while acting as a protective barrier to toxic metals, in particular for plants growing on ultramafic substrate with extreme soil conditions. Isolation of indigenous and stress-adapted beneficial ECM fungi could serve as a potential tool for inoculation of ECM endemic plants for the successful restoration of ultramafic ecosystems degraded by mining activities.     

Effect of salt stress on the metabolism of ethanolamine and choline in leaves of the betaine-producing mangrove species Avicennia marina

Glycinebetaine synthesis from [methyl-14C]choline and [1,2-14C]ethanolamine in leaf disks of Avicennia marina, was increased by salt stress (250 and 500 mM NaCl). After 18 h incubation with [methyl-14C]choline, phosphocholine and CO(2) were found to be heavily labelled. Phosphocholine contained 39% of the total radioactivity taken up by non-salinised (control) leaf disks and 15% of the total for salinised leaf disks stressed with 500 mM NaCl. Eighteen and 49% of the radioactivity absorbed by control and salinised disks, respectively, were released as CO(2). Metabolic studies of [1,2-14C]ethanolamine revealed that the radioactivity taken up by the leaf disks was recovered as the following compounds after 18 h: phosphorylated compounds (mainly phosphoethanolamine, phosphodimethylethanolamine and phosphocholine) (40-50%); choline (1-2%); glycinebetaine (3-5%); lipids (20-28%); CO(2) (6-10%). Unlike glycinebetaine, incorporation into phosphorylated compounds and lipids were reduced by salt stress. Incorporation of [methyl-14C]S-adenosyl-L-methionine (SAM) into choline, phosphocholine and glycinebetaine in leaf disks was stimulated by salt stress. In vitro activities of adenosine kinase and adenosine nucleosidase, which are implicated in stimulating the SAM regeneration cycle, increased after the leaf disks were incubated with 250 and 500 mM NaCl for 18 h. Changes in metabolism involving choline and glycinebetaine due to salt stress are discussed.     

Species-specific effects of elevated CO2 on resource allocation in Plantago maritima and Armeria maritima

We investigated the effects of increased atmospheric CO₂ on the biomass, photosynthesis, protein and phenolic concentrations and content of Plantago maritima and Armeria maritima. This enabled us to test the protein competition model (PCM) for predicting C allocation to phenolics. Three contrasting responses to elevated CO₂ (600 μmol CO₂ mol⁻¹) between the two study species were observed. (1) In P. maritima, plant biomass increased and the maximum carboxylation rate of Rubisco (V_c,max) was decreased. However, in A. maritima, shoot biomass decreased and the V_c,max of Rubisco was unchanged. (2) The total phenolic content increased in P. maritima but decreased in A. maritima. (3) Protein concentrations and content decreased in P. maritima and root protein concentrations and content increased in A. maritima. We conclude that C and N allocation to phenolics and proteins is species- and organ-specific and the PCM predictions were correct when phenolics and proteins were expressed on a per plant content basis.     

Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1^−/− mice lack any obvious limb or skeletal defects, Sost^−/− mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost^−/−; Sostdc1^−/− mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost^−/− and Sost^−/−; Sostdc1^−/− mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.     

Inhibitory effects of leachate from Eupatorium adenophorum on germination and growth of Amaranthus retroflexus and Chenopodium glaucum

Amaranthus retroflexus L. and Chenopodium glaucum L. are two widely distributed destructive weeds. Their strong adaptability and massive seed production make them the hardest weeds to deal with. This present study intended to investigate the effect of leachate from Eupatorium adenophorum on the growth of these weeds and explore the potential to develop an environmental friendly strategy to use the leachate to control the weeds. Seeds of A. retroflexus L. and C. glaucum L. were soaked in solutions containing 0%, 0.6%, 1.25%, 2.5%, and 5% leachate from E. adenophorum leaves. A. retroflexus and C. glaucum seedlings grown in pots were sprayed with leachate solutions in the same concentration range. The effects of these leachate solutions on membrane permeability and germination of seeds, and growth and physiological characteristics of the seedlings were investigated. The highest concentration of leachate (5%) caused significant damage to the cell membrane of seeds of both weed species, whereas lower concentrations (0.6%) promoted repair of the membrane system, as reflected by higher and lower than control in relative conductivity (RC), respectively. Different concentrations of leachate showed distinct allelopathic inhibitory effects on the two weed species; lower concentrations showed weak inhibitory or even positive effects, whereas higher concentrations showed stronger inhibitory effects. Higher concentrations of leachate (2.5% and 5%) delayed germination and significantly decreased the emergence rate of the seeds, survival rate, and dry matter accumulation of the seedlings. When treated by 5% leachate, the emergence date of A. retroflexus was delayed by 3.6 d, emergence rate of the seeds and survival rate was 69.1% and 70.6% of the control, respectively, seedling dry matter was 48.6% less than the control; In the case of C. glaucum, the emergence date was delayed by 2.7 d, emergence rate of the seeds and survival rate was 45.1% and 58.6% of the control, respectively, seedling dry matter was 44.7% less than the control. There were significant interactions among the different concentrations of leachate and the length of treatment period with respect to activities of antioxidant enzymes, malondialdehyde (MDA) contents, and chlorophyll contents. Seedlings treated with 0.6%, 1.25%, or 2.5% leachate solution for 24–72 h showed increased superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. When seedlings were treated with leachate solutions for 96 h, antioxidant enzyme activities and chlorophyll content decreased in A. retroflexus, but only CAT activity decreased in C. glaucum. When seedlings of the two weed species were treated with 5% leachate solution, CAT activity and chlorophyll content decreased and MDA content gradually increased with longer treatment times (from 24 to 96 h). The two weed species showed different allelopathic responses to E. adenophorum; A. retroflexus was more sensitive than C. glaucum. Based on the investigation, it could be speculated that the delayed germination and low germination rate of the weeds after treatment by leachate could be due to the fact that leachate damaged the membrane system of the seeds. By delaying germination, lowering the germination rate of the weeds and inhibiting seedling growth, leachate from E. adenophorum could provide an effective way of controlling the weeds.     

Isolation and characterization of a novel peroxisomal choline monooxygenase in barley

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [¹⁴C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [¹⁴C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.     

Otx2 and Otx1 protect diencephalon and mesencephalon from caudalization into metencephalon during early brain regionalization

Otx2 is expressed in each step and site of head development. To dissect each Otx2 function we have identified a series of Otx2 enhancers. The Otx2 expression in the anterior neuroectoderm is regulated by the AN enhancer and the subsequent expression in forebrain and midbrain later than E8.5 by FM1 and FM2 enhancers; the Otx1 expression takes place at E8.0. In telencephalon later than E9.5 Otx1 continues to be expressed in the entire pallium, while the Otx2 expression is confined to the most medial pallium. To determine the Otx functions in forebrain and midbrain development we have generated mouse mutants that lack both FM1 and FM2 enhancers (DKO: Otx2^{ΔFM1ΔFM2/ΔFM1ΔFM2}) and examined the TKO (Otx1^−/−Otx2^{ΔFM1ΔFM2/ΔFM1ΔFM2}) phenotype. The mutants develop normally until E8.0, but subsequently by E9.5 the diencephalon, including thalamic eminence and prethalamus, and the mesencephalon are caudalized into metencephalon consisting of isthmus and rhombomere 1; the caudalization does not extend to rhombomere 2 and more caudal rhombomeres. In rostral forebrain, neopallium, ganglionic eminences and hypothalamus in front of prethalamus develop; we propose that they become insensitive to the caudalization with the switch from the Otx2 expression under the AN enhancer to that under FM1 and FM2 enhancers. In contrast, the medial pallium requires Otx1 and Otx2 for its development later than E9.5, and the Otx2 expression in diencepalon and mesencephalon later than E9.5 is also directed by an enhancer other than FM1 and FM2 enhancers.     

Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses

Transgenic potato plants (Solanum tuberosum L. cv. Superior) with the ability to synthesize glycinebetaine (GB) in chloroplasts (referred to as SC plants) were developed via the introduction of the bacterial choline oxidase (codA) gene under the control of an oxidative stress-inducible SWPA2 promoter. SC1 and SC2 plants were selected via the evaluation of methyl viologen (MV)-mediated oxidative stress tolerance, using leaf discs for further characterization. The GB contents in the leaves of SC1 and SC2 plants following MV treatment were found to be 0.9 and 1.43 μmol/g fresh weight by HPLC analysis, respectively. In addition to reduced membrane damage after oxidative stress, the SC plants evidenced enhanced tolerance to NaCl and drought stress on the whole plant level. When the SC plants were subjected to two weeks of 150 mM NaCl stress, the photosynthetic activity of the SC1 and SC2 plants was attenuated by 38 and 27%, respectively, whereas that of non-transgenic (NT) plants was decreased by 58%. Under drought stress conditions, the SC plants maintained higher water contents and accumulated higher levels of vegetative biomass than was observed in the NT plants. These results indicate that stress-induced GB production in the chloroplasts of GB non-accumulating plants may prove useful in the development of industrial transgenic plants with increased tolerance to a variety of environmental stresses for sustainable agriculture applications.     

Effect of a formulation of Bacillus firmus on root-knot nematode Meloidogyne incognita infestation and the growth of tomato plants in the greenhouse and nursery

Bacillus firmus, commercial WP formulation (BioNem) was evaluated against the root-knot nematode Meloidogyne incognita in a laboratory, greenhouse and under field conditions on tomato plants. In the laboratory tests, an aqueous suspension of BioNem at 0.5%, 1%, 1.5% and 2% concentration reduced egg hatching from 98% to 100%, 24-days after treatment. Treatment of second-stage juveniles with 2.5% and 3% concentration of BioNem, caused 100% inhibition of mobility, 24 h after treatment. In the green house trials, BioNem applied at 8 g/pot (1200 cc soil) planted with a tomato seedlings reduced gall formation by 91%, final nematode populations by 76% and the number of eggs by 45%. Consequently, plant height and biomass was increased by 71% and 50%, respectively, compared to the untreated control, 50-days after treatment application. Application of BioNem at 16 g/pot was phytotoxic to plants. In the field trails, BioNem applied at 200 and 400 kg ha⁻¹ was effective in reducing the number of galls (75-84%), and increased shoot height (29-31%) and weight (20-24%) over the untreated control, 45-days after treatment. Our results indicate that B. firmus is a promising microorganism for the biological control of M. incognita in tomato pots.     

Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice

Choline kinase in mice is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneously occurring genomic deletion in murine Chkb results in neonatal bone deformity and hindlimb muscular dystrophy. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, causes hindlimb muscular dystrophy. The biosynthesis of phosphatidylcholine (PC) is impaired in the hindlimbs of Chkb^−/− mice, with an accumulation of choline and decreased amount of phosphocholine. The activity of CTP:phosphocholine cytidylyltransferase is also decreased in the hindlimb muscle of mutant mice. Concomitantly, the activities of PC phospholipase C and phospholipase A₂ are increased. The mitochondria in Chkb^−/− mice are abnormally large and exhibit decreased inner membrane potential. Despite the muscular dystrophy in Chkb^−/− mice, we observed increased expression of insulin like growth factor 1 and proliferating cell nuclear antigen. However, regeneration of hindlimb muscles of Chkb^−/− mice was impaired when challenged with cardiotoxin. Injection of CDP-choline increased PC content of hindlimb muscle and decreased creatine kinase activity in plasma of Chkb^−/− mice. We conclude that the hindlimb muscular dystrophy in Chkb^−/− mice is due to attenuated PC biosynthesis and enhanced catabolism of PC.