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Orthophosphate (P(i)) is an essential but limiting macronutrient for plant growth. Extensive soil P reserves exist in the form of organic P (P(o)), which is unavailable for root uptake until hydrolysed by secretory acid phosphatases (APases). The predominant purple APase (PAP) isozymes secreted by roots of P(i)-deficient (-P(i)) Arabidopsis thaliana were recently identified as AtPAP12 (At2g27190) and AtPAP26 (At5g34850). The present study demonstrated that exogenous P(o) compounds such as glycerol-3-phosphate or herring sperm DNA: (i) effectively substituted for P(i) in supporting the P nutrition of Arabidopsis seedlings, and (ii) caused upregulation and secretion of AtPAP12 and AtPAP26 into the growth medium. When cultivated under -P(i) conditions or supplied with P(o) as its sole source of P nutrition, an atpap26/atpap12 T-DNA double insertion mutant exhibited impaired growth coupled with60 and30% decreases in root secretory APase activity and rosette total P(i) concentration, respectively. Development of the atpap12/atpap26 mutant was unaffected during growth on P(i)-replete medium but was completely arrested when 7-day-old P(i)-sufficient seedlings were transplanted into a -P(i), P(o)-containing soil mix. Both PAPs were also strongly upregulated on root surfaces and in shoot cell-wall extracts of -P(i) seedlings. It is hypothesized that secreted AtPAP12 and AtPAP26 facilitate the acclimation of Arabidopsis to nutritional Pi deficiency by: (i) functioning in the rhizosphere to scavenge P(i) from the soil's accessible P(o) pool, while (ii) recycling P(i) from endogenous phosphomonoesters that have been leaked into cell walls from the cytoplasm. Thus, AtPAP12 and AtPAP26 are promising targets for improving crop P-use efficiency.

Valerian (Valeriana officinalis) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Among dozens of chemical constituents (e.g. alkaloids, iridoids, flavonoids, and terpenoids) found in valerian root, valerena-4,7(11)-diene and valerenic acid (C15 sesquiterpenoid) have been suggested as the active ingredients responsible for the sedative effect. However, the biosynthesis of the valerena-4,7(11)-diene hydrocarbon skeleton in valerian remains unknown to date. To identify the responsible terpene synthase, next-generation sequencing (Roche 454 pyrosequencing) was used to generate ∼ 1 million transcript reads from valerian root. From the assembled transcripts, two sesquiterpene synthases were identified (VoTPS1 and VoTPS2), both of which showed predominant expression patterns in root. Transgenic yeast expressing VoTPS1 and VoTPS2 produced germacrene C/germacrene D and valerena-4,7(11)-diene, respectively, as major terpene products. Purified VoTPS1 and VoTPS2 recombinant enzymes confirmed these activities in vitro, with competent kinetic properties (K(m) of ∼ 10 μm and k(cat) of 0.01 s(-1) for both enzymes). The structure of the valerena-4,7(11)-diene produced from the yeast expressing VoTPS2 was further substantiated by (13) C-NMR and GC-MS in comparison with the synthetic standard. This study demonstrates an integrative approach involving next-generation sequencing and metabolically engineered microbes to expand our knowledge of terpenoid diversity in medicinal plants.

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/β-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.

Phosphate (Pi) is an essential, but limiting macronutrient that plays critical roles in plant metabolism and development. Plants have evolved an intricate array of adaptations to enhance Pi acquisition and utilization from their environment. The availability of the complete genome sequence of the model plant Arabidopsis thaliana , together with a wide assortment of related genomic resources, has significantly advanced our understanding of the adaptations of Pi-starved plants. Information on the genetic identity, subcellular location, biochemical properties, and probable functions of acid phosphatases involved in the Pi metabolism of Pi-starved Arabidopsis is beginning to emerge. Acid phosphatases catalyze the hydrolysis of Pi from a broad range of phosphomonoesters with an acidic pH optimum. The Arabidopsis genome encodes 29 different purple acid phosphatases whose expression is influenced by various developmental and environmental factors. Pi starvation induces de novo synthesis of several extra- and intracellular Arabidopsis purple acid phosphatase isozymes; AtPAP12 and AtPAP26 appear to be the principal root-secreted acid phosphatases that scavenge Pi from extracellular Pi-esters, whereas the dual-targeted AtPAP26 is the predominant intracellular acid phosphatase that functions in vacuolar Pi recycling by Pi-starved Arabidopsis . The identification and functional characterization of intracellular and secreted purple acid phosphatase isozymes upregulated by Pi-deprived plants may help develop strategies for engineering Pi-efficient crops, thereby minimizing the use of unsustainable Pi fertilizers in agriculture.

Plant purple acid phosphatases (PAPs) belong to a large multigene family whose specific functions in Pi metabolism are poorly understood. Two PAP isozymes secreted by Pi-deficient (−Pi) Arabidopsis thaliana were purified from culture filtrates of −Pi suspension cells. They correspond to an AtPAP12 (At2g27190) homodimer and AtPAP26 (At5g34850) monomer composed of glycosylated 60 and 55 kDa subunit(s), respectively. Each PAP exhibited broad pH activity profiles centred at pH 5.6, and overlapping substrate specificities. Concanavalin-A chromatography resolved a pair of secreted AtPAP26 glycoforms. AtPAP26 is dual targeted during Pi stress because it is also the principal intracellular (vacuolar) PAP up-regulated by −Pi Arabidopsis. Differential glycosylation appears to influence the subcellular targeting and substrate selectivity of AtPAP26. The significant increase in secreted acid phosphatase activity of −Pi seedlings was correlated with the appearance of immunoreactive AtPAP12 and AtPAP26 polypeptides. Analysis of atpap12 and atpap26 T-DNA mutants verified that AtPAP12 and AtPAP26 account for most of the secreted acid phosphatase activity of −Pi wild-type seedlings. Semi-quantitative RT-PCR confirmed that transcriptional controls exert little influence on the up-regulation of AtPAP26 during Pi stress, whereas AtPAP12 transcripts correlate well with relative levels of secreted AtPAP12 polypeptides. We hypothesize that AtPAP12 and AtPAP26 facilitate Pi scavenging from soil-localized organophosphates during nutritional Pi deprivation.

Induction of intracellular and secreted acid phosphatases (APases) is a widespread response of orthophosphate (Pi)-starved (−Pi) plants. APases catalyze Pi hydrolysis from a broad range of phosphomonoesters at an acidic pH. The largest class of nonspecific plant APases is comprised of the purple APases (PAPs). Although the biochemical properties, subcellular location, and expression of several plant PAPs have been described, their physiological functions have not been fully resolved. Recent biochemical studies indicated that AtPAP26, one of 29 PAPs encoded by the Arabidopsis (Arabidopsis thaliana) genome, is the predominant intracellular APase, as well as a major secreted APase isozyme up-regulated by −Pi Arabidopsis. An atpap26 T-DNA insertion mutant lacking AtPAP26 transcripts and 55-kD immunoreactive AtPAP26 polypeptides exhibited: (1) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (2) a 40% decrease in secreted APase activity during Pi deprivation, (3) 35% and 50% reductions in free and total Pi concentration, respectively, as well as 5-fold higher anthocyanin levels in shoots of soil-grown −Pi plants, and (4) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function occurred under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. Transient expression of AtPAP26-mCherry in Arabidopsis suspension cells verified that AtPAP26 is targeted to the cell vacuole. Our results confirm that AtPAP26 is a principal contributor to Pi stress-inducible APase activity, and that it plays an important role in the Pi metabolism of −Pi Arabidopsis.

PEPC [PEP(phosphoenolpyruvate) carboxylase] is a tightly controlled cytosolic enzyme situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PPCK (PEPC kinase) in plant acclimation to nutritional Pi deprivation. However, little is known about the genetic origin or phosphorylation status of native PEPCs from −Pi (Pi-deficient) plants. The transfer of Arabidopsis suspension cells or seedlings to −Pi growth media resulted in: (i) the marked transcriptional upregulation of genes encoding the PEPC isoenzyme AtPPC1 ( Arabidopsis thaliana PEPC1), and PPCK isoenzymes AtPPCK1 and AtPPCK2; (ii) >2-fold increases in PEPC specific activity and in the amount of an immunoreactive 107-kDa PEPC polypeptide (p107); and (iii) In vivo p107 phosphorylation as revealed by immunoblotting of clarified extracts with phosphosite-specific antibodies to Ser-11 (which could be reversed following Pi resupply). Approx. 1.3 mg of PEPC was purified 660-fold from −Pi suspension cells to apparent homogeneity with a specific activity of 22.3 units · mg−1 of protein. Gel filtration, SDS/PAGE and immunoblotting demonstrated that purified PEPC exists as a 440-kDa homotetramer composed of identical p107 subunits. Sequencing of p107 tryptic and Asp-N peptides by tandem MS established that this PEPC is encoded by AtPPC1 . Pi-affinity PAGE coupled with immunoblotting indicated stoichiometric phosphorylation of the p107 subunits of AtPPC1 at its conserved Ser-11 phosphorylation site. Phosphorylation activated AtPPC1 at pH 7.3 by lowering its K m(PEP) and its sensitivity to inhibition by L-malate and L-aspartate, while enhancing activation by glucose 6-phosphate. Our results indicate that the simultaneous induction and In vivo phosphorylation activation of AtPPC1 contribute to the metabolic adaptations of −Pi Arabidopsis . Abbreviations: Ab, antibody; anti-RcPEPC IgG, anti-(Ricinus communis PEPC) IgG; AtPPC1, Arabidopsis thaliana PEPC1; CAM, crassulacean acid metabolism; DTT, dithiothreitol; Glc-6-P, glucose 6-phosphate; MALDI, matrix-assisted laser-desorption ionization; MS medium, Murashige and Skoog medium; MS/MS, tandem MS; p107, 107-kDa PEPC polypeptide; oMALDI 2, orthogonal MALDI 2; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; +Pi, Pi-sufficient; −Pi, Pi-deficient; PP2A, protein phosphatase type-2A; PPCK, PEPC kinase; Q-TOF, quadrupole time-of-flight; QqTOF, quadrupole/quadrupole TOF; RT, reverse transcription

This paper proposes a threshold algorithm to extract features and then create standard thresholds of EEG online signals using eye movements. Based on the standard thresholds, an electrical wheelchair can be controlled to reach the desired target. In this project, a Hamming band-pass filter is applied to detect noise of EEG signal. Moreover, calculation of blink-times and detection of confusion between left glance and right glance signals program are built and added to increase the accuracy of the wheelchair control. Additionally, a quality testing program is also built in Labview environment to improve effort of data collection. Finally, experimental results of controlling the electrical wheelchair in indoor environments demonstrated the effectiveness of the proposed method.

Dual-specificity protein phosphatases (DSPs) are important regulators of a wide variety of protein kinase signaling cascades in animals, fungi and plants. We previously identified a cluster of putative DSPs in Arabidopsis (including At3g52180 and At3g01510) in which the phosphatase domain is related to that of laforin, the human protein mutated in Lafora epilepsy. In animal and fungal systems, the laforin DSP and the beta-regulatory subunits of AMP-regulated protein kinase (AMPK) and Snf-1 have all been demonstrated to bind to glycogen by a glycogen-binding domain (GBD). We present a bioinformatic analysis which shows that these DSPs from Arabidopsis, together with other related plant DSPs, share with the above animal and fungal proteins a widespread and ancient carbohydrate-binding domain. We demonstrate that DSP At3g52180 binds to purified starch through its predicted carbohydrate-binding region, and that mutation of key conserved residues reduces this binding. Consistent with its ability to bind exogenous starch, DSP At3g52180 was found associated with starch purified from Arabidopsis plants and suspension cells. Immunolocalization experiments revealed a co-localization with chlorophyll, placing DSP At3g52180 in the chloroplast. Gene-expression data from different stages of the light-dark cycle and across a wide variety of tissues show a strong correlation between the pattern displayed by transcripts of the At3g52180 locus and that of genes encoding key starch degradative enzymes. Taken together, these data suggest the hypothesis that plant DSPs could be part of a protein assemblage at the starch granule, where they would be ideally situated to regulate starch metabolism through reversible phosphorylation events.

Natural rubber (cis-1,4-polyisoprene) is an indispensable biopolymer used to manufacture diverse consumer products. Although a major source of natural rubber is the rubber tree (Hevea brasiliensis), lettuce (Lactuca sativa) is also known to synthesize natural rubber. Here, we report that an unusual cis-prenyltransferase-like 2 (CPTL2) that lacks the conserved motifs of conventional cis-prenyltransferase is required for natural rubber biosynthesis in lettuce. CPTL2, identified from the lettuce rubber particle proteome, displays homology to a human NogoB receptor and is predominantly expressed in latex. Multiple transgenic lettuces expressing CPTL2-RNAi constructs showed that a decrease of CPTL2 transcripts (3-15% CPTL2 expression relative to controls) coincided with the reduction of natural rubber as low as 5%. We also identified a conventional cis-prenyltransferase 3 (CPT3), exclusively expressed in latex. In subcellular localization studies using fluorescent proteins, cytosolic CPT3 was relocalized to endoplasmic reticulum by co-occurrence of CPTL2 in tobacco and yeast at the log phase. Furthermore, yeast two-hybrid data showed that CPTL2 and CPT3 interact. Yeast microsomes containing CPTL2/CPT3 showed enhanced synthesis of short cis-polyisoprenes, but natural rubber could not be synthesized in vitro. Intriguingly, a homologous pair CPTL1/CPT1, which displays ubiquitous expressions in lettuce, showed a potent dolichol biosynthetic activity in vitro. Taken together, our data suggest that CPTL2 is a scaffolding protein that tethers CPT3 on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta, but yeast-expressed CPTL2 and CPT3 alone could not synthesize high molecular weight natural rubber in vitro.

Our knowledge of the serine/threonine protein phosphatases of the mammalian nucleus is limited compared with their cytosolic counterparts. Microcystin-Sepharose chromatography and mass spectrometry were utilized to affinity purify and identify protein phosphatase-associated proteins from isolated rat liver nuclei. Far Western analysis with labeled protein phosphatase 1 (PP1) showed that many more PP1 binding proteins exist in the nucleus than were previously demonstrated. Mass spectrometry confirmed the presence in the nucleus of the mammalian PP1 isoforms alpha1, alpha2, beta, and gamma1, plus the Aalpha and several of the B and B' subunits that are complexed to PP2A. Other proteins enriched on the microcystin matrix include the spliceosomal proteins known as the U2 snRNPs SAP145 and SAP155 and the U5 snRNPs p116 and p200, myosin heavy chain, and a nuclear PP1 myosin-targeting subunit related to M110. The putative RNA binding protein ZAP was also established as a nuclear PP1 binding protein using the criteria of co-purification with PP1 on microcystin-Sepharose, co-immunoprecipation, binding PP1 in an overlay assay, and presence of a putative PP1 binding site (KKRVRWAD). These results further support a key role for protein phosphatases in several nuclear functions, including the regulation of pre-mRNA splicing.

Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature.

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.

Valerian (Valeriana officinalis) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Among dozens of chemical constituents (e.g. alkaloids, iridoids, flavonoids, and terpenoids) found in valerian root, valerena-4,7(11)-diene and valerenic acid (C15 sesquiterpenoid) have been suggested as the active ingredients responsible for the sedative effect. However, the biosynthesis of the valerena-4,7(11)-diene hydrocarbon skeleton in valerian remains unknown to date. To identify the responsible terpene synthase, next-generation sequencing (Roche 454 pyrosequencing) was used to generate ∼ 1 million transcript reads from valerian root. From the assembled transcripts, two sesquiterpene synthases were identified (VoTPS1 and VoTPS2), both of which showed predominant expression patterns in root. Transgenic yeast expressing VoTPS1 and VoTPS2 produced germacrene C/germacrene D and valerena-4,7(11)-diene, respectively, as major terpene products. Purified VoTPS1 and VoTPS2 recombinant enzymes confirmed these activities in vitro, with competent kinetic properties (K(m) of ∼ 10 μm and k(cat) of 0.01 s(-1) for both enzymes). The structure of the valerena-4,7(11)-diene produced from the yeast expressing VoTPS2 was further substantiated by (13) C-NMR and GC-MS in comparison with the synthetic standard. This study demonstrates an integrative approach involving next-generation sequencing and metabolically engineered microbes to expand our knowledge of terpenoid diversity in medicinal plants.

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/β-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.

Orthophosphate (Pi) is an essential macronutrient that plays a central role in virtually all major metabolic processes in plants, particularly photosynthesis and respiration. Many metabolites are Pi monoesters, whereas the phosphoanhydride bonds of compounds such as ATP function to transfer energy

A proteomic approach was applied to compare the secretome (culture filtrate proteome) of phosphate-sufficient (+Pi) and Pi-deficient (-Pi) Arabidopsis thaliana suspension cell cultures. Secretomes harvested from the +Pi and -Pi cells yielded dissimilar 2-DE maps. PMF via MALDI-TOF MS resulted in the identification of 50 protein spots representing 37 discrete proteins having unique gene identities. A total of 24 Pi-starvation responsive proteins were identified, with 18 of these being up-regulated and six down-regulated. Secreted proteins up-regulated by the -Pi cells included a ribonuclease involved in Pi scavenging from extracellular nucleic acids, as well as enzymes of cell wall modification, proteolysis, pathogen responses, and ROS metabolism. Enzyme activity assays and immunoblotting demonstrated that a pair of purple acid phosphatase isoforms having subunit M(r)s of 65 and 55 kDa was also secreted by the -Pi cells. Semiquantitative RT-PCR was used to assess the relationship between mRNA levels and relative amounts of selected secretome proteins. The results indicate that transcriptional control is but one of many factors contributing to Arabidopsis Pi starvation responses, and highlight the importance of parallel biochemical/proteomic studies of -Pi plants.

The catalytic subunit of PP2A (PP2Ac) can be purified in milligram quantities from bovine heart using ethanol precipitation, ammonium sulfate precipitation, ion exchange and size exclusion chromatography. The detailed procedure is described to purify PP2Ac over 4 d.

The catalytic subunit of PP2A (PP2Ac) can be purified in milligram quantities from bovine heart using ethanol precipitation, ammonium sulfate precipitation, ion exchange and size exclusion chromatography. The detailed procedure is described to purify PP2Ac over 4 d.

Antipeptide antibodies generated against the N terminus of the protein phosphatase 1 (PP1) binding protein sds22 detected at least four forms of the protein in a rat liver nuclear extract. Four of these immunoreactive bands likely correspond to four predicted forms of sds22 that are generated by alternative splicing. These four proteins are expressed at different levels and appear to be localized exclusively in the nucleus, and two of these proteins copurify with PP1 on the protein phosphatase affinity matrix microcystin–Sepharose. Two higher molecular mass nuclear proteins that are immunoreactive with the sds22 antibodies also copurify on microcystin–Sepharose and may be novel forms of sds22 expressed in mammalian cells.Key words: sds22, protein phosphatase 1 (PP1), nucleus, microcystin–Sepharose.

Excimer laser ablation is an excellent means of depositing high temperature superconducting (HTS) thin films because of high quality electrical properties of the resulting films and also because of the relative simplicity of the deposition system. The principal disadvantage of laser ablation is the contamination of the deposited film by particles, leading to a rough film surface. The influence of substrate temperature on surface morphology in YBa2Cu3O7 thin films deposited by excimer laser on r-plane sapphire with a CeO2 buffer layer has been investigated. The substrate temperature was measured using gold-nickel thermocouples on the substrate surface during deposition. The optimal deposition temperatures were found to be lower in comparison with films deposited on LaAlO3 because damage occurs to the buffer layer. Special attention was paid to the occurrence of droplets and outgrowths in the films. The ablation process produces droplets that range from 0.1 micrometers to a maximum of 1 micrometers in size. In the course of film growth these droplets undergo metamorphosis and form the basis of outgrowths.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.