A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated alpha-glucans and is involved in starch degradation in Arabidopsis

An Arabidopsis thaliana gene encoding a homologue of the potato alpha-glucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C-3 position of the glucose unit of preferably pre-phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild-type leaf starch, the level of C-3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C-3 linked phospho-ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.     

Suppression of glucan,water dikinase in the endosperm alters wheat grain properties,germination and coleoptile growth

Starch phosphate ester content is known to alter the physicochemical properties of starch, including its susceptibility to degradation. Previous work producing wheat (Triticum aestivum) with down‐regulated glucan, water dikinase, the primary gene responsible for addition of phosphate groups to starch, in a grain‐specific manner found unexpected phenotypic alteration in grain and growth. Here, we report on further characterization of these lines focussing on mature grain and early growth. We find that coleoptile length has been increased in these transgenic lines independently of grain size increases. No changes in starch degradation rates during germination could be identified, or any major alteration in soluble sugar levels that may explain the coleoptile growth modification. We identify some alteration in hormones in the tissues in question. Mature grain size is examined, as is Hardness Index and starch conformation. We find no evidence that the increased growth of coleoptiles in these lines is connected to starch conformation or degradation or soluble sugar content and suggest these findings provide a novel means of increasing coleoptile growth and early seedling establishment in cereal crop species.     

EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.     

Light-induced degradation of storage starch in turions of Spirodela polyrhiza depends on nitrate

Light induces both the germination of turions of the duckweed Spirodela polyrhiza and the degradation of the reserve starch stored in the turions. The germination photoresponse requires nitrate, and we show here that nitrate is also needed for the light-induced degradation of the turion starch. Ammonium cannot substitute for nitrate in this regard, and nitrate thus acts specifically as signal to promote starch degradation in the turions. Irradiation with continuous red light leads to starch degradation via auto-phosphorylation of starch-associated glucan, water dikinase (GWD), phosphorylation of the turion starch and enhanced binding of alpha-amylase to starch granules. The present study shows that all of these processes require the presence of nitrate, and that nitrate exerts its effect on starch degradation at a point between the absorption of light by phytochrome and the auto-phosphorylation of the GWD. Nitrate acts to coordinate carbon and nitrogen metabolism in germinating turions: starch will only be broken down when sufficient nitrogen is present to ensure appropriate utilization of the released carbohydrate. These data constitute the first report of control over the initiation of reserve starch degradation by nitrate.     

A CBM20 low-affinity starch-binding domain from glucan, water dikinase

The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.     

Down-regulation of Glucan, Water-Dikinase activity in wheat endosperm increases vegetative biomass and yield

A novel mechanism for increasing vegetative biomass and grain yield has been identified in wheat (Triticum aestivum). RNAi-mediated down-regulation of Glucan, Water-Dikinase (GWD), the primary enzyme required for starch phosphorylation, under the control of an endosperm-specific promoter, resulted in a decrease in starch phosphate content and an increase in grain size. Unexpectedly, consistent increases in vegetative biomass and grain yield were observed in subsequent generations. In lines where GWD expression was decreased, germination rate was slightly reduced. However, significant increases in vegetative growth from the two leaf stage were observed. In glasshouse pot trials, down-regulation of GWD led to a 29% increase in grain yield while in glasshouse tub trials simulating field row spacing and canopy development, GWD down-regulation resulted in a grain yield increase of 26%. The enhanced yield resulted from a combination of increases in seed weight, tiller number, spikelets per head and seed number per spike. In field trials, all vegetative phenotypes were reproduced with the exception of increased tiller number. The expression of the transgene and suppression of endogenous GWD RNA levels were demonstrated to be grain specific. In addition to the direct effects of GWD down-regulation, an increased level of α-amylase activity was present in the aleurone layer during grain maturation. These findings provide a potentially important novel mechanism to increase biomass and grain yield in crop improvement programmes.     

Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene

Changes in carbon metabolism and δ¹³C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO₂ in the C₄ photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r²= 0.81), and was positively correlated with malate content (r²= 0.88). A significant increase in the δ¹³C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO₂. These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C₄‐type carbon metabolism. However, since parameters associated with CO₂ gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants.     

Primed phosphorylation of tau at Thr231 by glycogen synthase kinase 3beta (GSK3beta) plays a critical role in regulating tau's ability to bind and stabilize microtubules

Site-specific phosphorylation of tau negatively regulates its ability to bind and stabilize microtubule structure. Although tau is a substrate of glycogen synthase kinase 3beta (GSK3beta), the exact sites on tau that are phosphorylated by this kinase in situ have not yet been established, and the effect of these phosphorylation events on tau-microtubule interactions have not been fully elucidated. GSK3beta phosphorylates both primed and unprimed sites on tau, but only primed phosphorylation events significantly decrease the ability of tau to bind microtubules. The focus of the present study is on determining the importance of the GSK3beta-mediated phosphorylation of a specific primed site, Thr231, in regulating tau's function. Pre-phosphorylation of Ser235 primes tau for phosphorylation by GSK3beta at Thr231. Phosphorylation by GSK3beta of wild-type tau or tau with Ser235 mutated to Ala decreases tau-microtubule interactions. However, when Thr231 alone or Thr231 and Ser235 in tau were mutated to Ala, phosphorylation by GSK3beta did not decrease the association of tau with the cytoskeleton. Further, T231A tau was still able to efficiently bind microtubules after phosphorylation by GSK3beta. Expression of each tau construct alone increased tubulin acetylation, a marker of microtubule stability. However, when cells were cotransfected with wild-type tau and GSK3beta, the level of tubulin acetylation was decreased to vector-transfected levels. In contrast, coexpression of GSK3beta with mutated tau (T231A/S235A) did not significantly decrease the levels of acetylated tubulin. These results strongly indicate that phosphorylation of Thr231 in tau by GSK3beta plays a critical role in regulating tau's ability to bind and stabilize microtubules.     

Expression of C3 and C4 photosynthetic characteristics in the amphibious plant Eleocharis vivipara: structure and analysis of the expression of isogenes for pyruvate,orthophosphate dikinase

Eleocharis vivipara, a unique leafless amphibious sedge, adopts the C4 mode of photosynthesis under terrestrial conditions and the C3 mode under submerged aquatic conditions. To analyze the molecular basis of these responses to the contrasting environments, we isolated and characterized two full-length cDNAs for a key C4 enzyme, pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1). The isogenes for PPDK, designated ppdk1 and ppdk2, were highly homologous to one another but not identical. The PPDK1 protein, deduced from the nucleotide sequence of the cDNA, contained an extra domain at the amino terminus which, presumably, serves as a chloroplast transit peptide, while PPDK2 lacked this extra domain. It seems likely, therefore, that the ppdk1 and ppdk2 genes encode a chloroplastic and a cytosolic PPDK, respectively. Genomic Southern blot analysis revealed the existence of a small family of genes for PPDK in the genome of E. vivipara. Northern blot analysis indicate that both chloroplastic and cytosolic genes for PPDK are expressed simultaneously in the culms, a photosynthetic organ, of E. vivipara and that the pattern of expression of these genes differs between the growth forms.     

Autophosphorylation-dependent protein kinase phosphorylates Ser25, Ser38, Ser65, Ser71, and Ser411 in vimentin and thereby inhibits cytoskeletal intermediate filament assembly

The autophosphorylation-dependent protein kinase has been identified as a potent vimentin kinase that incorporates 2 mol of phosphates per mol of protein and generates five major phosphorylation sites in vimentin. Tryptic phosphopeptide mapping by high-performance liquid chromatography followed by sequential manual Edman degradation and direct peptide sequence analysis revealed that Ser-25, Ser-38, Ser-65, and Ser-71 in the amino-terminal domain and Ser-411 in the carboxyl-terminal domain are the phosphorylation sites in vimentin phosphorylated by this kinase, indicating that autophosphorylation-dependent protein kinase is a potent and unique vimentin kinase. Functional study further revealed that phosphorylation of vimentin by autophosphorylation-dependent protein kinase can completely inhibit polymerization and assembly of the cytoskeletal intermediate filament as demonstrated by electron microscopic analysis. Taken together, the results provide initial evidence that the autophosphorylation-dependent protein kinase may function as a vimentin kinase involved in the structure-function regulation of the cytoskeletal system. The results also support the notion that this cyclic nucleotide- and calcium-independent protein kinase may function as a multisubstrate/multifunctional protein kinase involved in the regulation of diverse cell functions.     

Failure to support a genetic contribution of AKT1 polymorphisms and altered AKT signaling in schizophrenia

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT) gene family comprises three human homologs that phosphorylate and inactivate glycogen synthase kinase 3beta (GSK3beta). Studies have reported the genetic association of AKT1 with schizophrenia. Additionally, decreased AKT1 protein expression and the reduced phosphorylation of GSK3beta were reported in this disease, leading to a new theory of attenuated AKT1-GSK3beta signaling in schizophrenia pathogenesis. We have evaluated this theory by performing both genetic and protein expression analyses. A family based association test of AKT1 did not show association with schizophrenia in Japanese subjects. The expression levels of total AKT, AKT1 and phosphorylated GSK3beta detected in the schizophrenic brains from two different brain banks also failed to support the theory. In addition, no attenuated AKT-GSK3beta signaling was observed in the lymphocytes from Japanese schizophrenics, contrasting with previous findings. Importantly, we found that the level of phosphorylated GSK3beta at Ser9 tended to be inversely correlated with postmortem intervals, and that the phosphorylation levels of AKT were inversely correlated with brain pH, issues not assessed in the previous study. These data introduce a note of caution when estimating the phosphorylation levels of GSK3beta and AKT in postmortem brains. Collectively, this study failed to support reduced signaling of the AKT-GSK3beta molecular cascade in schizophrenia.     

Effects of Two Convulsant β-Carboline Derivatives, DMCM and β-CCM, on Regional Neurotransmitter Amino Acid Levels and on In Vitro D-[3H]Aspartate Release in Rodents

Clonic seizures were induced in Swiss or DBA/2 mice by methyl-6-7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), 0.048 mmol/kg i.p., or by methyl-beta-carboline-3-carboxylate (beta-CCM), 0.044 mmol/kg i.p. Measurement of regional brain (cortex, hippocampus, striatum, and cerebellum) amino acid levels after 15 min of seizure activity showed increases in gamma-aminobutyric acid (GABA) (in all regions after beta-CCM, and in cortex and hippocampus after DMCM), and an increase in glycine in the striatum after beta-CCM. Aspartate levels fell (in cortex and hippocampus) after DMCM, but were unchanged in all regions after beta-CCM. Glutamate levels fell in cortex after beta-CCM and in striatum after DMCM. Pretreatment with the excitatory amino acid receptor antagonist, 2-amino-7-phosphonoheptanoic acid, 0.5 mmol/kg i.p., 45 min prior to the beta-carboline, significantly increased the ED50 for DMCM-induced clonic seizures (4.68 mumol/kg vs. 9.39 mumol/kg). Similar pretreatment did not significantly alter the ED50 for beta-CCM (4.22 mumol/kg vs. 6.6 mumol/kg). Pretreatment with 2-amino-7-phosphonoheptanoic acid, 1.0 mmol/kg, blocked the increase in GABA content produced by DMCM but not the fall in cortical aspartate content. Potassium-induced release of preloaded D-[3H]aspartate from rat cortical or hippocampal minislices was enhanced in the presence of DMCM (100 microM). In contrast, stimulated release of D-[3H]aspartate (from cortex or hippocampus) was not altered in the presence of beta-CCM (100 microM). Although DMCM and beta-CCM are both considered to induce convulsion by acting at the GABA--benzodiazepine receptor complex, the convulsions differ in several pharmacological and biochemical respects. It is suggested that enhanced release of excitatory amino acid neurotransmitters plays a more important role in seizures induced by DMCM.     

Influence of amylopectin structure and degree of phosphorylation on the molecular composition of potato starch lintners

Morphology, molecular structure, and thermal properties of potato starch granules with low to high phosphate content were studied as an effect of mild acid hydrolysis (lintnerization) to 80% solubilization at two temperatures (25 and 45°C). Light microscopy showed that the lintners contained apparently intact granules, which disintegrated into fragments upon dehydration. Transmission electron microscopy of rehydrated lintners revealed lacy networks of smaller subunits. The molecular composition of the lintners suggested that they largely consisted of remnants of crystalline lamellae. When lintnerization was performed at 45°C, the lintners contained more of branched dextrins compared to 25°C in both low and intermediate phosphate‐containing samples. High‐phosphate‐containing starch was, however, unaffected by temperature and this was probably due to an altered amylopectin structure rather than the phosphate content. After lintnerization, the melting endotherms were broad with decreased onset and increased peak melting temperatures. The relative crystallinity was lower in lintners prepared at 45°C. A hypothesis that combines the kinetics of lintnerization with the molecular and thermal characteristics of the lintners is presented. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 257–271, 2014.     

Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression

The RIC-3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC-3 (hRIC-3) protein enhances expression of α7 receptors and abolishes expression of α4β2 receptors. In vitro translation of hRIC-3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC-3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for α7 expression enhancement but not for α4β2 inhibition. A combination of increased α7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in α7 membrane expression induced by hRIC-3. Antibodies against hRIC-3 showed its expression in SH-SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC-3 in rat brain localized, in general, in places where α7 nAChRs were found.     

Isolation and characterisation of 8-hydroxy-3Z,5Z-tetradecadienoic acid,a putative intermediate in Pichia guilliermondii gamma-decalactone biosynthesis from ricinoleic acid

During a screening procedure for the discovery of a strong gamma-decalactone producer from ricinoleic acid, we observed that the yeast Pichia guilliermondii accumulated transiently 8-hydroxy-3Z,5Z-tetradecadienoic acid 1 during gamma-decalactone biosynthesis in the stationary phase of growth. The structural elucidation of 1 was based on nuclear magnetic resonance, infrared, ultraviolet and gas chromatography-mass spectrometry experiments. The occurrence of 1 is discussed in relation with previously proposed gamma-decalactone biosynthetic pathways.