Mechanism Underlying Heat Stability of the Rice Endosperm Cytosolic ADP-Glucose Pyrophosphorylase

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme’s regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature

Plant growth promotion and Penicillium citrinum

<p>Abstract</p> <p>Background</p> <p>Endophytic fungi are known plant symbionts. They produce a variety of beneficial metabolites for plant growth and survival, as well as defend their hosts from attack of certain pathogens. Coastal dunes are nutrient deficient and offer harsh, saline environment for the existing flora and fauna. Endophytic fungi may play an important role in plant survival by enhancing nutrient uptake and producing growth-promoting metabolites such as gibberellins and auxins. We screened roots of <it>Ixeris repenes </it>(L.) A. Gray, a common dune plant, for the isolation of gibberellin secreting endophytic fungi.</p> <p>Results</p> <p>We isolated 15 endophytic fungi from the roots of <it>Ixeris repenes </it>and screened them for growth promoting secondary metabolites. The fungal isolate IR-3-3 gave maximum plant growth when applied to waito-c rice and <it>Atriplex gemelinii </it>seedlings. Analysis of the culture filtrate of IR-3-3 showed the presence of physiologically active gibberellins, GA₁, GA₃, GA₄and GA₇(1.95 ng/ml, 3.83 ng/ml, 6.03 ng/ml and 2.35 ng/ml, respectively) along with other physiologically inactive GA₅, GA₉, GA₁₂, GA₁₅, GA₁₉, GA₂₀and, GA₂₄. The plant growth promotion and gibberellin producing capacity of IR-3-3 was much higher than the wild type <it>Gibberella fujikuroi</it>, which was taken as control during present study. GA₅, a precursor of bioactive GA₃was reported for the first time in fungi. The fungal isolate IR-3-3 was identified as a new strain of <it>Penicillium citrinum </it>(named as <it>P. citrinum </it>KACC43900) through phylogenetic analysis of 18S rDNA sequence.</p> <p>Conclusion</p> <p>Isolation of new strain of <it>Penicillium citrinum </it>from the sand dune flora is interesting as information on the presence of <it>Pencillium </it>species in coastal sand dunes is limited. The plant growth promoting ability of this fungal strain may help in conservation and revegetation of the rapidly eroding sand dune flora. <it>Penicillium citrinum </it>is already known for producing mycotoxin citrinin and cellulose digesting enzymes like cellulase and endoglucanase, as well as xylulase. Gibberellins producing ability of this fungus and the discovery about the presence of GA₅will open new aspects of research and investigations.</p

Korean Shock Society septic shock registry: a preliminary report

Objective To evaluate the clinical characteristics, therapeutic interventions, and outcomes of patients with septic shock admitted to the emergency department (ED). Methods This study was a preliminary, descriptive analysis of a prospective, multi-center, observational registry of the EDs of 10 hospitals participating in the Korean Shock Society. Patients aged 19 years or older who had a suspected or confirmed infection and evidence of refractory hypotension or hypoperfusion were included. Results A total of 468 patients were enrolled (median age, 71.3 years; male, 55.1%; refractory hypotension, 82.9%; hyperlactatemia without hypotension, 17.1%). Respiratory infection was the most common source of infection (31.0%). The median Sepsis-related Organ Failure Assessment score was 7.5. The sepsis bundle compliance was 91.2% for lactate measurement, 70.3% for blood culture, 68.4% for antibiotic administration, 80.3% for fluid resuscitation, 97.8% for vasopressor application, 68.0% for central venous pressure measurement, 22.0% for central venous oxygen saturation measurement, and 59.2% for repeated lactate measurement. Among patients who underwent interventions for source control (n=117, 25.1%), 43 (36.8%) received interventions within 12 hours of ED arrival. The in-hospital, 28-day, and 90-day mortality rates were 22.9%, 21.8%, and 27.1%, respectively. The median ED and hospital lengths of stay were 6.8 hours and 12 days, respectively. Conclusion This preliminary report revealed a mortality of over 20% in patients with septic shock, which suggests that there are areas for improvement in terms of the quality of initial resuscitation and outcomes of septic shock patients in the ED

ATP binding site in the plant ADP-glucose pyrophosphorylase large subunit

The ATP binding region in the catalytically inactive large subunit (LS) of the potato tuber ADP-glucose pyrophosphorylase was identified and investigated. Mutations at the ATP binding significantly affected not only the apparent affinities for ATP and Glc-1-P, and catalytic rate but also in many instances, sensitivity to 3-phosphoglycerate. The catalytic rates of the LS mutant enzymes correlated most strongly with changes in the affinity toward ATP, a relationship substantiated by photoaffinity labeling studies with azido-ATP analog. These results indicate that the LS, although catalytically defective, interacts cooperatively with the catalytic small subunit in binding substrates and effectors and, in turn, influencing net catalysis

Allosteric regulation of the higher plant ADP-glucose pyrophosphorylase is a product of synergy between the two subunits

The higher plant ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer consisting of two regulatory large subunits (LSs) and two catalytic small subunits (SSs). To further characterize the roles of these subunits in determining enzyme function, different combinations of wildtype LS (LWT) and variant forms (LUpReg1, LM345) were co-expressed with wildtype SS (SWT) and variant forms (STG-15 and Sdevo330) and their enzyme properties compared to those measured for the heterotetrameric wildtype enzyme and SS homotetrameric enzymes. Analysis of the allosteric regulatory properties of the various enzymes indicates that although the LS is required for optimal activation by 3-phosphoglyceric acid and resistance to Pi, the overall allosteric regulatory and kinetic properties are specified by both subunits. Our results show that the regulatory and kinetic properties of AGPase are not simply due to the LS modulating the properties of the SS but, instead, are a product of synergistic interaction between the two subunits

Direct appraisal of the potato tuber ADP-glucose pyrophosphorylase large subunit in enzyme function by study of a novel mutant form

The higher plant ADP-glucose pyrophosphorylase is a heterotetramer consisting of two subunit types, which have evolved at different rates from a common ancestral gene. The potato tuber small subunit (SS) displays both catalytic and regulatory properties, whereas the exact role of the large subunit (LS), which contains substrate and effector binding sites, remains unresolved. We identified a mutation, S302N, which increased the solubility of the recombinant potato tuber LS and, in turn, enabling it to form a homotetrameric structure. The LS302N homotetramer possesses very little enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS302N homotetramer enzyme is neither activated by the effector 3-phosphoglycerate nor inhibited by P(i). When combined with the catalytically silenced SS, S D143N, however, the LS302N-containing enzyme shows significantly enhanced catalytic activity and restored 3-PGA activation. This unmasking of catalytic and regulatory potential of the LS is conspicuously evident when the activities of the resurrected L(K41R.T51K.S302N) homotetramer are compared with its heterotetrameric form assembled with S D143N. Overall, these results indicate that the LS possesses catalytic and regulatory properties only when assembled with SS and that the net properties of the heterotetrameric enzyme is a product of subunit synergy

RNA-Binding Protein RBP-P Is Required for Glutelin and Prolamine mRNA Localization in Rice Endosperm Cells

In developing rice ( ) endosperm, mRNAs of the major storage proteins, glutelin and prolamine, are transported and anchored to distinct subdomains of the cortical endoplasmic reticulum. RNA binding protein RBP-P binds to both glutelin and prolamine mRNAs, suggesting a role in some aspect of their RNA metabolism. Here, we show that rice lines expressing mutant RBP-P mislocalize both glutelin and prolamine mRNAs. Different mutant RBP-P proteins exhibited varying degrees of reduced RNA binding and/or protein-protein interaction properties, which may account for the mislocalization of storage protein RNAs. In addition, partial loss of RBP-P function conferred a broad phenotypic variation ranging from dwarfism, chlorophyll deficiency, and sterility to late flowering and low spikelet fertility. Transcriptome analysis highlighted the essential role of RBP-P in regulating storage protein genes and several essential biological processes during grain development. Overall, our data demonstrate the significant roles of RBP-P in glutelin and prolamine mRNA localization and in the regulation of genes important for plant growth and development through its RNA binding activity and cooperative regulation with interacting proteins