Glycerol-3-phosphate and systemic immunity

Glycerol-3-phosphate (G3P), a conserved three-carbon sugar, is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis. G3P can be derived via the glycerol kinase-mediated phosphorylation of glycerol or G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate. Previously, we showed G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Inoculation of Arabidopsis with C. higginsianum correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in GLY1 encoded G3Pdh accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Recently, we showed that G3P is also a potent inducer of systemic acquired resistance (SAR) in plants. SAR is initiated after a localized infection and confers whole-plant immunity to secondary infections. SAR involves generation of a signal at the site of primary infection, which travels throughout the plants and alerts the un-infected distal portions of the plant against secondary infections. Plants unable to synthesize G3P are defective in SAR and exogenous G3P complements this defect. Exogenous G3P also induces SAR in the absence of a primary pathogen. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues. Together, these observations suggest that the cooperative interaction of DIR1 and G3P mediates the induction of SAR in plants.Glycerol-3-phosphate (G3P) is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis.^1,2 G3P levels in the plant are regulated by enzymes directly/indirectly involved in G3P biosynthesis, as well as those involved in G3P catabolism. G3P is synthesized via the glycerol kinase (GK)-mediated phosphorylation of glycerol,³ or the G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP)⁴ (Fig. 1). DHAP is derived from glycolysis via triosephosphate isomerase activity on glyceraldehyde-3-phosphate, or from the conversion of glycerol to dihydroxacetone (DHA) by glycerol dehydrogenase (Glydh) followed by phosphorylation of DHA to DHAP by DHA kinase (DHAK). G3P is catabolized either upon its conversion to glycerol by glycerol-3-phoshatase (GPP) or its utilization in glycerolipid/triacylglycerol biosynthesis. In Arabidopsis, the total G3P pool is derived from the activities of five G3Pdh isoforms and one GK isoform present in three cellular locations^5-9; GK and two of the G3Pdh isoforms are present in the cytoplasm, two other G3Pdh isoforms localize to plastids, and one to the mitochondria. One of the plastid localized G3Pdh isoforms, designated GLY1, was previously shown to be required for glycerolipid biosynthesis; a mutation in GLY1 compromised lipids synthesized via the plastidal pathway of lipid biosynthesis. The fact that exogenous application of glycerol to gly1 plants normalizes plastidal lipid levels¹⁰ and that GLY1 encodes a G3Pdh⁴ suggests that the G3P pool generated via the GLY1 catalyzed reaction is required for the biosynthesis of plastidal lipids. Intriguingly, unlike GLY1, neither the chloroplastic, nor the two cytosolic isoforms of G3Pdh, contribute to plastidal and/or extraplastidal lipid biosynthesis.⁹Open in a separate windowFigure 1.A condensed scheme of glycerol-3-phosphate metabolism in plants. Glycerol is phosphorylated to glycerol-3-phosphate (G3P) by glycerol kinase (GK; GLI1). G3P can also be generated by G3P dehydrogenase (G3Pdh) via the reduction of dihydroxyacetone phosphate (DHAP). DHAP is derived from glycolysis via triosephosphate isomerase (TPI) activity on glyceraldehyde-3-phosphate (Gld-3-P), or from the conversion of glycerol to dihydroxacetone (DHA) by glycerol dehydrogenase (Glydh) followed by phosphorylation of DHA to DHAP by DHA kinase (DHAK). G3Pdh isoforms are present in both the cytosol and the plastids (represented by the oval). GLY1 is one of the two plastidial G3Pdh isoforms that plays an important role in plastidial glycerolipid biosynthesis. In the plastids, G3P is acylated with oleic acid (18:1) by the ACT1-encoded G3P acyltransferase. This ACT1-utilized 18:1 is derived from the stearoyl-acyl carrier protein (ACP)-desaturase (SACPD)-catalyzed desaturation of stearic acid (18:0). The 18:1-ACP generated by SACPD either enters the prokaryotic lipid biosynthetic pathway through acylation of G3P or is exported out (dotted line) of the plastids as a coenzyme A (CoA)-thioester to enter the eukaryotic lipid biosynthetic pathway. Membranous fatty acid desaturases (FAD) catalyze desaturation of FAs present on membranous glycerolipids. Other abbreviations used are: GL, glycerolipid; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; Lyso-PA, acyl-G3P; PA, phosphatidic acid; PG, phosphatidylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; SL, sulfolipid; DAG, diacylglycerol.For glycerolipid biosynthesis, G3P is first acylated with the fatty acid (FA) oleic acid (18:1), to form lyso-phosphatidic acid (lyso-PA) via the activity of the soluble G3P acyltransferase (GPAT) encoded by the ACT1 gene in Arabidopsis¹¹ (Fig. 1). 18:1 in turn is derived from the saturated FA, stearic acid (18:0), via the activity of soluble stearoyl-acyl carrier protein desaturases (SACPD),¹² which introduce a single cis double bond in 18:0. The 18:1 generated via this reaction is either exported out of the plastids or acylated at the sn-1 position of G3P. Previously, we have shown that 18:1 levels are important regulators of plant defense signaling. In Arabidopsis, 18:1 is synthesized via the SSI2/FAB2-encoded SACPD,¹² which uses 18:0 as a substrate. A mutation in SSI2 results in the accumulation of 18:0 and a reduction in 18:1 levels. The mutant plants show stunting, spontaneous lesion formation, constitutive PR gene expression, and enhanced resistance to bacterial and oomycete pathogens.^4,12-17 Characterization of ssi2 suppressor mutants has shown that the altered defense-related phenotypes are the result of the reduction in the levels of the unsaturated FA, 18:1, which causes induction of several resistance (R) genes.^4,14,18,19 Restoration of 18:1 levels, via mutations in ACT1,¹⁴ GLY1⁴ or ACP4,¹⁸ normalizes R gene expression in ssi2 plants. The low 18:1-mediated induction of R gene expression and the associated defense signaling can also be suppressed by simultaneous mutations in EDS1 and the genes governing salicylic acid (SA) biosynthesis (SID2, EDS5).¹⁹ Furthermore, the functional redundancy between EDS1 and SA likely masks the requirement for EDS1 by several coiled coil (CC)- nucleotide binding site (NBS)- leucine rich repeat (LRR) proteins,¹⁹ previously thought to function independent of EDS1.²⁰ Thus, the reliance on EDS1 for signaling mediated by CC-NBS-LRR proteins becomes evident only in the absence of SA.The plastidal 18:1 levels are also regulated via the chloroplastic G3P pool and vice-versa. However, 18:1 and G3P appear to function distinctly in defense signaling. For example, G3P levels are important for basal defense against the hemibiotrophic fungus, Colletotrichum higginsianum.^21,22 Genetic mutations affecting G3P synthesis in Arabidopsis enhance susceptibility to C. higginsianum. Conversely, plants accumulating increased G3P show enhanced resistance. More recently, we demonstrated roles for G3P in R-mediated defense leading to systemic acquired resistance (SAR).⁹ R-mediated defense against the avirulent bacterial pathogen P. syringae is associated with a rapid increase in G3P levels; G3P levels peak within 6 h of inoculation with avirulent bacteria (avrRpt2), in resistant plants expressing the R gene RPS2. Strikingly, accumulation of G3P, in the infected and systemic tissues, precedes the accumulation of other metabolites known to be essential for SAR; SA,^23,24 jasmonic acid (JA)²⁵ and azelaic acid (AA)²⁶ accumulated at least 24 h post pathogen inoculation. Furthermore, mutants defective in G3P synthesis are compromised in SAR but accumulated normal levels of SA, AA, and JA. Compromised SAR in G3P deficient mutants was restored by exogenous application of G3P, thus arguing a role for G3P in SAR. This was further supported by the fact that exogenous G3P induced SAR in the absence of the primary pathogen in both Arabidopsis and soybean.⁹ That fact that G3P is a conserved metabolite common to prokaryotes, plants, and humans further corroborates the conserved nature of SAR signaling. Interestingly, although exogenous G3P did not induce SA biosynthesis, SAR conferred by exogenous G3P was dependent on SA. These results suggest that the onset and/or establishment of SAR likely requires basal, but not induced levels of SA, in the distal tissues. It is possible that the relatively small increase in SA observed in the systemic tissues during SAR is an indirect response that contributes to generalized resistance, rather than SAR itself. Interestingly, both G3P conferred SAR, and the systemic movement of G3P were dependent on the lipid transfer protein, DIR1, a well-known positive regulator of SAR.²⁷ Conversely, systemic movement of DIR1 required G3P. These findings did not correlate with the fact that G3P is cytosolic while DIR1 was a predicted apoplastic protein. To resolve this issue, we studied the localization of DIR1, and found that it is in fact a symplastic protein. The symplastic location of DIR1 was further corroborated when GFP fused to the signal peptide from DIR1 localized to the endoplasmic reticulum, rather than the typical cytoplasmic and nuclear location of GFP (Fig. 2). These results suggested that the symplastic movement of DIR1 is likely critical for SAR, and supported the facts that G3P and DIR1 are interdependent for translocation to systemic tissues. However, these findings could not explain how a lipid transfer-like protein might associate with the phosphorylated sugar G3P, to move systemically. Analysis of G3P in the leaf extracts showed that it was derivatized into an unknown compound before/during translocation. It is likely that the G3P derivative has a lipid moiety via which it associates with DIR1 for transfer. In summary, we showed that DIR1 together with a G3P-derived compound are sufficient for the induction of SAR in wild type plants. Our findings provide strong evidence in support of a direct defense-signaling role for G3P and warranty further analysis of its metabolic pathway(s) for their role(s) in various modes of plant defense.Open in a separate windowFigure 2.Confocal micrograph showing localization of GFP fused to DIR1 transit peptide (TP) or GFP alone in Nicotiana benthamiana plants expressing RFP-tagged nuclear histone protein H2B. Arrow indicates nucleus, arrowhead indicates endoplasmic reticulum.     

Step-up/step-down perfusion approach for increased mAb 520C9 production by a hybridoma cell line

The hybridoma cell line, HB-8696, produces a monoclonal antibody, 520C9 (mouse IgG₁) that recognizes the breast cancer oncoprotein, c-erbB2. The effect of perfusion rate (volume of fresh feed/working volume of reactor/day) on cell growth and mAb production was investigated but perfusion at a constant rate and at an arbitrarily increased rate could not maintain exponential cell growth or a higher specific mAb production rate. An optimum step-up/step-down perfusion strategy is therefore proposed for maintaining a steady state production phase at high cell density for ten days. The optimum step-up perfusion could achieve fast cell growth by avoiding any nutrient limited condition and the following optimum step-down perfusion could potentially maintain high live cell density and reduced product dilution as well. The maximum viable cell achieved under optimum perfusion strategy was 2.3 × 10⁷ cells/ml which was 19-fold higher than in optimum batch culture. The mAb yield and volumetric productivity were significantly improved to 52 and 50 mg/l day compared to 25 and 3.8 mg/l day in optimum batch, respectively, and could be maintained for up to ten days.     

Regulation of physiological aspects in plants by hydrogen sulfide and nitric oxide under challenging environment

Plants are exposed to a plethora of abiotic stresses such as drought, salinity, heavy metal and temperature stresses at different stages of their life cycle, from germination to seedling till the reproductive phase. As protective mechanisms, plants release signaling molecules that initiate a cascade of stress-signaling events, leading either to programmed cell death or plant acclimation. Hydrogen sulfide (H₂S) and nitric oxide (NO) are considered as new ‘gasotransmitter’ molecules that play key roles in regulating gene expression, posttranslational modification (PTM), as well as cross-talk with other hormones. Although the exact role of NO in plants remains unclear and is species dependent, various studies have suggested a positive correlation between NO accumulation and environmental stress in plants. These molecules are also involved in a large array of stress responses and act synergistically or antagonistically as signaling components, depending on their respective concentration. This study provides a comprehensive update on the signaling interplay between H₂S and NO in the regulation of various physiological processes under multiple abiotic stresses, modes of action and effects of exogenous application of these two molecules under drought, salt, heat and heavy metal stresses. However, the complete picture of the signaling cascades mediated by H₂S and NO is still elusive. Recent researches indicate that during certain plant processes, such as stomatal closure, H₂S could act upstream of NO signaling or downstream of NO in response to abiotic stresses by improving antioxidant activity in most plant species. In addition, PTMs of antioxidative pathways by these two molecules are also discussed.     

Gibberellic Acid-Priming Promotes Fluoride Tolerance in a Susceptible Indica Rice Cultivar by Regulating the Antioxidant and Phytohormone Homeostasis

Journal of Plant Growth Regulation - Excessive utilization of groundwater for anthropogenic purposes has led to severe depletion of the water table, resulting in contamination of fluorides from the...     

Single units in the medial prefrontal cortex with anxiety-related firing patterns are preferentially influenced by ventral hippocampal activity

The medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC) functionally interact during innate anxiety tasks. To explore the consequences of this interaction, we examined task-related firing of single units from the mPFC of mice exploring standard and modified versions of the elevated plus maze (EPM), an innate anxiety paradigm. Hippocampal local field potentials (LFPs) were simultaneously monitored. The population of mPFC units distinguished between safe and aversive locations within the maze, regardless of the nature of the anxiogenic stimulus. Strikingly, mPFC units with stronger task-related activity were more strongly coupled to theta-frequency activity in the vHPC LFP. Lastly, task-related activity was inversely correlated with behavioral measures of anxiety. These results clarify the role of the vHPC-mPFC circuit in innate anxiety and underscore how specific inputs may be involved in the generation of behaviorally relevant neural activity within the mPFC.     

Discovery of +(2-{4-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy]phenyl}-cyclopropyl)acetic acid as potent and selective alphavbeta3 inhibitor: design, synthesis, and optimization

The integrin alpha(v)beta(3) is expressed in a number of cell types and is thought to play a major role in several pathological conditions. Various small molecules that inhibit the integrin have been shown to suppress tumor growth and retinal angiogenesis. The tripeptide Arg-Gly-Asp (RGD), a common binding motif in several ligands that bind to alpha(v)beta(3), has been depeptidized and optimized in our efforts toward discovering a small molecule inhibitor. We recently disclosed the synthesis and biological activity of several small molecules that did not contain any peptide bond and mimic the tripeptide RGD. The phenethyl group in one of the lead compounds was successfully replaced with a cyclopropyl moiety. The new lead compound was optimized for potency, selectivity, and for its ADME properties. We describe herein the discovery, synthesis, and optimization of cyclopropyl containing analogs that are potent and selective inhibitors of alpha(v)beta(3).     

The karrikin ‘calisthenics’: Can compounds derived from smoke help in stress tolerance?

Karrikins (KARs) are unique butenolides derived as a by‐product of incomplete combustion during wildfire. Some receptive plant species respond to KARs in the form of accelerated germination. These molecules originate from stress to mediate tolerance against different sub‐optimal conditions like oxidative stress, drought and low‐light intensity (shade stress). KARs promote seed germination, seedling establishment and ecological diversity by accelerating the abundance of selective communities of plants. The signaling pathway is closely related, yet unique from strigolactones (SLs). Due to the structural relatedness with SLs, KARs have potential roles in mediating abiotic stress tolerance in plants. In addition, the KAR directly/indirectly interact with crucial phytohormones like abscisic acid, gibberellic acid, auxins and ethylene. This article is a summarized update on KAR research in recent times. The exhaustive discussions would be beneficial for understanding the extraordinary strategy devised by nature to protect plants from stress using a molecule which is itself generated out of stress.     

Targeting Glycinebetaine for Abiotic Stress Tolerance in Crop Plants: Physiological Mechanism,Molecular Interaction and Signaling

In the era of climate change, abiotic stresses (e.g., salinity, drought, extreme temperature, flooding, metal/metalloid(s), UV radiation, ozone, etc.) are considered as one of the most complex environmental constraints that restricts crop production worldwide. Introduction of stress-tolerant crop cultivars is the most auspicious way of surviving this constraint, and to produce these types of tolerant crops. Several bioengineering mechanisms involved in stress signaling are being adopted in this regard. One example of this kind of manipulation is the osmotic adjustment. The quarternary ammonium compound glycinebetaine (GB), also originally referred to as betaine is a methylated glycine derivative. Among the betaines, GB is the most abundant one in plants, which is mostly produced in response to dehydration caused by different abiotic stresses like drought, salinity, and extreme temperature. Glycinebetaine helps in decreased accumulation and detoxification of ROS, thereby restoring photosynthesis and reducing oxidative stress. It takes part in stabilizing membranes and macromolecules. It is also involved in the stabilization and protection of photosynthetic components, such as ribulose-1, 5-bisphosphate carboxylase/oxygenase, photosystem II and quarternary enzyme and protein complex structures under environmental stresses. Glycinebetaine was found to perform in chaperone-induced protein disaggregation. In addition, GB can confer stress tolerance in very low concentrations, and it acts in activating defense responsive genes with stress protection. Recently, field application of GB has also shown protective effects against environmental adversities increasing crop yield and quality. In this review, we will focus on the role of GB in conferring abiotic stress tolerance and the possible ways to engineer GB biosynthesis in plants.     

Novel Point and Combo-Mutations in the Genome of Hepatitis B Virus-Genotype D: Characterization and Impact on Liver Disease Progression to Hepatocellular Carcinoma

Background

The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC.

Methods

The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence.

Results

Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively.

Conclusions

Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.