Subcellular distribution of glutathione precursors in Arabidopsis thaliana

Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) - the first enzyme of glutathione synthesis - causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells.     

Suppression of phospholipase Dalpha1 induces freezing tolerance in Arabidopsis: response of cold-responsive genes and osmolyte accumulation

Phospholipase D (PLD; EC 3.1.4.4) plays an important role in membrane lipid hydrolysis and in mediation of plant responses to a wide range of stresses. PLDalpha1 abrogation through antisense suppression in Arabidopsis thaliana resulted in a significant increase in freezing tolerance of both non-acclimated and cold-acclimated plants. Although non-acclimated PLDalpha1-deficient plants did not show the activation of cold-responsive C-repeat/dehydration-responsive element binding factors (CBFs) and their target genes (COR47 and COR78), they did accumulate osmolytes to much higher levels than did the non-acclimated wild-type plants. However, a stronger expression of COR47 and COR78 in response to cold acclimation and to especially freezing was observed in PLDalpha1-deficient plants. Furthermore, a slower activation of CBF1 was observed in response to cold acclimation in these plants compared to the wild-type plants. Typically, cold acclimation resulted in a higher accumulation of osmolytes in PLDalpha1-deficient plants than in wild-type plants. Inhibition of PLD activity by using lysophosphatidylethanolamine (LPE) also increased freezing tolerance of Arabidopsis, albeit to a lesser extent than did the PLD antisense suppression. Exogenous LPE induced expression of COR15a and COR47 in the absence of cold stimulus. These results suggest that PLDalpha1 plays a key role in freezing tolerance of Arabidopsis by modulating the cold-responsive genes and accumulation of osmolytes.     

Quantitative profiling of polar glycerolipid species from organs of wild-type Arabidopsis and a phospholipase Dalpha1 knockout mutant

Lipid profiling is a targeted metabolomics platform that provides a comprehensive analysis of lipid species with high sensitivity. Profiling based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides quantitative data and is adaptable to high throughput analyses. Here we report the profiling of 140 apparent molecular species of polar glycerolipids in Arabidopsis leaves, flower stalks, flowers, siliques, roots, and seeds. Considerable differences in lipid species occur among these organs, providing insights into the different lipid metabolic activities in a specific organ. In addition, comparative profiling between wild-type and a knockout mutant pldalpha1 (locus ID: AT3G15730) provides insight into the metabolic function of phospholipase D (PLD) in different organs. PLDalpha1 contributes significantly to phosphatidic acid (PA) levels in roots, seeds, flowers, and flower stalks, but little to basal PA levels in siliques and leaves. In seeds of the pldalpha1 mutant plants, levels of PA, lysophosphatidylcholine, and lysophosphatidylethanolamine were significantly lower than those of wild-type seeds, suggesting a role for PLDalpha1 in membrane lipid degradation in seeds.     

Evidence for and characterization of Ca2+ binding to the catalytic region of Arabidopsis thaliana phospholipase Dbeta

Most types of plant phospholipase D (PLD) require Ca(2+) for activity, but how Ca(2+) affects PLD activity is not well understood. We reported previously that Ca(2+) binds to the regulatory C2 domain that occurs in the N terminus of the Ca(2+)-requiring PLDs. Using Arabidopsis thaliana PLDbeta and C2-deleted PLDbeta (PLDbetacat), we now show that Ca(2+) also interacts with the catalytic regions of PLD. PLDbetacat exhibited Ca(2+)-dependent activity, was much less active, and required a higher level of Ca(2+) than the full-length PLDbeta. Ca(2+) binding of the proteins was stimulated by phospholipids; phosphatidylserine was the most effective among those tested. Scatchard plot analysis of Ca(2+) binding data yielded an estimate of 3.6 high affinity (K(d) = 29 mum) binding sites on PLDbeta. The Ca(2+)-PLDbetacat interaction increased the affinity of the protein for the activator, phosphatidylinositol 4,5-bisphosphate, but not for the substrate, phosphatidylcholine. This is in contrast to the effect of Ca(2+) binding to the C2 domain, which stimulates phosphatidylcholine binding but inhibits phosphatidylinositol 4,5-bisphosphate binding of the domain. These results demonstrate the contrasting and complementary effects of the Ca(2+)- and lipid-binding properties of the C2 and catalytic domains of plant PLD and provide insight into the mechanism by which Ca(2+) regulates PLD activity.     

Phospholipase Dalpha3 is involved in the hyperosmotic response in Arabidopsis

Rapid activation of phospholipase D (PLD), which hydrolyzes membrane lipids to generate phosphatidic acid (PA), occurs under various hyperosmotic conditions, including salinity and water deficiency. The Arabidopsis thaliana PLD family has 12 members, and the function of PLD activation in hyperosmotic stress responses has remained elusive. Here, we show that knockout (KO) and overexpression (OE) of previously uncharacterized PLDalpha3 alter plant response to salinity and water deficit. PLDalpha3 uses multiple phospholipids as substrates with distinguishable preferences, and alterations of PLDalpha3 result in changes in PA level and membrane lipid composition. PLDalpha3-KO plants display increased sensitivities to salinity and water deficiency and also tend to induce abscisic acid-responsive genes more readily than wild-type plants, whereas PLDalpha3-OE plants have decreased sensitivities. In addition, PLDalpha3-KO plants flower later than wild-type plants in slightly dry conditions, whereas PLDalpha3-OE plants flower earlier. These data suggest that PLDalpha3 positively mediates plant responses to hyperosmotic stresses and that increased PLDalpha3 expression and associated lipid changes promote root growth, flowering, and stress avoidance.     

Arabidopsis phospholipase Dalpha1 interacts with the heterotrimeric G-protein alpha-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors

Phospholipase D (PLD) and heterotrimeric G-protein both play important, diverse roles in cellular regulation and signal transduction. Here we have determined the physical interaction between plant PLD and the only canonical alpha-subunit (Galpha) of the G-protein in Arabidopsis thaliana and the molecular basis for the interaction. PLDalpha1 expressed in either Escherichia coli or Arabidopsis was co-precipitated with Galpha. PLDalpha1 contains a sequence motif analogous to the G alpha-interacting DRY motif normally conserved in G-protein-coupled receptors. Mutation of the central Lys residue PLD(K564A) of this motif abolished the PLDalpha1-Galpha binding, whereas mutation of the two flanking residues PLD(E563A) and PLD(F565A) decreased the binding. Addition of Galpha to PLDalpha1 inhibited PLDalpha1 activity, whereas the PLD(K564A) mutation that disrupted the Galpha-PLDalpha1 binding abolished the inhibition. GTP relieved the Galpha inhibition of PLDalpha1 activity and also inhibited the binding between PLDalpha1 and Galpha. Meanwhile, the PLDalpha1-Galpha interaction stimulated the intrinsic GTPase activity of Galpha. Therefore, these results have demonstrated the direct binding between Galpha and PLDalpha1, identified the DRY motif on PLDalpha1 as the site for the interaction, and indicated that the interaction modulates reciprocally the activities of PLDalpha1 and Galpha.     

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.     

Regional distribution and age-dependent expression of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D in rat brain

The endocannabinoid anandamide (N-arachidonoylethanolamine) and other bioactive long-chain N-acylethanolamines are thought to be formed from their corresponding N-acylphosphatidylethanolamines by a specific phospholipase D (NAPE-PLD) in the brain as well as other tissues. However, regional distribution of NAPE-PLD in the brain has not been examined. In the present study, we investigated the expression levels of NAPE-PLD in nine different regions of rat brain by enzyme assay, western blotting and real-time PCR. The NAPE-PLD activity was detected in all the tested brain regions with the highest activity in thalamus. Similar distribution patterns of NAPE-PLD were observed at protein and mRNA levels. We also found a remarkable increase in the expression levels of protein and mRNA of the brain NAPE-PLD with development, which was in good agreement with the increase in the activity. The age-dependent increase was also seen with several brain regions and other NAPE-PLD-enriched organs (heart and testis). p-Chloromercuribenzoic acid and cetyltrimethylammonium chloride, which inhibited recombinant NAPE-PLD dose-dependently, strongly inhibited the enzyme of all the brain regions. These results demonstrated wide distribution of NAPE-PLD in various brain regions and its age-dependent expression, suggesting the central role of this enzyme in the formation of anandamide and other N-acylethanolamines in the brain.     

拟南芥血红蛋白1的亚细胞定位

为研究拟南芥的血红蛋白1(AtGLB1)基因的亚细胞定位,该实验构建了拟南芥血红蛋白1基因与绿色荧光蛋白基因融合的植物表达载体pUCGFP/ AtGLB1.利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,通过检测融合蛋白在洋葱表皮细胞中的分布来确定拟南芥血红蛋白1在细胞中的定位.荧光显微镜检测结果表明,AtGLB1基因表达产物主要定位在细胞核中,少量定位在细胞质中.