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The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is a member of the ETX_MTX family of beta-pore forming proteins (β-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this manuscript, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to have any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence approach including evaluation of the history of safe use (HOSU) of ETX_MTX2 proteins and Breviballus laterosporus. Comparisons between purified Mpp75Aa1.1 protein and a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55°C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. These results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins. Taken together, we report that Mpp75Aa1.1 is the first ETX-MTX2 insecticidal protein from B. laterosporus and displays a similar safety profile as typical Cry proteins from Bacillus thuringiensis. [ABSTRACT FROM AUTHOR]

Western corn rootworm (WCR), Diabrotica virgifera virgifera, LeConte, is an insect pest that poses a significant threat to the productivity of modern agriculture, causing significant economic and crop losses. The development of genetically modified (GM) crops expressing one or more proteins that confer tolerance to specific insect pests, such as WCR, was a historic breakthrough in agricultural biotechnology and continues to serve as an invaluable tool in pest management. Despite this, evolving resistance to existing insect control proteins expressed in current generation GM crops requires continued identification of new proteins with distinct modes of action while retaining targeted insecticidal efficacy. GM crops expressing insecticidal proteins must undergo extensive safety assessments prior to commercialization to ensure that they pose no increased risk to the health of humans or other animals relative to their non-GM conventional counterparts. As part of these safety evaluations, a weight of evidence approach is utilized to assess the safety of the expressed insecticidal proteins to evaluate any potential risk in the context of dietary exposure. This study describes the food and feed safety assessment of Vpb4Da2, a new Bacillus thuringiensis insecticidal protein that confers in planta tolerance to WCR. Vpb4Da2 exhibits structural and functional similarities to other insect control proteins expressed in commercialized GM crops. In addition, the lack of homology to known toxins or allergens, a lack of acute toxicity in mice, inactivation by conditions commonly experienced in the human gut or during cooking/food processing, and the extremely low expected dietary exposure to Vpb4Da2 provide a substantial weight of evidence to demonstrate that the Vpb4Da2 protein poses no indication of a risk to the health of humans or other animals. [ABSTRACT FROM AUTHOR]

Highlights: [•] One step purification of phosphinothricin acetyltransferase (PAT) is developed. [•] The purification employs an affinity chromatography using Reactive brown 10. [•] The method is simple, time-saving, inexpensive and reproducible. [•] The method enables to obtain pure PAT from various sources. [•] Other applications of the method are also discussed. [Copyright &y& Elsevier]

Pathogen infection of higher plants often induces rapid production of phosphatidic acid ( PA) and changes in lipid profiles, but the enzymatic basis and the function of the lipid change in pathogen-plant interactions are not well understood., Infection of phospholipase D β1 ( PLDβ1)-deficient plants by Pseudomonas syringae tomato pv DC3000 ( Pst DC30000) resulted in less bacterial growth than in wild-type plants, and the effect was more profound in virulent Pst DC3000 than avirulent Pst DC3000 (carrying the avirulence gene avrRpt2) infection. The expression levels of salicylic acid ( SA)-inducible genes were higher, but those inducible by jasmonic acid ( JA) showed lower expression in PLDβ1 mutants than in wild-type plants., However, PLDβ1-deficient plants were more susceptible than wild-type plants to the fungus Botrytis cinerea. The PLDβ1-deficient plants had lower levels of PA, JA and JA-related defense gene expression after B. cinerea inoculation., PLDβ1 plays a positive role in pathogen-induced JA production and plant resistance to the necrotrophic fungal pathogen B. cinerea, but a negative role in the SA-dependent signaling pathway and plant tolerance to infection with biotrophic Pst DC3000. PLDβ1 is responsible for most of the increase in PA production in response to necrotrophic B. cinerea and virulent Pst DC3000 infection, but contributes less to avirulent Pst DC3000 ( avrRpt2)-induced PA production. [ABSTRACT FROM AUTHOR]

Abstract: Plants possess multiple genes encoding calcium sensor proteins that are members of the penta-EF-hand (PEF) family. Characterized PEF proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit function in diverse cellular processes in a calcium-dependent manner by interacting with their target proteins at either their N-terminal extension or Ca2+ binding domains. We have identified a previously unreported class of PEF proteins in plants that are notable because they do not possess the hydrophobic amino acid rich N-terminal extension that is typical of these PEF proteins. We demonstrate that the maize PEF protein without the N-terminal extension has the characteristics of known PEF proteins; the protein binds calcium in the 100 nM range and, as a result of calcium binding, displays an increase in hydrophobicity. Characterization of the truncated maize PEF protein provides insights into the role of the N-terminal extension in PEF protein signaling. In the context of the current model of how PEF proteins are activated by calcium binding, these results demonstrate that this distinctive class of PEF proteins could function as calcium sensor proteins in plants even in the absence of the N-terminal extension. [Copyright &y& Elsevier]

Abstract: The phospholipase D (PLD) family of Arabidopsis thaliana has 12 identified members, including three highly homologous PLDγs. The enzymatic and molecular properties of PLDγ2 were characterized and compared with those of PLDγ1. Two variants of PLDγ2 cDNAs, designated PLDγ2a and PLDγ2b, were isolated, and they differ in the presence of a 96-nucleotide fragment at the beginning of the open reading frame. Catalytically active PLDγ2a was expressed in E. coli. PLDγ2a and γ1 both require phosphatidylinositol 4,5-bisphosphate (PIP2) and calcium for activity, but they differ in the effect of PIP2 and Triton X-100 on their activities. While Triton X-100 could greatly activate PLDγ1 activity and served only as a neutral diluent in the substrate vesicles, it totally abolished PLDγ2a activity and prohibited any stimulation effect from PIP2. PLDγ2a misses one of the basic, PIP2-interacting residues, which may weaken the binding of PIP2 to PLDγ2a. In addition, PLDγ2 and PLDγ1 displayed different patterns of expression in different tissues, and the transcript of PLDγ2a differs from that of PLDγ1 by having a longer 5′-UTR. These differences in biochemical and molecular properties suggest that the highly homologous PLDγs are subjected to unique regulations and might have distinguishable functions. [Copyright &y& Elsevier]

Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency.

Aluminum (Al) toxicity is the major stress in acidic soil that comprises about 50% of the world's arable land. The complex molecular mechanisms of Al toxicity have yet to be fully determined. As a barrier to Al entrance, plant cell membranes play essential roles in plant interaction with Al, and lipid composition and membrane integrity change significantly under Al stress. Here, we show that phospholipase Dγs (PLDγs) are induced by Al stress and contribute to Al-induced membrane lipid alterations. RNAi suppression of PLDγ resulted in a decrease in both PLDγ1 and PLDγ2 expression and an increase in Al resistance. Genetic disruption of PLDγ1 also led to an increased tolerance to Al while knockout of PLDγ2 did not. Both RNAi-suppressed and pldγ1-1 mutants displayed better root growth than wild-type under Al stress conditions, and PLDγ1-deficient plants had less accumulation of callose, less oxidative damage, and less lipid peroxidation compared to wild-type plants. Most phospholipids and glycolipids were altered in response to Al treatment of wild-type plants, whereas fewer changes in lipids occurred in response to Al stress in PLDγ mutant lines. Our results suggest that PLDγs play a role in membrane lipid modulation under Al stress and that high activities of PLDγs negatively modulate plant tolerance to Al.

It has been known that lipoxygenase (LOX) isozymes exhibit differences in product formation, but most product information to date is for LOX 1 among soybean (Glycine max) LOX isozymes. In this study, LOXs 2 and 3 were purified and used to generate hydroperoxide (HPOD) products in an in vitro system using linoleic acid as a substrate in the presence of either air or O2. The products were analyzed to determine their stereoisomeric characteristics. The control (no enzyme) showed only low levels of hydroperoxide production and no stereoisomeric specificity. LOX 2 shows high specificity in product formation, producing roughly 4 times more 13-HPOD than 9-HPOD, nearly all of which was 13-S(Z,E)-HPOD. LOX 3 produced more 9-HPOD than 13-HPOD at approximately a 2:1 ratio. No single stereoisomer was predominant among LOX 3 products. These results demonstrate that different isozymes of LOX have characteristic product profiles in in vitro reactions.

Development of the aleurone layer of maize grains requires the activity of the Defective kernel 1 (Dek1) gene, encoding a predicted 240-kDa membrane-anchored protein with a C terminus similar to animal calpain domain II&III. Three-dimensional modeling shows that DEK1 domain II contains a conserved calpain catalytic triad and that domain II&III has a predicted structure similar to m-calpain. Recombinant DEK1 domain II&III exhibits activity in the caseinolytic assay in the absence of calcium, although the activity is enhanced by calcium. This is in sharp contrast to animal calpains, which require Ca2+ to be active. Bacterially expressed DEK1 domain II does not display caseinolytic activity, suggesting an important role for DEK1 domain III. Mutation of the catalytic Cys residue to Ser leads to a loss of caseinolytic activity of DEK1 domain II&III. Two features of DEK1 calpain may contribute to maintaining the active site triad in an "active" configuration in the absence of Ca2+, both of which are predicted to keep m-calpain domains IIa and IIb apart. First, DEK1 lacks key charged residues in the basic loop of domain II, and secondly, the absence of an acidic loop in domain III, both of which are predicted to be neutralized upon Ca2+ binding. The Dek1 transcript is present in all cell types in developing maize endosperm, suggesting that the activity of the DEK1 calpain is regulated at the post-transcription level. The role of DEK1 in aleurone signaling is discussed.

Phospholipase D (PLD) is a major plant phospholipase family involved in many cellular processes such as signal transduction, membrane remodeling, and lipid degradation. Five classes of PLDs have been identified in Arabidopsis thaliana, and Ca(2+) and polyphosphoinositides have been suggested as key regulators for these enzymes. To investigate the catalysis and regulation mechanism of individual PLDs, surface-dilution kinetics studies were carried out on the newly identified PLDdelta from Arabidopsis. PLDdelta activity was dependent on both bulk concentration and surface concentration of substrate phospholipids in the Triton X-100/phospholipid mixed micelles. V(max), K(s)(A), and K(m)(B) values for PLDdelta toward phosphatidylcholine or phosphatidylethanolamine were determined; phosphatidylethanolamine was the preferred substrate. PLDdelta activity was stimulated greatly by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Maximal activation was observed at a PIP(2) molar ratio around 0.01. Kinetic analysis indicates that PIP(2) activates PLD by promoting substrate binding to the enzyme, without altering the bulk binding of the enzyme to the micelle surface. Ca(2+) is required for PLDdelta activity, and it significantly decreased the interfacial Michaelis constant K(m)(B). This indicates that Ca(2+) activates PLD by promoting the binding of phospholipid substrate to the catalytic site of the enzyme.

Phospholipases are activated in response to various cellular and environmental cues. Their activation can affect many cellular processes through their roles in signal transduction. Recent advances in the biochemical and molecular understanding of phospholipase D (PLD) have provided insights into potential networks of PLDs and other phospholipases in plants. PLDs are a family of heterogeneous enzymes, and the activities of the multiple types of PLDs are regulated in distinctly different manners. Phosphoinositides, free fatty acids, lysophospholipids, and calcium are differential modulators of PLDs. Since these modulators are substrates, products, or downstream targets of phospholipase As and phospholipase Cs, there are many potential regulatory and metabolic interrelationships among the various PLDs and other phospholipases.