Ana Rita Matos

A novel patatin-like gene stimulated by drought stress encodes a galactolipid acyl hydrolase.

A cDNA (Vupat1) encoding a predicted 43 kDa protein was isolated from drought‐stressed cowpea (Vigna unguiculata) leaves. It has homology with patatin, a potato tuber storage protein with lipolytic acyl hydrolase activity. The recombinant protein VUPAT1 expressed in the baculovirus system displays preferentially galactolipid acyl hydrolase activity. Phospholipids are very slowly hydrolyzed and apparently triacylglycerols are not deacylated. Vupat1 promoter contains putative drought‐inducible sequences. Northern blots showed that gene expression is stimulated by drought stress and is more pronounced in a drought‐sensitive cultivar than in a drought‐tolerant one. An involvement in drought‐induced galactolipid degradation is proposed for VUPAT1.

Patatin-related phospholipase A: nomenclature, subfamilies and functions in plants

The release of fatty acids from membrane glycerolipids has been implicated in a variety of cellular processes, but the enzymes involved and their regulation are poorly understood in plants. One large group of acyl-hydrolyzing enzymes is structurally related to patatins. Patatins are potato tuber proteins with acyl-hydrolyzing activity, and the patatin catalytic domain is widely spread in bacterial, yeast, plant and animal enzymes. Recent results have indicated that patatin-related enzymes are involved in different cellular functions, including plant responses to auxin, elicitors or pathogens, and abiotic stresses and lipid mobilization during seed germination. In this review, we highlight recent developments regarding these enzymes and propose the nomenclature pPLA for the patatin-related phospholipase A enzyme.

Lipid deacylating enzymes in plants: old activities, new genes.

Because lipids are major components of cellular membranes, their degradation under stress conditions compromises compartmentalization. However, in addition to having structural roles, membrane lipids are also implicated in signalling processes involving the activity of lipolytic enzymes. Phospholipases D and C, acting on the polar heads of phospholipids, have been relatively well characterized in plants. In contrast, knowledge of lipid deacylating enzymes remains limited. Lipid acyl hydrolases (LAH) are able to hydrolyse both fatty acid moieties of polar lipids. They differ from phospholipases A(1) or A(2) (PLA) acting on sn-1 or sn-2 positions of phospholipids, respectively, as well as from lipases which de-esterify triacylglycerols. The free polyunsaturated fatty acids generated by deacylating enzymes can be used in the biosynthesis of oxylipins and the lysophospholipids, provided by PLAs, are also bioactive molecules. In the four decades that have passed since the first description of LAH activities in plants some enzymes have been purified. In recent years, the widespread use of molecular approaches together with the attention paid to lipid signalling has contributed to a renewed interest in LAH and has led to the identification of different gene families and the characterization of new enzymes. Additionally, several proteins with putative lipase/esterase signatures have been identified. In the present paper we review currently available data on LAHs, PLAs, triacylglycerol lipases and other putative deacylating enzymes. The roles of lipid deacylating enzymes in plant growth, development and stress responses are discussed in the context of their involvement in membrane deterioration, lipid turnover and cellular signalling.

Response of the Diatom Phaeodactylum tricornutum to Photooxidative Stress Resulting from High Light Exposure

The response of microalgae to photooxidative stress resulting from high light exposure is a well-studied phenomenon. However, direct analyses of photosystem II (PSII) D1 protein (the main target of photoinhibition) in diatoms are scarce. In this study, the response of the diatom model species Phaeodactylum tricornutum to short-term exposure to high light was examined and the levels of D1 protein determined immunochemically. Low light (LL) acclimated cells (40 µmol photons m−2 s−1) subjected to high light (HL, 1,250 µmol photons m−2 s−1) showed rapid induction of non-photochemical quenching (NPQ) and ca. 20-fold increase in diatoxanthin (DT) concentration. This resulted from the conversion of diadinoxanthin (DD) to DT through the activation of the DD-cycle. D1 protein levels under LL decreased about 30% after 1 h of the addition of lincomycin (LINC), a chloroplast protein synthesis inhibitor, showing significant D1 degradation and repair under low irradiance. Exposure to HL lead to a 3.2-fold increase in D1 degradation rate, whereas average D1 repair rate was 1.3-x higher under HL than LL, leading to decreased levels of D1 protein under HL. There were significant effects of both HL and LINC on P. tricornutum maximum quantum yield of PSII (F v/F m), showing a reduction of active PSII reaction centres. Partial recovery of F v/F m in the dark demonstrates the photosynthetic resilience of this diatom to changes in the light regime. P. tricornutum showed high allocation of total protein to D1 and an active D1-repair cycle to limit photoinhibition.

Study of the effects of salicylic acid on soybean mitochondrial lipids and respiratory properties using the alternative oxidase as a stress-reporter protein.

Biotic and abiotic stresses can lead to modifications in the lipid composition of cell membranes. Although mitochondria appear to be implicated in stress responses, little is known about the membrane lipid changes that occur in these organelles in plants. Besides cytochrome c oxidase, plant mitochondria have an alternative oxidase (AOX) that accepts electrons directly from ubiquinol, dissipating energy as heat. AOX upregulation occurs under a variety of stresses and its induction by salicylic acid (SA) has been observed in different plant species. AOX was also suggested to be used as a functional marker for cell reprogramming under stress. In the present study, we have used etiolated soybean (Glycine max (L.) Merr. cv Cresir) seedlings to study the effects of SA treatment on the lipid composition and the respiratory properties of hypocotyl mitochondria. AOX expression was studied in detail, as a reporter protein, to evaluate whether modifications in mitochondrial energy metabolism were occurring. In mitochondria extracted from SA-treated seedlings, AOX capacity and protein contents increased. Both AOX1 and AOX2b transcripts accumulated in response to SA, but with different kinetics. A reduction in external NADH oxidation capacity was observed, whereas succinate respiration remained unchanged. The phospholipid composition of mitochondria remained similar in control and SA-treated plants, but a reduction in the relative amount of linolenic acid was observed in phosphatidylcholine, phosphatidylethanolamine and cardiolipin. The possible causes of the fatty acid modifications observed, and the implications for mitochondrial metabolism are discussed.

Cloning and characterization of drought-stimulated phosphatidic acid phosphatase genes from Vigna unguiculata

Under environmental stresses, several lipolytic enzymes are known to be activated and to contribute to membrane lipid turnover and generation of second messengers. In animal cells, phosphatidic acid phosphatase (PAP, EC 3.1.3.4), which dephosphorylates phosphatidic acid generating diacylglycerol, is long known as an enzyme involved in lipid synthesis and cell signalling. However, knowledge on PAP in plants remains very limited. The aim of this work was to isolate and characterize PAP genes in the tropical legume Vigna unguiculata (cowpea), and to study their expression under different stress conditions. Two cDNAs designated as VuPAPalpha and VuPAPbeta were cloned from the leaves of cowpea. Both proteins share sequence homology to animal type 2 PAP, namely, the six transmembrane regions and the consensus sequences corresponding to the catalytic domain of the phosphatase family, like the recently described Arabidopsis LPP (Lipid Phosphate Phosphatase) proteins. The recombinant protein VuPAPalpha expressed in Escherichia coli cells was able to convert phosphatidic acid into diacylglycerol. Unlike VuPAPbeta, VuPAPalpha has an N-terminal transit peptide and was addressed to chloroplast in vitro. Both genes are expressed in several cowpea organs and their transcripts accumulate in leaves in response to water deficit, including progressive dehydration of whole plants and rapid desiccation of detached leaves. No changes in expression of both genes were observed after wounding or by treatment with jasmonic acid. Furthermore, the in silico analysis of VuPAPalpha promoter allowed the identification of several putative drought-related regulatory elements. The possible physiological role of the two cloned PAPs is discussed.

Specific adjustments in grapevine leaf proteome discriminating resistant and susceptible grapevine genotypes to Plasmopara viticola

Grapevine downy mildew is an important disease affecting crop production leading to severe yield losses. This study aims to identify the grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry. In addition, we analysed ROS production, antioxidant capacity, lipid peroxidation and gene expression. Proteins related to photosynthesis and metabolism allowed the discrimination of resistant and susceptible grapevine cultivars prior to P. viticola inoculation. Following inoculation increase of hydrogen peroxide levels, cellular redox regulation, establishment of ROS signalling and plant cell death seem to be key points differentiating the resistant genotype. Lipid associated signalling events, particularly related to jasmonates appear also to play a major role in the establishment of resistance. The findings from this study contribute to a better understanding of genotype-specific differences that account for a successful establishment of a defence response to the downy mildew pathogen. BIOLOGICAL SIGNIFICANCE Here, we present for the first time grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). We have highlighted that, following inoculation, the major factors differentiating the resistant from the susceptible grapevine cultivars are the establishment of effective ROS signalling together with lipid-associated signalling events, particularly related to jasmonates. It is believed that plants infected with biotrophic pathogens suppress JA-mediated responses, however recent evidences shown that jasmonic acid signalling pathway in grapevine resistance against Plasmopara viticola. Our results corroborate those evidences and highlight the importance of lipid- signalling for an effective resistance response against the downy mildew pathogen.

Incorporation of tocopherol acetate-containing particles in acrylic bone cement

Acrylic bone cement (BC) is used in orthopaedic surgery to anchor cemented prostheses to bone. Association of antioxidant molecules to BC may suppress reactive species injury which contributes to implant failure. Tocopherol acetate (ATA)-loaded polymethylmethacrylate (PMMA) particles (ATA(PMMA)) were prepared by single emulsion solvent evaporation technique and were incorporated into BC. An encapsulation efficiency of 84% (w/w) was obtained and drug release studies showed distinct ATA release profiles and mechanisms before and after particle incorporation into BC. Experimental data, analysed using first-order, Higuchi and Korsmeyer-Peppas models revealed that ATA was released from particles by a Fickian diffusion mechanism while a non-Fickian transport was observed upon particle incorporation in BC. There were no changes in the mechanical properties of BC specimens containing ATA(PMMA) particles, in contrast to what was observed when ATA was loaded directly into BC. Overall, ATA(PMMA) particles are potential carriers for the incorporation of an antioxidant drug into BC.

A minocycline-releasing PMMA system as a space maintainer for staged bone reconstructions?in vitro antibacterial, cytocompatibility and anti-inflammatory characterization

In the present work, we study the development and biological characterization of a polymethyl methacrylate (PMMA)-based minocycline delivery system, to be used as a space maintainer within craniofacial staged regenerative interventions. The developed delivery systems were characterized regarding solid state characteristics and assayed in vitro for antibacterial and anti-inflammatory activity, and cytocompatibility with human bone cells. A drug release profile allowed for an initial burst release and a more sustained and controlled release over time, with minimum inhibitory concentrations for the assayed and relevant pathogenic bacteria (i.e., Staphylococcus aureus, slime-producer Staphylococcus epidermidis and Escherichia coli) being easily attained in the early time points, and sustained up to 72 h. Furthermore, an improved osteoblastic cell response-with enhancement of cell adhesion and cell proliferation-and increased anti-inflammatory activity were verified in developed systems, compared to a control (non minocycline-loaded PMMA cement). The obtained results converge to support the possible efficacy of the developed PMMA-based minocycline delivery systems for the clinical management of complex craniofacial trauma. Here, biomaterials with space maintenance properties are necessary for the management of staged reconstructive approaches, thus minimizing the risk of peri-operative infections and enhancing the local tissue healing and early stages of regeneration.

Laser induced fluorescence technique for environmental applications

We discuss the development of laser induced fluorescence sensors and their application in the evaluation of water pollution and physiological status of higher plants and algae. The sensors were built on the basis of reliable and robust solid-state Nd:YAG lasers. They demonstrated good efficiency in: i) detecting and characterizing oil spills and dissolved organic matter; ii) evaluating the impact of stress on higher plants (cork oak, maritime pine, and genetically modified Arabidopsis); iii) tracking biomass changes in intertidal microphytobenthos; and iv) mapping macroalgal communities in the Tagus Estuary.