Comparative Transcriptomics of Multi-Stress Responses in Pachycladon cheesemanii and Arabidopsis thaliana.

The environment is seldom optimal for plant growth and changes in abiotic and biotic signals, including temperature, water availability, radiation and pests, induce plant responses to optimise survival. The New Zealand native plant species and close relative to Arabidopsis thaliana, Pachycladon cheesemanii, grows under environmental conditions that are unsustainable for many plant species. Here, we compare the responses of both species to different stressors (low temperature, salt and UV-B radiation) to help understand how P. cheesemanii can grow in such harsh environments. The stress transcriptomes were determined and comparative transcriptome and network analyses discovered similar and unique responses within species, and between the two plant species. A number of widely studied plant stress processes were highly conserved in A. thaliana and P. cheesemanii. However, in response to cold stress, Gene Ontology terms related to glycosinolate metabolism were only enriched in P. cheesemanii. Salt stress was associated with alteration of the cuticle and proline biosynthesis in A. thaliana and P. cheesemanii, respectively. Anthocyanin production may be a more important strategy to contribute to the UV-B radiation tolerance in P. cheesemanii. These results allowed us to define broad stress response pathways in A. thaliana and P. cheesemanii and suggested that regulation of glycosinolate, proline and anthocyanin metabolism are strategies that help mitigate environmental stress.

Current Insights into the Molecular Mode of Action of Seaweed-Based Biostimulants and the Sustainability of Seaweeds as Raw Material Resources.

Natural biostimulants, such as seaweed extracts, can stimulate plant growth and development in both model and crop plants. Due to the increasing demands for their use in agriculture, it is crucial to ensure the sustainability of the sources from which they are produced. Furthermore, some seaweed extracts were recently shown to prime and protect from adverse environmental factors such as drought, salinity and extreme temperatures, as well as from oxidative stress. The molecular mode of action of these biostimulants has still not been fully elucidated, but there has been significant progress in this direction in the last years. Firstly, this review examines the sustainability aspects of harvesting seaweed resources as raw materials for manufacturing biostimulants and provides an overview of the regulatory landscape pertaining to seaweed-based biostimulants. The review then summarises the recent advances in determining the genetic and molecular mechanisms activated by seaweed-based biostimulants, their influence on transcriptome reconfiguration, metabolite adjustment, and ultimately stress protection, improved nutrient uptake, and plant growth and performance. This knowledge is important for deciphering the intricate stress signalling network modulated by seaweed-based biostimulants and can aid in designing molecular priming technologies for crop improvement.

Putative alternative translation start site-encoding nucleotides of CPR5 regulate growth and resistance.

BACKGROUND: The Arabidopsis CONSTITUTIVE EXPRESSER of PATHOGENESIS-RELATED GENES 5 (CPR5) has recently been shown to play a role in gating as part of the nuclear pore complex (NPC). Mutations in CPR5 cause multiple defects, including aberrant trichomes, reduced ploidy levels, reduced growth and enhanced resistance to bacterial and fungal pathogens. The pleiotropic nature of cpr5 mutations implicates that the CPR5 protein affects multiple pathways. However, little is known about the structural features that allow CPR5 to affect the different pathways. RESULTS: Our in silico studies suggest that in addition to three clusters of putative nuclear localization signals and four or five transmembrane domains, CPR5 contains two putative alternative translation start sites. To test the role of the methionine-encoding nucleotides implicated in those sites, metCPR5 cDNAs, in which the relevant nucleotides were changed to encode glutamine, were fused to the CPR5 native promoter and the constructs transformed to cpr5-2 plants to complement cpr5-compromised phenotypes. The control and metCPR5 constructs were able to complement all cpr5 phenotypes, although the extent of complementation depended on the specific complementing plant lines. Remarkably, plants transformed with metCPR5 constructs showed larger leaves and displayed reduced resistance when challenged to Pseudomonas syringae pv Pst DC3000, as compared to control plants. Thus, the methionine-encoding nucleotides regulate growth and resistance. We propose that structural features of the CPR5 N-terminus are implicated in selective gating of proteins involved in regulating the balance between growth and resistance. CONCLUSION: Plants need to carefully balance the amount of resources used for growth and resistance. The Arabidopsis CPR5 protein regulates plant growth and immunity. Here we show that N-terminal features of CPR5 are involved in the regulation of the balance between growth and resistance. These findings may benefit efforts to improve plant yield, while maintaining optimal levels of disease resistance.

A Biostimulant Obtained from the Seaweed Ascophyllum nodosum Protects Arabidopsis thaliana from Severe Oxidative Stress.

Abiotic stresses cause oxidative damage in plants. Here, we demonstrate that foliar application of an extract from the seaweed Ascophyllum nodosum, SuperFifty (SF), largely prevents paraquat (PQ)-induced oxidative stress in Arabidopsis thaliana. While PQ-stressed plants develop necrotic lesions, plants pre-treated with SF (i.e., primed plants) were unaffected by PQ. Transcriptome analysis revealed induction of reactive oxygen species (ROS) marker genes, genes involved in ROS-induced programmed cell death, and autophagy-related genes after PQ treatment. These changes did not occur in PQ-stressed plants primed with SF. In contrast, upregulation of several carbohydrate metabolism genes, growth, and hormone signaling as well as antioxidant-related genes were specific to SF-primed plants. Metabolomic analyses revealed accumulation of the stress-protective metabolite maltose and the tricarboxylic acid cycle intermediates fumarate and malate in SF-primed plants. Lipidome analysis indicated that those lipids associated with oxidative stress-induced cell death and chloroplast degradation, such as triacylglycerols (TAGs), declined upon SF priming. Our study demonstrated that SF confers tolerance to PQ-induced oxidative stress in A. thaliana, an effect achieved by modulating a range of processes at the transcriptomic, metabolic, and lipid levels.

An Ascophyllum nodosum-Derived Biostimulant Protects Model and Crop Plants from Oxidative Stress.

Abiotic stresses, which at the molecular level leads to oxidative damage, are major determinants of crop yield loss worldwide. Therefore, considerable efforts are directed towards developing strategies for their limitation and mitigation. Here the superoxide-inducing agent paraquat (PQ) was used to induce oxidative stress in the model species Arabidopsis thaliana and the crops tomato and pepper. Pre-treatment with the biostimulant SuperFifty (SF) effectively and universally suppressed PQ-induced leaf lesions, H2O2 build up, cell destruction and photosynthesis inhibition. To further investigate the stress responses and SF-induced protection at the molecular level, we investigated the metabolites by GC-MS metabolomics. PQ induced specific metabolic changes such as accumulation of free amino acids (AA) and stress metabolites. These changes were fully prevented by the SF pre-treatment. Moreover, the metabolic changes of the specific groups were tightly correlating with their phenotypic characteristics. Overall, this study presents physiological and metabolomics data which shows that SF protects against oxidative stress in all three plant species.

A novel seed plants gene regulates oxidative stress tolerance in Arabidopsis thaliana.

Oxidative stress can lead to plant growth retardation, yield loss, and death. The atr7 mutant of Arabidopsis thaliana exhibits pronounced tolerance to oxidative stress. Using positional cloning, confirmed by knockout and RNA interference (RNAi) lines, we identified the atr7 mutation and revealed that ATR7 is a previously uncharacterized gene with orthologs in other seed plants but with no homology to genes in lower plants, fungi or animals. Expression of ATR7-GFP fusion shows that ATR7 is a nuclear-localized protein. RNA-seq analysis reveals that transcript levels of genes encoding abiotic- and oxidative stress-related transcription factors (DREB19, HSFA2, ZAT10), chromatin remodelers (CHR34), and unknown or uncharacterized proteins (AT5G59390, AT1G30170, AT1G21520) are elevated in atr7. This indicates that atr7 is primed for an upcoming oxidative stress via pathways involving genes of unknown functions. Collectively, the data reveal ATR7 as a novel seed plants-specific nuclear regulator of oxidative stress response.

Genome-Wide Analysis of ROS Antioxidant Genes in Resurrection Species Suggest an Involvement of Distinct ROS Detoxification Systems during Desiccation.

Abiotic stress is one of the major threats to plant crop yield and productivity. When plants are exposed to stress, production of reactive oxygen species (ROS) increases, which could lead to extensive cellular damage and hence crop loss. During evolution, plants have acquired antioxidant defense systems which can not only detoxify ROS but also adjust ROS levels required for proper cell signaling. Ascorbate peroxidase (APX), glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) are crucial enzymes involved in ROS detoxification. In this study, 40 putative APX, 28 GPX, 16 CAT, and 41 SOD genes were identified from genomes of the resurrection species Boea hygrometrica, Selaginella lepidophylla, Xerophyta viscosa, and Oropetium thomaeum, and the mesophile Selaginella moellendorffii. Phylogenetic analyses classified the APX, GPX, and SOD proteins into five clades each, and CAT proteins into three clades. Using co-expression network analysis, various regulatory modules were discovered, mainly involving glutathione, that likely work together to maintain ROS homeostasis upon desiccation stress in resurrection species. These regulatory modules also support the existence of species-specific ROS detoxification systems. The results suggest molecular pathways that regulate ROS in resurrection species and the role of APX, GPX, CAT and SOD genes in resurrection species during stress.

Molecular Mechanisms Preventing Senescence in Response to Prolonged Darkness in a Desiccation-Tolerant Plant.

The desiccation-tolerant plant Haberlea rhodopensis can withstand months of darkness without any visible senescence. Here, we investigated the molecular mechanisms of this adaptation to prolonged (30 d) darkness and subsequent return to light. H. rhodopensis plants remained green and viable throughout the dark treatment. Transcriptomic analysis revealed that darkness regulated several transcription factor (TF) genes. Stress- and autophagy-related TFs such as ERF8, HSFA2b, RD26, TGA1, and WRKY33 were up-regulated, while chloroplast- and flowering-related TFs such as ATH1, COL2, COL4, RL1, and PTAC7 were repressed. PHYTOCHROME INTERACTING FACTOR4, a negative regulator of photomorphogenesis and promoter of senescence, also was down-regulated. In response to darkness, most of the photosynthesis- and photorespiratory-related genes were strongly down-regulated, while genes related to autophagy were up-regulated. This occurred concomitant with the induction of SUCROSE NON-FERMENTING1-RELATED PROTEIN KINASES (SnRK1) signaling pathway genes, which regulate responses to stress-induced starvation and autophagy. Most of the genes associated with chlorophyll catabolism, which are induced by darkness in dark-senescing species, were either unregulated (PHEOPHORBIDE A OXYGENASE, PAO; RED CHLOROPHYLL CATABOLITE REDUCTASE, RCCR) or repressed (STAY GREEN-LIKE, PHEOPHYTINASE, and NON-YELLOW COLORING1). Metabolite profiling revealed increases in the levels of many amino acids in darkness, suggesting increased protein degradation. In darkness, levels of the chloroplastic lipids digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylglycerol, and sulfoquinovosyldiacylglycerol decreased, while those of storage triacylglycerols increased, suggesting degradation of chloroplast membrane lipids and their conversion to triacylglycerols for use as energy and carbon sources. Collectively, these data show a coordinated response to darkness, including repression of photosynthetic, photorespiratory, flowering, and chlorophyll catabolic genes, induction of autophagy and SnRK1 pathways, and metabolic reconfigurations that enable survival under prolonged darkness.

ROS-mediated abiotic stress-induced programmed cell death in plants.

During the course of their ontogenesis plants are continuously exposed to a large variety of abiotic stress factors which can damage tissues and jeopardize the survival of the organism unless properly countered. While animals can simply escape and thus evade stressors, plants as sessile organisms have developed complex strategies to withstand them. When the intensity of a detrimental factor is high, one of the defense programs employed by plants is the induction of programmed cell death (PCD). This is an active, genetically controlled process which is initiated to isolate and remove damaged tissues thereby ensuring the survival of the organism. The mechanism of PCD induction usually includes an increase in the levels of reactive oxygen species (ROS) which are utilized as mediators of the stress signal. Abiotic stress-induced PCD is not only a process of fundamental biological importance, but also of considerable interest to agricultural practice as it has the potential to significantly influence crop yield. Therefore, numerous scientific enterprises have focused on elucidating the mechanisms leading to and controlling PCD in response to adverse conditions in plants. This knowledge may help develop novel strategies to obtain more resilient crop varieties with improved tolerance and enhanced productivity. The aim of the present review is to summarize the recent advances in research on ROS-induced PCD related to abiotic stress and the role of the organelles in the process.

A cell type-specific view on the translation of mRNAs from ROS-responsive genes upon paraquat treatment of Arabidopsis thaliana leaves.

Oxidative stress causes dramatic changes in the expression levels of many genes. The formation of a functional protein through successful mRNA translation is central to a coordinated cellular response. To what extent the response towards reactive oxygen species (ROS) is regulated at the translational level is poorly understood. Here we analysed leaf- and tissue-specific translatomes using a set of transgenic Arabidopsis thaliana lines expressing a FLAG-tagged ribosomal protein to immunopurify polysome-bound mRNAs before and after oxidative stress. We determined transcript levels of 171 ROS-responsive genes upon paraquat treatment, which causes formation of superoxide radicals, at the whole-organ level. Furthermore, the translation of mRNAs was determined for five cell types: mesophyll, bundle sheath, phloem companion, epidermal and guard cells. Mesophyll and bundle sheath cells showed the strongest response to paraquat treatment. Interestingly, several ROS-responsive transcription factors displayed cell type-specific translation patterns, while others were translated in all cell types. In part, cell type-specific translation could be explained by the length of the 5'-untranslated region (5'-UTR) and the presence of upstream open reading frames (uORFs). Our analysis reveals insights into the translational regulation of ROS-responsive genes, which is important to understanding cell-specific responses and functions during oxidative stress.

Natural products from resurrection plants: potential for medical applications.

Resurrection species are a group of land plants that can tolerate extreme desiccation of their vegetative tissues during harsh drought stress, and still quickly - often within hours - regain normal physiological and metabolic functions following rehydration. At the molecular level, this desiccation tolerance is attributed to basal cellular mechanisms including the constitutive expression of stress-associated genes and high levels of protective metabolites present already in the absence of stress, as well as to transcriptome and metabolome reconfigurations rapidly occurring during the initial phases of drought stress. Parts of this response are conferred by unique metabolites, including a diverse array of sugars, phenolic compounds, and polyols, some of which accumulate to high concentrations within the plant cell. In addition to drought stress, these metabolites are proposed to contribute to the protection against other abiotic stresses and to an increased oxidative stress tolerance. Recently, extracts of resurrection species and particular secondary metabolites therein were reported to display biological activities of importance to medicine, with e.g. antibacterial, anticancer, antifungal, and antiviral activities, rendering them possible candidates for the development of novel drug substances as well as for cosmetics. Herein, we provide an overview of the metabolite composition of resurrection species, summarize the latest reports related to the use of natural products from resurrection plants, and outline their potential for medical applications.

Comparative metabolic profiling of Haberlea rhodopensis, Thellungiella halophyla, and Arabidopsis thaliana exposed to low temperature.

Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desiccation but also with ability to withstand low temperatures and freezing stress. In order to identify biochemical strategies which contribute to Haberlea's remarkable stress tolerance, the metabolic reconfiguration of H. rhodopensis during low temperature (4°C) and subsequent return to optimal temperatures (21°C) was investigated and compared with that of the stress tolerant Thellungiella halophyla and the stress sensitive Arabidopsis thaliana. Metabolic analysis by GC-MS revealed intrinsic differences in the metabolite levels of the three species even at 21°C. H. rhodopensis had significantly more raffinose, melibiose, trehalose, rhamnose, myo-inositol, sorbitol, galactinol, erythronate, threonate, 2-oxoglutarate, citrate, and glycerol than the other two species. A. thaliana had the highest levels of putrescine and fumarate, while T. halophila had much higher levels of several amino acids, including alanine, asparagine, beta-alanine, histidine, isoleucine, phenylalanine, serine, threonine, and valine. In addition, the three species responded differently to the low temperature treatment and the subsequent recovery, especially with regard to the sugar metabolism. Chilling induced accumulation of maltose in H. rhodopensis and raffinose in A. thaliana but the raffinose levels in low temperature exposed Arabidopsis were still much lower than these in unstressed Haberlea. While all species accumulated sucrose during chilling, that accumulation was transient in H. rhodopensis and A. thaliana but sustained in T. halophila after the return to optimal temperature. Thus, Haberlea's metabolome appeared primed for chilling stress but the low temperature acclimation induced additional stress-protective mechanisms. A diverse array of sugars, organic acids, and polyols constitute Haberlea's main metabolic defence mechanisms against chilling, while accumulation of amino acids and amino acid derivatives contribute to the low temperature acclimation in Arabidopsis and Thellungiella. Collectively, these results show inherent differences in the metabolomes under the ambient temperature and the strategies to respond to low temperature in the three species.

Molecular mechanisms of desiccation tolerance in the resurrection glacial relic Haberlea rhodopensis.

Haberlea rhodopensis is a resurrection plant with remarkable tolerance to desiccation. Haberlea exposed to drought stress, desiccation, and subsequent rehydration showed no signs of damage or severe oxidative stress compared to untreated control plants. Transcriptome analysis by next-generation sequencing revealed a drought-induced reprogramming, which redirected resources from growth towards cell protection. Repression of photosynthetic and growth-related genes during water deficiency was concomitant with induction of transcription factors (members of the NAC, NF-YA, MADS box, HSF, GRAS, and WRKY families) presumably acting as master switches of the genetic reprogramming, as well as with an upregulation of genes related to sugar metabolism, signaling, and genes encoding early light-inducible (ELIP), late embryogenesis abundant (LEA), and heat shock (HSP) proteins. At the same time, genes encoding other LEA, HSP, and stress protective proteins were constitutively expressed at high levels even in unstressed controls. Genes normally involved in tolerance to salinity, chilling, and pathogens were also highly induced, suggesting a possible cross-tolerance against a number of abiotic and biotic stress factors. A notable percentage of the genes highly regulated in dehydration and subsequent rehydration were novel, with no sequence homology to genes from other plant genomes. Additionally, an extensive antioxidant gene network was identified with several gene families possessing a greater number of antioxidant genes than most other species with sequenced genomes. Two of the transcripts most abundant during all conditions encoded catalases and five more catalases were induced in water-deficient samples. Using the pharmacological inhibitor 3-aminotriazole (AT) to compromise catalase activity resulted in increased sensitivity to desiccation. Metabolome analysis by GC or LC-MS revealed accumulation of sucrose, verbascose, spermidine, and γ-aminobutyric acid during drought, as well as particular secondary metabolites accumulating during rehydration. This observation, together with the complex antioxidant system and the constitutive expression of stress protective genes suggests that both constitutive and inducible mechanisms contribute to the extreme desiccation tolerance of H. rhodopensis.

Molecular mechanisms of desiccation tolerance in resurrection plants.

Resurrection plants are a small but diverse group of land plants characterized by their tolerance to extreme drought or desiccation. They have the unique ability to survive months to years without water, lose most of the free water in their vegetative tissues, fall into anabiosis, and, upon rewatering, quickly regain normal activity. Thus, they are fundamentally different from other drought-surviving plants such as succulents or ephemerals, which cope with drought by maintaining higher steady state water potential or via a short life cycle, respectively. This review describes the unique physiological and molecular adaptations of resurrection plants enabling them to withstand long periods of desiccation. The recent transcriptome analysis of Craterostigma plantagineum and Haberlea rhodopensis under drought, desiccation, and subsequent rehydration revealed common genetic pathways with other desiccation-tolerant species as well as unique genes that might contribute to the outstanding desiccation tolerance of the two resurrection species. While some of the molecular responses appear to be common for both drought stress and desiccation, resurrection plants also possess genes that are highly induced or repressed during desiccation with no apparent sequence homologies to genes of other species. Thus, resurrection plants are potential sources for gene discovery. Further proteome and metabolome analyses of the resurrection plants contributed to a better understanding of molecular mechanisms that are involved in surviving severe water loss. Understanding the cellular mechanisms of desiccation tolerance in this unique group of plants may enable future molecular improvement of drought tolerance in crop plants.

Identification of cis-regulatory elements specific for different types of reactive oxygen species in Arabidopsis thaliana.

The type of reactive oxygen species (ROS) is a major factor that determines the specificity of biological responses. These responses may be elicited by activation of transcription factors that recognize ROS-specific cis-regulatory elements in target genes. In search for Arabidopsis promoter motifs specific for particular types of ROS, genome-wide microarray expression profiles for 283 abiotic stress-related conditions were subjected to cluster analysis to identify gene groups induced by singlet oxygen, superoxide radicals, and H(2)O(2). Promoters of these gene groups were analyzed to identify cis-regulatory elements that are associated with specific types of ROS. Eleven ROS-specific de novo identified elements, seven known promoter motifs and several sequences enriched in ROS-responsive clusters but lacking in specificity are reported. The conservation of the identified motifs was determined in orthologous genes in C. papaya, V. vinifera and P. trichocarpa. Finally, biological functions were attributed to the motifs by calculation of GO-term enrichment for genes with conserved ROS-responsive elements.

A universal microscope manipulator / Um manipulador microscópico universal

A modified and improved model of a mechanical manipulator for observation of pinned and mounted insects is described. This device allows movement of the observed object around three perpendicular axes in the field of vision at all magnifications of stereomicroscopes. The main improvement of this new model is positioning of the guiding knobs for rotating around two of the axes next to each other, allowing faster and easier manipulation of the studied object. Thus, one of the main advantages of this device is the possibility to rotate the specimen without the need to refocus. The device enables easily reaching a precession deviation in the intersection point of axes up to 0.5 mm in the process of assembling.

Um modelo modificado e melhorado de um manipulador mecânico para observação de insetos fixados e montados é descrito. Este dispositivo permite o movimento do objeto observado em torno de três eixos perpendiculares no campo de visão para todas as ampliações do microscópio. A principal melhoria deste novo modelo é o posicionamento dos botões de rotação em torno dos dois eixos ao lado do outro, permitindo uma rápida e fácil manipulação do objeto estudado. Assim, uma das principais vantagens deste dispositivo é a possibilidade de girar o espécime sem a necessidade de mudar o foco. O dispositivo permite facilmente chegar a um desvio de precisão no ponto de interseção dos eixos de até 0,5 mm no processo de montagem.

Programmed cell death in plants: new insights into redox regulation and the role of hydrogen peroxide.

Programmed cell death (PCD), the highly regulated dismantling of cells, is essential for plant growth and survival. PCD plays key roles in embryo development, formation and maturation of many cell types and tissues, and plant reaction/adaptation to environmental conditions. Reactive oxygen species (ROS) are not only toxic by products of aerobic metabolism with strictly controlled cellular levels, but they also function as signaling agents regulating many biological processes and producing pleiotropic effects. Over the last decade, ROS have become recognized as important modulators of plant PCD. Molecular genetic approaches using plant mutants and transcriptome studies related to ROS-mediated PCD have revealed a wide array of plant-specific cell death regulators and have contributed to unraveling the elaborate redox signaling network. This review summarizes the biological processes, in which plant PCD participates and discusses the signaling functions of ROS with emphasis on hydrogen peroxide.

Arabidopsis AAL-toxin-resistant mutant atr1 shows enhanced tolerance to programmed cell death induced by reactive oxygen species.

The fungal AAL-toxin triggers programmed cell death (PCD) through perturbations of sphingolipid metabolism in AAL-toxin-sensitive plants. While Arabidopsis is relatively insensitive to the toxin, the loh2 mutant exhibits increased susceptibility to AAL-toxin due to the knockout of a gene involved in sphingolipid metabolism. Genetic screening of mutagenized loh2 seeds resulted in the isolation of AAL-toxin-resistant mutant atr1.Atr1 displays a wild type phenotype when grown on soil but it develops less biomass than loh2 on media supplemented with 2% and 3% sucrose. Atr1 was also more tolerant to the reactive oxygen species-generating herbicides aminotriazole (AT) and paraquat. Microarray analyses of atr1 and loh2 under AT-treatment conditions that trigger cell death in loh2 and no visible damage in atr1 revealed genes specifically regulated in atr1 or loh2. In addition, most of the genes strongly downregulated in both mutants were related to cell wall extension and cell growth, consistent with the apparent and similar AT-induced cessation of growth in both mutants. This indicates that two different pathways, a first controlling growth inhibition and a second triggering cell death, are associated with AT-induced oxidative stress.

A comparison of quantitative and qualitative superoxide dismutase assays for application to low temperature microalgae.

Antioxidant enzymes such as superoxide dismutase (SOD) play a key role in the removal of reactive oxygen species produced during visible and ultraviolet irradiance stress in microalgae and plants. However, little is known about the enzymatic antioxidative stress responses in ecologically important Antarctic marine microalgae. SOD in particular is difficult to analyze, possibly due to problems in obtaining sufficient quantities necessary for reliable and reproducible enzymatic assays. The aim of the present work was to create a sensitive, easy-to-use and reliable method for SOD determination in Antarctic microalgal material by comparing and optimizing existing protein extraction procedures and SOD assays in the marine Antarctic diatom Chaetoceros brevis. Optimization was achieved in cell disruption (sonication) and protein extraction procedures, extraction buffers, SOD assay methods (xanthine/xanthine oxidase and NBT/riboflavin photometric quantitative methods and native gel electrophoresis qualitative method) and the assay temperature. Protein extraction was optimal at low sonication amplitudes after a few pulses, irrespective of the type of buffer used. Extraction efficiency varied highly between the tested buffers; most protein was extracted in the presence of 1% of Triton X-100. SOD activity was best quantified using the NBT/riboflavin method in combination with a buffer containing potassium phosphate and Triton X-100. Moreover, the NBT/riboflavin method was demonstrated to be the most reliable and sensitive method at low temperatures (5 degrees C).