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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 plant 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 then determined and comparative transcriptome and network analyses discovered similar and unique responses within species between different stresses, 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 strategy to cope with UV-B radiation stress in P. cheesemanii only. These results allowed us to construct broad stress response pathways in A. thaliana and P. cheesemanii and identify possible novel plant strategies that help mitigate environmental stress.

Plant species that exhibit vegetative desiccation tolerance can survive extreme desiccation for months and resume normal physiological activities upon re-watering. Here we survey the recent knowledge gathered from the sequenced genomes of angiosperm and non-angiosperm desiccation-tolerant plants (resurrection plants) and highlight some distinct genes and gene families that are central to the desiccation response. Furthermore, we review the vast amount of data accumulated from analyses of transcriptomes and metabolomes of resurrection species exposed to desiccation and subsequent rehydration, which allows us to build a systems biology view on the molecular and genetic mechanisms of desiccation tolerance in plants.

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.

Drought represents a major threat to plants in natural ecosystems and agricultural settings. The biostimulant Super Fifty (SF), produced from the brown alga Ascophyllum nodosum, enables ecologically friendly stress mitigation. We investigated the physiological and whole-genome transcriptome responses of Arabidopsis thaliana to drought stress after a treatment with SF. SF strongly decreased drought-induced damage. Accumulation of reactive oxygen species (ROS), which typically stifle plant growth during drought, was reduced in SF-primed plants. Relative water content remained high in SF-treated plants, whilst ion leakage, a measure of cell damage, was reduced compared to controls. Plant growth requires a functional shoot apical meristem (SAM). Expression of a stress-responsive negative growth regulator, RESPONSIVE TO DESICCATION 26 (RD26), was repressed by SF treatment at the SAM, consistent with the model that SF priming maintains the function of the SAM during drought stress. Accordingly, expression of the cell cycle marker gene HISTONE H4 (HIS4) was maintained at the SAMs of SF-primed plants, revealing active cell cycle progression after SF priming during drought. In accordance with this, CYCP2, 1, which promotes meristem cell division, was repressed by drought but enhanced by SF. SF also positively affected stomatal behavior to support the tolerance to drought stress. Collectively, our data show that SF priming mitigates multiple cellular processes that otherwise impair plant growth under drought stress, thereby providing a knowledge basis for future research on crops.

Abiotic stresses cause plant growth inhibition, damage, and in the most severe cases, cell death, resulting in major crop yield losses worldwide. Many abiotic stresses lead also to oxidative stress. Recent genetic and genomics studies have revealed highly complex and integrated gene networks which are responsible for stress adaptation. Here we summarize the main findings of the papers published in the Special Issue “ROS and Abiotic Stress in Plants”, providing a global picture of the link between reactive oxygen species and various abiotic stresses such as acid toxicity, drought, heat, heavy metals, osmotic stress, oxidative stress, and salinity.

We describe the principles of PlantaSYST as an example for the novel European Teaming projects, funded under the ‘Spreading Excellence and Widening Participation’ Horizon 2020 program. The goal of the Teaming projects is to bridge the gap between research centers with low and high participation in the EU framework program.

The reactive oxygen species (ROS) gene network, consisting of both ROS-generating and detoxifying enzymes, adjusts ROS levels in response to various stimuli. We performed a cross-kingdom comparison of ROS gene networks to investigate how they have evolved across all Eukaryotes, including protists, fungi, plants and animals. We included the genomes of 16 extremotolerant Eukaryotes to gain insight into ROS gene evolution in organisms that experience extreme stress conditions. Our analysis focused on ROS genes found in all Eukaryotes (such as catalases, superoxide dismutases, glutathione reductases, peroxidases and glutathione peroxidase/peroxiredoxins) as well as those specific to certain groups, such as ascorbate peroxidases, dehydroascorbate/monodehydroascorbate reductases in plants and other photosynthetic organisms. ROS-producing NADPH oxidases (NOX) were found in most multicellular organisms, although several NOX-like genes were identified in unicellular or filamentous species. However, despite the extreme conditions experienced by extremophile species, we found no evidence for expansion of ROS-related gene families in these species compared to other Eukaryotes. Tardigrades and rotifers do show ROS gene expansions that could be related to their extreme lifestyles, although a high rate of lineage-specific horizontal gene transfer events, coupled with recent tetraploidy in rotifers, could explain this observation. This suggests that the basal Eukaryotic ROS scavenging systems are sufficient to maintain ROS homeostasis even under the most extreme conditions.

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.

Background Pachycladon cheesemanii is a close relative of Arabidopsis thaliana and is an allotetraploid perennial herb which is widespread in the South Island of New Zealand. It grows at altitudes of up to 1000 m where it is subject to relatively high levels of ultraviolet (UV)-B radiation. To gain first insights into how Pachycladon copes with UV-B stress, we sequenced its genome and compared the UV-B tolerance of two Pachycladon accessions with those of two A. thaliana accessions from different altitudes. Results A high-quality draft genome of P. cheesemanii was assembled with a high percentage of conserved single-copy plant orthologs. Synteny analysis with genomes from other species of the Brassicaceae family found a close phylogenetic relationship of P. cheesemanii with Boechera stricta from Brassicaceae lineage I. While UV-B radiation caused a greater growth reduction in the A. thaliana accessions than in the P. cheesemanii accessions, growth was not reduced in one P. cheesemanii accession. The homologues of A. thaliana UV-B radiation response genes were duplicated in P. cheesemanii, and an expression analysis of those genes indicated that the tolerance mechanism in P. cheesemanii appears to differ from that in A. thaliana. Conclusion Although the P. cheesemanii genome shows close similarity with that of A. thaliana, it appears to have evolved novel strategies allowing the plant to tolerate relatively high UV-B radiation.

Abiotic stresses, including drought, salinity, extreme temperature, and pollutants, are the main cause of crop losses worldwide. Novel climate-adapted crops and stress tolerance-enhancing compounds are needed increasingly to counteract the negative effects of unfavorable stressful environments. A number of natural products and synthetic chemicals can protect model and crop plants against abiotic stresses through the ectopic induction of molecular and physiological defense mechanisms, a process known as molecular priming. In addition to their stress-protective effect, some of these compounds can also stimulate plant growth. Here, we provide an overview of the known physiological and molecular mechanisms behind the compounds that induce molecular priming, together with a survey of approaches to discover and functionally study new stress-alleviating chemicals.

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, confrmed by knockout and RNA interference (RNAi) lines, we identifed 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-specifc nuclear regulator of oxidative stress response., Published online: 27 June 2019

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.

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.

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.

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.

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 gamma-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.

The Arabidopsis thaliana atr7 mutant is tolerant to oxidative stress induced by paraquat (PQ) or the catalase inhibitor aminotriazole (AT), while its original background loh2 and wild-type plants are sensitive. Both, AT and PQ which stimulate the intracellular formation of H2O2 or superoxide anions, respectively, trigger cell death in loh2 but do not lead to visible damage in atr7. To study gene expression during oxidative stress and ROS-induced programmed cell death, two platforms for multi-parallel quantitative real-time PCR (qRT-PCR) analysis of 217 antioxidant and 180 ROS marker genes were employed. The qRT-PCR analyses revealed AT- and PQ-induced expression of many ROS-responsive genes mainly in loh2, confirming that an oxidative burst plays a role in the activation of the cell death in this mutant. Some of the genes were specifically regulated by either AT or PQ serving as markers for particular types of ROS. Genes significantly induced by both AT and PQ in loh2 included transcription factors (ANAC042/JUB1, ANAC102, DREB19, HSFA2, RRTF1, ZAT10, ZAT12, ethylene-responsive factors), signaling compounds, ferritins, alternative oxidases, and antioxidant enzymes. Many of these genes were upregulated in atr7 compared to loh2 under non-stress conditions at the first time point, indicating that higher basal levels of ROS and higher antioxidant capacity in atr7 are responsible for the enhanced tolerance to oxidative stress and suggesting a possible tolerance against multiple stresses of this mutant. (c) 2012 Elsevier Masson SAS. All rights reserved.

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.

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 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.

We have previously reported a method for isolation of mutants with enhanced tolerance to the fungal AAL toxin and given a detailed characterization of atr1 (AAL toxin resistant, Gechev et al. in Biochem Biophys Res Commun 375:639–644, 2008). Herewith, we report eight more mutants with enhanced tolerance to the AAL toxin. Phenotypic analysis showed that six of the mutants were reduced in size compared with their original background loh2. Furthermore, atr2 showed delayed flowering and senescence. The mutants were also evaluated for oxidative stress tolerance by growing them on ROS-inducing media supplemented with either aminotriazole or paraquat, generating, respectively, H2O2 or superoxide radicals. Oxidative stress, confirmed by induction of the marker genes, HIGH AFFINITY NITRATE TRANSPORTER At1G08090 and HEAT SHOCK PROTEIN 17 At3G46230, inhibited growth of all lines. However, while the original background loh2 developed necrotic lesions and died rapidly on ROS-inducing plant growth media, atr1, atr2, atr7 and atr9 remained green and viable. The tolerance against oxidative stress-induced cell death was confirmed by fresh weight and chlorophyll measurements. Real-time PCR analysis revealed that the expression of the EXTENSIN gene At5G46890, previously shown to be downregulated by aminotriazole in atr1, was repressed in all lines, consistent with the growth inhibition induced by oxidative stress. Taken together, the data indicate a complex link between growth, development and oxidative stress tolerance and indicates that growth inhibition can be uncoupled from oxidative stress-induced cell death.

In this paper we investigate the changes in fresh weight, chlorophyll content, catalase and guiacol peroxidase activities of nine oxidative stress-tolerant atr (AAL-toxin resistant) Arabidopsis thaliana mutants subjected to cadmium stress. We show that atr2, atr7 and atr9 are more tolerant to CdCl2. There was no significant change in catalase activity under cadmium treatment in all atr lines including the parental line loh2 compared with untreated seedlings. All lines but atr9 showed reduction in guaiacol peroxidase activity. This suggests that the sensitivity to the cadmium-induced oxidative stress at least in some lines is linked to impaired reactive oxygen species.

Due to their sessile lifestyle, plants are exposed to a variety of stresses and have to develop ingenious mechanisms to avoid or cope with the consequences of extreme stress factors. Plant genomes have therefore evolved to meet environmental challenges and many plant genes are dedi- cated to stress protective mechanisms. Examples of adverse environmental factors include drought, salinity, solar radiation (excess light or high light intensities, UV-light), extreme temperatures (heat and low temperature/freezing stress), and pollutants (heavy metals, herbicides). In addition, low concentrations of essential macro- and micronutrients or conditions that result in poor uptake of these nutrients are also perceived as stress by plants. For example, phosphate deficiency is one of the most common reasons for poor plant growth and reduced crop yield [1]. Sometimes, two and even more abiotic and/ or biotic stress factors impose their effects simultaneously, so that plants have to deal with a multitude of challenges [2]. Oxidative stress is a common consequence of many of these factors.

Tolerance against oxidative stress generated by high light intensities or the catalase inhibitor aminotriazole (AT) was induced in intact tobacco plants by spraying them with hydrogen peroxide (H2O2). Stress tolerance was concomitant with an enhanced antioxidant status as reflected by higher activity and/or protein levels of catalase, ascorbate peroxidase, guaiacol peroxidases, and glutathione peroxidase, as well as an increased glutathione pool. The induced stress tolerance was dependent on the dose of H2O2 applied. Moderate doses of H2O2 enhanced the antioxidant status and induced stress tolerance, while higher concentrations caused oxidative stress and symptoms resembling a hypersensitive response. In stress-tolerant plants, induction of catalase was 1.5-fold, that of ascorbate peroxidase and glutathione peroxidase was 2-fold, and that of guaiacol peroxidases was approximately 3-fold. Stress resistance was monitored by measuring levels of malondialdehyde, an indicator of lipid peroxidation. The levels of malondialdehyde in all H2O2-treated plants exposed to subsequent high light or AT stress were similar to those of unstressed plants, whereas lipid peroxidation in H2O2-untreated plants stressed with either high light or AT was 1.5- or 2-fold higher, respectively. Although all stress factors caused increases in the levels of reduced glutathione, its levels were much higher in all H2O2-pretreated plants. Moreover, significant accumulation of oxidized glutathione was observed only in plants that were not pretreated with H2O2. Extending the AT stress period from 1 to 7 days resulted in death of tobacco plants that were not pretreated with H2O2, while all H2O2-pretreated plants remained little affected by the prolonged treatment. Thus, activation of the plant antioxidant system by H2O2 plays an important role in the induced tolerance against oxidative stress.

Summary In greening etiolated primary leaves of barley (Hordeum vulgare L.), Mn2+ ions have been shown to inhibit chlorophyll (Chl) accumulation in a dose dependent manner and to lead to an accumulation of protoporphyrin IX (Proto) and Mg-protoporphyrin IX monomethyl ester (MgPE). The amount of MgPE that accumulated, was 2 times higher than Proto. In the dark, Proto and MgPE were observed to have accumulated to high levels in seven-day old green and etiolated leaves in the presence of 5 mmol/L Mn2+, but only if 5 mmol/L δ-aminolevulinic acid (ALA) was present. The 24 hours of irradiation of the green barley leaves treated in this way, resulted in a photodynamic destruction of Proto and MgPE as well as of Chl and carotenoids (Car). The observed porphyrin accumulation caused by the Mn2+ ions was reversed in the presence of active iron (Fe2+). This effect was observed when the iron concentration in incubation solutions was half the Mn2+ concentration, most effective for porphyrin synthesis, i.e. 5 mmol/L. The action of Mn2+ on porphyrin accumulation is also discussed.

The photodynamic damage of the sensitive plants wheat and mustard, treated with chlorophyll (Chl) precursors 5-aminolevulinic acid (ALA) and glutamic acid (Glu) and with 1,10-phenanthroline (Phen), was caused by tetrapyrroles, which accumulated after 17 h in the dark period, followed by 12 h of irradiation with "white light". The effect of accumulated Chl in mustard plants was accompanied by changes in the amounts of the Chls and carotenoids and by dehydration of the tissues, partial chlorosis, and necrosis. The molecular nature of the specific photodynamic sensitivity of the mustard and wheat plants under the influence of Phen and Chl precursors was important: accumulation of tetrapyrroles was a necessary, but not only reason for photodynamic damage of the plants. The degree of leaf damage was related to the amount and chemical nature of accumulated tetrapyrroles and to the greening group to which the investigated plant belongs.

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.

Plants use programmed cell death (PCD) as a tool in their growth and development. PCD is also involved in defense against different kinds of stresses including pathogen attack. In both types of PCD, reactive oxygen species (ROS) play an important role. ROS is not only a toxic by-product but also acts as signaling molecule that can trigger defense mechanisms in plant. Earlier transcriptome studies indicated the activation of ROS responsive and/or generating genes. Eight genes were selected with a potential role in ROS-induced PCD pathway and one of them, encoding the zinc finger protein ZAT11 (Zinc Arabidopsis thaliana11) exhibited an altered cell death phenotype. Two independent zat11 mutants in a loh2 (LAG one homologue2) genetic background showed enhanced tolerance to paraquat-induced oxidative stress and PCD; whereas, these double mutants exhibited cell death triggered by Alternaria alternata f. sp. lycopersici-toxin (AAL-toxin) or 3-aminotriazole (AT). This indicates that ZAT11 is involved in an intricate oxidative stress-induced PCD network, and the final outcome depends on ZAT11 interactions with other players specific for the particular types of oxidative stress.

A genetic approach is described to isolate mutants more tolerant to oxidative stress. A collection of T-DNA activation tag Arabidopsis thaliana mutant lines was screened for survivors under conditions that trigger H2O2-induced cell death. Oxidative stress was induced by applying the catalase (CAT) inhibitor aminotriazole (AT) in the growth media, which results in decrease in CAT enzyme activity, H2O2 accumulation, and subsequent plant death. One mutant was recovered from the screening and named oxr1 (oxidative stress resistant 1). The location of the T-DNA insertion was identified by TAIL-PCR. Oxr1 exhibited lack of cell death symptoms and more fresh weight and chlorophyll content compared to wild type. The lack of cell death correlated with more prominent induction of anthocyanins synthesis in oxr1. These results demonstrate the feasibility of AT as a screening agent for the isolation of oxidative stress-tolerant mutants and indicate a possible protective role for anthocyanins against AT-induced cell death. The chapter includes protocols for ethyl methanesulfonate mutagenesis, mutant screening using AT, T-DNA identification by TAIL-PCR, CAT activity measurements, and determination of malondialdehyde, chlorophyll, and anthocyanins.

Haberlea rhodopensis is a glacial relic with impressive tolerance to desiccation and freezing stress. It is mainly known as an ornamental plant and its other potential uses were largely neglected. Transcriptome analyses by next generation sequencing and cDNA–AFLP technologies identified transcription factors, stress-related and novel genes that could contribute to drought tolerance. These recent molecular studies have raised possibilities of gene discovery for crop improvement and established H. rhodopensis as one of the models to study desiccation tolerance. Furthermore, the abundant secondary metabolites of H. rhodopensis are rich sources of compounds with medicinal properties. Extracts from Haberlea possess radioprotective, anticlastogenic and antioxidant activities and can stimulate regeneration of human fibroblasts in vitro. All this has rejuvenated the interest in H. rhodopensis and indicated potential applications of this species in biology and medicine.

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.

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 down-regulated 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. (c) 2008 Elsevier Inc. All rights reserved.

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.

Reactive oxygen species (ROS) are known as toxic metabolic products in plants and other aerobic organisms. An elaborate and highly redundant plant ROS network, composed of antioxidant enzymes, antioxidants and ROS-producing enzymes, is responsible for maintaining ROS levels under tight control. This allows ROS to serve as signaling molecules that coordinate an astonishing range of diverse plant processes. The specificity of the biological response to ROS depends on the chemical identity of ROS, intensity of the signal, sites of production, plant developmental stage, previous stresses encountered and interactions with other signaling molecules such as nitric oxide, lipid messengers and plant hormones. Although many components of the ROS signaling network have recently been identified, the challenge remains to understand how ROS-derived signals are integrated to eventually regulate such biological processes as plant growth, development, stress adaptation and programmed cell death.

Transcriptomic Footprints Disclose Specificity of Reactive Oxygen Species Signaling in Arabidopsis1,[W] Ilya Gadjev2, Sandy Vanderauwera2, Tsanko S. Gechev, Christophe Laloi, Ivan N. Minkov, Vladimir Shulaev, Klaus Apel, Dirk Inzé, Ron Mittler and Frank Van Breusegem* Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, B–9052 Gent, Belgium (I.G., S.V., D.I., F.V.B.); Department of Plant Physiology and Plant Molecular Biology, University of Plovdiv, Plovdiv 4000, Bulgaria (I.G., T.S.G., I.N.M.); Department Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, NL–9751 NN Haren, The Netherlands (T.S.G.); Institute of Plant Sciences, Plant Genetics, Swiss Federal Institute of Technology, CH–8092 Zurich, Switzerland (C.L., K.A.); Department of Biochemistry, University of Nevada, Reno, Nevada 89557 (R.M.); and Virginia Bioinformatics Institute, Blacksburg, Virginia 24061 (V.S.) Reactive oxygen species (ROS) are key players in the regulation of plant development, stress responses, and programmed cell death. Previous studies indicated that depending on the type of ROS (hydrogen peroxide, superoxide, or singlet oxygen) or its subcellular production site (plastidic, cytosolic, peroxisomal, or apoplastic), a different physiological, biochemical, and molecular response is provoked. We used transcriptome data generated from ROS-related microarray experiments to assess the specificity of ROS-driven transcript expression. Data sets obtained by exogenous application of oxidative stress-causing agents (methyl viologen, Alternaria alternata toxin, 3-aminotriazole, and ozone) and from a mutant (fluorescent) and transgenic plants, in which the activity of an individual antioxidant enzyme was perturbed (catalase, cytosolic ascorbate peroxidase, and copper/zinc superoxide dismutase), were compared. In total, the abundance of nearly 26,000 transcripts of Arabidopsis (Arabidopsis thaliana) was monitored in response to different ROS. Overall, 8,056, 5,312, and 3,925 transcripts showed at least a 3-, 4-, or 5-fold change in expression, respectively. In addition to marker transcripts that were specifically regulated by hydrogen peroxide, superoxide, or singlet oxygen, several transcripts were identified as general oxidative stress response markers because their steady-state levels were at least 5-fold elevated in most experiments. We also assessed the expression characteristics of all annotated transcription factors and inferred new candidate regulatory transcripts that could be responsible for orchestrating the specific transcriptomic signatures triggered by different ROS. Our analysis provides a framework that will assist future efforts to address the impact of ROS signals within environmental stress conditions and elucidate the molecular mechanisms of the oxidative stress response in plants.

Hydrogen peroxide is a major regulator of plant programmed cell death (PCD) but little is known about the downstream genes from the H(2)O(2)-signaling network that mediate the cell death. To address this question, a novel system for studying H(2)O(2)-induced programmed cell death in Arabidopsis thaliana was used. The catalase inhibitor aminotriazole (AT) reduced the catalase activity and caused endogenous accumulation of hydrogen peroxide that eventually triggered cell death. Microarray analysis with a DNA chip representing 21500 genes and subsequent comparison with other PCD-related expression studies revealed a set of new H(2)O(2)-responsive genes that were highly regulated in a common fashion during different types of PCD. These included an oxoglutarate-dependent dioxygenase and various oxidoreductases, the transcription factors Zat11, WRKY75 and NAM, proteasomal components, a heterologous group of genes with diverse functions, and genes encoding proteins with unknown functions. Knockout lines of the oxoglutarate-dependent dioxygenase exhibited significantly reduced death symptoms and chlorophyll loss upon H(2)O(2)-induced cell death, indicating a role for this gene in the cell death network.

Host-selective mycotoxins (HSTs) are various secondary metabolites or proteinaceous compounds secreted by pathogenic necrotrophic fungi that feed off on dead tissues of certain plants. Research on the HSTs has not only fundamental but also practical importance. On one hand they are implicated in the onset of devastating crop diseases. On the other hand, they have been studied as a good model for revealing the intricate mechanisms of plant-pathogen interactions. At the cellular level, HSTs target different compartments and in most instances induce programmed cell death (PCD) by a wide range of mechanisms. Often the responses provoked by HSTs resemble the effector-triggered immunity used by plant cells to combat biotrophic pathogens, which suggests that HST-producing fungi exploit the plants' own defensive systems to derive benefits. Although by definition HSTs are active only in tissues of susceptible plant genotypes, it has been demonstrated that some of them are able to influence animal cells as well. The possible effects, like cytotoxicity or cytostasis, can be harmful or beneficial and thus HSTs may either pose a health risk for humans and livestock, or be of prospective use in the fields of pharmacology, medicine and agriculture.

Leaf senescence is the final stage of leaf development and is induced by the gradual occurrence of age-related changes (ARCs). The process of leaf senescence has been well described, but the cellular events leading to this process are still poorly understood. By analysis of progressively ageing, but not yet senescing, Arabidopsis thaliana rosette leaves, we aimed to better understand processes occurring prior to the onset of senescence. Using gene expression analysis, we found that as leaves mature, genes responding to oxidative stress and genes involved in stress hormone biosynthesis and signalling were up-regulated. A decrease in primary metabolites that provide protection against oxidative stress was a possible explanation for the increased stress signature. The gene expression and metabolomics changes occurred concomitantly to a decrease in drought, salinity, and dark stress tolerance of individual leaves. Importantly, stress-related genes showed elevated expression in the early ageing mutant old5 and decreased expression in the delayed ageing mutant ore9. We propose that the decreased stress tolerance with age results from the occurrence of senescence-inducing ARCs that is integrated into the leaf developmental programme, and that this ensures a timely and certain death., Intrinsic stress levels increase as part of the normal leaf developmental programme and this helps the plant to decide which leaves to sacrifice for whole-plant survival during environmental stress.

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.

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.

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.