Assessment of oxidative stress response genes in Avicennia marina exposed to oil contamination - Polyphenol oxidase (PPOA) as a biomarker.

Mangrove plants, which inhabit and form sensitive ecosystems in the intertidal zones of tropical and subtropical coastlines, though vulnerable to petroleum pollution, still maintain their growth under oil contamination. To elucidate the molecular response of mangrove plants to crude oil-sediment mixture, seeds of Avicennia marina were planted and grown on 0, 2.5, 5.0, 7.5 and10 % (w/w) oil-contaminated soil. Plant biomass was highly affected from 3.05 ± 0.28 (Control) to 0.50 ± .07 (10 %) and from 3.47 ± 0.12 to 1.88 ± 0.08 in 2 and 4 months old plants respectively. The expression analysis of 11genes belonging to detoxification pathways in the roots and leaves of 2 and 4 month-old plants was evaluated by qRT-PCR. Our results showed changes in expression levels of Fe-SOD, Mn-SOD, CAT, PRX, PPOs, GSTs, and NAP2 whose products are involved in reactive oxygen species (ROS) and xenobiotic detoxification. PPOA showed the highest expression induction of 43 ± 1.15, followed by CAT (12.61 ± 3.25) and PPOB (6.38 ± 1.34) in leaves of 2 months old seedlings grown on 7.5, 10 and 7.5 % oil contaminated soil respectively. PPOA (39.23 ± 2.1), PRX (32.13 ± 1.2) as well as PPOB (26.11 ± 1.3) showed the highest expression induction in leaves of 4 months old plants grown in 2.5 % oil contaminated soil. Our data indicated that PPOA can be a good biomarker candidate gene for long term exposure to oil contamination in A. marina.

Arabidopsis mutants impaired in glutathione biosynthesis exhibit higher sensitivity towards the glucosinolate hydrolysis product allyl-isothiocyanate.

Upon tissue damage the plant secondary metabolites glucosinolates can generate various hydrolysis products, including isothiocyanates (ITCs). Their role in plant defence against insects and pest and their potential health benefits have been well documented, but our knowledge regarding the endogenous molecular mechanisms of their effect in plants is limited. Here we investigated the effect of allyl-isothiocyanate (AITC) on Arabidopsis thaliana mutants impaired in homeostasis of the low-molecular weight thiol glutathione. We show that glutathione is important for the AITC-induced physiological responses, since mutants deficient in glutathione biosynthesis displayed a lower biomass and higher root growth inhibition than WT seedlings. These mutants were also more susceptible than WT to another ITC, sulforaphane. Sulforaphane was however more potent in inhibiting root growth than AITC. Combining AITC with the glutathione biosynthesis inhibitor L-buthionine-sulfoximine (BSO) led to an even stronger phenotype than observed for the single treatments. Furthermore, transgenic plants expressing the redox-sensitive fluorescent biomarker roGFP2 indicated more oxidative conditions during AITC treatment. Taken together, we provide genetic evidence that glutathione plays an important role in AITC-induced growth inhibition, although further studies need to be conducted to reveal the underlying mechanisms.

Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation.

In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

Allyl-isothiocyanate treatment induces a complex transcriptional reprogramming including heat stress, oxidative stress and plant defence responses in Arabidopsis thaliana.

BACKGROUND: Isothiocyanates (ITCs) are degradation products of the plant secondary metabolites glucosinolates (GSLs) and are known to affect human health as well as plant herbivores and pathogens. To investigate the processes engaged in plants upon exposure to isothiocyanate we performed a genome scale transcriptional profiling of Arabidopsis thaliana at different time points in response to an exogenous treatment with allyl-isothiocyanate. RESULTS: The treatment triggered a substantial response with the expression of 431 genes affected (P < 0.05 and log2 ≥ 1 or ≤ -1) already after 30 min and that of 3915 genes affected after 9 h of exposure, most of the affected genes being upregulated. These are involved in a considerable number of different biological processes, some of which are described in detail: glucosinolate metabolism, sulphate uptake and assimilation, heat stress response, oxidative stress response, elicitor perception, plant defence and cell death mechanisms. CONCLUSION: Exposure of Arabidopsis thaliana to vapours of allyl-isothiocyanate triggered a rapid and substantial transcriptional response affecting numerous biological processes. These include multiple stress stimuli such as heat stress response and oxidative stress response, cell death and sulphur secondary defence metabolism. Hence, effects of isothiocyanates on plants previously reported in the literature were found to be regulated at the gene expression level. This opens some avenues for further investigations to decipher the molecular mechanisms underlying the effects of isothiocyanates on plants.

The IDA/IDA-LIKE and PIP/PIP-LIKE gene families in Arabidopsis: phylogenetic relationship, expression patterns, and transcriptional effect of the PIPL3 peptide.

Peptide ligands play crucial roles in the life cycle of plants by modulating the innate immunity against pathogens and regulating growth and developmental processes. One well-studied example is INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which controls floral organ abscission and lateral root emergence in Arabidopsis thaliana. IDA belongs to a family of five additional IDA-LIKE (IDL) members that have all been suggested to be involved in regulation of Arabidopsis development. Here we present three novel members of the IDL subfamily and show that two of them are strongly and rapidly induced by different biotic and abiotic stresses. Furthermore, we provide data that the recently identified PAMP-INDUCED SECRETED PEPTIDE (PIP) and PIP-LIKE (PIPL) peptides, which show similarity to the IDL and C-TERMINALLY ENCODED PEPTIDE (CEP) peptides, are not only involved in innate immune response in Arabidopsis but are also induced by abiotic stress. Expression patterns of the IDA/IDL and PIP/PIPL genes were analysed using in silico data, qRT-PCR and GUS promoter lines. Transcriptomic responses to PIPL3 peptide treatment suggested a role in regulation of biotic stress responses and cell wall modification.

Disintegration of microtubules in Arabidopsis thaliana and bladder cancer cells by isothiocyanates.

Isothiocyanates (ITCs) from biodegradation of glucosinolates comprise a group of electrophiles associated with growth-inhibitory effects in plant- and mammalian cells. The underlying modes of action of this feature are not fully understood. Clarifying this has involved mammalian cancer cells due to ITCs' chemopreventive potential. The binding of ITCs to tubulins has been reported as a mechanism by which ITCs induce cell cycle arrest and apoptosis. In the present study we demonstrate that ITCs disrupt microtubules in Arabidopsis thaliana contributing to the observed inhibited growth phenotype. We also confirmed this in rat bladder cancer cells (AY-27) suggesting that cells from plant and animals share mechanisms by which ITCs affect growth. Exposure of A. thaliana to vapor-phase of allyl ITC (AITC) inhibited growth and induced a concurrent bleaching of leaves in a dose-dependent manner. Transcriptional analysis was used to show an upregulation of heat shock-genes upon AITC-treatment. Transgenic A. thaliana expressing GFP-marked α-tubulin was employed to show a time- and dose-dependent disintegration of microtubules by AITC. Treatment of AY-27 with ITCs resulted in a time- and dose-dependent decrease of cell proliferation and G2/M-arrest. AY-27 transiently transfected to express GFP-tagged α-tubulin were treated with ITCs resulting in a loss of microtubular filaments and the subsequent formation of apoptotic bodies. In conclusion, our data demonstrate an ITC-induced mechanism leading to growth inhibition in A. thaliana and rat bladder cancer cells, and expose clues to the mechanisms underlying the physiological role of glucosinolates in vivo.

System responses to equal doses of photosynthetically usable radiation of blue, green, and red light in the marine diatom Phaeodactylum tricornutum.

Due to the selective attenuation of solar light and the absorption properties of seawater and seawater constituents, free-floating photosynthetic organisms have to cope with rapid and unpredictable changes in both intensity and spectral quality. We have studied the transcriptional, metabolic and photo-physiological responses to light of different spectral quality in the marine diatom Phaeodactylum tricornutum through time-series studies of cultures exposed to equal doses of photosynthetically usable radiation of blue, green and red light. The experiments showed that short-term differences in gene expression and profiles are mainly light quality-dependent. Transcription of photosynthesis-associated nuclear genes was activated mainly through a light quality-independent mechanism likely to rely on chloroplast-to-nucleus signaling. In contrast, genes encoding proteins important for photoprotection and PSII repair were highly dependent on a blue light receptor-mediated signal. Changes in energy transfer efficiency by light-harvesting pigments were spectrally dependent; furthermore, a declining trend in photosynthetic efficiency was observed in red light. The combined results suggest that diatoms possess a light quality-dependent ability to activate photoprotection and efficient repair of photodamaged PSII. In spite of approximately equal numbers of PSII-absorbed quanta in blue, green and red light, the spectral quality of light is important for diatom responses to ambient light conditions.

NEVERSHED and INFLORESCENCE DEFICIENT IN ABSCISSION are differentially required for cell expansion and cell separation during floral organ abscission in Arabidopsis thaliana.

Floral organ shedding is a cell separation event preceded by cell-wall loosening and generally accompanied by cell expansion. Mutations in NEVERSHED (NEV) or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) block floral organ abscission in Arabidopsis thaliana. NEV encodes an ADP-ribosylation factor GTPase-activating protein, and cells of nev mutant flowers display membrane-trafficking defects. IDA encodes a secreted peptide that signals through the receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2). Analyses of single and double mutants revealed unique features of the nev and ida phenotypes. Cell-wall loosening was delayed in ida flowers. In contrast, nev and nev ida mutants displayed ectopic enlargement of abscission zone (AZ) cells, indicating that cell expansion alone is not sufficient to trigger organ loss. These results suggest that NEV initially prevents precocious cell expansion but is later integral for cell separation. IDA is involved primarily in the final cell separation step. A mutation in KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1), a suppressor of the ida mutant, could not rescue the abscission defects of nev mutant flowers, indicating that NEV-dependent activity downstream of KNAT1 is required. Transcriptional profiling of mutant AZs identified gene clusters regulated by IDA-HAE/HSL2. Several genes were more strongly downregulated in nev-7 compared with ida and hae hsl2 mutants, consistent with the rapid inhibition of organ loosening in nev mutants, and the overlapping roles of NEV and IDA in cell separation. A model of the crosstalk between the IDA signalling pathway and NEV-mediated membrane traffic during floral organ abscission is presented.

Genome-wide profiling of responses to cadmium in the diatom Phaeodactylum tricornutum.

The only group of organisms in which a biological function for cadmium has been shown is the diatoms, which are unicellular phytoplankton. Yet diatoms exhibit similar sensitivity to Cd as other groups of phytoplankton. We have investigated responses of Cd on molecular, metabolic, and physiological levels in the diatom Phaeodactylum tricornutum. P. tricornutum apparently has a high tolerance to Cd; only minor responses were observed on growth, pigment, and transcriptional changes at cadmium concentrations of 123 µg/L. No significant changes in chlorophyll and xanthophyll levels were observed, and the very few transcripts affected strongly indicate that the cells were able to respond to the increased Cd(2+) levels without changing proteins levels. At 10 times this concentration, 1230 µg/L, a much clearer response was observed, including transcripts encoding proteins involved in metal transport, cell signaling, and detoxification processes. Our results point toward putative pathways for the removal or detoxification of Cd and its metabolites as well as a possible Cd uptake mechanism. We predict that ATPase5-1B is involved in removal of Cd by pumping Cd(2+) ions out of the cell, whereas VIT1/CCC1 sequesters Cd(2+) in the vacuole.

Phytochemicals of Brassicaceae in plant protection and human health--influences of climate, environment and agronomic practice.

In this review, we provide an overview of the role of glucosinolates and other phytochemical compounds present in the Brassicaceae in relation to plant protection and human health. Current knowledge of the factors that influence phytochemical content and profile in the Brassicaceae is also summarized and multi-factorial approaches are briefly discussed. Variation in agronomic conditions (plant species, cultivar, developmental stage, plant organ, plant competition, fertilization, pH), season, climatic factors, water availability, light (intensity, quality, duration) and CO(2) are known to significantly affect content and profile of phytochemicals. Phytochemicals such as the glucosinolates and leaf surface waxes play an important role in interactions with pests and pathogens. Factors that affect production of phytochemicals are important when designing plant protection strategies that exploit these compounds to minimize crop damage caused by plant pests and pathogens. Brassicaceous plants are consumed increasingly for possible health benefits, for example, glucosinolate-derived effects on degenerative diseases such as cancer, cardiovascular and neurodegenerative diseases. Thus, factors influencing phytochemical content and profile in the production of brassicaceous plants are worth considering both for plant and human health. Even though it is known that factors that influence phytochemical content and profile may interact, studies of plant compounds were, until recently, restricted by methods allowing only a reductionistic approach. It is now possible to design multi-factorial experiments that simulate their combined effects. This will provide important information to ecologists, plant breeders and agronomists.

Plant peptides in signalling: looking for new partners.

A novel candidate ligand-receptor system, INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and the related receptor-like kinases (RLKs) HAESA (HAE) and HAESA-LIKE (HSL)2, has been shown to control floral abscission in Arabidopsis thaliana. Furthermore, several IDA-LIKE (IDL) proteins, which contain a conserved C-terminal domain resembling that of the CLAVATA (CLV)3-ENDOSPERM SURROUNDING REGION (ESR)-RELATED (CLE) protein family, have been shown to be partially redundant with IDA. Here, we use the genetic similarities between the IDA and CLV3 signalling systems to hypothesize that closely related peptide ligands are likely to interact with families of closely related RLKs. Guided by this hypothesis and with the aid of genetics and novel methods, ligand-receptor systems can be identified to improve our understanding of developmental processes in plants.

The FRO2 ferric reductase is required for glycine betaine's effect on chilling tolerance in Arabidopsis roots.

FRO2 (At1g01580) codes for an NADPH-dependent ferric reductase in plasma membranes of root epidermal cells with a demonstrated role in iron uptake by plants. Ferric reductase activity has been shown to be the rate-limiting step for iron uptake in strategy I plants like Arabidopsis and in rice, but it has been unclear whether FRO genes have other physiological functions. We hypothesized that FRO2 was involved in chilling stress tolerance because its expression was upregulated by treatment of plants with glycine betaine (GB), a chemical that prevents reactive oxygen species (ROS) signaling in chilling stress. This idea was confirmed by showing that the FRO2 null mutant frd1-1 failed to respond to GB in chilling assays either in relation to root growth recovery or inhibition of ROS accumulation. Measurements of ferric reductase activity in wild-type plants treated with GB before chilling showed no significant GB effect compared with controls. In addition, 35S-FRO2 transgenics with elevated mRNA levels did not have improved chilling tolerance. However, ferric reductase activity in wild-type plants or 35S-FRO2 transgenics pretreated with GB was several-fold higher after chilling compared with non-pretreated controls. These experiments identify a new physiological function for FRO2, i.e. blocking ROS accumulation during chilling. They also suggest that GB has a major effect on FRO2 activity posttranscriptionally in the cold.

The cabbage aphid: a walking mustard oil bomb.

The cabbage aphid, Brevicoryne brassicae, has developed a chemical defence system that exploits and mimics that of its host plants, involving sequestration of the major plant secondary metabolites (glucosinolates). Like its host plants, the aphid produces a myrosinase (beta-thioglucoside glucohydrolase) to catalyse the hydrolysis of glucosinolates, yielding biologically active products. Here, we demonstrate that aphid myrosinase expression in head/thoracic muscle starts during embryonic development and protein levels continue to accumulate after the nymphs are born. However, aphids are entirely dependent on the host plant for the glucosinolate substrate, which they store in the haemolymph. Uptake of a glucosinolate (sinigrin) was investigated when aphids fed on plants or an in vitro system and followed a different developmental pattern in winged and wingless aphid morphs. In nymphs of the wingless aphid morph, glucosinolate level continued to increase throughout the development to the adult stage, but the quantity in nymphs of the winged form peaked before eclosion (at day 7) and subsequently declined. Winged aphids excreted significantly higher amounts of glucosinolate in the honeydew when compared with wingless aphids, suggesting regulated transport across the gut. The higher level of sinigrin in wingless aphids had a significant negative impact on survival of a ladybird predator. Larvae of Adalia bipunctata were unable to survive when fed adult wingless aphids from a 1% sinigrin diet, but survived successfully when fed aphids from a glucosinolate-free diet (wingless or winged), or winged aphids from 1% sinigrin. The apparent lack of an effective chemical defence system in adult winged aphids possibly reflects their energetic investment in flight as an alternative predator avoidance mechanism.

The enzymic and chemically induced decomposition of glucosinolates.

While the myrosinase-glucosinolate system has been reviewed in recent years by a number of authors, little attention has been paid to the enzymic and non-enzymic degradation of glucosinolates. Non-enzymic degradation processes are particularly important in the processing of brassica vegetables with respect to both flavour and in the role of glucosinolates as precursors of anticancer compounds in the diet. This review highlights early empirical work on glucosinolate degradation along with more recent aspects related to current research on mechanism of glucosinolate degradation in plants, microbes and animals.

Expression and occurrence of uracil-DNA glycosylase in higher plants.

Uracil-DNA glycosylase (UDG) is the first enzyme in the base excision repair pathway for removal of uracil in DNA. DNA repair capacity is likely to be a critical factor in mutagenesis and thereby in the capacity to prevent genetic damage and unwanted variation. We have studied expression of UDG in 9 higher plant species. The highest expression of UDG was measured in Solanum tuberosum. A comparison of 6 Solanum tuberosum cultivars showed that the specific activity ranged from 30 pmol mg1 protein min-1 in the cultivar Laila to 80 pmol mg-1 protein min-1 in the cultivar Ostara. Measurement of UDG in Begonia X cheimantha gave no indications of enzyme activity. The possible effects of no or low UDG activity is discussed. In vitro cultures of Solanum tuberosum and Thymus vulgaris were used to examine the effect of auxin and cytokinin on the UDG activity. Axillary shoots of Solanum tuberosum were cultured on medium including 20 variations in hormone concentration. Auxin (1-naphtaleneacetic acid) increased the expression of UDG. Plants cultured on medium supplemented with 3 mg 1-1 1-naphtaleneacetic acid showed a specific UDG activity which was approximately 3-fold higher than the activity in controls. The cytokinin benzyladenine reduced the specific UDG activity at concentrations in the range 0.25-10 mg 1-1 . In vitro cultured Saintpaulia ionantha was used to examine UDG activity during initiation, conditioning and multiplication cycles. In general, highest expression of UDG was measured in the conditioning cycle on hormone free medium. Measurement of UDG expression during single subculture periods, clearly showed that UDG expression may vary over one culture period. Expression of UDG was in general highest three weeks after transfer to fresh medium. Of different seedling organs from 0- to 15-day-old Brassica napus L., roots and hypocotyls showed the highest UDG activities. In cotyledons a very low and nearly constant specific activity was observed. In 12-day-old seedlings the activity in roots was approximately 20 times higher than the activity in cotyledons.