Christophe Bailly

Active oxygen species and antioxidants in seed biology

Active oxygen species (AOS) are involved in various aspects of seed physiology. Their generation, which occurs during seed desiccation, germination and ageing, may lead to oxidative stress and cellular damage, resulting in seed deterioration. However, cells are endowed with detoxifying enzymes and antioxidant compounds that scavenge AOS and participate in seed survival. The detoxifying mechanisms play a key role in acquisition of desiccation tolerance of developing seeds, completion of seed germination and seed storability. However, AOS must also be regarded as molecules intervening in cellular signalling. They are involved in growth processes occurring at early embryogenesis during seed development, and participate in the mechanisms underlying radicle protrusion during seed germination. AOS might also have a regulatory function in the changes in gene expression during seed development, dormancy and germination. Their interplay with other molecules, particularly with hormones such as abscisic acid, suggests that they should be considered as key components of an integrated signalling network involved in many aspects of seed physiology.

Changes in activities of antioxidant enzymes and lipoxygenase during growth of sunflower seedlings from seeds of different vigour

The aim of this study was to investigate whether there was a relationship between growth of sunflower seedlings at 15°C in the dark and activities of enzymes involved in scavenging of reactive oxygen species (ROS), especially superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), or in production of free radicals, namely lipoxygenase (LOX). Untreated control seeds were compared with seeds exposed to accelerated ageing (5 d at 45°C and 100% relative humidity), osmopriming (7 d at 15°C with a polyethylene glycol (PEG) solution at –2 MPa) and accelerated ageing followed by priming. Accelerated ageing decreased seed germinability and slowed down hypocotyl growth, whereas priming resulted in an increase in germination rate and enhanced seedling development. Osmopriming of aged seeds almost completely restored the initial rate of germination and seedling growth. The activity of all the enzymes studied increased during seed germination and seedling development, except that of SOD. Seed imbibition or radicle protrusion were related mainly with an increase in CAT activity and, to a lesser extent, in GR activity. Increase of LOX activity was clearly associated with the onset of hypocotyl elongation. However, in all cases, malondialdehyde measurements did not reveal intense lipid peroxidation. Priming induced a marked stimulation of CAT and GR during seed imbibition or very early during seedling development, as compared to the control seedlings and particularly to the seedlings generated by aged seeds. Hydrogen peroxide (H2O2) contents of seeds and seedlings were closely correlated to the activities of CAT and GR and to the kinetics of seedling development. The results obtained establish a clear relationship between sunflower seed vigour and ROS scavenging.

Role of Reactive Oxygen Species in the Regulation of Arabidopsis Seed Dormancy

Freshly harvested seeds of Arabidopsis thaliana, Columbia (Col) accession were dormant when imbibed at 25°C in the dark. Their dormancy was alleviated by continuous light during imbibition or by 5 weeks of storage at 20°C (after-ripening). We investigated the possible role of reactive oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25°C, non-dormant seeds produced more ROS than dormant seeds, and their catalase activity was lower. In situ ROS localization revealed that germination was associated with an accumulation of superoxide and hydrogen peroxide in the radicle. ROS production was temporally and spatially regulated: ROS were first localized within the cytoplasm upon imbibition of non-dormant seeds, then in the nucleus and finally in the cell wall, which suggests that ROS play different roles during germination. Imbibition of dormant and non-dormant seeds in the presence of ROS scavengers or donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly harvested seeds of the mutants defective in catalase (cat2-1) and vitamin E (vte1-1) did not display dormancy; however, seeds of the NADPH oxidase mutants (rbohD) were deeply dormant. Expression of a set of genes related to dormancy upon imbibition in the cat2-1 and vet1-1 seeds revealed that their non-dormant phenotype was probably not related to ABA or gibberellin metabolism, but suggested that ROS could trigger germination through gibberellin signaling activation.

Antioxidant systems in sunflower (Helianthus annuus L.) seeds as affected by priming

Priming treatment of sunflower (Helianthus annuus L., cv Briosol) seeds for 7 days at 15 ° C with a polyethylene glycol solution at −2.0 MPa strongly improved their subsequent germination at 15 ° C on water. This stimulatory effect of priming remained after drying back the seeds at 20 ° C for 3 days. Malondialdehyde (MDA) and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were measured in control unprimed seeds, primed seeds, seeds primed then dried, and after 3 and 6 hours of imbibition of controland dried primed seeds in order to determine whether the cell antioxidant systems were involved in the beneficial effect of priming. The osmotreatment resulted in a strong increase in SOD and CAT activities but did not markedly affect MDA and GR activity. Following the 3 days of drying, MDA increased and the enzyme activities became similar to those measured in dry unprimed seeds, although the stimulatory effect of priming on germination remained. Imbibition of control dry seeds was associated with an increase in MDA and a decrease in CAT and GR activities, whereas reimbibition of dried primed seeds resulted in a decrease in MDA and an increase in SOD, CAT and GR activities. Isoform patterns on native gels showed no difference between treated (priming with or without subsequent drying) and control seeds for SOD (7 isoforms) and GR (5 isoforms), but the osmotreatment did induce a second isoform of CAT. The results obtained indicate that the CAT isoform pattern might be used as an indicato of the priming treatment that promotes germination. Involvement of theantioxidant systems in seed vigour is discussed

Ethylene, a key factor in the regulation of seed dormancy

Ethylene is an important component of the gaseous environment, and regulates numerous plant developmental processes including seed germination and seedling establishment. Dormancy, the inability to germinate in apparently favorable conditions, has been demonstrated to be regulated by the hormonal balance between abscisic acid (ABA) and gibberellins (GAs). Ethylene plays a key role in dormancy release in numerous species, the effective concentrations allowing the germination of dormant seeds ranging between 0.1 and 200 μL L-1. Studies using inhibitors of ethylene biosynthesis or of ethylene action and analysis of mutant lines altered in genes involved in the ethylene signaling pathway (etr1, ein2, ain1, etr1, and erf1) demonstrate the involvement of ethylene in the regulation of germination and dormancy. Ethylene counteracts ABA effects through a regulation of ABA metabolism and signaling pathways. Moreover, ethylene insensitive mutants in Arabidopsis are more sensitive to ABA and the seeds are more dormant. Numerous data also show an interaction between ABA, GAs and ethylene metabolism and signaling pathways. It has been increasingly demonstrated that reactive oxygen species (ROS) may play a significant role in the regulation of seed germination interacting with hormonal signaling pathways. In the present review the responsiveness of seeds to ethylene will be described, and the key role of ethylene in the regulation of seed dormancy via a crosstalk between hormones and other signals will be discussed.

Role of protein and mRNA oxidation in seed dormancy and germination.

Reactive oxygen species (ROS) are key players in the regulation of seed germination and dormancy. Although their regulated accumulation is a prerequisite for germination, the cellular basis of their action remains unknown, but very challenging to elucidate due to the lack of specificity of these compounds that can potentially react with all biomolecules. Among these, nucleic acids and proteins are very prone to oxidative damage. RNA is highly sensitive to oxidation because of its single-stranded structure and the absence of a repair system. Oxidation of mRNAs induces their decay through processing bodies or results in the synthesis of aberrant proteins through altered translation. Depending on the oxidized amino acid, ROS damage of proteins can be irreversible (i.e., carbonylation) thus triggering the degradation of the oxidized proteins by the cytosolic 20S proteasome or can be reversed through the action of thioredoxins, peroxiredoxins, or glutaredoxins (cysteine oxidation) or by methionine sulfoxide reductase (methionine oxidation). Seed dormancy alleviation in the dry state, referred to as after-ripening, requires both selective mRNA oxidation and protein carbonylation. Similarly, seed imbibition of non-dormant seeds is associated with targeted oxidation of a subset of proteins. Altogether, these specific features testify that such oxidative modifications play important role in commitment of the cellular functioning toward germination completion.

Use of the hydrothermal time model to analyse interacting effects of water and temperature on germination of three grass species

The temperate grass species, red fescue (Festuca rubra ssp. litoralis), perennial ryegrass (Lolium perenne) and Kentucky bluegrass (Poa pratensis), are often sown in mixtures for turfgrass. Differences in germination response to environmental conditions often result in different establishment success. The three species were compared in a study of imbibition at reduced water potential (Ψ) and in a study of the effects of Ψ and temperature on germination. In all three species, imbibition rate and extent were reduced with decreasing Ψ, causing a prolonged lag-phase before germination, particularly in Kentucky bluegrass. In perennial ryegrass and Kentucky bluegrass, water content at radicle emergence decreased non-linearly with decreasing Ψ, with a lower asymptote expressing the critical water content for germination. Seeds were germinated in factorial combinations of two temperatures (10 and 25°C) and nine Ψ levels (‐2 to 0 MPa). Kentucky bluegrass required a longer thermal time to germinate than the other species and exhibited conditional dormancy at 25°C, which affected the use of the thermal time model. The effects of Ψ were described by a hydrotime model. Kentucky bluegrass had higher base water potential (Ψ b ) and required a longer hydrotime for germination than the other species. However, Ψ b increased with temperature in Kentucky bluegrass and red fescue, but decreased in perennial ryegrass, and the base temperatures also changed with Ψ. The interacting effects of Ψ and temperature affected the ability of the hydrothermal time model to predict germination performance across all temperature and Ψ conditions.

ROS Signaling in Seed Dormancy Alleviation.

Reactive oxygen species have been suggested to play a signaling role in seed dormancy alleviation. When sunflower seeds become able to fully germinate during dry after-ripening, they accumulate high amount of hydrogen peroxide and exhibit a low detoxifying ability through catalase, resulting from the decrease in CATA1 transcript. ROS accumulation entails oxidative modification of soluble and storage proteins through carbonylation, which suggests that this process might play an important role in plant developmental processes. However other oxidative signaling pathways cannot be excluded. For example, a cDNA-AFLP study shows that seed after-ripening is also associated with changes in gene expression and that changes in ROS content during seed imbibition are also related to changes in expression pattern.

An Endosperm-Associated Cuticle Is Required for Arabidopsis Seed Viability, Dormancy and Early Control of Germination

Cuticular layers and seeds are prominent plant adaptations to terrestrial life that appeared early and late during plant evolution, respectively. The cuticle is a waterproof film covering plant aerial organs preventing excessive water loss and protecting against biotic and abiotic stresses. Cutin, consisting of crosslinked fatty acid monomers, is the most abundant and studied cuticular component. Seeds are dry, metabolically inert structures promoting plant dispersal by keeping the plant embryo in an arrested protected state. In Arabidopsis thaliana seeds, the embryo is surrounded by a single cell endosperm layer itself surrounded by a seed coat layer, the testa. Whole genome analyses lead us to identify cutin biosynthesis genes as regulatory targets of the phytohormones gibberellins (GA) and abscisic acid (ABA) signaling pathways that control seed germination. Cutin-containing layers are present in seed coats of numerous species, including Arabidopsis, where they regulate permeability to outer compounds. However, the role of cutin in mature seed physiology and germination remains poorly understood. Here we identify in mature seeds a thick cuticular film covering the entire outer surface of the endosperm. This seed cuticle is defective in cutin-deficient bodyguard1 seeds, which is associated with alterations in endospermic permeability. Furthermore, mutants affected in cutin biosynthesis display low seed dormancy and viability levels, which correlates with higher levels of seed lipid oxidative stress. Upon seed imbibition cutin biosynthesis genes are essential to prevent endosperm cellular expansion and testa rupture in response to low GA synthesis. Taken together, our findings suggest that in the course of land plant evolution cuticular structures were co-opted to achieve key physiological seed properties.

The effects of abscisic acid and methyl jasmonate on 1-aminocyclopropane 1-carboxylic acid conversion to ethylene in hypocotyl segments of sunflower seedlings, and their control by calcium and calmodulin

The conversion of 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene by hypocotyl segments of sunflower (Helianthus annuus L.) seedlings was inhibited by abscisic acid (ABA) and methyl jasmonate (Me-Ja), and this inhibitory effect increased with increasing concentration of both growth regulators. On the contrary, CaCl, enhanced ACC conversion to ethylene at the concentrations of 10-4 M and 5 x 10-4 M, however lower and higher concentrations had no significant action. CaCl, (5 x 10-4M) seemed to magnify the inhibition of the reaction induced by ABA, whereas it reduced (5 x 10-4M) and even abolished (10-3M) the inhibitory action of Me-Ja. The results obtained with a Ca2+ chelator (EGTA), a Ca2+ channel blocker (nifedipine) and calmodulin antagonists (W7 and TFP), given in association with ABA or Me-Ja, suggested that calcium was involved in the inhibition of ACC conversion to ethylene by ABA and Me-Ja through an interaction with calmodulin. However, the mechanism of action of the two growth regulators seemed to be different, since all treatments which resulted in a decrease in cytosolic Ca2+ concentration or in calmodulin action induced a decrease in the effect of ABA and an increase in the effect of Me-Ja.