Fate and ecotoxicological impact of new generation herbicides from the triketone family: An overview to assess the environmental risks.

Triketones, derived chemically from a natural phytotoxin (leptospermone), are a good example of allelochemicals as lead molecules for the development of new herbicides. Targeting a new and key enzyme involved in carotenoid biosynthesis, these latest-generation herbicides (sulcotrione, mesotrione and tembotrione) were designed to be eco-friendly and commercialized fifteen-twenty years ago. The mechanisms controlling their fate in different ecological niches as well as their toxicity and impact on different organisms or ecosystems are still under investigation. This review combines an overview of the results published in the literature on ß-triketones and more specifically, on the commercially-available herbicides and includes new results obtained in our interdisciplinary study aiming to understand all the processes involved (i) in their transfer from the soil to the connected aquatic compartments, (ii) in their transformation by photochemical and biological mechanisms but also to evaluate (iii) the impacts of the parent molecules and their transformation products on various target and non-target organisms (aquatic microorganisms, plants, soil microbial communities). Analysis of all the data on the fate and impact of these molecules, used pure, as formulation or in cocktails, give an overall guide for the assessment of their environmental risks.

Physiological impacts of soil pollution and arsenic uptake in three plant species: Agrostis capillaris, Solanum nigrum and Vicia faba.

In order to revegetate an industrial soil polluted by trace metals and metalloids (As, Pb, Cu, Cd, Sb), the impact of pollution on three plant species, Solanum nigrum and Agrostis capillaris, both native species in an industrial site, and Vicia faba, a plant model species, is studied. Following the study of soil pollution from the industrial wasteland of Auzon, it appears that the As is the principal pollutant. Particular attention is given to this metalloid, both in its content and its speciation in the soil that the level of its accumulation in plants. In V. faba and A. capillaris, the trace metals and metalloids inhibit the biomass production and involve a lipid peroxidation in the leaves. Furthermore, these pollutants cause a photosynthesis perturbation by stomatal limitations and a dysfunction of photosystem II. Whatever the plant, the As content is less than 0.1 percent of dry matter, the majority of As absorbed is stored in the roots which play the role of trap organ. In parallel, the culture of S. nigrum decreases significantly the exchangeable and weakly adsorbed fraction of As in rhizospheric soil. This study has highlighted the ability of tolerance to trace metals of S. nigrum and to a lesser extent A. capillaris. Our data indicate that V. faba is not tolerant to soil pollution and is not a metallophyte species.

Sequential effects of cadmium on genotoxicity and lipoperoxidation in Vicia faba roots.

Kinetics of stress responses to Cd exposure (50, 100 and 200 µM) expanding from 12 to 48 h were studied in roots of hydroponically cultivated-Vicia faba seedlings. The heavy metal induced toxicity symptoms and growth arrest of Vicia roots gradually to the Cd concentration and duration of the treatment. The intracellular oxidative stress was evaluated with the H(2)O(2) production. The H(2)O(2) content increased gradually with the sequestered Cd and root growth inhibition. Lipid peroxidation-evidenced by malondialdehyde (MDA) content and Evans blue uptake-and genotoxicity-evidenced by mitotic index (MI) and micronuclei (MCN) values-were concomitantly investigated in root tips. By 12 h, root meristematic cells lost 15% of their mitotic activity under 50 or 100 µM Cd treatment and 50% under 200 µM Cd treatment and led cells with MCN, while the MDA content and Evans blue absorption were not affected. The loss of membrane integrity occurred subsequently by 24 h. The increase in MDA content in root cells treated with 50, 100 and 200 µM Cd was significantly higher than the control. By 48 h, the MDA content increased 134, 178 or 208% in root cells treated with 50, 100 and 200 µM Cd, respectively. The Evans blue absorption was also affected by 24 h in roots when treated with 200 µM Cd and gradually increase by 48 h with the Cd concentration of the treatment. The decrease of mitotic activity triggered by 12 h was even higher by 24 h and the MI reduced to 44, 56 or 80% compared to the control in the three different Cd concentrations tested. The different kinetics of early in vivo physiological and cytogenetic responses to Cd might be relevant to the characterization of its toxicity mechanisms in disrupting primarily the mitosis process.

Exposure of Vicia faba and Pisum sativum to copper-induced genotoxicity.

The potential genotoxicity of Cu(2+) was investigated in Vicia faba and Pisum sativum seedlings in hydroponic culture conditions. Cu(2+) caused a dose-dependent increase in micronuclei frequencies in both plant models. Cytological analysis of root tips cells showed clastogenic and aneugenic effects of this heavy metal on V. faba root meristems. Cu(2+) induced chromosomal alterations at the lowest concentration used (2.5 mM) when incubated for 42 h, indicating the potent mutagenic effect of this ion. A spectrum of chromosomal abnormalities was observed in V. faba root meristems, illustrating the genotoxic events leading to micronuclei formation.

Stressed Jerusalem artichoke tubers (Helianthus tuberosus L.) excrete a protein fraction with specific cytotoxicity on plant and animal tumour cell.

Wounds from Jerusalem artichoke (Helianthus tuberosus L.) tubers excrete bioactive metabolites from a variety of structural classes, including proteins. Here we describe a protein specifically active against tumour cells arising either from human, animal or plant tissues. The non-tumour animal cells or the plant callus cells are not sensitive to these excreta. The active product was only obtained after a wound-drought stress of plant tubers. The cytotoxicity varies according to the tumour cell type. For instance, some human tumour cell lines and especially the human mammary tumour cells MDA-MB-231 were shown to be very susceptible to the active product. The active agent is shown to contain an 18-kDa polypeptide with homology to a superoxide dismutase (SOD). A 28-kDa polypeptide, related to an alkaline phosphatase (AP), was shown to be tightly linked to this 18-kDa polypeptide. The excreted 28-kDa polypeptide also displayed a consensus sequence similar to the group of DING proteins, but with a smaller molecular weight. The superoxide dismutase polypeptide was shown to be involved in the antitumour activity, but the presence of smaller factors (MW<10 kDa), such as salicylic acid, can enhance this activity.

Analysis of secreted protease inhibitors after water stress in potato tubers.

Potato tubers (Solanum tuberosum) secrete two kinds of proteinase inhibitors after a water stress. The polypeptides have differing inhibitory activities but are Kunitz-type inhibitors based on amino-terminal sequences homologies. A proteolysis maturation type of a cell protease inhibitor was observed. They can constitute high MW complex, sometimes with another type of protein. The function of these protease inhibitors is discussed in relation to plant defence.

Microwave irradiation affects gene expression in plants.

The physiological impact of nonionizing radiation has long been considered negligible. However, here we use a carefully calibrated stimulation system that mimics the characteristics (isotropy and homogeneity) of electromagnetic fields present in the environment to measure changes in a molecular marker (mRNA encoding the stress-related bZIP transcription factor), and show that low amplitude, short duration, 900 MHz EMF evokes the accumulation of this mRNA. Accumulation is rapid (peaking 5-15 min after stimulation) and strong (3.5-fold), and is similar to that evoked by mechanical stimulations.

Cloning, sequence and characterization of a sunflower (Helianthus annuus L.) pathogen-induced gene showing sequence homology with auxin-induced genes from plants.

The establishment of a plant-pathogen interaction involves changes in gene expressions in both organisms. To isolate Helianthus annuus genes whose expression is induced during processes of resistance to Plasmopara halstedii, a comparison of the expression pattern of healthy sunflowers was made with sunflowers infected with 2 races of P. halstedii, either virulent or avirulent, using differential display of mRNA. A full-length cDNA, HaAC1, representing a sunflower gene whose expression is enhanced during early stages of the incompatible interaction, was isolated. Different timing of RNA accumulation is observed between compatible and incompatible combinations. Sequence analysis and database search revealed significant homology with auxin-induced genes from plants. The expression of this gene, is also induced after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D), salicylic acid (SA) and wounding.

Is membrane potential involved in calmodulin gene expression after external stimulation in plants?

In Bidens pilosa (cv. radiata), a non-injurious stimulus induces a local and transient change in membrane potential, and an injurious stimulus induces a transmitted electrical signal described as the combination of an action potential and a slow wave. We have studied calmodulin gene expression after these stimuli. When the stimulus is non-injurious, calmodulin mRNA accumulation is only increased in the stimulated region. In contrast, when the stimulus is injurious, mRNA accumulation takes place in both wounded and distant, unwounded tissue. We propose that the slow wave plays a role in the long-distance transmission of a wound-induced information in plants.

Mitochondrial DNA RFLP and genetical studies of cytoplasmic male sterility in the sunflower (Helianthus annuus).

Fifteen sunflower (Helianthus annuus L.) cytoplasmic male-sterile, and a single male-fertile, cytotypes were studied by both mtDNA (mitochondrial DNA) restriction fragment length polymorphism (RFLP) and genetical analysis of male-fertility restoration patterns. It was found by multivariate analysis that the two methods of identification of cytoplasmic male sterility (CMS) should be of use in sunflower breeding programs. The RFLP study distinguished 13 groups based on differences in mtDNA organization. DNA molecular diversity occurs both within and between the Helianthus species from which the steriles originate. The mitochondrial genes analyzed present specific molecular configurations for each type of sterility studied. The analysis of male-fertility restoration separated the cytotypes into 12 groups. The associations of CMS and inbred restorer lines indicated the presence of specific nuclear genes involved in cytoplasmic male-sterility restoration.

Molecular cloning and physiological analysis of an invertase isoenzyme in Helianthus tissues.

A soluble acid invertase activity isolated from Helianthus tuberosus (Jerusalem artichoke) shoots and analyzed by immunochromatography using polyclonal yeast antibodies, represents around 5% of the total invertase activity. This invertase isoenzyme was also isolated from dormant tuber parenchyma. In these partially dormant tissues, the specific activity of this isoenzyme is low suggesting a partial inactivation of the invertase molecules. Polyacrylamide gel electrophoresis of immunopurified fractions yields similar levels of the 58 kDa polypeptide both in shoots and dormant tubers, but with much lower activity of the enzyme in the tubers. A cDNA library was constructed in pUEX 1 from poly (A)+ RNA extracted from Jerusalem artichoke tubers. This library was screened for invertase using (i) a Bacillus subtilis invertase DNA probe and (ii) anti-yeast invertase antibodies. A recombinant clone of approximately 1.8 kb size was selected by these two methods. Using Northern blots, a temporal sequence in the expression of invertase gene was observed during the breaking of dormancy with the main level after 8 weeks of cold treatment at 4 degrees C. A 2.5 kb transcript was detected, translation of which would yield a 97 kDa polypeptide representing the precursor of Jerusalem artichoke invertase.

Cytoplasmic male sterility in sunflower: comparison of molecular biology and genetic studies.

The genetics of male fertility restoration and the RFLP of mitochondrial DNA were studied for 16 sunflower cytoplasms (15 male-sterile and a male-fertile). Male fertility restoration/male sterility maintenance patterns distinguished 12 cytotypes. Four cytoplasms were completely unrestored so they were not distinguished genetically. The sunflower lines, tested for their restorer/maintenance reaction, showed that there was a continuous range between 0% and 100% of restorer genotypes according to the CMS considered. Restoration/maintenance patterns indicated that at least some restorer genes are specific to certain CMS. RFLP of mitochondrial DNA revealed specific differences between the cytotypes studied. Three restriction enzymes and 12 probes permitted distinction of 13 cytotypes. No relationship exists between CMS cytotypes and the species from which they originated. For genetical and mitochondrial RFLP studies, phenograms were constructed according to the similarity indexes between cytotypes. Most of the CMS defined by restoration patterns correspond with a restriction fragment pattern of mitochondrial DNA.

Nucleotide sequence and transcriptional analysis of a mitochondrial plasmid from a cytoplasmic male-sterile line of sunflower.

A mitochondrial plasmid of 1,939 bp (P2) from a cytoplasmic male-sterile line of sunflower has been cloned and sequenced. It presents 437 bp of near-perfect homology to the 1.4-kb mitochondrial plasmid P1 from sunflower. Sequences homologous to P2 were found in nuclear DNA. P2 was transcribed into a major 980-nucleotide (nt) RNA molecule and two minor transcripts of 570 and 520 nt. They were all transcribed from the same strand and within the region nonhomologous to P1. A single 5' boundary and three 3' termini were determined for P2 transcripts. The 5' end is similar to a consensus sequence for plant mitochondrial genes. No evidence of translation products can be provided.

Properties and nucleotide sequence of a mitochondrial plasmid from sunflower.

The 1.413 circular supercoiled mitochondrial DNA plasmid P1 from a fertile sunflower line was sequenced, and a series of 160 bp tandemly repeated sequences was observed. The P1 plasmid was detected in both fertile and cytoplasmic male-sterile (CMS) lines, but in different quantities. Two other circular plasmids, P2 and P3, each 1.8 kbp in length, were shown to share common sequences with P1. The mitochondrial plasmid P1 detected homologous sequences in the nuclear DNA of sunflower, but not in chloroplast DNA nor in main band mitochondrial DNA. RNA molecules of about 680 and 550 nucleotides were detected that were complementary to mt plasmid P1.

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny.

A circular supercoiled mitochondrial DNA plasmid P1 (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P2) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P1 was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P1 and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P1 and P2. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.

Translational regulation of protein synthesis during light-induced chloroplast development in Euglena.

Control of gene expression in Euglena was examined during light-induced chloroplast development. Greening was achieved under standard conditions which allowed the synthesis of all plastid proteins in both cytoplasmic and chloroplastic compartments, or under experimentally modified conditions inducing the preferential synthesis of the photosystem II (PSII) light-harvesting antenna or reaction centers. The relative composition of total mRNAs in cellular, cytoplasmic or chloroplastic fractions, as analyzed by their in-vitro translation products in cell-free systems did not significantly change during the in-vivo protein-synthesis processes which are specific to each greening system. By contrast, cytoplasmic polysomal mRNAs extracted during the selective recovery phase of PSII light-harvesting antennae provided a major in-vitro synthesis product of 28 kDa which could correspond to a precursor of the main 26-kDa apoprotein of the light-harvesting chlorophyll a/b protein complex. Similarly, the in-vivo selective synthesis of the 41-kDa and 51-kDa polypeptides of PSII reaction centers was concomitant with an enrichment of plastid polysomes in mRNA species coding for polypeptides of the same molecular weight. These observations confirm that protein synthesis during chloroplast development in Euglena is weakly regulated at the transcription level and they demonstrate that translational regulation occurs in both the cytoplasmic and the chloroplastic compartments.

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi.

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.

[Periodic, metabolic and structural phenomena in a protist, Euglena gracilis]. / Phénomènes périodiques, métaboliques et structuraux chez un protiste, Euglena gracilis.

Sychronous divisions of Euglena gracilis strain Z can be obtained by various methods. When the cells are cultivated in a medium containing lactate as the sole carbon source, synchronous divisions are observed, independent of the conditions of illumination. Nevertheless, there exists a relationship between the phase of cell division and ther periods of light and darkness applied to the culture. During the cell cycle, the synthesis of macromolecules is discontinuous--this is true of nuclear and mitochondrial DNA, ribosomal and nonribosomal RNA, and certain proteins (cytochrome c 558). Cyclic variations in the structure of mitochondria and chloroplasts are observed. In the course of the cell cycle, sequential metabolic processes accompany structural modifications of the organelles. Also, at the beginning of the cycle, at the start of phase G1, the cytoplasmic ribosomes are synthesized, and then, in green euglenids, nonribosomal RNAs are formed. These syntheses of RNA precede enlargement of the chondriome and plastids. In mid-G1 phase, a new synthesis of RNA begins, which precedes synthesis of nuclear and mitochondrial DNA. At the end of G1 phase, division of organelles starts, beginning with the chondriome and plastids, arranged in a network.

[Study of the regulation of plastid development in Euglena gracilis. II. Functional localisation and synthesis of ribosomal chloroplast particles (author's transl)]. / Etude de la regulation du développement plastidial chez Euglena gracilis. II. Localisation fonctionnelle et synthèse des particules ribosomiques chloroplastiques.

Chloroplast ribosomes in greening cells of Euglena gracilis are found either in the stroma or bound to thylakoid membranes. The membrane-bound chloroplast ribosomes are of two main types: those which can be released by 0.5 M KCl or by puromycin and 0.5 M KCl, and those which are released by detergent (deoxycholate or Triton X-100) and KCl. The ribosomes which are released by puromycin are presumably bound to chloroplast membrane by nascent peptide chains. Ribosomes released by puromycin are found only during the course of plastidial differentiation at the time of active thylacoid membrane synthesis. Following greening, those ribosomes remain bound to the membranes but can be removed by KCl alone. An analysis of RNA labelling showed that 30-S but not 53-S subunits of membrane-bound ribosomes are of uniform specific activity. This suggests that 30-S subunit exchange in a common pool while 53 S subunits remain membrane bound and do not exchange in a common pool. Membrane-bound chloroplast ribosomes which are released either by puromycin or by detergent are originally derived from loosely bound particles, released by 0.5 M KCl.