Investigating the remediation potential of iron oxide nanoparticles in Cu-polluted soil-plant systems: coupled geochemical, geophysical and biological approaches.

Although the use of iron oxide nanoparticles (IONPs) has high potential in remediation and agriculture, a major hindrance to their use includes the risk of contamination of soil and water resources with underexplored effects of IONPs on biota. The fate, phytotoxicity and remediation potential of IONPs are investigated with soil column experiments using 7 nm-sized magnetite (Fe3O4) nanoparticles (magnNPs) and sunflower (Helianthus annuus). Control soil, magnNP-containing soil (10 g magnNPs per kg soil), copper-polluted soil (500 mg Cu per kg soil) and copper-polluted soil containing magnNPs (10 g magnNPs per kg soil and 500 mg Cu per kg soil) support sunflower growth for 57 and 95 days. In magnNP-exposed plants, the occurrence of magnNPs does not affect the growth of the vegetative aerial parts and photosynthetic efficiency. Decreased lipid peroxidation indicates an enhanced antioxidant enzymatic response of magnNP-exposed plants. In plants grown in Cu- and magnNP-Cu-soils, the physiological and biochemical impacts of excess copper are clearly identified, resulting in growth retardation, decreased pigment contents and photosynthetic efficiency, and increased lipid peroxidation and peroxidase (POD) activities. Based on magnetic susceptibility, a higher amount of magnNPs is detected after 57 days in the roots of magnNP-exposed plants (1400 mg kg-1) than in the roots of magnNP-Cu-exposed plants (920 mg kg-1). In the latter, magnNP internalization is likely hampered because of the plants' physiological responses to Cu toxicity. At the working Cu and magnNP concentrations, magnNPs neither decrease Cu accumulation in the plant tissues nor alleviate the overall growth retardation of sunflowers and certain phytotoxic effects induced by excess Cu. However, this study highlights several positive environmental aspects relative to magnNP use, including the harmless effects of magnNPs on sunflowers (1% magnNPs in soil) and the ability of magnNPs to influence Cu mobility in the soil (which could be even more pronounced at lower Cu concentration).

DELLA family duplication events lead to different selective constraints in angiosperms.

Gibberellic acid (GA) is a major plant hormone involved in several biological processes from the flowering to the symbiosis with microorganisms. Thus, the GA regulation is crucial for plant biology. This regulation occurs via the DELLA proteins that belong to the GRAS transcription factor family. DELLA proteins are characterised by a DELLA N-terminal and a GRAS C-terminal domains. It is well known that DELLA activity appears after the bryophytes divergence and then evolved in the vascular plant lineages. Here we present the phylogeny of DELLA across 75 species belonging to various lineages from algae, liverworts and angiosperms. Our study confirmed two main duplication events, the first occurring before the angiosperms divergence and the other specific to the eudicots lineage. Comparative analysis of DELLA subclades in angiosperms revealed the loss in Poaceae and strong alteration in other species of the DELLA functional domain in the DELLA2 clade. In addition, molecular evolution analysis suggests that each of the clades (named DELLA1.1, DELLA1.2 and DELLA2) evolved differently but copies of each subclade are under strong purifying selection. This also suggests that, although the DELLA functional domain is altered in DELLA2, DELLA2 orthologs are still functional and operate in a different way compared to DELLA1 copies. In angiosperms, additional duplication events occurred and led to duplicate copies in species, genus or family such as in the Fabaceae subfamily Papilionoideae. This duplication led to the formation of additional paralogs in the DELLA1.2 subclade (DELLA1.2.1 and DELLA1.2.2). Interestingly, both copies appeared to be under relaxing selection revealing different evolutionary fate of the DELLA duplicated copies.

RNA sequencing and analysis of three Lupinus nodulomes provide new insights into specific host-symbiont relationships with compatible and incompatible Bradyrhizobium strains.

Nitrogen fixation in the legume root-nodule symbiosis has a critical importance in natural and agricultural ecosystems and depends on the proper choice of the symbiotic partners. However, the genetic determinism of symbiotic specificity remains unclear. To study this process, we inoculated three Lupinus species (L. albus, L. luteus, L. mariae-josephae), belonging to the under-investigated tribe of Genistoids, with two Bradyrhizobium strains (B. japonicum, B. valentinum) presenting contrasted degrees of symbiotic specificity depending on the host. We produced the first transcriptomes (RNA-Seq) from lupine nodules in a context of symbiotic specificity. For each lupine species, we compared gene expression between functional and non-functional interactions and determined differentially expressed (DE) genes. This revealed that L. luteus and L. mariae-josephae (nodulated by only one of the Bradyrhizobium strains) specific nodulomes were richest in DE genes than L. albus (nodulation with both microsymbionts, but non-functional with B. valentinum) and share a higher number of these genes between them than with L. albus. In addition, a functional analysis of DE genes highlighted the central role of the genetic pathways controlling infection and nodule organogenesis, hormones, secondary, carbon and nitrogen metabolisms, as well as the implication of plant defence in response to compatible or incompatible Bradyrhizobium strains.

The evolutionary fate of the chloroplast and nuclear rps16 genes as revealed through the sequencing and comparative analyses of four novel legume chloroplast genomes from Lupinus.

The Fabaceae family is considered as a model system for understanding chloroplast genome evolution due to the presence of extensive structural rearrangements, gene losses and localized hypermutable regions. Here, we provide sequences of four chloroplast genomes from the Lupinus genus, belonging to the underinvestigated Genistoid clade. Notably, we found in Lupinus species the functional loss of the essential rps16 gene, which was most likely replaced by the nuclear rps16 gene that encodes chloroplast and mitochondrion targeted RPS16 proteins. To study the evolutionary fate of the rps16 gene, we explored all available plant chloroplast, mitochondrial and nuclear genomes. Whereas no plant mitochondrial genomes carry an rps16 gene, many plants still have a functional nuclear and chloroplast rps16 gene. Ka/Ks ratios revealed that both chloroplast and nuclear rps16 copies were under purifying selection. However, due to the dual targeting of the nuclear rps16 gene product and the absence of a mitochondrial copy, the chloroplast gene may be lost. We also performed comparative analyses of lupine plastomes (SNPs, indels and repeat elements), identified the most variable regions and examined their phylogenetic utility. The markers identified here will help to reveal the evolutionary history of lupines, Genistoids and closely related clades.

Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase.

Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 microm) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate. For its structural identification, the chemically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by NMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species.

Potassium and sodium fluxes in the phytopathogenic fungus Fusarium oxysporum var. lini.

Rubidium uptake in potassium-starved cells followed biphasic kinetics in the micromolar and millimolar range and was independent of the temperature. In contrast, Rb(+) uptake in normal-K(+) cells followed a monophasic kinetics in the millimolar range and increased at temperatures higher than 30 degrees C. Differences in the K(m) values and in the Arrhenius plots of Rb(+) uptake suggest different uptake systems in K(+)-starved and in normal-K(+) cells. In addition, the substantial inhibition of Rb(+) uptake caused by carbonyl cyanide-m-chlorophenyl hydrazone indicates that these systems are strongly dependent on membrane voltage. Lithium (sodium) tolerance, influx, and efflux were separately studied. F. oxysporum was shown to be very tolerant to sodium, while lithium caused a specific toxic effect. Li(+) uptake in K(+)-starved cells exhibits a monophasic kinetics with low affinity. Li(+) efflux was not affected by external pH or addition of potassium to the medium, suggesting that a Na(+)/cation antiporter is not involved in this process.

Optimized expression and catalytic properties of a wheat obtusifoliol 14alpha-demethylase (CYP51) expressed in yeast. Complementation of erg11Delta yeast mutants by plant CYP51.

CYP51s form the only family of P450 proteins conserved in evolution from prokaryotes to fungi, plants and mammals. In all eukaryotes, CYP51s catalyse 14alpha-demethylation of sterols. We have recently isolated two CYP51 cDNAs from sorghum [Bak, S., Kahn, R.A., Olsen, C. E. & Halkier, B.A. (1997) Plant J. 11, 191-201] and wheat [Cabello-Hurtado, F., Zimmerlin, A., Rahier, A., Taton, M., DeRose, R., Nedelkina, S., Batard, Y., Durst, F., Pallett, K.E. & Werck-Reichhart, D. (1997) Biophys. Biochem. Res. Commun. 230, 381-385]. Wheat and sorghum CYP51 proteins show a high identity (92%) compared with their identity with their fungal and mammalian orthologues (32-39%). Data obtained with plant microsomes have previously suggested that differences in primary sequences reflect differences in sterol pathways and CYP51 substrate specificities between animals, fungi and plants. To investigate more thoroughly the properties of the plant CYP51, the wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences. The endogenous sterol demethylase gene (ERG11) was then disrupted. A sorghum-wheat fusion protein expressed with the Arabidopsis thaliana reductase ATR1 showed the highest level of expression and activity. The expression induced a marked proliferation of microsomal membranes so as to obtain 70 nmol P450.(L culture)-1, with CYP51 representing 1.5% of microsomal protein. Without disruption of the ERG11 gene, the expression level was fivefold reduced. CYP51 from wheat complemented the ERG11 disruption, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions. The fusion plant enzyme catalysed 14alpha-demethylation of obtusifoliol very actively (Km,app = 197 microm, kcat = 1.2 min-1) and with very strict substrate specificity. No metabolism of lanosterol and eburicol, the substrates of the fungal and mammalian CYP51s, nor metabolism of herbicides and fatty acids was detected in the recombinant yeast microsomes. Surprisingly lanosterol (Ks = 2.2 microM) and eburicol (Ks = 2.5 microm) were found to bind the active site of the plant enzyme with affinities higher than that for obtusifoliol (Ks = 289 microM), giving typical type-I spectra. The amplitudes of these spectra, however, suggested that lanosterol and eburicol were less favourably positioned to be metabolized than obtusifoliol. The recombinant enzyme was also used to test the relative binding constants of two azole compounds, LAB170250F and gamma-ketotriazole, which were previously reported to be potent inhibitors of the plant enzyme. The Ks of plant CYP51 for LAB170250F (0.29 microM) and gamma-ketotriazole (0.40 microM) calculated from the type-II sp2 nitrogen-binding spectra were in better agreement with their reported effects as plant CYP51 inhibitors than values previously determined with plant microsomes. This optimized expression system thus provides an excellent tool for detailed enzymological and mechanistic studies, and for improving the selectivity of inhibitory molecules.

The chemically inducible plant cytochrome P450 CYP76B1 actively metabolizes phenylureas and other xenobiotics

Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas. CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds. Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation.

Piperonylic acid, a selective, mechanism-based inactivator of the trans-cinnamate 4-hydroxylase: A new tool to control the flux of metabolites in the phenylpropanoid pathway

Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (KI = 17 &mgr;M, kinact = 0.064 min-1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.

Cloning, expression in yeast, and functional characterization of CYP81B1, a plant cytochrome P450 that catalyzes in-chain hydroxylation of fatty acids.

Several omega and in-chain fatty acid hydroxylases have been characterized in higher plants. In microsomes from Helianthus tuberosus tuber the omega-2, omega-3, and omega-4 hydroxylation of lauric acid is catalyzed by one or a few closely related aminopyrine- and MnCl2-inducible cytochrome P450(s). To isolate the cDNA and determine the sequences of the(se) enzyme(s), we used antibodies directed against a P450-enriched fraction purified from Mn2+-induced tissues. Screening of a cDNA expression library from aminopyrine-treated tubers led to the identification of a cDNA (CYP81B1) corresponding to a transcript induced by aminopyrine. CYP81B1 was expressed in yeast. A systematic exploration of its function revealed that it specifically catalyzes the hydroxylation of medium chain saturated fatty acids, capric (C10:0), lauric (C12:0), and myristic (C14:0) acids. The same metabolites were obtained with transgenic yeast and plant microsomes, a mixture of omega-1 to omega-5 monohydroxylated products. The three fatty acids were metabolized with high and similar efficiencies, the major position of attack depending on chain length. When lauric acid was the substrate, turnover was 30.7 +/- 1.4 min-1 and Km(app) 788 +/- 400 nM. No metabolism of long chain fatty acids, aromatic molecules, or herbicides was detected. This new fatty acid hydroxylase is typical from higher plants and differs from those already isolated from other living organisms.

Cloning and functional expression in yeast of a cDNA coding for an obtusifoliol 14alpha-demethylase (CYP51) in wheat.

Screening of a wheat cDNA library with an heterologous CYP81B1 probe from Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids. Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein. The corresponding segment from the yeast lanosterol 14alpha-demethylase was linked to the partial wheat cDNA and the chimera expressed in Saccharomyces cerevisiae. Compared to microsomes from control yeasts, membranes of yeast expressing the chimera catalysed 14alpha-demethylation of obtusifoliol with an increased efficiency relative to lanosterol demethylase activity. W516 is thus a plant member of the most ancient and conserved P450 family, CYP51.