Luc Didierjean

Cytochromes P450 for engineering herbicide tolerance

In recent years, genome sequencing has revealed that cytochromes P450 (P450s) constitute the largest family of enzymatic proteins in higher plants. P450s are mono-oxygenases that insert one atom of oxygen into inert hydrophobic molecules to make them more reactive and hydrosoluble. Besides their physiological functions in the biosynthesis of hormones, lipids and secondary metabolites, P450s help plants to cope with harmful exogenous chemicals including pesticides and industrial pollutants, making them less phytotoxic. The recovery of an increasing number of plant P450 genes in recombinant form has enabled their use in experimentation, which has revealed their extraordinary potential for engineering herbicide tolerance, biosafening, bioremediation and green chemistry.

Engineering Herbicide Metabolism in Tobacco and Arabidopsis with CYP76B1, a Cytochrome P450 Enzyme from Jerusalem Artichoke

The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites.

Heavy-metal-responsive genes in maize: identification and comparison of their expression upon various forms of abiotic stress

To identify genes involved in defense against heavy-metal stresses, a cDNA library originating from mercuric chloride-treated maize (Zea mays L. cv. INRA 258) leaves was constructed and analysed by differential screening using cDNAs derived from treated and untreated plants. Transcriptionally activated cDNA clones, designated CHEM (chemically-activated), were isolated and characterized. They represent various known proteins, such as glycine-rich proteins, pathogenesis-related proteins, chaperones and membrane proteins. The expression of the genes encoding these proteins was studied in maize subjected to other forms of abiotic stress. Expression of glycine-rich proteins was greatly enhanced by heat stress, and also stimulated by NaCl, polluted rainwater, wounding and cold stress. Pathogenesis-related proteins were strongly induced by ultraviolet light and to a lesser extent by NaCl, polluted rainwater and wounding. Heat-shock protein was mainly induced by heat and cold, and ubiquitin by wounding. Expression of the membrane channel protein was stimulated by heat stress, NaCl, polluted rainwater and ultraviolet-light irradiation.

Stress responses in maize: sequence analysis of cDNAs encoding glycine-rich proteins.

A number of sequences for plant glycine-rich proteins (GRPs) have been obtained by cDNA or genomic cloning. All the proteins encoded by these DNAs are characterized by a high glycine content with repetitive glycine stretches. Their role is not clearly established yet, some of these proteins are thought to be components of the cell wall, but so far the bean GRP 1.8 is the only one for which direct evidence exists for its location in the cell wall [8]. The expression of some of the GRP genes seems to be organ-specific and developmentally regulated [ 11 ]. These genes are also influenced by environmental stimuli, thus wounding induces the petunia [ 1 ] and bean genes [9]. Salicylic acid and virus infection induce the tobacco gene [7]. In maize, abscisic acid (ABA) and desiccation induce a GRP in embryo and leaves respectively [6]. Here we report on the isolation and sequence analysis of two cDNA clones encoding GRPs in chemically treated maize leaves. One GRP shows a complete identity with that induced by ABA, the second is structurally distinct. Maize plants (Zea mays) were treated when 12 days old by spraying a 0.2?o mercuric chloride solution onto the leaves. Polyadenylated RNA was extracted 6 hours after onset of the chemical treatment; details have been described elsewhere [3]. Doubled-stranded cDNA was synthesized with the Pharmacia kit and cloned into the Eco RI site of 2gtl0 cloning vector. About 40 000 recombinant clones (10 ~o of the total library) have been screened using [ 32p ]_labelled cDNAs synthesized by using RNAs isolated from treated and control plants as probes. Among the various positive clones obtained, two have been selected, designated CHEM1 and CHEM2, subcloned into the Bluescript KS( + ) plasmid vector and sequenced. The nucleotide sequence of the cDNAs was determined by the dideoxy method [ 14]. DNA sequence analysis was performed on VAX computer using the GCG package [ 5 ]. Maize genomic DNA was isolated from germinated embryos according to Dellaporta et al. [4]. Total RNA was isolated as previously described [3]. Southern and northern transfers and hybridization were performed according to Sambrook et al. [13] using Hybond N membranes. The cDNA clones CHEM1 and CHEM2 were obtained from a cDNA library prepared from

Differential expression of bean chitinase genes by virus infection, chemical treatment and UV irradiation

Three chitinases have been shown previously to be induced upon various stresses of bean leaves. Time course studies of mRNA accumulation of two of them (P3- and P4-chitinases) have been studied upon virus infection, mercuric chloride treatment and UV irradiation. In alfalfa mosaic virus (A1MV)-infected plants both mRNAs, absent in uninfected bean leaves, become detectable 36 h after inoculation. A maximum level of mRNAs is reached 84 h after inoculation and, whereas the amount of P3-ch mRNA decreases soon after having reached the maximum, the amount of P4-ch mRNA remains at high levels for several days. In mercuric chloride-treated leaves P4-ch mRNA becomes detectable 1–1.5 h after onset of treatment and a maximum level is observed between 6 h and 24 h after treatment; P3-ch mRNA becomes detectable later than P4-ch mRNA in treated leaves and reaches a maximum as late as 18 h after treatment has been applied. UV light also induces the synthesis of both mRNAs but, here again, important differences are observed in the accumulation rate of the two transcripts. The relative amounts of each mRNA induced by the different stresses have been compared. The most effective inducer of P3-ch mRNA is A1MV. In contrast, mercuric chloride induces P4-ch mRNA more efficiently than A1MV or UV light. We have also determined the complete nucleotide sequence of the cDNA encoding P3-chitinase that has been isolated from a cDNA library by using the cucumber lysozyme-chitinase cDNA as a probe. The 1072 bp P3-ch cDNA encodes a mature protein of 268 amino acid residues and the 25 residue NH2-terminal signal peptide of the precursor. Because of its high structural homology to the cucumber and Arabidopsis acidic chitinases as well as to the N-terminal amino acid sequence of the bifunctional lysozyme-chitinase from P. quinquifolia, bean P3-chitinase can be considered to belong to the class III chitinases. Southern blot analysis of bean genomic DNA revealed that P3-chitinase is encoded by a single gene.

Abiotic stresses induce a thaumatin-like protein in maize ; cDNA isolation and sequence analysis

The cDNA clone (CHEM4) coding for a stress-induced protein has been isolated from a mercuric chloride-treated maize (Zea mays L.) cDNA library by differential screening. Nucleotide sequence analysis of the 694-bp-cDNA insert predicts an open reading frame of 522 nucleotides encoding a mature protein of 149 amino acids and a signal peptide of 25 amino acid residues. The sequence of CHEM4-encoded protein shows 50 to 60% sequence identity with thaumatin, bifunctional α-amylase/trypsin inhibitor from maize and thaumatin-like (TL) proteins from dicotyledonous plants. Higher sequence identities are found to wheat PWIR2 and barley Hv-1b TL-proteins. CHEM4-mRNA is not only induced by mercuric chloride treatment but also by other stresses such as high salt concentration, high temperatures, UV light, wounding and polluted rainwater. CHEM4 genes appear to be highly inducible by UV light and mercuric chloride, moderately by wounding, salt stress, polluted rainwater and heat shock and very weakly by cooling.

Characterization of cadmium binding peptides from pepper (Capsicum annuum)

Abstract Pepper plants (Capsicum annuum), like many other plant species, respond when stressed with cadmium chloride by the synthesis of phytochelatins [(γGlu-Cys)nGly] (PCs) and desglycyl phytochelatins [(γGlu-Cys)n], where n=2–4. Higher molecular weight PCs with a chain length longer than four have also been detected; their synthesis is dependent upon the duration of the experiments and the concentrations of Cd used in the culture medium. The synthesis of PCs and related peptides in Cd-stressed pepper plants is also strongly suggested by the use of buthionine sulfoximine a specific inhibitor of the γ-glutamyl-cysteine synthetase (enzyme involved in the synthesis of glutathione, the precursor of PCs). Indeed no thiol-containing compounds were detected in crude extracts of Cd-treated pepper plants, when they were grown in the presence of BSO. In addition to the synthesis of PCs and PC derivatives, Cd treatment of pepper plants also leads to the synthesis of two 10-kDa proteins, which differ in their amino acid composition and are absent in untreated plants. The function and role of these two proteins is still unknown, but they might also be involved in defense mechanisms against heavy metals.

Expression of a green tissue-specific 11 kDa proline-rich protein gene in bean in response to heavy metals

Abstract A cDNA clone encoding a proline-rich protein specifically expressed in green tissues of bean has been isolated. This clone contains an open reading frame of 288 nucleotides coding for a putative protein of 96 amino acids with an estimated molecular mass of 11 kDa. This protein, named PvPRP2, contains several specific amino acid motifs such as PPVYK and shows a high degree of sequence identity with the soybean SbPRP3. The PvPRP2 gene is constitutively expressed in stems and to a lesser extent in primary leaf tissue. In both tissues the cDNA probe hybridized to a 600-nucleotide transcript. In root-tissues, where no 600-nucleotide transcript was detectable, the probe hybridized to a transcript of about 900 bp which was not found in green tissues, thus suggesting that both roots and green tissues contain specific PRPs. In contrast to the green tissues genes, the expression of the root-gene is not altered by heavy metal stresses. While transcripts were at low levels in unstressed bean leaves, most of the abiotic stresses we have investigated (metals, wounding, drought, elevated temperature, UV, salt and ABA) triggered a strong response by increasing the expression of this gene.