Two NADPH-cytochrome P450 reductase-encoding cDNAs were isolated from an Arabidopsis cDNA library by metabolic interference in a Saccharomyces cerevisiae mutant disrupted for its endogenous cpr1 gene. ATR1 encodes a protein of 692 amino acids, while ATR2 encodes either a 712-residue protein (ATR2-1), or a 702-residue protein (ATR2-2) depending on the choice of the initiation codon. Comparative analysis of ATR1 and ATR2-1 indicates 64% amino acid sequence identity and the absence of conservation in the third base of conserved amino acid codons. The two Arabidopsis reductases are encoded by distinct genes whose divergence is expected an early event in angiosperms evolution. A poly(Ser/Thr) stretch reminiscent of a plant chloroplastic targeting signal is present at the ATR2-1 N-terminal end but absent in ATR1. The cDNA open reading frames were expressed in yeast. The recombinant polypeptides were found present in the yeast endoplasmic reticulum membrane and exhibited a high specific NADPH-cytochrome c reductase activity. To gain more insight into the respective functions of the two reductases, the Arabidopsis cDNA encoding cinnamate 4-hydroxylase (CYP73A5) was cloned and co-expressed with ATR1 or ATR2 in yeast. Biochemical characterization of the Arabidopsis ATR1/CYP73A5 and ATR2-1/CYP73A5 systems demonstrates that the two distantly related Arabidopsis reductases similarly support the first oxidative step of the phenylpropanoid general pathway.

An efficient system for the in vitro assembly of U1 snRNPs is described. RNA‐protein interactions in a series of U1 snRNA mutants assembled both in vivo and in vitro were studied in order to verify the accuracy of the system. Two discrete protein binding sites are defined by immunoprecipitation with antibodies against different protein components of the U1 snRNP and a newly developed protein sequestering assay. The U1 snRNP‐specific proteins 70K and A require only the 5′‐most stem‐loop structure of U1 snRNA for binding, the common U snRNP proteins require the conserved Sm binding site (AUnG). Interactions between these two groups of proteins are detected. These results are combined to derive a model of the U1 snRNP structure. The potential use of the in vitro system in the functional analysis of U1 snRNP proteins is discussed.

Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Succharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S. cerevisk strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H. When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced ironkarbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4-hydroxylase activity upon coupling with the different P450 reductases and NADPH. Some CA4H protein was found also addressed to the yeast mitochondria but as a low-activity form. The spectral and kinetic characterizations of the yeast-produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constituted by the overexpressed yeast P450 reductase and CA4H is characterized by a 1 : 1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH-dependent P450 reaction. Based on spectral perturbation and inhibition studies, coumarate appeared to have no detectable affinity for the enzyme. A possible geometry of the substrate recognition pocket is discussed in the light of these data.

SummaryChanges in gene expression, by application of H 2 O 2 , O 2°-generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H 2 O 2 and gamma irradiation, but not to O 2°-generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H 2 O 2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H 2 O 2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H 2 O 2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.

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A novel Arabidopsis thaliana gene (AtNADK-1) was identified based on its response to radiation and oxidative stress. Levels of AtNADK-1 mRNA increase eight-fold following exposure to ionising radiation and are enhanced three-fold by treatment with hydrogen peroxide. The gene also appears to be differentially regulated during compatible and incompatible plant-pathogen interactions in response to Pseudomonas syringae pv. tomato. The full-length AtNADK-1 cDNA encodes a 58-kDa protein that shows high sequence homology to the recently defined family of NAD(H) kinases. Recombinant AtNADK-1 utilises ATP to phosphorylate both NAD and NADH, showing a two-fold preference for NADH. Using reverse genetics, we demonstrate that AtNADK-1 deficient plants display enhanced sensitivity to gamma irradiation and to paraquat-induced oxidative stress. Our results indicate that this novel NAD(H) kinase may contribute to the maintenance of redox status in Arabidopsis thaliana.

have been investigated. Microsomes from transformed yeast catalysed trans-cinnamate hydroxylation with high efficiency. CYP73 was highly specific for its natural substrate, and did not catalyse oxygenation of p-coumarate, benzoate, ferulate, naringenin or furanocoumarins. No metabolism of terpenoids or fatty acids, known substrates of plant P450s, was observed. CYP73 however demethylated the natural coumarin herniarin into umbelliferone. In addition, it was shown to oxygenate five xenobiotics and mechanism-based inactivators, including the herbicide chlorotoluron. All substrates of CYP73 were small planar aromatic molecules. Comparison of the kinetic parameters of CYP73 for its various substrates showed that, as expected, cinnamate was by far the best substrate of this P450. The physiological and toxicological significance of these observations are discussed.Cytochromes P450 form a large superfamily of several hundreds to several thousands of hemoproteins, involved in oxygen activation and oxygen tranfer into lipophilic molecules. They all share some sequence identity related to their common catalytic properties, i.e. heme and oxygen binding, electron transfer and oxygen activation [l]. Differences in their primary sequences usually reflect variations in specificity for substrates oxygenated. These differences may concern more than 80% of the amino acid sequence. However, the modification of a single amino acid residue can be sufficient to completely alter the substrate specificity of the enzyme 121.Full-length amino acid sequences presently available for plant P450s share less than 30% identity. This almost certainly implies great differences in the structures of their substrate binding sites. However, no relation between the structure of the enzymes and their catalytic activities has yet been established. CYP73 is the first DNA sequence coding for a plant P450 with an identified physiological function to have been isolated 131. It catalyses the 4-hydroxylation of trunscinnamic acid into p-coumaric acid. This hydroxylation is ductase (4-hydroxylating) (EC 1.14.13.11).the second reaction in the phenylpropanoid pathway. Cinnamate 4-hydroxylase (CA4H) is thus an obligatory step for the biosynthesis of lignin, a major component of the earth's total biomass. It is involved in the formation of most of the phenylpropanoid derivatives, essential for plant development, pigmentation and defense against both ultraviolet light and pathogens. Recent data [4-61 suggest that CA4H plays a central role in the regulation of the phenylpropanoid pathway. We, therefore, decided to establish more precisely which molecules are transformed by the enzyme or interfere with its catalytic activity. The determination of a population of substrates was also intended to provide information for analysis of the structurelfunction relationship of this plant P450. We devised an optimized system for the expression of CYP73 in Saccharomyces cerevisae [7]. This system provided yeast microsomes for which the only detectable P450 was CYP73. It routine...

By screening for Arabidopsis genes activated by ionising radiation (IR)-induced DNA damage, we have isolated a cDNA hybridising with a 3.2-kb mRNA that accumulates rapidly and strongly in irradiated cell suspensions or whole plants. The cDNA codes for a 110-kDa protein that is highly homologous to the 116-kDa vertebrate poly(ADP-ribose) polymerase (PARP-1). It is recognised by a human anti-PARP-1 antibody, binds efficiently to DNA strand interruptions in vitro, and catalyses DNA damage-dependent (ADP-ribose) polymer synthesis. We have named this protein AtPARP-1. We have also extended our observations to the Arabidopsis app (AtPARP-2) gene, demonstrating for the first time that IR-induced DNA strand interruptions induce rapid and massive accumulation of AtPARP-1 and AtPARP-2 transcripts, whereas dehydration and cadmium preferentially induce the accumulation of AtPARP-2 transcripts. The IR-induced PARP gene expression seen in Arabidopsis is in striking contrast to the post-translational activation of the PARP-1 protein that is associated with genotoxic stress in animal cells. AtPARP-1 transcripts accumulate in all plant organs after exposure to ionising radiation, but this is followed by an increase in AtPARP-1 protein levels only in tissues that contain large amounts of actively dividing cells. This cell-type specific accumulation of AtPARP-1 protein in response to DNA damage is compatible with a role for the AtPARP-1 protein in the maintenance of DNA integrity during replication, similar to the role of "guardian of the genome" attributed to its animal counterpart.