Progesterone 5ß-reductase genes of the Brassicaceae family as function-associated molecular markers.

This study aimed to define progesterone 5ß-reductases (P5ßR, EC 1.3.99.6, enone 1,4-reductases) as function-associated molecular markers at the plant family level. Therefore cDNAs were isolated from 25 Brassicaceae species, including two species, Erysimum crepidifolium and Draba aizoides, known to produce cardiac glycosides. The sequences were used in a molecular phylogeny study. The cladogram created is congruent to the existing molecular analyses. Recombinant His-tagged forms of the P5ßR cDNAs from Aethionema grandiflorum, Draba aizoides, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale were expressed in E. coli. Enone 1,4-reductase activity was demonstrated in vitro using progesterone and 2-cyclohexen-1-one as substrates. Evidence is provided that functional P5ßRs are ubiquitous in the Brassicaceae. The recombinant P5ßR enzymes showed different substrate preferences towards progesterone and 2-cyclohexen-1-one. Sequence comparison of the catalytic pocket of the P5ßR enzymes and homology modelling using Digitalis lanata P5ßR (PDB ID: 2V6G) as template highlighted the importance of the hydrophobicity of the binding pocket for substrate discrimination. It is concluded that P5ßR genes or P5ßR proteins can be used as valuable function-associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective.

The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-Delta4,5-steroid 5beta-reductase activity in Arabidopsis thaliana L.

The Arabidopsis thaliana VEP1 gene product shows about 70% sequence identity to Digitalis lanata progesterone 5beta-reductase, an enzyme considered to catalyze a key step in the biosynthesis of cardiac glycosides. A. thaliana does not accumulate cardenolides but protein extracts prepared from its leaves were capable of reducing progesterone to 5beta-pregnane-3,20-dione. A full-length cDNA clone encoding a Delta(4,5)-steroid 5beta-reductase (At5beta-StR, EC 1.1.1.145/1.3.1.23), a member of the short-chain dehydrogenase/reductase (SDR) family, was isolated from A. thaliana leaves. A SphI/SalI At5beta-StR gene fragment was cloned into the pQE vector system and transformed into Escherichia coli. The gene was functionally expressed and the recombinant His-tagged fusion protein was characterized. K(m) values and specific activities for putative 3-oxo-Delta(4,5)-steroid substrates such as progesterone, cortisol, cortexone and 4-androstene-3,17-dione, and for the co-substrate NADPH were determined. Progesterone was stereo-specifically reduced to 5beta-pregnane-3,20-dione and none of the 3-oxo-Delta(5,6)-steroids tested were accepted as a substrate. The gene encoding At5beta-StR was strongly transcribed in stems and leaves. A three-dimensional model of At5beta-StR highlights a close structural similarity to the related, previously described D. lanata progesterone 5beta-reductase. This homology extends to the active site where single amino acid substitutions might be responsible for the increased catalytic efficiency of At5beta-StR when compared to the activity of the recombinant form of the D. lanata enzyme.

Novel plant Ca(2+)-binding protein expressed in response to abscisic acid and osmotic stress.

A cDNA corresponding to an mRNA which accumulates in germinating rice seeds in response to the phytohormone abscisic acid was isolated by differential hybridization. Northern blotting indicated that the mRNA also accumulates in vegetative tissues in response to treatment with abscisic acid and to osmotic stress. Sequencing identified a major open reading frame encoding a novel protein of 27.4 kDa. The identity of the open reading frame was confirmed by comparing the translation products of cellular, hybrid-selected, and in vitro transcribed RNAs and by immunoprecipitation. Western blotting of cellular extracts indicated that the protein is associated with microsomal or membrane fractions. Data base searches indicated that it contains a conserved Ca(2+)-binding, EF-hand motif and that related proteins are similarly expressed in Arabidopsis thaliana. A fusion protein purified from Escherichia coli containing the putative EF-hand region was shown to bind Ca2+ in blot binding assays. These data identify a novel gene family encoding proteins involved in the response of plants to abscisic acid and osmotic stress.

The barley 60 kDa jasmonate-induced protein (JIP60) is a novel ribosome-inactivating protein.

The N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome-inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.

Structure and expression of the barley lipid transfer protein gene Ltp1.

We have characterized a gene (Ltp1) encoding a barley lipid transfer protein. Northern blot analysis showed that Ltp1 mRNA accumulates specifically in the aleurone layer of developing and germinating seeds. Southern blot analysis indicated that LTP1 protein is encoded by a single gene in barley. Sequence analysis of Ltp1 showed that it contains an open reading frame of 351 bp interrupted by a single intron of 133 bp. Transient expression assays indicated that 702 bp of the 5' upstream region of Ltp1 is sufficient to direct aleurone-specific expression during late seed development and early germination.