The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post‐embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk ) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C‐14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk‐like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C‐24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C‐8,7 isomerase. We describe genetic interactions between cph , hyd1 and fk , and studies with 15‐azasterol, an inhibitor of sterol C‐14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK , whereas BR receptor gene BRI1 acts downstream of FK to promote post‐embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC–MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.

In Hiyama−Nozaki reactions of allylchromium with aldehydes the expected products are homoallylalcohols. However, oxidation products derived from these, predominantly allyl ketones, can be common side products. This can be explained by an Oppenauer−(Meerwein−Ponndorf−Verley)-type mechanism (OMPV-reaction). The amount of oxidation is strongly dependent on the substitution pattern of the reaction partners and the reaction conditions. An appropriate choice of these can lead to preferential formation of ketones instead of the alcohols. In addition to its synthetic usefulness, the oxidation−reduction equilibrium is of the utmost importance for the design of enantioselective Hiyama−Nozaki reactions because it is also a potential racemization pathway.

[17‐2H2]GA20‐13‐O‐[6′‐2H2]glucoside was synthesized and applied to seedlings of Phaseolus coccineus L. After incubation for 72 h the conjugate metabolites were purified and shown by LC‐ESI‐tandem‐MS and GC‐MS to be [17‐2H2]GA1‐13‐O‐[6′‐2H2]glucoside and [17‐2H2]GA29‐13‐O‐[6′‐2H2]glucoside. This is the first evidence for the conversion of intact GA‐O‐glucosides, and represents an additional metabolic pathway of the gibberellin metabolism in P. coccineus L. The results indicate that intact GA‐O‐glucosides are accepted by 2‐ and 3‐oxidases in the plant.

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high α-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.

The enzymatic conversion of one chromogenic substrate, -glutamine-p-nitroanilide, and two fluorogenic substrates, -glutaminyl-2-naphthylamide and -glutaminyl-4-methylcoumarinylamide, into their respective pyroglutamic acid derivatives by glutaminyl cyclase (QC) was estimated by introducing a new coupled assay using pyroglutamyl aminopeptidase as the auxiliary enzyme. For the purified papaya QC, the kinetic parameters were found to be in the range of those previously reported for other glutaminyl peptides, such as Gln-Gln, Gln-Ala, or Gln-tert-butyl ester. The assay can be performed in the presence of ammonia up to a concentration of 50 mM. Increasing ionic strength, e.g., potassium chloride up to 300 mM, resulted in an increase in enzymatic activity of about 20%. This is the first report of a fast, continuous, and reliable determination of QC activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis.

Three cDNAs encoding very similar but unique isoforms of chalcone synthase (EC 2.3.1.74) were isolated from a cDNA library prepared from RNA from root tissue of the Thai medicinal plant Cassia alata L. (ringworm bush, Leguminosae). Gene transcript for these three type-III polyketide synthases was found to accumulate predominantly in roots. The heterologously expressed enzymes accepted acetyl-, n-butyryl-, isovaleryl-, n-hexanoyl-, benzoyl-, cinnamoyl-, and p-coumaroyl-CoA as starter molecules and together with the co-substrate malonyl-CoA, formed multiple products. With the exception of the assay in which acetyl-CoA was used as the starter molecule, all substrates yielded a phloroglucinol derivative resulting from three sequential condensations of acetate units derived from three malonyl-CoA decarboxylations. Every substrate tested also produced two pyrone derivatives, one resulting from two acetate unit condensations (a bis-noryangonin-type pyrone derailment product) and one resulting from three acetate unit condensations (a 4-coumaroyltriacetic acid lactone-type pyrone derailment). C. alata accumulates the flavonoids quercetin, naringenin and kaempferol in roots, suggesting that the in planta function of these enzymes is the biosynthesis of root flavonoids.

Three previously published resistance gene analogues (RGAs), pic13, pic21 and pic19, were mapped in relation to sugarcane mosaic virus (SCMV) resistance genes (Scmv1, Scmv2) in maize. We cloned these RGAs from six inbreds including three SCMV-resistant lines (D21, D32, FAP1360A) and three SCMV-susceptible lines (D145, D408, F7). Pairwise sequence alignments among the six inbreds revealed a frequency of one single nucleotide polymorphism (SNP) per 33 bp for the three RGAs, indicating a high degree of polymorphism and a high probability of success in converting RGAs into codominant cleaved amplified polymorphic sequence (CAPS) markers compared to other sequences. SNPs were used to develop CAPS markers for mapping of the three RGAs in relation to Scmv1 (chromosome 6) and Scmv2 (chromosome 3), and for pedigree analyses of resistant inbred lines. By genetic mapping pic21 was shown to be different from Scmv2, whereas pic19 and pic13 are still candidates for Scmv1 and Scmv2, respectively, due to genetic mapping and consistent restriction patterns of ancestral lines.

Infiltration of potato leaves with the phytopathogenic bacteria Pseudomonas syringae pv. maculicola induces local and systemic defense gene expression as well as increased resistance against subsequent pathogen attacks. By cDNA-AFLP a gene was identified that is activated locally in potato leaves in response to bacterial infiltration and after infection with Phytophthora infestans, the causal agent of late blight disease. The encoded protein has high homology to a phosphate starvation-induced acid phosphatase from tomato. Possibly, decreased phosphate availability after pathogen infection acts as a signal for the activation of the potato phosphatase gene.

The jasmonic acid (JA)-dependent regulation of the Thi2.1 gene had previously been exploited for setting up a genetic screen for the isolation of signal transduction mutants of Arabidopsis thaliana (L.) Heynh. that constitutively express the thionin gene. Several cet mutants had been isolated which showed a constitutive expression of the thionin gene. These cet mutants, except for one, also showed spontaneous leaf cell necrosis and were up-regulated in the expression of the PR1 gene, reactions often associated with the systemic acquired resistance (SAR) pathway. Four of these cet mutants, cet1, cet2, cet3 and cet4.1 were crossed with the fad triple and coi1 mutants that are blocked at two steps within the JA-dependent signaling pathway, and with transgenic NahG plants that are deficient in salicylic acid (SA) and are unable to activate SAR. Analysis of the various double-mutant lines revealed that the four cet genes act within a signaling cascade at or prior to branch points from which not only JA-dependent signals but also SA-dependent signaling and cell death pathways diverge.

The leaves of Luvunga sarmentosa (Bl.) Kurz. yielded eight apotirucallane triterpenoids named luvungins A–G, and 1α-acetoxyluvungin A. Characteristic of the structure are the seven-membered lactone-ring A, the α-hydroxyl or α-acetoxyl group at C-7 and an oxygen bridge in the side chain giving five-, six- or seven-membered rings, respectively. Because of a hemiacetal function at C-21, luvungin C occurred as a mixture of 21-epimers. The structures have been elucidated on the basis of MS and NMR spectral data. In addition, two known coumarins ostruthin (6-geranyl-7-hydroxycoumarin) and 8-geranyl-7-hydroxycoumarin as well as five known triterpenes friedelin, flindissone, melianone, niloticin and limonin were isolated.The leaves of Luvunga sarmentosa (Bl.) Kurz. yielded eight apotirucallane triterpenoids named luvungins A–G and 1α-acetoxyluvungin A. Characteristic for the luvungins were the seven-membered lactone-ring A, the α-hydroxyl or α-acetoxyl group at C-7 and an oxygen bridge in the side chain giving five-, six- or seven-membered rings.