No abstract

Elicitor-induced sanguinarine accumulation in opium poppy (Papaver somniferum) cell cultures provides a responsive model system to profile modulations in gene transcripts and metabolites related to alkaloid biosynthesis. An annotated expressed sequence tag (EST) database was assembled from 10,224 random clones isolated from an elicitor-treated opium poppy cell culture cDNA library. The most abundant ESTs encoded defense proteins, and enzymes involved in alkaloid metabolism and S-adenosylmethionine-dependent methyl transfer. ESTs corresponding to 40 enzymes involved in the conversion of sucrose to sanguinarine were identified. A corresponding DNA microarray was probed with RNA from cell cultures collected at various time-points after elicitor treatment, and compared with RNA from control cells. Several diverse transcript populations were coordinately induced, with alkaloid biosynthetic enzyme and defense protein transcripts displaying the most rapid and substantial increases. In addition to all known sanguinarine biosynthetic gene transcripts, mRNAs encoding several upstream primary metabolic enzymes were coordinately induced. Fourier transform-ion cyclotron resonance-mass spectrometry was used to characterize the metabolite profiles of control and elicitor-treated cell cultures. Principle component analysis revealed a significant and dynamic separation in the metabolome, represented by 992 independent detected analytes, in response to elicitor treatment. Identified metabolites included sanguinarine, dihydrosanguinarine, and the methoxylated derivatives dihydrochelirubine and chelirubine, and the alkaloid pathway intermediates N-methylcoclaurine, N-methylstylopine, and protopine. Some of the detected analytes showed temporal changes in abundance consistent with modulations in the profiles of alkaloid biosynthetic gene transcripts.

Azotobacter vinelandii grown in iron-limited medium containing 1 tiM molybdate released the catecholate siderophores azotochelin and aminochelin [bis(2,3-dihydroxybenzoyl-lysine) and 2,3-dihydroxybenzoyl-putrescine, respectively] into the culture fluid. However these catecholates were not observed when the medium contained 1 mM molybdate, but were replaced by another catecholate compound. The appearance of this new compound was not an artifact of extraction of the catecholates from the culture fluid in the presence of high molybdate. Full and partial acid hydrolysis and fast atom bombardment mass spectroscopy showed that the new compound was the tricatecholate protochelin, a product of the condensation of azotochelin and aminochelin. The production of protochelin was iron-repressible and protochelin very rapidly decolorized the Chrome AzuroI-S assay. Protochelin promoted the growth of the siderophore-deficient A. vinelandii strain PI00 under iron-restricted conditions and promoted 55Fe uptake into iron-limited cells, confirming that protochelin can be used as a siderophore by A. vinelandii.

In iron-limited medium, Azotobacter winelandii strain UW produces three catecholate siderophores : the tricatecholate protochelin, the dicatecholate azotochelin and the monocatecholate aminochelin. Each siderophore was found to bind Fe3+ preferentially to Fez+, in a 1igand:Fe ratio of 1 :1,3:2 and 3: 1 , respectively. Protochelin had the highest affinity for Fe3+, with a calculated proton-independent solubility coefficient of lWg, comparable to ferrioxamine B. Iron-limited wild-type strain UW grown under N,-fixing or nitrogen-sufficient conditions hyper-produced catecholate siderophores in response to oxidative stress caused by high aeration. In addition, superoxide dismutase activity was greatly diminished in iron-limited cells, whereas catalase activity was maintained. The ferredoxin I (Fdl)-negative A. vinelandii strain LM100 also hyper-produced catecholates, especially protochelin, under oxidative stress conditions, but had decreased activities of both superoxide dismutase and catalase, and was about 10 times more sensitive to paraquat than strain UW. Protochelin and azotochelin held Fe3+ firmly enough to prevent its reduction by -0; and did not promote the generation of hydroxyl radical by the Fenton reaction. Ferric-aminochelin was unable to resist reduction by -0; and was a Fenton catalyst. These data suggest that under iron-limited conditions, A. vinelandii suffers oxidative stress caused by .O;. The catecholate siderophores azotochelin, and especially protochelin, are hyper-produced to offer chemical protection from oxidative damage catalysed by -0; and Fe3+. The results are also consistent with Fdl being required for oxidative stress management in A. winelandii.

Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdosiderophore complexes were formed but low enough that the complexes were readily destabilized by Fe 3؉ . Thus, competition between Fe 3؉ and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn 2؉ , and Mn 2؉ . We found that all five of these metal ions partially inhibited uptake of 55 Fe-protochelin and 55 Feazotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.