Salim Al-Babili

Carotenoid oxygenases involved in plant branching catalyse a highly specific conserved apocarotenoid cleavage reaction

Recent studies with the high-tillering mutants in rice (Oryza sativa), the max (more axillary growth) mutants in Arabidopsis thaliana and the rms (ramosus) mutants in pea (Pisum sativum) have indicated the presence of a novel plant hormone that inhibits branching in an auxin-dependent manner. The synthesis of this inhibitor is initiated by the two CCDs [carotenoid-cleaving (di)oxygenases] OsCCD7/OsCCD8b, MAX3/MAX4 and RMS5/RMS1 in rice, Arabidopsis and pea respectively. MAX3 and MAX4 are thought to catalyse the successive cleavage of a carotenoid substrate yielding an apocarotenoid that, possibly after further modification, inhibits the outgrowth of axillary buds. To elucidate the substrate specificity of OsCCD8b, MAX4 and RMS1, we investigated their activities in vitro using naturally accumulated carotenoids and synthetic apocarotenoid substrates, and in vivo using carotenoid-accumulating Escherichia coli strains. The results obtained suggest that these enzymes are highly specific, converting the C27 compounds beta-apo-10-carotenal and its alcohol into beta-apo-13-carotenone in vitro. Our data suggest that the second cleavage step in the biosynthesis of the plant branching inhibitor is conserved in monocotyledonous and dicotyledonous species.

Deviation of the neurosporaxanthin pathway towards β-carotene biosynthesis in Fusarium fujikuroi by a point mutation in the phytoene desaturase gene

Carotenoids are widespread terpenoid pigments with applications in the food and feed industries. Upon illumination, the gibberellin‐producing fungus Fusariumu2003fujikuroi (Gibberellau2003fujikuroi mating population C) develops an orange pigmentation caused by an accumulation of the carboxylic apocarotenoid neurosporaxanthin. The synthesis of this xanthophyll includes five desaturation steps presumed to be catalysed by the carB‐encoded phytoene desaturase. In this study, we identified a yellow mutant (SF21) by mutagenesis of a carotenoid‐overproducing strain. HPLC analyses indicated a specific impairment in the ability of SF21‐CarB to perform the fifth desaturation, as implied by the accumulation of γ‐carotene and β‐carotene, which arise through four‐step desaturation. Sequencing of the SF21 carB allele revealed a single mutation resulting in an exchange of a residue conserved in other five‐step desaturases. Targeted carB allele replacement proved that this single mutation is the cause of the SF21 carotenoid pattern. In support, expression of SF21 CarB in engineered carotene‐producing Escherichiau2003coli strains demonstrated its reduced ability to catalyse the fifth desaturation step on both monocyclic and acyclic substrates. Further mutagenesis of SF21 led to the isolation of two mutants, SF73 and SF98, showing low desaturase activities, which mediated only two desaturation steps, resulting in accumulation of the intermediate ζ‐carotene at low levels. Both strains contained an additional mutation affecting a CarB domain tentatively associated with carotenoid binding. SF21 exhibited higher carotenoid amounts than its precursor strain or the SF73 and SF98 mutants, although carotenogenic mRNA levels were similar in the four strains.