A Cvtochrome P450 In Cotton Efficiently Hvdorxylates A Phytotoxic Intermediate During Gossypol Biosynthesis

Terpenoids constitute one of the largest families of natural products of plants,many of which function as signaling molecules in bio-interactions or as defense compounds(phytoalexins)to safeguard the plants.The huge complexity and diversity of sesquiterpenoids is due to the diversity of the enzymes responsible for their biosynthesis.Of these enzymes,the P450 monooxygenases(P450s)are among the most import players.Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds against pathogens and pests but pose health concerns in cottonseed products.The gossypol biosynthesis pathway has been elucidated only partially,to date four enzymes have been functionally characterized,including the sesquiterpene synthase CDNC,a short-chain alcohol dehydrogenase DH1 and two cytochrome P450 monooxygenases CYP706B1 and CYP82D113.To further characterize the pathway,we conducted transcriptome comparison and co-expression analyses of Gossypium hirsutum,which identified 146 candidates linked to gossypol biosynthesis.Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing(VIGS)let to the identification of a new P450 CYP71BE79 and its supposed substrate.In vitro enzymatic assay identified that CYP71BE79 transferred a new hydroxyl group to C-11 of 8-hydroxy-7-keto-δ-cadinene to form 8,11-dihydroxy-7-keto-δ-cadinene.Furthermore,pathway reconstitution by transient expression in tobacco leaves elucidated a series of oxidative reactions of gossypol biosynthesis pathway.Notably,kinetic analyses of the enzymes determined in vitro revealed that CYP71BE79 exhibited the highest catalytic activity among the five enzymes of the pathway assayed.In addition,despite their dispersed distribution in the cotton genome,all of the enzyme genes identified show a tight correlation of expression.These results suggest that the enzymatic steps in gossypol pathway are highly coordinated to ensure efficient substrate conversion.Of the six intermediates identified from the VIGS-treated leaves,8-hydroxy-7-keto-δ-cadinene(C234)exerted a deleterious effect in dampening plant disease resistance when accumulated upon silencing of CYP71BE79.C234 harbors anα,β-unsaturated carbonyl thus is a reactive electrophile species(RES).To see if the effect was plant-specific,we infiltrated C234 into Arabidopsis leaves,which also dampened the resistance to the bacterial pathogen Pseudomonas syringae pv.maculicola(Psm).We then treated Arabidopsis with C234,Psm and(Psm+C234),and analyzed the leaf transcriptomes and comparied the leaf transcriptomes.While C234 alone exerted a mild effect,it stimulated an overresponse of the plant to pathogen.Of the 7335 genes affected in the(Psm+C234)-treated leaves,3476 were unresponsive without the chemical,in which such functional categories as "nucleotides transport","vesicle transport","MAP kinases","G-proteins","protein assembly and cofactor ligation" and "light reaction" were enriched,suggesting that C234 disturbed certain physiological processes and the protein complex assembly,leading to distorted defense response and decreased disease resistance.In addition to the high catalytic activity,sequence analysis show that CYP71BE79 occurs only in in Gossypium,suggesting an adaptive selection to catalyze a highly controlled step to prevent the accumulation of the phytotoxic metabolite,along with gossypol pathway evolution.