A cytochrome P450 CYP71 enzyme expressed in Sorghum bicolor root hair cells participates in the biosynthesis of the benzoquinone allelochemical sorgoleone

Abstract

Summary Sorgoleone, a major component of the hydrophobic root exudates of Sorghum spp., is probably responsible for many of the allelopathic properties attributed to members of this genus. Much of the biosynthetic pathway for this compound has been elucidated, with the exception of the enzyme responsible for the catalysis of the addition of two hydroxyl groups to the resorcinol ring.A library prepared from isolated Sorghum bicolor root hair cells was first mined for P450‐like sequences, which were then analyzed by quantitative reverse transcription‐polymerase chain reaction (RT‐qPCR) to identify those preferentially expressed in root hairs. Full‐length open reading frames for each candidate were generated, and then analyzed biochemically using both a yeast expression system and transient expression in Nicotiana benthamiana leaves. RNA interference (RNAi)‐mediated repression in transgenic S. bicolor was used to confirm the roles of these candidates in the biosynthesis of sorgoleone in planta.A P450 enzyme, designated CYP71AM1, was found to be capable of catalyzing the formation of dihydrosorgoleone using 5‐pentadecatrienyl resorcinol‐3‐methyl ether as substrate, as determined by gas chromatography‐mass spectroscopy (GC‐MS). RNAi‐mediated repression of CYP71AM1 in S. bicolor resulted in decreased sorgoleone contents in multiple independent transformant events.Our results strongly suggest that CYP71AM1 participates in the biosynthetic pathway of the allelochemical sorgoleone.

OsCYP71D8L as a key regulator involved in growth and stress response by mediating gibberellins and cytokinins homeostasis in rice

“…Rice genome contains at least 356 P450 genes grouped into 10 different clans, and CYP71 clan is the largest set (Nelson and Werck-Reichhart, 2011; Nelson et al, 2004). In sunflowers and sorghum bicolor, several CYP71 members participated in the synthesis of eupatolide, inunolide and sorgoleone (Frey et al, 2018; Pan et al, 2018). In rice, eight CYP450 genes including three highly homologous proteins of CYP71 clan ( OsCYP71D8L, OsCYP71D8 and OsCYP71D7 ) are clustered as a tandem array on chromosome 2, but their characters and functions have been little known.…”

“…The CYP71 clan is the largest set of P450s and represents more than half of all CYPs in higher plants (Nelson and Werck-Reichhart, 2011; Nelson et al, 2004). In plants, CYP71BL1, CYP71DD6 and CYP71AM1 are involved in the synthesis of eupatolide, inunolide, and sorgoleone, respectively (Frey et al, 2018; Pan et al, 2018). We previous showed that CYP71D8 was differentially expressed in two rice varieties under drought stress (Zhang et al, 2012), but the functions of CYP71 members remain unknown in rice.…”

In vivo assembly of the sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana

“…The binary vectors for transient expression experiments (pLH‐Sorg, pLH‐G, pLH‐DES, pLH‐PQ, pLH‐GPQ and pLH‐PQD), as well as pCB404‐P19 (Pan et al ., 2018), were mobilized into the Agrobacterium tumefaciens strain EHA105 as described previously (Hofgen & Willmitzer, 1988). The cultures were maintained in LB (Luria‐Bertani) medium supplemented with appropriate antibiotics (25 mg l −1 rifampicin, 100 mg l −1 spectinomycin, and 300 mg l −1 streptomycin for strains harboring pLH vectors; 25 mg l −1 rifampicin, 300 mg l −1 streptomycin, and 50 mg l −1 kanamycin for the pCB vector).…”

“…The solution was heated at 100°C for 30 min and then cooled to room temperature. The silylated fractions were then analyzed by GC-MS according to previously described methods (Pan et al, 2018). Sorgoleone quantification was performed according to the methods described previously by Cook et al (2010).…”

Root traits for low input agroecosystems in Africa

“…Increasing exploration of shallow soil by increasing root length density or by increasing root hair length and density might be beneficial for P capture. In addition, benefits from more root hairs of sorghum could come from their contribution to the synthesis and exudation of sorgoleone that have demonstrated roles in growth inhibition of weeds (Netzly & Butler 1986;Pan et al 2018).…”