Strigolactone biosynthesis catalyzed by cytochrome P450 and sulfotransferase in sorghum

Abstract

Summary Root parasitic plants such as Striga, Orobanche, and Phelipanche spp. cause serious damage to crop production world‐wide. Deletion of the Low Germination Stimulant 1 (LGS1) gene gives a Striga‐resistance trait in sorghum (Sorghum bicolor). The LGS1 gene encodes a sulfotransferase‐like protein, but its function has not been elucidated. Since the profile of strigolactones (SLs) that induce seed germination in root parasitic plants is altered in the lgs1 mutant, LGS1 is thought to be an SL biosynthetic enzyme. In order to clarify the enzymatic function of LGS1, we looked for candidate SL substrates that accumulate in the lgs1 mutants and performed in vivo and in vitro metabolism experiments. We found the SL precursor 18‐hydroxycarlactonoic acid (18‐OH‐CLA) is a substrate for LGS1. CYP711A cytochrome P450 enzymes (SbMAX1 proteins) in sorghum produce 18‐OH‐CLA. When LGS1 and SbMAX1 coding sequences were co‐expressed in Nicotiana benthamiana with the upstream SL biosynthesis genes from sorghum, the canonical SLs 5‐deoxystrigol and 4‐deoxyorobanchol were produced. This finding showed that LGS1 in sorghum uses a sulfo group to catalyze leaving of a hydroxyl group and cyclization of 18‐OH‐CLA. A similar SL biosynthetic pathway has not been found in other plant species.

A Unique Sulfotransferase-Involving Strigolactone Biosynthetic Route in Sorghum

“…Introduction of LGS1 to ECL/YSL2a (resulting ECL/YSL8a, Supplementary Table 3 ) resulted in substantial decrease of 18-hydroxy-CLA and the appearance of 4DO and 5DS (ratio ∼ 1:1, Figure 3A ), though the amount is low in comparison to 18-hydroxy-CLA and OB ( Figure 3A ). This result is also consistent with the very recently reported characterization of LGS1 in converting 18-hydroxy-CLA to 5DS and 4DO in both the tobacco transient expression and in vitro assays ( Yoda et al, 2021 ). Similar to many previous SOT studies ( Hirschmann et al, 2014 ), the putative intermediate 18-sulfate-CLA was not detected from in vivo assays using SL-producing microbial consortia ( Supplementary Figure 7 ).…”

“…Likely SbMAX1a first catalyzes three-step oxidation on C19 to synthesize CLA, followed by additional oxidations on C18 to afford the synthesis of 18-hydroxy-CLA and subsequently 18-oxo-CLA, which than converts to OB ( Figure 1 ; Wakabayashi et al, 2019 ; Mori et al, 2020 ). This result is partially consistent with the very recent characterization of SbMAX1a as an 18-hydroxy-CLA synthase, except for the detection of OB as a side product in ECL/YSL2a ( Yoda et al, 2021 ). The conversion from 18-hydroxy-CLA to OB is catalyzed by SbMAX1a as shunt product or by endogenous enzymes in yeast or E. coli that remains to be investigated.…”

“…However, the enzyme catalyzing the exclusive conversion of 18-sulfate-CLA to 5DS is still missing and requires further investigation into sorghum (Figure 1). Out independent identification of LGS1 using SL-producing microbial consortium is consistent with the very recently published characterization of LGS1 heterologously in tobacco and in vitro (Yoda et al, 2021).…”

“…transient expression and in vitro assays (Yoda et al, 2021). Similar to many previous SOT studies (Hirschmann et al, 2014), the putative intermediate 18-sulfate-CLA was not detected from in vivo assays using SL-producing microbial consortia (Supplementary Figure 7).…”

“…18-hydroxy-CLA has been identified as a general precursor to the synthesis of canonical SL such as 4DO, 5DS, and OB ( Zhang et al, 2014 ; Wakabayashi et al, 2019 , 2020 ). Since the amount of 18-hydroxy-CLA is substantially higher in the lgs1 mutant compared with the wild-type sorghum ( Yoda et al, 2021 ), it is likely that LGS1 also employs 18-hydroxy-CLA as the substrate. LGS1 contains sulfotransferase (SOT) domain and may sulfate 18-hydroxy-CLA, similar to as some plant SOTs sulfate phytohormones [e.g., AtSOT10 sulfate brassinosteroids and AtSOT15 sulfate jasmonates ( Hirschmann et al, 2014 ; Figure 3B )].…”