Unveiling the inequivalent biochemical functions of OsSMO2 ‐1 and OsSMO2 ‐2 in rice phytosterol biosynthesis using a customized sterol standards mixture

SUMMARY Phytosterols, natural active components widely distributed in plants, commonly include β ‐sitosterol, stigmasterol, and campesterol. However, identifying their synthesis intermediates can be challenging because access to corresponding commercial standard compounds is not always possible. To overcome these limitations, we synthesized phytosterol intermediates by co‐infecting Nicotiana benthamiana with verified phytosterol synthases and identified them via gas chromatography–mass spectrometry analysis. Then, we mixed the identified intermediates, re‐analyzed them by GC–MS to get a customized standard mixture, and established the corresponding standard spectrum library and characteristic ion database. To prove the usability of this standard mixture, we analyzed sterol intermediate types and contents in OsSMO2‐1 and OsSMO2‐2 rice mutants with this tool. Using the standard mixture, we analyzed sterolomics in wild‐type rice buds at different germination days. As buds grew, detectable sterol intermediates decreased; only campesterol, sitosterol, and stigmasterol were found at the 9‐day‐old bud. When we compared mutants ( ossmo2‐1/ossmo2‐2 ) with the wild type, we noted an obvious peak of 24‐ethylidenelophenol accumulation in the ossmo2‐1 mutant, indicating non‐equivalent functions of OsSMO2‐1 and OsSMO2‐2 in sterol synthesis. OsSMO2‐1 is more crucial for stigmasterol and sitosterol synthesis. This validates the standard mixture's utility.