Biosynthesis of ecdysone and ecdysone phosphates in spinach

Abstract

The polar ecdysteroid conjugate, ecdysone phosphate (2β,3β,14α,22 R,25-pentahydroxy-7-en-6-one-3- phosphate) was identified in excised first leaves of spinach, where it is endogenously produced during 20- hydroxyecdysone biosynthesis. Radiolabelled [ 14C]ecdysone phosphate was isolated from several excised leaf assays and was hydrolysed with wheat germ acid phosphatase to yield [ 14C]ecdysone. Incorporation of ecdysone into excised first leaves followed by 32P exposure produced a compound with 32P activity, with chromatographic properties identical to those of the isolated [ 14C]ecdysone phosphate and upon hydrolysis released ecdysone. In spinach first leaves with active ecdysteroid biosynthesis, ecdysone is present at 0.004% of the total free ecdysteroid and contained 6% of the total radioactivity from [2- 14C]mevalonic acid (MVA). These biosynthetically active tissues also produce radiolabelled lathosterol, ecdysone-3-phosphate and 20-hydroxyecdysone. In biosynthetically inactive tissue (immature apical organs) no radiolabelled lathosterol, ecdysone-3-phosphate, ecdysone or 20-hydroxyecdysone was produced from [2- 14C]MVA despite an active biosynthesis of C 29-sterols. Several intermediate and end product ecdysteroids, when incorporated into excised first leaves of spinach produced conjugates which were readily cleaved by wheat germ acid phosphatase. The ecdysteroid pathway appears to be regulated by the presence of ecdysteroid substrates.