tion of a trace level of (Z)-3-hexenal that was equivalent to that with the reaction mixture with all the heatdenatured membrane fraction. LC-MS/MS analysis indicated the formation of 12-oxo-phytodienoic acid (OPDA), 13hydroxy-12-oxo-(9Z,15Z)-octadecadienoic acid (-ketol), and 9-hydroxy-12-oxo-(10E,15Z)-octadecadienoic acid (-ketol) within the reaction of 13HPOT using the membrane fraction (Yanagi et al., 2011; Figure 4). No other peak corresponding to divinyl ether (m/z of 291.2) or epoxy alcohol (m/z of 309.two) that would be formed via DES or EAS activity on 13HPOT was detected. Accordingly, we named Smo133317 (SmCYP74J1) as SmAOS4. Recombinant IDH1 Inhibitor MedChemExpress SmAOS4 showed the highest activity at pH five.5. Under representative reaction conditions with 40 in the substrate, the recombinant SmAOS4 showed the highest activity with 13HPOD, followed by 13HPOT (Table 1), whereas 9HPOD showed only 14 activity with 13HPOD.Frontiers in Plant Science | frontiersin.orgOctober 2021 | Volume 12 | ArticleTanaka et al.Green Leaf Volatile-Burst in Selaginella moellendorffiiFIGURE 4 | LC-MS/MS analyses of goods formed by (A) recombinant SmHPL1a, (B) recombinant SmHPL1b, (C) recombinant SmAOS4, (E) recombinant bell pepper HPL (CaHPL) from 13HPOT. Because the empty vector control, the membrane fraction of your E. coli cells harboring pET15b was reacted with 13HPOT (D). Genuine standards of 12-oxo-(Z)-9-dodecenoic acid (peak a) (F) and 12-oxo-phytodienoic acid (OPDA) (peak b) (G) were also analyzed. The substrate, 13HPOT, is shown with peak c. The red trace shows extracted ion chromatograms with m/z of 291.two 0.5 for hydroperoxides of linolenic acid [M-H3 O+ ]- , 12-oxo-phytodienoic acid [M-H+ ], or colnelenic acid [M-H+ ]- . The blue trace is with m/z of 211.1 0.5 for 12-oxo-(Z)-9-dodecenoic acid [M-H+ ]- . The green trace is with m/z of 309.two 0.five for hydroperoxides of linolenic acid [M-H+ ]- , – and -ketols [M-H+ ]- , or epoxy alcohol [M-H+ ]- .TABLE 1 | Substrate specificities of recombinant SmHPL1b and SmAOS4. SmHPL1b Substrate 13HPOT 13HPOD 9HPOT 9HPOT Relative activity ( ) 100 6.ten 1.22 two.18 0.98 2.32 0.85 SmAOS4 Relative activity ( ) 52.six two.63 100 14.9 0.88 14.0 0.have been only slightly catalyzed by recombinant SmHPL1b. The recombinant enzyme followed Michaelis-Menten kinetics, as well as the Km worth with 13HPOT was estimated to be 31.four (Supplementary Figure 6).Gene ExpressionRT-qPCR analyses showed that the transcription levels of SmAOS2 and SmAOS4 within the COX-2 Modulator Gene ID shoots were greater than those inside the roots, although that of SmAOS3 inside the roots was larger than that inside the shoots (Figure six). The level of SmAOS3 within the shoots slightly improved immediately after mechanical wounding. The expression amount of SmAOS4 right away dropped following mechanical wounding and gradually returned for the original level right after 60 min of wounding. Expression of SmHPL1a/b was rather distinct for the shoots and the expression inside the roots was not detected. The expression level varied slightly soon after mechanical wounding on the shoots, but no apparent induction or suppression right after wounding was observed.FIGURE five | GC-MS analyses from the volatile goods formed by recombinant SmHPL1a (blue) and SmHPL1b (brown) from 13HPOT. Because the constructive manage, volatile goods formed by bell pepper HPL (CaHPL) from 13HPOT are also shown (magenta). The vector manage was run using the membrane fraction of E. coli cells harboring pET15b (black).The transcription degree of SmOPR5 elevated immediately after mechanical wounding as reported previously (Pratiwi et al.,