consisting of pyridine/hexamethyldisilazane/trimethylchlorosilane
Plant Seeds and Conditions of Plant Growth
(2:1:2, v/v/v).
Wheat (Triticum aestivum L., var. Kazan Jubilee), barley (Hordeum
vulgare L., var. Svyatich), oat (Avena sativa L., var. Allure), and
proso millet (Panicum miliaceum L., var. Tatar red) seeds were pur-
chased from SSI (State Scientific Institution) Tatar Scientific Re-
search Institute of Agriculture of the Russian Academy of Sciences
(Kazan, Russia). Seeds of three sorghum species, namely, Sudan-
grass [Sorghum bicolor (L.) Moench ssp. drummondii (Nees ex
Steud.) de Wet & Harlan, var. Udacha/Luck], technical sorghum
[Sorghum vulgare Pers. var. technicum (Koern.) Jµv. , var. Master],
and Chinese sorghum (Sorghum chinense Jakusch., var. Cream)
were generously provided by the Agricultural Research Institute of
South-East Region (Saratov, Russia). Rice (Oryza sativa L., var.
Renar) seeds were purchased from All-Russia Rice Research Insti-
tute (Krasnodar, Russia). Seeds were germinated on tap water with-
out supplements at 238C for 5–7 days.
Methanol-Trapping Experiments
The protein pellet obtained by (NH4)2SO4 precipitation as described
above from 5 g (fresh weight) of wheat roots was dissolved in
0.05m Tris·HCl buffer (1 mL, pH 7.5). The resulting protein solution
was combined with 9-HPOD (200 mg) dissolved in cold hexane
(7 mL). This biphasic hexane/buffer system was vigorously vortexed
for 5 min at 08C. The phases were separated by centrifugation at
5000 g for 1 min. The upper hexane layer was aspirated, and the
main part of it was evacuated in vacuo. The products were methy-
lated with ethereal diazomethane. Ether was evacuated in vacuo,
the ice-cold MeOH (3 mL) was added, and the solution was left for
15 min at 08C. Then, methanol was evacuated, and the products
(Me esters) were trimethylsilylated as described above.
Cell-Free Preparations from Cereal Roots
Extraction of the Endogenous Oxylipins from Cereal Roots
Roots of germinating cereal (wheat, sorghum, barley, oat, or proso
millet) seeds were cut after 5–7 days of germination. Cut roots
(10 g, fresh weight) were suspended in cold (0–48C) 0.05m Tris·HCl
buffer (20 mL, pH 7.5) and homogenized with Ultra-Turrax. The ho-
mogenate was filtered through cheesecloth and centrifuged at
15000 g for 15 min. The supernatant was decanted and used for
incubations with a-linolenic acid, linoleic acid, or hydroperoxides
of the latter. Alternatively, the 15000 g supernatant was adjusted
to 60% (NH4)2SO4 saturation, stirred for 30 min at 0–48C, and cen-
trifuged at 15000 g for 15 min. The supernatant was decanted, and
the pellet obtained after the addition of (NH4)2SO4 was frozen with
liquid nitrogen and stored at 858C prior to use for incubations.
Cut roots (10 g, by fresh weight) were homogenized and extracted
at 0–48C with ice-cold hexane/ethyl acetate (1:1, v/v). The extract
was concentrated, and acidic lipids were purified with Supelclean
LC-NH2 (3 mL) cartridges as described above. Then, the free fatty
acids were esterified with diazomethane and trimethylsilylated.
General Schemes of Product Analyses
The methyl esters of the products (or their methyl ester TMS deriv-
atives) were subjected to direct GC–MS analysis after amino car-
tridge purification and derivatization. Alternatively, the methyl
esters of the products were preliminarily separated by NP-HPLC.
Products were separated as methyl esters by NP-HPLC on a Ma-
cherey–Nagel Nucleodur 100-3 column (2504.6 mm, 3 mm) by
using a mixture of hexane/2-propanol (98:2, v/v) at a flow rate of
0.4 mLminÀ1. Products were collected and rechromatographed by
NP-HPLC, eluting with hexane/2-propanol (99.8:0.2, v/v) at a flow
Incubation of Enzyme Preparations with Linoleic Acid or
Linolenic Acid and their Hydroperoxides
Standard incubations were initiated by the addition of either lino-
leic acid or linolenic acid (100 mg) or fatty-acid hydroperoxide (9-
HPOD or 13-HPOD, 100 mg) to an aliquot of 15000 supernatant
preparations, corresponding to 1 g of the roots (fresh weight). The
mixture was stirred for 30 min with continuous oxygen bubbling
for 15 min or without oxygen bubbling (for 9-HPOD or 13-HPOD
incubation). Alternatively, the protein pellet obtained by (NH4)2SO4
precipitation as described above from 12.8 g (fresh weight) of
wheat roots was dissolved in 0.05m Tris·HCl buffer (3.6 mL, pH 7.5).
The resulting protein solution was incubated with 9-HPOD or 13-
HPOD (100 mg) for 15 min at 238C. Then, the products were ex-
tracted and derivatized as described below.
rate of 0.4 mLminÀ1
.
Spectral Studies
UV spectra were recorded online during the HPLC separations with
a SPD-M20A diode array detector (Shimadzu). GC–MS analyses
were performed by using a Shimadzu QP5050A mass spectrometer
connected to a Shimadzu GC-17A gas chromatograph equipped
with an MDN-5S (5% phenyl, 95% methylpolysiloxane) fused capil-
lary column (length, 30 m; inner diameter, 0.25 mm; film thickness,
0.25 mm). Helium at a flow rate of 30 cmsÀ1 was used as the carrier
gas. Injections were made in the split mode by using an initial
column temperature of 1208C. The temperature was raised at
108CminÀ1 until 2408C. Full-scan GC–MS analyses were performed
Extraction, Preliminary Purification and Derivatization of
Products
1
1
1
by using an ionization energy of 70 eV. The H NMR, H– H COSY,
1
1
13
13
1H– H TOCSY, 1H– H NOESY, 1H– C HSQC, and 1H– C HMBC spectra
were recorded with a Bruker Avance III 600 instrument (600 MHz,
[2H6]benzene, 296 K).
The incubation mixture was acidified with acetic acid (CH3COOH)
to pH 5–6 and was extracted with hexane/ethyl acetate (1:1, v/v).
Acidic lipids were separated and purified for analysis by using Su-
pelclean LC-NH2 (3 mL) cartridges (Supelco, Bellefonte, PA, USA) as
described before.[30] First, the total lipid extract dissolved in a mix-
ture of chloroform/2-propanol (2:1, v/v) was passed through the
cartridges. Then, free carboxylic acids were eluted with a mixture
of ethyl acetate/acetic acid (98:2, v/v). Free fatty acids were esteri-
fied with diazomethane. If specified, the methyl esters of the prod-
ucts were trimethylsilylated by treatment with a silylating mixture
Abbreviations
AOS, allene oxide synthase; DES, divinyl ether synthase; HPL, hy-
droperoxides lyase; 9-H(P)OD, (9S,10E,12Z)-9-hydro(pero)xy-10,12-
octadecadienoic acid; 9-HPOT, (9S,10E,12Z,15Z)-9-hydroperoxy-
10,12,15-octadecatrienoic acid; 13-H(P)OD, (9Z,11E,13S)-13-hydro-
ChemistryOpen 2018, 7, 336 –343
342
ꢀ 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim