5.3. ABA (11) quantificationSamples

5.3. ABA (11) quantification
Samples of 100 mg leaf fr. wt were homogenized in a mortar
with liquid nitrogen and 2 mL of MeOH:twice-distilled H2O:H3PO4
(80:19:1, v/v/v). The extract was maintained over-night at 4 C,
and then centrifuged 10 min at 10,000g. The supernatant was
added with [2H6]-ABA (100 ng; gift of J.D. Cohen, Department of
Horticulture, University of Minnesota, Saint Paul, MN, USA) dissolved
in MeOH (100 lL) as internal standard, and allowed 1 h in
darkness and 4 C for isotope equilibration. The solvent was evaporated
in a Speed Vac at room temperature (Eppendorf-Concentrator
Plus; Westbury, NY, USA). The residue was dissolved in 3 mL of
twice-distilled H2O pH 3 (1% H3PO4) and passed through a Sep-
Pack C18 reversed phase cartridge (500 mg of material, Waters
Associates, Milford, MA, USA). This elution was performed using
n-hexane (2 mL) and MeOH (2 mL):twice distilled H2O:H3PO4
(80:19:1, v/v/v). The last fraction was collected and after solvent
evaporation in vacuo at room temperature, the residue was dissolved
in twice-distilled H2O (1 mL) at pH 7.0 (1% NH4OH) and
transferred to Oasis WAX (weak anion exchanger) cartridges
(60 mg of material; Waters Associates). Then column was washed
with 5% NH4OH in MeOH (2 mL), MeOH (2 mL) and 3.5% H–CO2H in
MeOH (3 mL). The acidic eluate (which contained ABA) was evaporated
at 35 C and then converted to methyl-ester derivatives
with MeOH (3 lL) plus fresh ethereal CH2N2 (5 lL) (30 min at room
temperature). After organic solvents had been eliminated in a vacuum
concentrator, samples were dissolved in n-hexane (50 lL),
and 1 lL was injected split–splitless into a Perkin–Elmer Elite-
5MS, crosslinked methyl silicone capillary column (30 m length,
0.25 mm inner diameter, and 0.25 lm film thickness) fitted in a
capillary gas chromatograph–electron impact mass spectrometer
(GC–EIMS; Clarus 500, PerkinElmer, Shelton, CT, USA). The GC column
was eluted with He (0.7 mL min1). The GC temperature program
was 100–200 C at 20 C min1, then augmented to 280 C at
4 C min1 and held for 15 min. The mass spectrometer was operated
with electron impact ionization energy of 70 eV. The injector
temperature was 230 C, ion source temperature was 120 C and
the interface temperature was 150 C. After performing selected
ion monitoring (SIM) the amount of ABA was calculated by comparison
of the peak areas of the major ions for the methyl-ester
derivative of the deuterated internal standard [2H6]-ABA(194/
166) relative to its non-labeled counterpart (190/162).
5.4. Diterpenes and triterpenes derivatives quantification
Samples of leaf fr. wt (100 mg) were ground to a fine powder
using a mortar and pestle, and then macerated with CHCl3:MeOH
(2 mL, 1:1, v/v). The suspension was transferred to glass vials with
Teflon-coated screw caps and allowed to extract over-night in
darkness at 4 C. The macerated residue was shaken and centrifuged
15 min at 10,000g. The supernatant was collected and evaporated
to dryness in Speed Vac at room temperature. The residue
was dissolved with n-hexane (500 lL) of and then, 1 lL was injected
in split–splitless mode in a GC–EIMS system for analysis of
terpenes. The analysis was carried out with the same column and
equipment described above. The oven temperature program was:
initial temperature at 60 C for 1 min followed by an increase of
10 C min1 to 280 C and held at 280 C for 22 min. To estimate
the concentration of the different metabolites, 50 ng lL1 of nhexadecane
as internal standard (Supelco, Bellefonte, PA, USA)
was added to each sample. The identities of compounds were confirmed
by comparison of their retention times and full scan mass
spectra with those of authentic standards, and with mass spectra
of the National Institute of Standards and Technology (NIST)
library.
5.5. Monoterpenes and E-nerolidol determinations
Samples of CHCl3:MeOH (500 lL, 1:1, v/v) extract of above was
placed in glass vials with Teflon-coated screw caps (1.5 mL), added
with 500 lL of n-hexane, shaken and left over-night in darkness at
4 C. From the hexane extract, an aliquot of 1 lL was injected in
split–splitless mode in the GC–EIMS system, with the same conditions
as for sterols and tocopherols determinations, except that the
oven temperature program was: initial temperature at 45 C for
1 min, followed by an increase of 2 C min1 to 130 C, then from
130 to 250 C at a rate of 20 C min1 and held for 10 min at
250 C. The ionization potential was 70 eV and a range of
40–500 amu was scanned. Compounds were identified by comparison
of retention times with a set of authentic standards, E-nerolidol
(7), pinene (8), terpinolene (9), carene (10) obtained from
Fluka (Sigma–Aldrich, Steinheim, Switzerland), and peak areas
were referred to the standard n-hexadecane for quantification.
5.6. Terpene synthase activity measurements
Samples of leaf fr. wt. (100 mg) were homogenized in a coldice
mortar with pestle and 1 M potassium phosphate buffer
(800 lL, pH 6.5–7), 20% (w/v) glycerol, 10 mM sodium metabisulfite,
10 mM ascorbic acid, 15 mM MgCl2, 0.5% PVP (insoluble polyvinyl
polypirrolidone, Sigma Chem. Co., St. Louis, MO, USA, MW
40.000) and 1.47 mM 2-b mercapthoethanol. Each total protein
homogenate was centrifuged 5 min at 14,000g and supernatant
(10 lL) was incubated with 0.2 lM of radioactive trans, trans-farnesyl
pyro-phosphate triammonium salt (40 lL, [1-3H]-FPP, specific
activity 20.5 Ci mM1, Perkin Elmer, Boston, MA, USA), FPP
(12) (0.14 lM of Sigma Chem. Co., St. Louis, MO, USA) as carrier,
and reaction buffer containing 250 mM Tris:HCl (pH 6.5–7),
50 mM MgCl2. The mixture was incubated at 30 C and after
20 min the reaction was stopped with H3PO4 (10 lL, 85%). The
reaction products were partitioned with n-hexane (150 lL) and
reacted with silica gel powder (5 mg, 240–300 Mesh, Sigma
Chem. Co., St. Louis, MO, USA). An aliquot of n-hexane (50 lL)
was located in a scintillation vial with Fluka cocktail (4 mL) (Sigma
Chem. Co., St. Louis, MO, USA) and radioactivity was determined
by using a Tricarb liquid scintillation analyzer (Perkin
Elmer, Illinois, USA). TPS activity was expressed as nmol [3H]-
FPP transformed mg of protein1 h1 according to Vögeli and
Chapell (1988). The protein concentration of the extract was
determined by the method of Bradford (Bradford, 1976) using bovine
serum albumin (Bio-Rad Laboratories, Philadelphia, PA, USA)
as standard.