Experiment 2. Time-course of changes in nicotine concentration in tobacco plants after removal of the shoot apex
: The transfer and plant culture were the same as inexperiment 1. In this experiment, the plants were grown in a growthroom with a 14 h photoperiod. The photosynthetically activeradiation at the surface of the pots was 220–270 mmol m
provided by reﬂector sunlight dysprosium lamps (DDF 400,Nanjing, China). The treatments started 90 d after sowing (
30 dafter transfer into the quartz sand).
Treatments and harvest procedures
: Four treatments were conductedin this experiment: (i) control; (ii) removing apex; (iii) in treatment (ii), lateral buds began to grow out about 7 d after excision of theshoot apex. In some of these plants, the lateral buds were removedbetween days 7 and 11 after removal of the apex. One lateral budwas removed each day (removing apex and lateral buds). (iv) Apaste consisting of 2-chloro-9-hydroxyﬂuorenecarboxylic acid-(9)-methylester (CFM), an auxin transport inhibitor (Beyer andQuebedeaux, 1974), and Tween in lanolin (10 mg CFM+1.0 gTween+5.5 g lanolin) was applied annularly on the stem directlyunder the shoot apex of intact plants (CFM).The control plants were harvested on the day of treatment, and at 3, 9, and 15 d after the treatments. The decapitated plantswere harvested at 3, 6, 9, 12, and 15 d after removal of the shoot apex. The plants in treatment (iii) were harvested 15 d after removalof the shoot apex. The plants in treatment (iv) were harvested 3 dand 9 d after application of CFM. The harvest procedure was thesame as that described for experiment 1.
Experiment 3. Time-course of changes in nicotine concentration in tobacco plants after removal of the shoot apex and leaf damage
To compare the inﬂuences of removing the shoot apex and leaf damage on nicotine production, a complementary study wasperformed. The plant culture conditions were the same as describedin experiment 2. When the plants had six unfolded leaves, includingtwo smaller already senesced leaves, the following treatments werecommenced: (i) control; (ii) leaves 1–3 were wounded by the plasticcomb-like brush as in experiment 1 (leaf damage). The total punc-tures in leaf 1, and in leaves 2 and 3 were 432 and 1152, respectively.(iii) Removing the apex. The lateral buds that emerged after remov-ing the apex were not excised. On the day of, and on different daysafter the treatments started (for details, see Fig. 4), plants wereharvested. At harvest, plants were separated into roots, stems, tops(apex and new leaves), the upper stratum of leaves nos 1–3, andthe lower stratum of leaves nos 4–6. Other harvest procedureswere the same as in experiment 1.
Experiment 4. Changes in JA concentrations in tobacco plants after removal of the shoot apex and leaf damage
: The hydroponics culture was used to facilitate theharvest of roots for JA measurement. Tobacco seedlings weretransferred to 2.0 l pots (one plant one pot) with aerated nutrient solution, which was the same as used for experiment 1. The initialpH of the nutrient solution was adjusted to 6.0. The nutrient solutionwas renewed every day during the experimental period. The plantswere grown in a growth room, and the growth conditions were thesame as described for experiment 2. The treatments started80 d after sowing (20 d after transferring to hydroponics condition).The leaves were numbered in the same way as in experiment 1.
Treatments and harvest procedures
: Plants were divided into four groups of three plants, each of a similar size and developmental stage:(i) control; (ii) removing the apex; (iii) apices were removed in thesame way as in (ii), and immediately a paste consisting of NAA wasapplied onto the decapitated stem stump (removing apex+NAA); (iv)leaves nos 1 and 2 were damaged by a comb-like brush with a total of 720 punctures (leaf damage). Intact plants were harvested at 0, 90,and 210 min; decapitated plants were harvested at 30, 90, 210, and360 min; and leaves of damaged plants and NAA-treated plants wereharvested at 90 and 210 min after the start of the treatments. At harvest, leaves nos 1 and 2, stem segment (internode between leaf nos 1 and 3), and root tips (3 cm in length) were harvested, andfrozen immediately in liquid nitrogen, then stored at –30
C untilanalysis of JA.
Analysis of JA
The methods of Baldwin
. (1997) and Schittko
. (2000) wereused for extraction, puriﬁcation and determination of JA. Frozentissues (
0.5 g fresh weight) were homogenized in an ice-coldmixture of 70% acetone and 30% 50 mM citric acid (v/v). To themixture, 100 ng of 9,10-dihydrojasmonic acid (Olchemim Ltd.,Czech Republic) was added as an internal standard (Mueller andBrodschelm, 1994; Engelberth
., 2003). In the pre-experiment,9,10-dihydrojasmonic acid was not detectable in the samples (resultsnot shown). The samples were extracted overnight at 4
C. After centrifugation at 40 000
C for 20 min, the supernatant wasevaporated under a N
stream to remove the acetone. The remainingaqueous solution was extracted with 3
3 ml of diethyl ether. Theether extracts were puriﬁed by passing them through individualaminopropyl solid-phase extraction cartridges (500 mg of sorbent;Supelco, USA). After a further washing with 7 ml of chloroform:isopropanol (3:1, v/v), JA was eluted with 6 ml of diethyl ether:acetic acid (98:2, v/v). The eluate was then evaporated to drynessunder a N
stream and derivatized with ethereal diazomethane. After derivatization, samples were dissolved in 50 ml of ethyl acetate.A 1 ml aliquot was injected into a GC–MS (Trace 2000-Voyager,Finnigan, Thermo-Quest, USA). The methyl esters of JA andDHJA were separated by gas chromatography on a DB-5 column(30 m long, 0.25 mm i.d.) with a 0.8 ml min
He ﬂow, anddetected by MS in selective ion monitoring (electron impact of 70 eV) at
224 and 226, respectively. The mass spectra of Me-JA and the methyl ester of 9,10-dihydrojasmonic acid areshown in Fig. 1.
Measurement of nicotine concentration
The nicotine concentration was analysed by the ultraviolet absorp-tion method (Willits
, 1950). In brief,
0.5 g of dry sample wasweighed in a dry, clean glass tube of 5 cm inner diameter, and 20 mlof distilled water and 10 ml of 30% (w/v) NaOH solution were added.The tube was placed in a distillation device and a 250 ml ﬂat-bottomed ﬂask was used to collect the distilled nicotine solution.Distilled water was added to make the solution up to 250 ml and thenit was measured colorimetrically at 236, 259, and 282 nm using a spectrophotometer (Shimadzu UV-2201, Japan). The nicotine con-centration was expressed as a percentage of the tissue dry weight.
Effects of different treatments on growth of tobacco plants
Removal of the shoot apex stimulatedgrowth of the upper leaves and outgrowth of lateral buds,which was inhibited by the immediate application of theauxin analogue NAA, which appeared also to havea signiﬁcantly positive effect on root growth (Table 1).
Auxin regulation of nicotine synthesis