Abstract
Mechanical wounding stimulates nicotine synthesis in tobacco plants. In the practice of tobacco production, most nitrogen (N) is taken up before removal of the shoot apex, while nicotine is mainly synthesized afterwards. Since N is required for nicotine synthesis, it is interesting to know whether plants can use N taken up before removal of the shoot apex to synthesize nicotine after wounding. To address this question, a hydroponics culture experiment was carried out, in which N was supplied as NH4NO3 at two levels (1 mM and 6 mM) in pre-culture, and N was either withdrawn or replaced by 15N after removing the shoot apex for the next seven days. Removal of the shoot apex caused a marked increase in nicotine concentration in various organs, also when plants grew under low-N conditions and showed symptoms of N deficiency. Increased nicotine accumulation even occurred when N was withdrawn from the growth medium before the apex was removed, indicating that tobacco plants can use N taken up previously to synthesize nicotine after mechanical wounding. The amount of N used for nicotine synthesis accounted for 5–6% of the total N, irrespective of treatment. Although most of the nicotine in intact plants and plants with the apex removed was synthesized de novo, as evidenced by the data when N was replaced by 15N-labeled NH4NO3, a large amount of the N absorbed before the N replacement was incorporated into the newly formed nicotine. The proportion of nicotine-15N to total nicotine-N was almost the same as that of 15N to total N in various organs. The results show the utilization of remobilized N taken up before excision of the shoot apex for nicotine synthesis afterwards, and highlight the importance of N cycling within plants, both when grown under N-sufficient and N-deficient conditions.
Key words: 15N-isotope nitrogen, mechanical wounding, nicotine concentration, nicotine synthesis, nitrogen deficiency, removal of the shoot apex, tobacco (Nicotiana tabacum L.)
Introduction
Nicotine is an alkaloid, which is only produced in tobacco, and accounts for about 95% of its total alkaloid content.1,2 In undamaged tobacco plants, the nicotine concentration in leaves is only 0.1–1% of its dry mass. Nicotine is a defense compound,3 and its synthesis is stimulated by herbivore attack and mechanical wounding.4 Since nicotine is synthesized in the roots and transported in the xylem to the shoot,5,6 a signal molecule between the stimulus in the shoot and the response in the roots is needed. Both jasmonic acid and auxin can act as long-distance signals between the wounding stimulus and the nicotine response in tobacco.2,4,7 However, it is still not clear, whether tobacco plants can use N taken up before mechanical wounding to synthesize nicotine afterwards, for instance, after excision of the shoot apex. The aim of this study was to investigate the contribution of N taken up before wounding, especially when plants were grown under N-deficient conditions and showed symptoms of N deficiency, to nicotine synthesis after removing the shoot apex.
Results and Discussion
Effects on Plant Growth, Nitrogen Uptake and 15N Abundance in Each Organ.
After 9 d pre-culture at 1 mM N, typical N-deficiency symptoms were observed, such as small plant size and decreased shoot/root ratio (calculated from the results in Table 1), small and senesced lower leaves. The total N content in whole plant was also lower than in those grown at 6 mM N (Table 2). After an additional 7 d growth under different treatments, the highest total net increments of both dry weight and N content were found in intact plants, irrespective of N supply. Removal of the shoot apex changed the pattern of dry matter distribution within plants, and stimulated the growth of upper leaves and roots (Table 1).12,13 The net N gain in upper leaves and roots of the plants whose apex was removed grown at either N level was also enhanced at the second harvest, compared with that of the intact plants (Table 2). However, regardless of the shift of the dry matter distribution within plants to roots, resulting in a larger root system (Table 1), the total net N increment of the plants whose apex was removed grown under either N level for 7 d was less than that of the respective intact plants, especially in the case of the plants grown at high N level (Table 2). The results indicate that the amount of nitrogen taken up was not determined by the root size, but by the presence of the shoot apex. The higher N uptake by the intact plants would be explained by a demand-driven regulatory mechanism.14,15 The plant apex is a center of growth and metabolism, and imports assimilates from source leaves16 and mineral nutrients taken up by the root system. A sink-dependent stimulation of nutrient uptake in plants has been reported before.17,18 Withdrawing N from the growth medium after removal of the shoot apex reduced net dry matter gains in the plants grown at both N levels (Table 1); N taken up before N withdrawal was remobilized from the lower leaves and recycled to the vigorously growing parts of the plants, such as upper leaves and roots (Table 2). N is a mobile nutrient in plants, moving easily between organs.19
Table 1.
Initial values and net increments in dry weights of component organs and of whole tobacco plant as affected by two N levels and removing apex over a 7 d study period
| Organs | 1 mM N | 6 mM N | |||||||
| Net increments in DW (g organ-1 Plant-1) | Net increments in DW (g organ-1 Plant-1) | ||||||||
| Initial Values | Removing apex - N | Removing a pex +15N | Intact Plant +15N | Initial Values | Removing apex - N | Removing apex +15N | Intact Plant +15N | ||
| Top* | 1.4 b | 5.5 a | |||||||
| Upper leaves | 1.4 ± 0.2 | 1.2 cd | 2.1 c | 1.1 d | 1.9 | 3.8 b | 5.5 a | 1.7 cd | |
| Lower leaves | 1.0 ± 0.2 | 0.5 a | 0.4 ab | 0.6 a | 1.4 | 0.3 ab | 0.5 a | - 0.1 b | |
| Stem | 0.3 ± 0.1 | 0.5 c | 0.6 c | 0.8 b | 0.4 | 0.6 c | 0.9 b | 1.9 a | |
| Roots | 0.5 ± 0.1 | 0.8 c | 1.0 b | 0.9 bc | 0.6‘ | 1.5 a | 1.1 b | 0.9 bc | |
| Whole plant | 3.3 ± 0.5 | 2.9 e | 4.1 de | 4.7 d | 4.3 | 6.2 c | 8.0 b | 10.0 a | |
Including apex and newly formed leaves. Values in rows (excepting initial value) followed by different letters are significantly different (LSD Test p < 0.05).
Table 2.
Initial values and net increments in nitrogen contents in component organs and of whole tobacco plant as affected by two N levels and removing apex over a 7 d study period
| 1 mM N | 6 mM N | |||||||
| Position | Net Increments in N (mg organ-1 plant-1) | Net Increments in N (mg organ-1 plant-1) | ||||||
| Initial Values | Removing apex-N | Removing apex +15N | Intact Plant +15N | Initial Values | Removing apex-N | Removing apex +15N | Intact Plant +15N | |
| Top* | 43.9 b | 279.3 a | ||||||
| Upper leaves | 27.8 ± 2.8 | 2.0 e | 56.6 bc | 26.5 c | 95.9 | 12.5 d | 239.1 a | 64.9 b |
| Lower leaves | 15.0 ± 2.2 | - 1.5 c | 6.3 c | 10.9 b | 56.8 | - 20.4 d | 27.2 a | - 3.6 c |
| Stem | 2.5 ± 0.7 | 0.7 d | 10.0 c | 9.1 c | 10.6 | 1.5 d | 32.5 b | 47.0 a |
| Roots | 10.9 ± 1.9 | 1.1 d | 25.3 c | 20.8 c | 22.7 | 11.1 c | 45.9 a | 33.6 b |
| Whole plant | 56.1 ± 6.0 | 2.3 d | 98.2 c | 111.2 c | 186.0 | 4.7 d | 344.7 b | 421.1 a |
Including apex and newly formed leaves. Values in rows (excepting initial value) followed by different letters are significantly different (LSD Test, p < 0.05).
The results of the N replacement by 15N-labeled NH4NO3 showed that the amount of 15N in different leaves increased from the bottom to the top (Table 3), indicating that the recently absorbed N was distributed to all parts of the plant, especially to the rapid growth tissues, such as top and roots. On the other hand, a certain amount of the N taken up before the N replacement was also incorporated into the newly formed top tissue during the 7-d study period. A high N supply enhanced both the 15N abundance in all plant organs (Table 3) and the total N content in all plant organs (Table 2).
Table 3.
The proportion of 15N to total N in various organs of tobacco plants grown under two N levels over a 7 d study period
| 15N Abundances (%) | ||||
| Position | 1 mM N | 6 mM N | ||
| Removing apex +15N | Intact plant +15N | Removing apex +15N | Intact plant +15N | |
| Top* | 65.2 b | 72.6 a | ||
| Upper leaves | 56.1 b | 47.9 c | 67.3 a | 50.1 c |
| Lower leaves | 22.3 b | 20.4 b | 34.5 a | 23.9 b |
| Stem | 44.6 c | 43.5c | 60.3 b | 63.5 a |
| Roots | 54.5 b | 50.4 c | 66.0 a | 63.1 a |
Including apex and newly formed leaves. Note: Values in rows followed by different letter are significantly different (LSD Test, p < 0.05).
Effects on Nicotine Concentration and nicotine-15N Abundance in Total nicotine-N in each Organ.
The present results demonstrate that removing the shoot apex markedly increased the nicotine concentration in tobacco, especially in leaves, independent of the N level supplied (Table 4) which supports the conclusion that mechanical wounding stimulates nicotine synthesis in tobacco plants.4,7 As shown in Table 4, the plants could use previously absorbed N to synthesis nicotine, even when N was withdrawn from the growth medium after removing the apex, in spite of its lower nicotine concentration, compared with that in plants whose apex was removed grown at either N level. Furthermore, since the plants supplied with l mM N already suffered from N deficiency before removal of the shoot apex (Table 1), and there was no free NO3- detected in any organ of the tobacco plants supplied with 2 mM N in one of our previous studies (ref. 20) which was at the same growth stage as the plants in the present study, the N used for nicotine synthesis after the treatment could only come from remobilization in plant tissues. The amount of N used for nicotine synthesis in the plants whose apex was removed and whose N supply was withdrawn accounted for 5%–6% of the total N (Fig. 1), and most of the nicotine was synthesized de novo during the 7-d study period after removing the shoot apex (calculated from the results in Tables 1 and 4). Continuous supply of N after excision of the shoot apex enhanced the nicotine concentration in leaves. The more N was supplied, the higher the nicotine concentration in leaves (Table 4), and the proportion of N used for nicotine synthesis to total N in plants decreased, especially when plants grew at a high N level, compared with that in plants whose apex was excised and N was withdrawn from the growth medium (Fig. 1). Although most of the nicotine in plants was newly synthesized after N replacement, there was still a large amount of N incorporated into nicotine that was absorbed before the N replacement, and the proportion of nicotine-15N to total nicotine-N increased, when more N was supplied in the growth medium. Comparing the results in Tables 3 and 5 shows that the proportion of nicotine-15N to total nicotine-N was almost that of 15N to total N in various organs. The results suggest strong N cycling within plants, even when they suffered from N deficiency. The importance of cycling of nitrogen within both N-sufficient and N-deficient plants has been described highlighted before by Lambers et al., (ref. 21).
Table 4.
Initial and final nicotine concentration in various organs of tobacco plants grown under two N levels over a 7 d study period
| Nicotine concentration (mg g-1) | ||||||||
| Position | 1 mM N | 6 mM N | ||||||
| Initial Values | Removing apex-N | Removing apex +15N | Intact Plant +15N | Initial Values | Removing apex-N | Removing apex +15N | Intact Plant +15N | |
| Top* | 1.4 b | 2.5 a | ||||||
| Upper leaves | 1.1 d | 3.6 c | 7.5 b | 1.6 d | 1.6 d | 7.0 b | 8.5 a | 3.0 c |
| Lower leaves | 1.7 e | 2.7 d | 4.1 bc | 1.9 e | 2.6 d | 4.4 b | 9.5 a | 3.4 c |
| Stem | 1.3 d | 3.0 b | 3.2 b | 1.4 d | 1.9 cd | 4.8 a | 4.4 a | 2.3 c |
| Roots | 2.3 d | 4.0 b | 3.7 bc | 2.3 d | 2.3 d | 4.9 a | 3.4 bc | 2.8 c |
Including apex and newly formed leaves. Note: Values in rows (excepting initial value) followed by different letter are significantly different (LSD Test, p < 0.05).
Figure 1.
Ratios of nicotine-N to total-N in whole tobacco plants grown under two N levels over a 7 days study period.
Table 5.
The proportion of nicotine-15N to total nicotine-N in various organs of tobacco plants grown under two N levels over a 7 d study period
| Amount of nicotine-15N (%) | ||||
| Position | 1 mM N | 6 mM N | ||
| Removing apex +15N | Intact plant +15N | Removing apex +15N | Intact plant +15N | |
| Top* | 60.8 b | 72.1 a | ||
| Upper leaves | 55.9 b | 42.8 d | 67.6 a | 50.7 c |
| Lower leaves | 27.8 b | 19.7 c | 39.8 a | 24.0 bc |
| Stem | 37.8 b | 37.7 b | 64.4 a | 66.0 a |
| Roots | 51.6 b | 46.9 c | 67.1 a | 65.8 a |
Including apex and newly formed leaves. Note: Values in rows followed by different letter are significantly different (LSD Test, p < 0.05).
Although a high N supply enhanced the nicotine concentration in leaves after removal of the shoot apex, there is no direct relation between leaf N and nicotine concentration, since nicotine is synthesized in the roots and transported in the xylem to the shoot.5,6
In conclusion, the results from the present study indicated that removal of the shoot apex of tobacco plants grown at either N level reduced net increases in dry matter and N content, and shifted dry mass and N distribution to the upper leaves and roots. However, the amount of N taken up was not determined by the size of the root system, but by the presence of the shoot apex. On the other hand, removal of the shoot apex stimulated nicotine synthesis in tobacco plants. The results highlight the utilization of remobilized N taken up before for nicotine synthesis after excision of the shoot apex, even when plants grew under N-deficient condition.
Materials and Methods
Plant cultivation.
Tobacco seeds (Nicotiana tabacum L. K 326) were germinated in a mixture consisting of 60% (w/w) peat culture substrate, 20% (w/w) ground maize stalk and 20% (w/w) perlite, and grown in a seedbed in a naturally illuminated greenhouse for 60 d. Afterwards, the seedlings were washed with tap water to remove all substrates from the roots, and then transferred to 2.1 L porcelain pots (one plant per pot) with 1/4 strength aerated nutrient solution. The solution consisted of (in mM for full strength): 3 NH4NO3; 1 KH2PO4; 2 K2SO4; 2 CaCl2; 2.5 x 10-1 MgSO4; 2.5 x 10-2 KCl; 1.25 x 10-2 H3BO3; 1 x 10-3 MnSO4; 1 x 10-3 ZnSO4; 2.5 x 10-4 CuSO4; 1 x 10-1 Fe-EDTA; 2.5 x 10-4(NH4)6Mo7O24. Plants were grown in a growth room with a 14-h photoperiod. The photosynthetically active radiation at the surface of the pots was 220–270 µmol m-2 s-1 provided by reflector sunlight metal halide lamps (Philip Hpiplus, 250W, Belgium).
Treatments and harvest procedures.
After 5 d growth in 1/4 strength nutrient solution, the plants were divided into two groups of 20 plants, each of similar size and development, and full-strength nutrient solution with two N levels (1 mM and 6 mM) was supplied. Nitrogen was provided in the form of NH4NO3. The first harvest of 5 plants in each group was at 9 d after supply of two N levels. On the same day, the remaining 15 plants of the two groups were divided into three sub-groups and treated as follows: 1) intact plants, N was replaced by 15N-labeled NH4NO3 with the same concentration (control). 15N was provided as 15NH415NO3, produced in Research Institute of Chemical Industry in Shanghai, China; 2) apex was excised above the youngest unfolded leaf, number eight from its base (removing apex), and N was withdrawn from the nutrient solution; 3) apex was excised in the same way as in 2), and N was replaced by 15N-labeled NH4NO3. The second harvest was at 7 d after the replacement by 15N-labeled NH4NO3. There were 5 replicates in each treatment.
Plant leaves were numbered in ascending order, starting with the lowest mature leaf, which was designed as leaf 1. Smaller leaves, which had already senesced, were removed. At the first harvest, the youngest unfolded leaf was no. 8, and the apical part was incorporated into the upper leaves because of its small size. At harvest, plants were separated into roots, stems, tops (for the second harvest, including apex and newly formed leaves after the treatments), the lower stratum of leaves no. 1–5 and the upper stratum of leaves no. 6–8. The lateral buds of the plants whose apex was removed were removed immediately after their emergence and incorporated into upper leaves by harvest. All plants parts were dried (70°C) until constant dry weight and weighed. They were finely ground (<5 mm) and used for N and nicotine determinations.
Measurement of total N content and 15N abundance.
To determine total N content, a sample of about 0.3 g dry mass was digested, distilled and titrated according to the semi-micro-Kjeldahl method.8 To determine 15N abundance, 0.5–1 g sample was used. After titration, the solution was condensed to 1–3 mL in a water bath at 100°C. 15N abundance was determined using the method of Buresh et al9 in the Institute of Soil Science, Chinese Academy of Sciences, Nanjing, using a mass spectrometer (F2.32innigan-Mat-251, Mass-Spectrometers, Finnigan, Germany).
Nicotine concentration and 15N abundance in nicotine-N.
The nicotine concentration was analyzed by the ultraviolet-absorption method.10 In brief, about 0.5 g of dry sample was weighed in a dry, clean glass tube with 5 cm inner diameter, and 20 mL 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 then it was measured colorimetrically at 236 nm, 259 nm, and 282 nm using a spectrophotometer (Shimadzu UV-2201, Japan).
To measure the 15N abundance in nicotine-N, 1–5 g of the dry samples was weighed, and the nicotine distillate was obtained as mentioned above. The distillate was concentrated in about 10 mL on a water bath at 100°C, and the total nicotine-N and the amount of 15N in nicotine-N was analyzed by the same method described above for determining total N content and 15N abundance.
Statistical analyses.
Dry weight, total N content, 15N abundance in total N, nicotine concentration and 15N abundance in nicotine-N were obtained from five replicates of each treatment at harvest. All further analyses were made with five individual samples for each organ. For statistical analysis of the data, the program SAS for Windows (version 6.12) was used.11 Differences between data in all tables were subjected to ANOVA.
Acknowledgements
The authors are grateful to Dr. H. Lambers for valuable comments and careful correction of the manuscript, the National Natural Science Foundation of China (30370842) and Program for Changjiang Scholars and Innovative Research Team in University (IRT0511) for financial supports.
Footnotes
Previously published online as a Plant Signaling & Behavior E-publication: www.landesbioscience.com/journals/psb/article/5121
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