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AOBPreview originally published online on August 12, 2005
Annals of Botany 2005 96(5):793-797; doi:10.1093/aob/mci229
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© The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Nitrogen Supply after Removing the Shoot Apex Increases the Nicotine Concentration and Nitrogen Content of Tobacco Plants

X. Y. XI, C. J. LI* and F. S. ZHANG

The Key Laboratory of Plant Nutrition, Ministry of Agriculture, Key Laboratory of Plant–Soil Interactions, Ministry of Education, China Agricultural University, Beijing 100094, China

* For correspondence. E-mail lichj{at}cau.edu.cn

Received: 18 January 2005    Returned for revision: 9 May 2005    Accepted: 1 June 2005    Published electronically: 12 August 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

• Background and Aims High nicotine concentrations in leaves, especially in the upper leaves, offer a serious problem for the cultivation of tobacco (Nicotiana tabacum). Preliminary field experiments showed that rapid mineralization of soil N during late stages of growth may contribute to high nicotine concentrations in leaves.

• Methods A sand-culture experiment was carried out in the greenhouse. The N supply was controlled during the experiment, and different amounts of 15N were supplied during late stages of growth (after removal of the shoot apex), to investigate the contribution of the N taken up at this time to the N content of and nicotine concentration in tobacco plants.

• Key Results Addition of 1·6 g or 4 g 15N-labelled NH4NO3 after removing the shoot apex and flushing out the 14N did not increase leaf dry weights; however, it did result in delayed leaf senescence, more lateral bud formation, and an increase in 15N as a proportion of total N, and nicotine-15N as a proportion of total nicotine-N in each organ. The nicotine concentration, 15N and nicotine-15N abundances were increased from the bottom to the top leaves. When more 15N-labelled NH4NO3 was supplied, the nicotine concentration in leaves increased, and so did the 15N abundance in nicotine-N.

• Conclusion Enhanced N supply in the later growth stages (after removing the apex) increased N content and nicotine concentration in tobacco plants. Nicotine was synthesized de novo during the late growth stages.

Key words: Flue-cured tobacco, 15N-isotope, nitrogen, nicotine concentration, removal of the shoot apex


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Nicotine concentration is a key index for evaluating the quality of tobacco, and is closely correlated with the amount of nitrogen (N) supplied since N is 17·3 % of the molecular weight of nicotine (Weybrew and Woltz, 1975Go; Campell et al., 1982Go; Collins and Hawks, 1994Go). In tobacco, N uptake is low during the first 3 weeks after transplanting, and then sharply increases between 3 and 8 weeks. About 80 % of total N is taken up by 8 weeks (Collins and Hawks, 1994Go). Less N uptake after excising the apex is beneficial to maintain a low nicotine concentration in the leaves, since nicotine accumulation in tobacco leaves occurs mainly during late stages of growth, especially during the period after removing the apex (Mumba and Banda, 1990Go; Hu et al., 1999Go). As shown by the results of a previous field experiment using 15N, tobacco plants take up a substantial amount of N after removal of the apex and >50 % of N taken up was from the soil, e.g. mineralized N (Li et al., unpublished results). In other tobacco-producing countries, where leaves have lower nicotine concentrations, light-textured soils such as sandy soils with quite low amounts of organic matter are chosen for tobacco production (Chari et al., 1994Go; Collins and Hawks, 1994Go). It seems that mineralization of soil N during late growth stages might contribute to high N contents and nicotine concentrations in flue-cured tobacco. To test this hypothesis, a sand-culture experiment was carried out, in which the N supply was controlled, and different amounts of 15N were supplied during the late stages of growth (after removal of the shoot apex), to investigate the contribution of the N taken up at this time to the amount of N and nicotine concentration of leaves of flue-cured tobacco plants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Plant culture and growth conditions
Tobacco (Nicotiana tabacum L. ‘K 326’) seeds 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 were grown on in a seed bed in a naturally lit glasshouse for 60 d. On 23 May 2003, the seedlings were washed with tap water until all substrate was removed from the roots, and then they were transplanted into pots (one plant per pot 25 cm in diameter and 40 cm in height) containing 16 kg quartz sand (0·25–0·5 mm in diameter). The plants were grown in a glasshouse at China Agricultural University, Beijing.

Watering, treatments and harvest procedures
The plants were watered daily according to transpiration (weight lost) by weighing the pots. There were four treatments in the experiment: (1) 8 g 14N-NH4NO3 was applied until removal of the apex, the pot was then washed with 23 L water and 10 L N-free nutrient solution, and watered with the same N-free solution afterwards (8 g 14N-I); (2) 8 g 14N-NH4NO3 was supplied until 10 d after removal of the apex, and then the same procedure as in (1) was applied to flush out the remaining N (8 g 14N-II); (3) and (4) 8 g 14N-NH4NO3 was supplied until the apex was removed and then, after washing out the remainder of the 14N with tap water, 1·6 g and 4 g, respectively, 15N-NH4NO3 was supplied until harvest (8 g 14N + 1·6 g15N and 8 g 14N + 4 g 15N). 15N was provided as 15NH415NO3, produced in Research Institute of Chemical Industry in Shanghai, China. Before removal of the apex, N was provided daily according to the demand of the plants for N as shown in Fig. 1. There were four replicates of each treatment. Except when the pots were washed to remove 14N, the hole in the bottom of the pot was plugged tightly during the whole experimental period to avoid loss of N and water. Other nutrients were supplied throughout the experiment with the same dose for each treatment, and at the same time as N additions, as follows: (in mM for full strength) 1 KH2PO4; 1·5 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 10–2 CuSO4; 2·5 x 10–2 (NH4)6Mo7O24; 1 x 10–1 Fe-EDTA.



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  		FIG. 1. Amounts of nitrogen supplied weekly and cumulative curves (inserted figure) for tobacco plants subjected to four N treatments.

 
As is the practice in tobacco production, the shoot apex was excised when the first flower of the inflorescence was blossoming and 23 leaves were left. The leaves were harvested from bottom to top when they turned yellow. The times for excision of the apex and the first and last harvest of the leaves are shown in Table 1. Leaves were numbered, starting with the lowest mature leaf, which was called leaf 1. After harvest, leaves were separated into six groups with three or four leaves each (details in tables). Excision of the apex releases lateral shoot buds (Cline, 1994Go); these were removed and collected as one sample. After harvesting the last leaf, stem and roots were separated. Roots were washed free of sand with water. All plant parts were weighed (fresh weight), dried (70 °C) and weighed again (dry weight). The dry samples were finely ground (<0·5 mm) for N determinations.


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  		TABLE 1. Times of topping and the first and the last harvests of the leaves after transplanting tobacco seedlings to Wagner pots for different N treatments

 
Measurement of total N content and 15N abundance
To determine total N content, a sample of about 0·3 g was digested, distilled and titrated according to the semi-micro-Kjeldahl method (Shen and Xu, 1983Go). To determine 15N abundance, 0·5–1 g samples were 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 al. (1982)Go in the Institute of Soil Science, Chinese Academy of Sciences, Nanjing, using mass spectrometry (F2.32innigan-Mat-251, Mass-Spectrometers, Finnigan, Germany).

Nicotine concentration and 15N abundance in nicotine-N
The nicotine concentration was analysed by the ultraviolet-absorption method (Willits et al., 1950Go). In brief, about 0·5 g of dry sample was weighed in a dry, clean glass tube of 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). The nicotine concentration was expressed as a percentage of the tissue dry weight.

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 analysed 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 four replicates of each treatment at harvest. All further analyses were made with four individual samples for each organ. For statistical analysis of the data, the program SAS for Windows (version 6.12) was used (SAS Institute, 1987Go). Differences between data in all tables were subjected to ANOVA.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Effects of N supply during late growth stages on overall plant growth
Despite the greater total dry weights of the plants supplied with 1·6 or 4 g of 15N-labelled NH4NO3 after removal of the apex, withdrawal of N supply during the late growth stages did not influence final dry weights of the harvested leaves (Table 2). Unlike other crops, it is the senescing leaves of tobacco plants which are of economic value (Zuo, 1993Go, translated by Zhu, 1993Go). The present results suggested that N taken up before removal of the apex was sufficient to meet the plant's demand for growth and development until harvest. Extra supply of N after excision of the apex led to some negative effects, such as delayed leaf maturation and harvest time (Table 1), and more outgrowths of lateral buds (results not shown). It has been suggested that removal of the shoot apex of tobacco plants could stimulate root growth because of the changed pattern of assimilate distribution within the plants (Goenaga et al., 1989Go; Jiang et al., 2001Go). Extra supply of N after topping further enhanced root growth (Table 2), which might be one reason for the increased nicotine concentration in different leaves, since nicotine is synthesized in the roots of tobacco, especially in the root tips (Tso and Jeffrey, 1956Go; Solt, 1957Go).


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  		TABLE 2. Dry weights of different organs and whole tobacco plant at harvest as affected by different amounts of N supplied after topping

 
Effects on N content and 15N abundance and their distribution within the plant
There were no differences in total N uptake and distribution within the plants between the 8 g 14N-I and 8 g 14N-II treatments (Table 3), although in the second treatment the N supply was prolonged for 10 d more. Extra supply of 1·6 g 15N after washing out the rest of the 14N also did not change the N content of each leaf; however, the source of the N in the leaves was totally different. The amount of 15N in different leaves increased from the bottom to the top: in leaves 1–3, only 1 % of the total N was 15N, while in leaves 20–23, the proportion of 15N reached 21·3 % (Table 4). These results indicated that (a) when no N was supplied after removal of the apex, N taken up before topping was translocated from the lower parts of the plant such as stems and roots to the young leaves (N is a relatively mobile element in plants, moving easily between organs; Marschner, 1995Go); and (b) when extra N was supplied after removing the apex, the recently absorbed N was distributed to all parts of the plant, especially to the developing leaves. Increasing the amount of extra 15N (4 g) supplied after topping enhanced both the 15N abundance in all plant organs and the total N content in all plant organs (Tables 3 and 4).


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  		TABLE 3. Nitrogen contents of various organs and of whole tobacco plants at harvest as affected by different amounts of N supplied after topping

 

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  		TABLE 4. The 15N contents and proportion of 15N to total N in various organs of tobacco plants at harvest

 
Effects on nicotine concentration and nicotine-15N abundance in total nicotine-N in each organ
Nicotine concentrations in lateral buds, stems and roots were lower than those in leaves, irrespective of N regime. In leaves, the nicotine concentration increased from bottom to top in all of the treated plants. An extra supply of 1·6 g 15N after washing out the remaining 14N markedly increased the nicotine concentration in middle and upper leaves; increasing the 15N supply (4 g) further increased the nicotine concentration in leaves, but not in stems and roots (Table 5). Although extra 15N supplied after excision of the apex increased 15N abundance, both in total N content and in nicotine-N in different leaves (Tables 4 and 6), it should be pointed out that there is no direct relationship between leaf N content and nicotine concentration, since nicotine is synthesized in the roots and transported in the xylem to the shoot (Tso and Jeffrey, 1956Go; Solt, 1957Go; Alworth and Rapoport, 1965Go). N cannot be used directly to synthesize nicotine in leaves.


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  		TABLE 5. Nicotine concentrations in various organs of tobacco plants at harvest as affected by different amount of N supplied after topping

 

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  		TABLE 6. Amount of nicotine-15N and proportion of nicotine-15N to total nicotine-N in various organs of tobacco plants at harvest

 
The results indicate that the more N was supplied after removing the apex, the higher the nicotine concentration in the leaves, and the higher the 15N abundance in nicotine-N (Tables 5 and 6). The results show that more N was taken up during the late growth stages and was used to synthesize nicotine. Therefore, by controlling N supply during the late stages of growth in tobacco plants it is possible to reduce the nicotine concentration in the leaves. However, the nicotine concentration in leaves 20–23 of the plants not supplied with extra N still reached 1·8 % after topping (Table 5), meaning that plants could use the N taken up before removing the apex to synthesize nicotine.

It has been reported that mechanical wounding of tobacco leaves can induce a 10-fold local increase in jasmonic acid concentration within 90 min of them being damaged and, systemically, a 3·5-fold increase in the roots 180 min after wounding, with nicotine concentrations in the leaves reaching their highest levels 5 d after wounding (Baldwin et al., 1997Go; Ohnmeiss et al., 1997Go). These results indicated that nicotine accumulation is a response to wounding, and jasmonic acid functions as an intermediary between stimulus and response. It would be interesting to know whether removing the apex before the leaves are harvested, as is the practice in tobacco production, also stimulates jasmonic acid synthesis, and if this results in increased nicotine concentration in the leaves, and, if so, how this is regulated.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Professor Dr H. Lambers for valuable comments and careful correction of the manuscript, the NNSFC (No. 30370842), the Ministry of Agriculture 948 program (No. 2003-Z53) and the State Company for Tobacco Production and Selling for financial support.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

    Alworth WL, Rapoport H. 1965. Biosynthesis of the nicotine alkaloids in Nicotiana glutinosa among nicotine, nornicotine, anabasine, and anatabine. Archives of Biochemistry and Biophysics 112: 45–53.[CrossRef][Web of Science][Medline]

    Baldwin IT, Zhang ZP, Diab N, Ohnmeiss TE, McCloud ES, Lynds GY, et al. 1997. Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris. Planta 201: 397–404.[CrossRef][Web of Science]

    Buresh RJ, Austin ER, Craswell ET. 1982. Analytical methods in 15N research. Fertilizer Research 3: 37–62.

    Campbell CR, Chaplin JF, Johnson WH. 1982. Cultural factors affecting yield, alkaloids, and sugars of close-grown tobacco. Agronomy Journal 74: 279–283.[Abstract/Free Full Text]

    Chari MS, Rao J, Reddy PR, Rao CC, Murthy KS. 1994. Nitrogen requirement of flue-cured tobacco in northern light soils of Andhra Pradesh. Tobacco Research 20: 53–57.

    Cline MG. 1994. The role of hormones in apical dominance. New approaches to an old problem in plant development. Physiologia Plantarum 90: 230–237.[CrossRef]

    Collins WK, Hawks Jr SN. 1994. Principles of flue-cured tobacco production. Raleigh, NC: North Carolina State University, 23–98.

    Goenaga RJ, Volk RJ, Long RC. 1989. Uptake of nitrogen by flue-cured tobacco during maturation and senescence. I. Partitioning of nitrogen derived from soil and fertilizer sources. Plant and Soil 120: 11–139.[CrossRef]

    Hu GS, Han JF, Mu L. 1999. Study on accumulation characteristics of nicotine in flue-cured tobacco, Tobacco in Fujian 2: 31–32 [In Chinese].

    Jiang F, Li CJ, Jeschke WD, Zhang FS. 2001. Effect of top excision and replacement by 1-naphthylacetic acid on partition and flow of potassium in tobacco plants. Journal of Experimental Botany 52: 2143–2150.[Abstract/Free Full Text]

    Marschner H. 1995. Mineral nutrition of higher plants, 2nd edn. London: Academic Press.

    Mumba PP, Banda HL. 1990. Nicotine content of flue tobacco (Nicotiana tabacum L.) at different stages of growth. Tobacco Science 30: 179–183.

    Ohnmeiss TE, McCloud ES, Lynds GY, Baldwin IT. 1997. Within-plant relationships among wounding, jasmonic acid and nicotine: implications for defence in Nicotiana sylvestris. New Phytologist 137: 441–452.[CrossRef][Web of Science]

    SAS Institute 1987. SAS/STAT guide for personal computers, 6th edn. Cary, NC: SAS Institute.

    Shen D, Xu H. 1983. Digestion of plant samples and determination of total N. In: Analysis methods for soil and agricultural chemistry. Beijing: Soil Science Society of China/Science Press, pp. 272–275 [in Chinese].

    Solt ML. 1957. Nicotine production and growth of excised tobacco root cultures. Plant Physiology 32: 480–484.[Free Full Text]

    Tso TC, Jeffrey RN. 1956. Studied on tobacco alkaloids. II. The formation of nicotine and nornicotine in tobacco supplied with N15,12. Plant Physiology 31: 86–93.

    Weybrew JA, Woltz WG. 1975. Production factors affecting chemical properties of the flued-cured leaf. IV. Influence of management and water. Tobacco International 177: 46–48.

    Willits CO, Swan Margaret L, Connelly JA, Brice BA. 1950. Spectrophotometric determination of nicotine. Analytical Chemistry 22: 430–433.[CrossRef]

    Zhu ZQ. 1993. Production, physiology and biochemistry of tobacco. Shanghai: Fareast Press (translation of book written by Zuo TJ).


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