Nicotine concentration in leaves of flue-cured tobacco plants as affected by removal of the shoot apex and lateral buds

It is believed that the nicotine concentration in tobacco is closely correlated with the amount of nitrogen (N) supplied.On the other hand,N uptake mainly occurs at the early growth stage,whereas nicotine concentration increases at the late growth stage,especially after removing the shoot apex.To identify the causes of the increased nicotine concentration in tobacco plants,and to compare the effects of different ways of mechanical wounding on nicotine concentration,field experiments were carried out in Fuzhou,Fujian Province in 2003 and 2004.Excision of the shoot apex had almost no influence on N content in the plant;however,it caused dramatic increases in nicotine concentration in leaves,especially in the middle and upper leaves.An additional increase of the nicotine concentration was obtained by removal of axillary buds.The wounding caused by routine leaf harvests,however,did not change the leaf nicotine concentration,and neither did reducing leaf harvest times.The present results revealed no direct relationship between N supply and nicotine concentration in tobacco leaves,and indicate that not all kinds of mechanical wounding were capable of stimulating nicotine synthesis in tobacco plants.Since nicotine production is highly dependent on the removal of apical meristems and hence on the major sources of auxin in the plant,and application of 1-naphthylacetic acid onto the cut surface of the stem after removing the shoot apex markedly decreased the nicotine concentration in different leaves and the total nicotine content in the plant,the results suggest that decreased auxin supply caused by removal of the shoot apex as a kind of mechanical wounding might regulate nicotine synthesis in the roots of tobacco plants.     

Tobacco plants can use nitrogen taken up before mechanical wounding to synthesize nicotine afterwards

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.)     

Shoot Apex Demand Determines Assimilate and Nutrients Partitioning and Nutrient-uptake Rate in Tobacco Plants

Our previous experiment revealed that apex-removed plants have larger root systems but a lower K＋-uptake rates than intact tobacco plants. Since the apex is not only a center of growth and metabolism, but also an important place of auxin synthesis and export, the aims of this study were to distinguish whether the apex demand or auxin synthesized in the apex regulates assimilate and nutrients partitioning within plant, and to explain the reason for the lower K＋-uptake rate of the apex-removed plant. In comparison with the control plant, covering the shoot apex with a black transparent plastic bag reduced net increases in dry matter and nutrients; however, the distribution of the dry matter and nutrients between shoot and roots and nutrient-uptake rates were not changed. Removal of the shoot apex shifted the dry mass and nutrients distributions to roots, and reduced the rate of nutrient uptake. Application of 1-naphthylacetic acid （NAA） could partly replace the role of the removed apex, stimulated assimilate and nutrient deposition into the treated tissue, and enhanced the reduced plasma membrane ATPase activity of roots to the control level. However, treatment of the apex-removed plants with NAA could not rescue the reduced nutrient uptake rate and the shifted assimilates and nutrients partitioning caused by excision of the apex. Higher nutrient uptake rate of the intact plants could not be explained by root growth parameters, such as total root surface area and number of root tips. The results from the present study indicate that strong apex demand determined assimilates and nutrients partitioning and nutrient-uptake rate in tobacco （Nicotiana tabacum） plants.     

Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris

Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-¹³C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this long-distance signal-transduction pathway. Received: 24 April 1996 / Accepted: 18 July 1996     

Transport of [2-14C]jasmonic acid from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris

Jasmonic acid (JA) is part of a long-distance signal-transduction pathway that effects increases in de-novo nicotine synthesis in the roots of Nicotiana sylvestris Speg et Comes (Solanaceae) after leaf wounding. Elevated nicotine synthesis increases whole-plant nicotine pools and makes plants more resistant to herbivores. Leaf wounding rapidly increases JA pools in damaged leaves, and after a 90-min delay, root JA pools also increase. The systemic response in the roots could result from either: (i) the direct transport of JA from wounded leaves, or (ii) JA synthesis or its release from conjugates in roots in response to a second, systemic signal. We synthesized [2-¹⁴C]JA, and applied it to a single leaf in a quantity (189 μg) known to elicit both a whole-plant nicotine and root JA response equivalent to that found in plants subjected to leaf wounding. We quantified radioactive material in JA, and in metabolites both more and less polar than JA, from treated and untreated leaves and roots of plants in eight harvests after JA application. [2-¹⁴C]Jasmonic acid was transported from treated leaves to roots at rates and in quantities equivalent to the wound-induced changes in endogenous JA pools. The [2-¹⁴C]JA that had been transported to the roots declined at the same rate as endogenous JA pools in the roots of plants after leaf wounding. Most of the labeled material applied to leaves was metabolized or otherwise immobilized at the application site, and the levels of [2-¹⁴C]JA in untreated leaves did not increase over time. We measured the free JA pools before and after four different hydrolytic extractions of root and shoot tissues to estimate the size of the potential JA conjugate pools, and found them to be 10% or less of the free JA pool. We conclude that the direct transport of wound-induced JA from leaves to roots can account for the systemic increase in root JA pools after leaf wounding, and that metabolism into less polar structures determines the duration of this systemic increase. However, the conclusive falsification of this hypothesis will require the suppression of all other signalling pathways which could have shoot-to-root transport kinetics similar to that of endogenous JA. Received: 14 April 1997 / Accepted: 9 June 1997     

Growth Regulators in Populus tremula IV. Apical Dominance and Suckering in Young Plants

Experiments with small plants of Populus tremula L. growing in solution culture indicate that polarly transported auxin is an important factor in the control of axillary bud growth. If the auxin supply from the growing apex is eliminated, the number of buds released is influenced by factors translocated in the transpiration stream from the roots. Suckers may be induced to develop from aspen roots, the age of which is six weeks or more. Removal of the growing apex and the axillary buds or stoppage of shoot growth by short day treatment were effective in inducing abundant suckering in small aspen plants. Some mature leaves had to be maintained, indicating the dependence of sucker formation on carbohydrate supply. These treatments are known to decrease auxin production in the shoots. Extraction and biological assay showed a decrease in the content of auxin in the roots as a consequence of removal of growing shoot parts. The results indicate that suckering in roots of intact aspen plants is prevented by auxin transported into the roots from growing shoot parts.     

Nitrogen supply after removing the shoot apex increases the nicotine concentration and nitrogen content of tobacco plants

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.     

Induction of root Fe(lll) reductase activity and proton extrusion by iron deficiency is mediated by auxin-based systemic signalling in Malus xiaojinensis

Iron is a critical cofactor for a number of metalloenzymes involved in respiration and photosynthesis, but plants often suffer from iron deficiency due to limited supplies of soluble iron in the soil. Iron deficiency induces a series of adaptive responses in various plant species, but the mechanisms by which they are triggered remain largely unknown. Using pH imaging and hormone localization techniques, it has been demonstrated here that root Fe(III) reductase activity and proton extrusion upon iron deficiency are up-regulated by systemic auxin signalling in a Fe-efficient woody plant, Malus xiaojinensis. Split-root experiments demonstrated that Fe-deprivation in a portion of the root system induced a dramatic increase in Fe(III) reductase activity and proton extrusion in the Fe-supplied portion, suggesting that the iron deficiency responses were mediated by a systemic signalling. Reciprocal grafting experiments of M. xiaojinensis with Malus baccata, a plant with no capability to produce the corresponding responses, indicate that the initiation of the systemic signalling is likely to be determined by roots rather than shoots. Iron deficiency induced a substantial increase in the IAA content in the shoot apex and supplying exogenous IAA analogues (NAA) to the shoot apex could mimic the iron deficiency to trigger the corresponding responses. Conversely, preventing IAA transport from shoot to roots blocked the iron deficiency responses. These results strongly indicate that the iron deficiency-induced physiological responses are mediated by systemic auxin signalling.     

Herbivory and caterpillar regurgitants amplify the wound-induced increases in jasmonic acid but not nicotine in Nicotiana sylvestris

Both herbivory and mechanical damage result in increases in the concentration of the wound-signal molecule, jasmonic acid (JA), and the defense metabolite, nicotine, in native tobacco plants, Nicotiana sylvestris Speg. et Comes (Solanaceae). We found that higher concentrations of JA resulted from herbivory by Manduca sexta (L.) larvae than from the mechanical damage designed to mimic the herbivory. While both herbivory and mechanical damage increased JA concentrations in roots of wounded plants, herbivory did not induce either higher root JA or nicotine responses than mechanical damage. In a separate experiment in which mechanical damage was not designed to mimic herbivory, JA responses to herbivory were higher than those to mechanical damage, but the whole-plant (WP) nicotine responses were smaller. Furthermore, when regurgitants from M. sexta larvae were applied to standardized mechanical leaf wounds, leaf JA responses were dramatically amplified. However, neither the root JA response nor the WP nicotine response was comparably amplified by application of regurgitants. Our findings demonstrate that the response of N. sylvestris to herbivory is different from its response to mechanical damage; moreover, oral secretions from larvae may be partly responsible for the difference. During feeding, M. sexta larvae appear to modify the plant's normal defensive response to leaf wounding by reducing the systemic increase in root JA after leaf damage and the subsequent WP nicotine response. Received: 28 February 1997 / Accepted: 9 June 1997     

Effect of apex excision and replacement by 1-naphthylacetic acid on cytokinin concentration and apical dominance in pea plants

As known from literature lateral buds from pea ( Pisum sativum ) plants are released from apical dominance when repeatedly treated with exogenous cytokinins. Little is known, however, about the endogenous role of cytokinins in this process and whether they interact with basipolar transported IAA, generally regarded as the main signal controlling apical dominance. This paper presents evidence that such an interaction exists.
The excision of the apex of pea plants resulted in the release of inhibited lateral buds from apical dominance (AD). This could be entirely prevented by applying 1-naphthylacetic acid (NAA) to the cut end of the shoot. Removal of the apex also resulted in a rapid and rather large increase in the endogenous concentrations of zeatin riboside (ZR), isopentenyladenosine (iAdo) and an as yet unidentified polar zeatin derivative in the node and internode below the point of decapitation. This accumulation of ZR and iAdo, was strongly reduced by the application of NAA. The observed increase in cytokinin concentration preceded the elongation of the lateral buds, suggesting that endogenous cytokinins play a significant role in the release of lateral buds from AD. However, the effect of NAA on the concentration of cytokinins clearly demonstrated the dominant role of the polar basipetally transported auxin in AD. The results suggest a mutual interaction between the basipolar IAA transport system and cytokinins obviously produced in the roots and transported via the xylem into the stem of the pea plants.     

Plants can grow on internal water

Abstract. Water uptake is required when plants enlarge but growth may also occur when no external water is present. To determine whether this growth also depends on water, we studied etiolated seedlings of soybean ( Glycine max L. Merr.) deprived of external water by transplanting to vermiculite of low water content or by transferring to water-saturated air. When the external water supply was decreased or removed, the roots continued to grow rapidly but the stems abruptly decreased in growth (in 1 min) and continued to grow slowly. The stem tissues gained water content in the upper elongating region and lost water content in the basal region. Removal of the basal stem caused growth to slow further. When all tissues surrounding the stem elongating region were removed simultaneously, stem growth decreased abruptly to near zero. Control experiments showed that the decreased growth was not caused by wounding or the removal of the auxin or nutrient supply. It is concluded that growth always depended on a source of water and, when external supplies were absent, internal water was mobilized to enlarging tissues of shoots and roots mostly from the basal stem tissues in these seedlings.     

Immunomodulation of jasmonate to manipulate the wound response

Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation.     

Wounding Nicotiana tabacum Leaves Causes a Decline in Endogenous Indole-3-Acetic Acid

We have previously observed that auxin can act as a repressor of the wound-inducible activation of a chimeric potato proteinase inhibitor II-CAT chimeric gene (pin2-CAT) in transgenic tobacco (Nicotiana tobacum) callus and in whole plants. Therefore, this study was designed to examine endogenous levels of indole-3-acetic acid (IAA) in plant tissues both before and after wounding. Endogenous IAA was measured in whole plant tissues by gas chromatography-mass spectrometry using an isotope dilution technique. ¹³C-Labeled IAA was used as an internal standard. The endogenous levels of IAA declined two- to threefold within 6 hours after a wound. The kinetics of auxin decline are consistent with the kinetics of activation of the pin2-CAT construction in the foliage of transgenic tobacco.     

Saturated humidity accelerates lateral root development in rice (Oryza sativa L.) seedlings by increasing phloem-based auxin transport

Auxin transport plays a significant role modifying plant growth and development in response to environmental signals such as light and gravity. However, the effect of humidity on auxin transport is rarely documented. It is shown here that the transport of labelled indole-3-acetic acid (IAA) from the shoot to the root is accelerated in rice (Oryza sativa L. ssp. indica cv. IR8) seedlings grown under saturated humidity (SH-seedlings) compared with plants grown under normal humidity (NH-seedlings). The development of lateral roots in SH-seedlings was greatly enhanced compared with NH-seedlings. Removal of the shoot from SH-seedlings reduced the density of lateral roots, and the application of IAA to the cut stem restored the lateral root density, while the decapitation of NH-seedlings did not alter lateral root development. Phloem-based auxin transport appeared responsible for enhanced lateral root formation in SH-seedlings since (i) the rate of IAA transport from the shoot to the root tip was greater than 3.5 cm h-1 and (ii) naphthylphthalamic acid (NPA)-induced reduction of polar auxin transport in the shoot did not influence the number of lateral roots in SH-seedlings. It is proposed that high humidity conditions accelerate the phloem-based transport of IAA from the leaf to the root, resulting in an increase in the number of lateral roots.     

Jasmonate mediates salt induced nicotine biosynthesis in tobacco (Nicotiana tabacum L.)

Jasmonate (JA), as an important signal, plays a key role in multiple processes of plant growth, deve lopment and stress response. Nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L.) are essential secondary metabolites. Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported. Here, we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco. We found that salt stress induced the biosynthesis of JA, which subsequently triggered the activation of JA responsive gene expression and, ultimately, nicotine synthesis. Bioinformatics analysis revealed the existence of many NtMYC2a recognized G box motifs in the promoter regions of NtLOX, NtAOS, NtAOC and NtOPR genes. Applying exogenous JA increased nicotine content, while suppressing JA biosynthesis reduced nicotine biosynthesis. Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti COI1 tobacco plants. These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.     

Jasmonic acid treatment to part of the root system is consistent with simulated leaf herbivory, diverting recently assimilated carbon towards untreated roots within an hour

It is known that shoot application of jasmonic acid (JA) leads to an increased carbon export from leaves to stem and roots, and that root treatment with JA inhibits root growth. Using the radioisotope ¹¹C, we measured JA effects on carbon partitioning in sterile, split-root, barley plants. JA applied to one root half reduced carbon partitioning to the JA-treated tissue within minutes, whereas the untreated side showed a corresponding – but slower – increase. This response was not observed when instead of applying JA, the sink strength of one root half was reduced by cooling it: there was no enhanced partitioning to the untreated roots. The slower response in the JA-untreated roots, and the difference between the effect of JA and temperature, suggest that root JA treatment caused transduction of a signal from the treated roots to the shoot, leading to an increase in carbon allocation from the leaves to the untreated root tissue, as was indeed observed 10 min after the shoot application of JA. This supports the hypothesis that the response of some plant species to both leaf and root herbivores may be the diversion of resources to safer locations.