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Keywords:

  • Manduca sexta;
  • fatty acid–amino acid conjugates (FACs);
  • image analysis;
  • nicotine;
  • plant defence;
  • plant–insect interactions;
  • trichomes

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Many studies demonstrate resource-based trade-offs between growth and defence on a large timescale. Yet, the short-term dynamics of this growth reaction are still completely unclear, making it difficult to explain growth-defence trade-offs mechanistically. In this study, image-based non-destructive methods were used to quantify root growth reactions happening within hours following simulated herbivore attack. The induction of wound reactions in Nicotiana attenuata in the seedling stage led to transiently decreased root growth rates. Application of the oral secretion of the specialist herbivore Manduca sexta to the leaves led to a transient decrease in root growth that was more pronounced than if a mere mechanical wounding was imposed. Root growth reduction was more pronounced than leaf growth reduction. When fatty acid–amino acid conjugates (FACs) were applied to wounds, root growth reduction occurred in the same intensity as when oral secretion was applied. Timing of the transient growth reduction coincided with endogenous bursts of jasmonate (JA) and ethylene emissions reported in literature. Simulation of a wound response by applying methyl jasmonate (MeJA) led to more prolonged negative effects on root growth. Increased nicotine concentrations, trichome lengths and densities were observed within 72 h in seedlings that were treated with MeJA or that were mechanically wounded. Overall, these reactions indicate that even in a very early developmental stage, the diversion of plant metabolism from primary (growth-sustaining) to secondary (defence-related) metabolism can cause profound alterations of plant growth performance.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Throughout their entire life, plants need to acclimate to a suite of fluctuating biotic and abiotic factors. Within the framework of their genetically determined response options that vary throughout ontogeny, they react towards stress situations by maximizing protection against stress while minimizing deviations from optimal growth and development. Herbivore attack is probably the prime biotic stress factor for a wide range of plants. It has been well investigated that acclimation occurs both on the biochemical and morphological level, for example, by increasing defence compounds (Karban & Baldwin 1997), by synthesizing defence-related proteins such as protease inhibitors (PI), by emitting volatiles to attract predators and parasites of herbivores (De Moraes et al. 1998), and by altering plant morphology via increased formation of trichomes, thorns or scleromorphy (Kudo 1996; Traw & Dawson 2002; Dalin & Bjorkman 2003). Those defence reactions are necessarily associated with ecological and genetic costs or allocation trade-offs (Heil & Baldwin 2002).

Many studies have demonstrated resource-based trade-offs between growth and defence on a large timescale (Ohnmeiss & Baldwin 1994; Zangerl, Arntz & Berenbaum 1997; Collantes, Gianoli & Niemeyer 1998; Smith & Schowalter 2001; Zavala et al. 2004; Walls et al. 2005). Yet, it is unclear how fast a trade-off-linked reorganization of plant metabolism, diverting resources away from growth or development towards defence, can occur. In recent years, development of growth imaging methods (Walter & Schurr 2005) has allowed the study of short-term growth responses of above- and belowground sink organs towards fluctuations of environmental factors, such as alterations of atmospheric CO2 (Walter et al. 2005) or light climate (Lai et al. 2005; Nagel, Schurr & Walter 2006). Clear responses are often seen best in young seedlings as they are characterized by highest relative growth rates (RGRs) throughout plant ontogeny. There, primary root growth can be monitored relatively easy by cultivation in translucent agar-filled Petri dishes (Walter & Schurr 2005; Nagel et al. 2006), while growth of the small leaves is somewhat difficult to analyse because leaves need to be forced mechanically into the focal plane of a camera for image acquisition (Schmundt et al. 1998; Walter & Schurr 2005).

In seedlings, defence reactions such as induced formation of PIs and glucosinolates after mechanical wounding of cotyledons or first true leaves in 1-week-old Brassica napus plants are reported (Bodnaryk 1992; Cipollini & Bergelson 2000). Yet, for PIs, it has been reported that in Nicotiana attenuata, defence-related induction is only possible in the rosette, but not in the seedling or flowering stage (van Dam et al. 2001). Hanley & Felton (2007) recently reported that cotyledon removal in seedling stage strongly affectedgrowth and flowering in mature plants; however, the short-term growth dynamics following wounding remains still unclear. As N. attenuata has become a model system for studying herbivory-induced defence reactions, it would be interesting to elucidate its defence system in the seedling stage in more detail.

When N. attenuata is attacked by the specialist lepidopteran herbivore Manduca sexta, a diverse set of plant hormones like jasmonate (JA), methyl jasmonate (MeJA) and ethylene are rapidly induced immediately following wounding or herbivore attack (McCloud & Baldwin 1997; Zhang & Baldwin 1997; von Dahl & Baldwin 2004). JA is known to reduce root growth (Staswick, Su & Howell 1992; Uppalapati et al. 2005), albeit root growth-promoting effects at low JA and MeJA concentrations have also been reported in literature (Tung et al. 1996; Toro, Martin-Closas & Pelacho 2003). However, in theses studies, JA has been directly applied to roots, which does not reflect the situation of herbivory, because JA bursts occur in the wounded leaf tissue and JA is subsequently transported downwards to the root system (Zhang & Baldwin 1997). Ethylene concentrations and emissions have also been reported to increase strongly when M. sexta is feeding on N. attenuata (Kahl et al. 2000), and growth-inhibiting effects of ethylene have been reported in numerous studies (Abeles 1972). Yet, apart from a hormonal-induced decrease of growth activity, an increase of root growth as immediate reaction towards herbivore attack in N. attenuata is conceivable as well. Increased export of carbohydrates from shoots to roots within 2 h following simulated herbivore attack or the application of JA to leaves has been reported recently in two studies utilizing the short-lived istotope 11C to monitor carbon flux within the plant (Babst et al. 2005; Schwachtje et al. 2006). Such a re-allocation of carbohydrates to the root would ecologically make sense, as carbohydrates would then be retrieved from leaf-consuming herbivores and could be used for regrowth and/or reproduction after the threat has passed by. Increasing carbohydrate import to the root can lead to immediate and strong induction of root growth (Aguirrezabal, Deleens & Tardieu 1994; Nagel et al. 2006).

Hence, the aims of our study were (1) to monitor whether young seedlings of N. attenuata were able to induce defence-related reactions within a short time upon a simulated herbivore attack and (2) to assess the dynamics of root growth reaction following such an attack to provide more insight into the temporal coordination of resource allocation trade-offs during defence reactions.

MATERIAL AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plants and cultivation systems

Seeds of Nicotiana attenuata Torr. ex Wats. (Solanaceae) from an inbred line originating from a natural population in Utah were smoke treated and sterilized as described by Krügel et al. (2002). Plants were raised in square Petri dishes (120 × 120 × 17 mm). Five seeds were placed in line in the Petri dishes containing 125 mL sterile 1% Phytagel (w/v) with full-strength Gamborg B5 Medium (Duchefa, Haarlem, the Netherlands). Seeds were pushed approximately 1 mm into the agar to ensure that roots were growing within the agar. The Petri dishes were sealed with fabric tape (Micropore; 3M Health Care, Neuss, Germany) to facilitate gas exchange. They were set almost horizontal until germination (5 d after sowing). Afterwards, the Petri dishes were put almost vertical (85°) to guarantee that the roots grew along the lid of the Petri dish. The shoots grew in the air-filled volume of the Petri dish. In this system, treatments were applied as specified further below. The plants were grown in cultivation rooms under 26 °C during the light phase and under 22 °C during the dark phase with a photoperiod of 14 h. They were exposed to a photon flux density of 85 µmol m−2 s−1. Light was switched on at 0600 h and switched off at 2000 h.

A slightly different cultivation system than the conventional one described earlier was used for application of treatments in high-resolution growth monitoring experiments (Fig. 1a) and for application of MeJA. In this ‘microrhizotron’ set-up, which is described in more detail elsewhere (Nagel et al. 2006), shoots grew outside the Petri dish, which was almost completely filled with Phytagel (200 mL) (Duchefa). After the Phytagel (Duchefa) had cooled down, three small holes were melted into the side of the Petri dish using a glowing bolt to guarantee sterility. Immediately after piercing, 2-day-old sterile seedlings from the conventional system were transplanted into each hole. The holes were sealed with sterilized silicon fat to prevent contamination (Baysilone-Paste; Bayer, Leverkusen, Germany). The Petri dishes were then set almost vertical and were exposed to the same experimental conditions as described earlier.

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Figure 1. Experimental set-up. (a) Original image of a root tip acquired by a CCD camera and used for digital image sequence processing. (b) Wounding procedure with sterile tweezers. (c) Nicotiana attenuata at rosette stage before wounding treatments were applied. Numbers depict successive leaf developmental stages. T, transition leaf (see text for explanation).

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Wounding treatments

The inducibility of wound reactions in seedlings was tested by applying the wound-signalling substance MeJA (experiments 1 and 3). MeJA was dissolved in 1 µL lanolin paste (Sigma-Aldrich, Steinheim, Germany). This solution was applied directly on the primary leaf (the first true leaf appearing after cotyledons) of N. attenuata 16 d after germination. Control plants were treated with lanolin paste only. In this developmental stage, the third leaf just emerged and shoots were about 15 mm high. For these experiments, shoots were grown outside of the Petri dishes (as described previously) to avoid a direct effect of volatile MeJA on the roots. In experiment 1, plants treated with 500 ng MeJA were harvested 4 d after application for nicotine and trichome analysis. In experiment 3, plants were treated with 50, 500 or 5000 ng MeJA, respectively.

For the other wounding experiments of seedlings (experiments 2, 4–6), approximately one-third of the primary leaf tissue was squeezed 16 d after germination using sterile tweezers to simulate herbivore attack (Fig. 1b). By this procedure, 13 ± 3% of the total leaf area (including the cotyledons) was damaged. Immediately after wounding, one of the following substances was applied: (1) 1 µL H2O; (2) 20 µL oral secretions and regurgitants of the natural herbivore M. sexta (diluted 1:5 with phosphate buffer; spit); (3) fatty acid–amino acid conjugates [FACs, N-linolenoyl-L-Gln (50 ng µL−1, 0.12 mM) and N-linolenoyl-L-Glu (138 ng µL−1, 0.34 mM)], which are known to be the main elicitors of plant defence responses in M. sexta regurgitant. To monitor the basic reaction of root growth (experiments 4 and 5), the plants were grown inside Petri dishes; therefore, the treatments were performed within sterile clean benches to avoid any contamination. For monitoring high-resolution effects on root growth, shoots were grown outside the Petri dish and treatments were applied in the microrhizotron set-up (experiment 6). All treatments were performed at 1300 h.

To test the effect of simulated herbivory on leaf growth (experiment 7), two leaves (developmental stages T-3 to T-1; see further below) were wounded and growth reaction was either monitored in one of those leaves or by characterizing growth of the entire leaf rosette area or by characterizing ‘systemic’ effects on younger, strongly expanding leaves (+1 or +2). Wounding was applied by a pattern wheel as described in Halitschke et al. (2000); the wounded area was treated with 20 µL of water (buffered to 7.8 pH by 50 mM phosphate buffer; H2O) or 20 µL oral secretions and regurgitants of the natural herbivore M. sexta (diluted 1:5 with phosphate buffer; spit).

Trichome quantification

Glandular trichomes of N. attenuata contain high amounts of nicotine and are hence chemical and physical defence systems (Duffey 1986; Baldwin & Karb 1995). Trichomes emerge from predisposed epidermal cells usually in an early developmental stage but can also develop during secondary growth of hypocotyl and stem. For quantification of trichome length and number, complete hypocotyls were photographed using a binocular microscope immediately prior to the destructive harvests for nicotine analysis in experiments 1 and 2. The contrast of the images was improved with Corel Photo Paint (Corel Corporation, New York, NY, USA). The projection of trichomes was used to determine their length and their number per millimetre hypocotyl length. Their densities were therefore underestimated as trichomes pointing towards the microscope were not visible. Because all plants were treated equally, the determined ‘quasi-lateral’ trichome density was a good proxy for the total trichome density. Moreover, in this study, we were not interested in the absolute trichome density, but rather in the induction of trichome formation per se by wounding. The total lengths of hypocotyls were also determined to test whether the induced trichome density was due to reduction in growth or to an increase in absolute trichome number. However, no significant difference in hypocotyl length and diameter was found between treatments.

Nicotine analysis

The entire shoot was harvested in experiments 1 and 2, weighed and immediately shock frozen in liquid N2. Plant material was ground frozen with a pestle in a 2 mL tube. The samples were extracted with 400 µL of 40% MeOH (v/v) containing 0.5% acetic acid (v/v) and were shaken at 23 °C for 1 h. After extraction, the samples were centrifuged (10 min, 9 300 g), filtered (0.45 µM) and transferred into a vial for the HPLC analysis. Nicotine was separated by a Merck–Hitachi HPLC system (Darmstadt, Germany) on a Multospher column (120 RP 18-HP 3 µm, 250 × 4 mm; CS-Chromatographie Service, Langerwehe, Germany) attached to a Multospher pre-column (Multospher 100 RP 18; 5 µm, CS-Chromatographie Service) and detected with a diode array detector at 254 nm. The mobile phases were the following: A: water (0.1% H3PO4); B: acetonitrile. The flow rate was 0.5 mL min−1, and the following gradient programme was performed: 0–28 min, linear gradient 95–70% A; 28–38 min, isocratic 70% A; 38–60 min, linear gradient 70–40% A (remaining fraction for B, respectively).

Basic root growth monitoring

Growth velocity of the primary root tip (VTip) of each individual seedling was quantified using a ruler in experiments 1–6. Each day, the position of the root tip was marked with a pen on the rear side of the Petri dish. For root growth analysis, we included only plants with a root length between 35 and 50 mm at treatment day. All suitable plants were treated and VTip was averaged within each Petri dish. The number of Petri dishes was taken as the replicate number for each treatment in experiments 3–5. Temperature of the Phytagel (Duchefa) of one representative dish was recorded once per minute using a thermocouple attached to a datalogger (Delta-t Devices Ltd, Cambridge, England).

Root growth data were normalized in Figs 4–7 by calculating the ratio between the population mean values of growth at the depicted point in time and growth at the time of treatment.

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Figure 4. Normalized values of root tip growth (VTip) after application of different amounts of methyl jasmonate (MeJA) dissolved in lanolin paste (experiment 3, mean values ± SE, n = 15).

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Figure 5. Normalized values of root tip growth (VTip) after wounding (experiment 4). Primary leaf was wounded with tweezers and immediately supplied with 1 µL water (H2O) or oral secretions and regurgitant of Manduca sexta (spit) diluted 1:5 with phosphate buffer (mean values ± SE, n = 10). Different letters indicate significant differences at P < 0.05 level.

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Figure 6. Normalized values of root growth (VTip) 24 h after wounding (experiment 5). Primary leaf was wounded with tweezers and immediately supplied with 1 µL phosphate buffer (H2O) or oral secretions of Manduca sexta (spit) diluted 1:5 with phosphate buffer or fatty acid–amino acid conjugates (FACs) (mean values ± SE, n = 10). Different letters indicate significant differences at P < 0.05 level.

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Figure 7. Time series of high-resolution root growth analysis (experiment 6). (a) Temperature course over 24 h during the experiments. Light was switched on at 0600 h and switched off at 2000 h (night period, gray shaped). Treatments were performed at 1300 h. (b) Normalized values of root growth velocities (VTip) with high resolution after wounding and the application of water (H2O) or 1 µL oral secretions of Manduca sexta (spit) diluted 1:5 (mean values ± SE, n = 5). (c) For each specific time point, values of control and treatment were subtracted and differences are displayed.

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High-resolution root growth monitoring

Five days after germination, the Petri dishes were positioned into the microrhizotron set-up. Every 20 s, an image of the root growth zone was taken with a CCD camera (Sony XC-ST50; Sony, Köln, Germany; Fig. 1a) with a resolution of 700 × 480 pixels. Each pixel corresponded to a real area of 3.6 × 2.5 mm. Infrared illumination (λ = 940 nm) enabled image acquisition also during the dark phase. The camera was equipped with a low-pass infrared filter (RG; Schott, Mainz, Germany) to block visible irradiation. Each replicate was measured for at least 24 h. The root tips were followed via a tracking algorithm that controlled a set of x-y moving stages that repositioned the entire Petri dish and hence the root tip, when it approached the borderline of the image. The custom-made algorithms for root tracking and image sequence acquisition are based on a digital image sequence processing software package (Heurisko; Aeon, Hanau, Germany) (Schmundt et al. 1998; Walter et al. 2002; Walter, Feil & Schurr 2003). The image sequences were used to calculate the root tip velocity (VTip) and the distribution of relative elemental growth rate (REGR) along the root growth zone via the structure tensor method (Schmundt et al. 1998; Haußecker & Spies 1999). Nicotiana attenuata grew at this developmental stage with an average VTip of 550 ± 20 µm h−1 and 310 ± 20 µm h−1 during light and dark phases, respectively.

Leaf growth measurements

To assess whether the observed growth effects were specific for roots or whether they might have a more general impact on overall growth performance of the plant, leaf growth analyses were performed with N. attenuata plants in a developmental stage typical for herbivory experiments in this plant (experiment 7). It was not possible to analyse leaf growth in seedlings of the developmental stage used throughout the rest of this study, as leaves were too small. On the other hand, it was also impossible to monitor root growth in the ‘usual stage’ of herbivory experiments because of the extended root system of the plant in this developmental stage and because it is not possible to raise plants to this size in the Petri dish system.

For leaf growth analysis, the plants were raised in the greenhouse at 16 h/8 h light/dark cycles with 28 °C/22 °C, respectively. Mean midday irradiance [photosynthetically active radiation (PAR)] averaged 800 µmol m−2 s−1, with maximal values of 1200 µmol m−2 s−1. When sunlight was lower than 130 µmol m−2 s−1, artificial illumination was provided by SON-T Agro 400 W (Philips, Köln, Germany) lamps. The plants were potted in soil and watered and provided with nutrient solution as needed. Approximately 4 weeks after germination, the plants were assigned to the wounding treatments (Fig. 1c). At this stage, leaves were characterized according to the terminology of Wait, Jones & Coleman (1998) and Halitschke et al. (2000), where the sink–source transition leaf T is defined as the youngest, completely unrolled leaf. All other leaves were numbered in chronological order of appearance relative to T (Fig. 1c).

Growth of the entire leaf rosette area (total leaf area) was recorded by measuring the length and width of all leaves each day with a ruler. Individual leaf area A is given by A = 0.64 × length × width. The allometric factor of 0.64 was determined empirically in a pre-experiment by sketching the outline of 50 leaves of different developmental stages on paper (density: 80 g m−2), cutting and weighing the paper and relating its area to the product of length and width. Total leaf area was calculated by taking the sum of all individual leaf areas. RGR of the total leaf area was then calculated (unit: % d−1) by RGR = 100 × ln (area at day 2/area at day 1), assuming an exponential growth model.

The increase of individual leaf area was monitored in high temporal resolution by attaching a thread to the leaf tip, guiding it over a custom-made rotary displacement transducer (F. Gilmer, Jülich, Germany) and straining it with a weight of 12 g. The rotation of the displacement transducer was proportional to the increase in leaf length and was detected as the electrical signal of a variable resistor. The length increase was analysed with a temporal resolution of 10 min, and the RGR of the leaf length was calculated as described earlier (unit: % h−1).

Statistical analysis

Data were analysed with Statistica, version 6.0 (StatSoft Inc., Tulsa, USA). Trichome density, trichome length and nicotine concentration following MeJA applications were each tested by t-tests. Two-way analyses of variance (anovas) with treatments and harvest days as main effects were performed for the destructive wounding experiment. VTip data were normalized by dividing all values by VTip at treatment day and were analysed with repeated measures one-way anova. The experiment with FACs was tested with one-way anova. All anovas were followed by Fisher's protected least significant difference (LSD) post hoc comparisons.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Trichome and nicotine analysis in seedlings

Plants treated with 500 ng MeJA (experiment 1) significantly increased trichome density 1.3-fold (P = 0.003, Fig. 2a) and trichome length 1.6-fold (P < 0.001, Fig. 2b) within 4 d. Moreover, nicotine concentrations increased 8.8-fold compared to the controls (P < 0.001) showing that both chemical and physical defence mechanisms were strongly induced (Fig. 2c). Wounding treatments (experiment 2) significantly affected trichome densities (F2,39 = 5.633, P = 0.007; Fig. 3a). The application of H2O to wounds increased trichome density significantly within 72 h compared to the control plants (P = 0.006). Spit-treated plants did not significantly increase their trichome density within 72 h compared to control plants (P = 0.066); however, we found a significant increase in trichome densities when compared to the treatment day (P = 0.001). Whole plant nicotine contents were significantly increased by wounding treatments (F2,39 = 19.672, P < 0.001; Fig. 3b). The application of H2O and spit to mechanically wounded leaves significantly increased nicotine concentrations within 24 h compared to control plants (1.7-fold, P < 0.001; 1.6-fold, P < 0.001, respectively). After 72 h, nicotine concentrations of H2O- and spit-treated plants still remained significantly higher compared to controls, but no further increase was found (Fig. 3b). These results demonstrate clearly that N. attenuata is already inducible in very early developmental stages, and hence the seedling represents a convenient model to be used in the sterile Petri dish system for root growth analysis.

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Figure 2. Defence properties of Nicotiana attenuata 4 d after application of 500 ng methyl jasmonate (MeJA) on primary leaf (experiment 1). (a) Trichome density (mean values ± SE; control, n = 6; MeJA, n = 8). (b) Trichome length (mean values ± SE; control, n = 6; MeJA, n = 8). (c) Nicotine concentration of fresh weight (mean values ± SE; control, n = 4; MeJA, n = 5). Asterisks indicate significant differences between control and MeJA (**P < 0.01, ***P < 0.001).

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Figure 3. Defence dynamics of Nicotiana attenuata after wounding and application of 1 µL water (H2O) or 1 µL oral secretions of Manduca sexta (spit) diluted 1:5 with water (experiment 2). (a) trichome densities and (b) nicotine concentration of fresh weight (mean values ± SE, n = 5). Asterisks indicate significant differences between treatments and control (*P < 0.05, **P < 0.01, ***P < 0.001).

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To test whether cultivation of N. attenuata seedlings in low light conditions necessary for root growth monitoring leads to erroneous nicotine concentrations, a side experiment with four Petri dishes in the greenhouse was performed. There, very similar nicotine concentrations were reached as in the growth room conditions (88 ± 7 ng mg−1, n = 4; data not represented graphically).

Basic root growth analysis

Plants treated with MeJA (experiment 3) significantly reduced VTip in a dose–response relationship within 24 h (F3,63 = 68.112, P < 0.001; Fig. 4). Moreover, different wounding treatments (experiments 4 and 5) significantly affected VTip, and statistical differences were found among the factors: treatment, time and the interaction of both factors (F3,36 Treatment = 15.334, P < 0.001; F2,72 Time = 17.248, P < 0.001; F6,72 Time×Treatment = 4.258, P = 0.001; Fig. 5). Wounding of leaves with the application of water (H2O) significantly reduced root growth by about 27% compared to the control plants (P = 0.009), but growth started to recover already during the second day (Fig. 5, experiment 4). To understand whether the reduction of root growth was only due to a reduction in photosynthetic active leaf area, we excised the complete primary leaf (‘excised’ treatment) representing more than three times the leaf area which was wounded in the H2O treatment. No significant reduction of VTip was found compared to the H2O treatments (P = 0.425, Fig. 5). These results demonstrate that the short-term reduction of root growth induced by wounding cannot exclusively be explained through the diminution of the photosynthetic leaf area, but other factors must play a role. Strongest reduction of root growth was observed for the treatment with oral secretions and regurgitants of M. sexta. VTip decreased to less than 50% of the initial value and was significantly lower than VTip of H2O-treated plants (P = 0.006, Fig. 5). However, the spit-treated plants increased VTip significantly over time and had significantly higher values during the second and the third day compared to the first day (second day, P = 0.001; third day, P < 0.001). These results demonstrate that the application of spit to wounds decreases root growth stronger than mechanical damage alone. Yet, the growth reduction induced by a single wounding event is only transient; the recovering phase is started during the second day after treatments.

The effect of spit could be reproduced when FACs occurring naturally in oral secretions of M. sexta were applied to the wounded leaves (F3,36 = 80.354, P < 0.001; Fig. 6; experiment 5). These results indicate that spit transiently reduces root growth for about 24 h and demonstrates that FACs are the elicitors of this transient reduction in root growth.

High-resolution root growth analysis

To elucidate root growth dynamics within the first 24 h after wounding, we analysed root growth by digital image sequence processing with high spatial and temporal resolution (experiment 6). Control plants displayed a diel variation of root growth activity (VTip) that was correlated to the temperature observed in the Phytagel (Duchefa) (Fig. 7a,b): from 1300 h to the end of the day, temperature and VTip of control plants remained largely constant. At night, VTip and temperature decreased monotonically, and from dawn until 1300 h, they rose again to their initial values. As temperature strongly affects root growth (Pahlavanian & Silk 1988; Walter et al. 2002), the diel fluctuations affecting root growth in all treatments can be considered to be caused by the prevailing temperature regime and will not be discussed later on. In contrast to this parallel behavior of temperature and root growth, wounded plants treated with H2O showed an immediate decrease of VTip throughout 90 min following wounding at 1300 h. Spit-treated plants decreased growth for more than 150 min down to 50% of the initial value of VTip and remained 10% below the H2O-treated plants. At night, VTip of plants from both treatments approached VTip of control plants gradually, which is best displayed by differences between control and treatment values (Fig. 7c). Seventeen hours after treatment, exactly when light was switched on again (Fig. 7b), all three populations showed the same VTip.

Two hours after wounding, both treatments showed reduced REGR distributions compared to control plants throughout the entire growth zone (Fig. 8a). The strongest difference between H2O and spit treatment was present between 1.0 and 1.2 mm behind the root tip. Maximal REGR was decreased in both treatments to about 60% of that of control plants. In the middle of the night, maximal REGR was almost comparable to that of control plants again, but growth zone length was much shorter in both treatments (Fig. 8b). Now, strongest differences between H2O and spit-treated plants were present in the basal flank of the REGR distribution, while the apical flanks were practically identical.

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Figure 8. Distribution of relative elemental growth rate (REGR) along the root growth zone (a) 4 h (day) and (b) 11 h (night) after wounding of Nicotiana attenuata and application of water (H2O) or 1 µL oral secretions of Manduca sexta (spit) diluted 1:5 (experiment 6, mean values ± SE, n = 5). REGR distributions were averaged over 1 h, from 3–4 h and 11–12 h after treatment, respectively.

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Leaf growth analysis

For leaf growth, diel variation was even more pronounced than for root growth and was not directly related to the variation of temperature (Fig. 9a, experiment 7). Highest growth rates were reached in the early morning (when temperature was low; compare Fig. 7a) and growth rate decreased throughout the afternoon, reaching zero growth at night. Because of this peculiarity of the diel leaf growth cycle, wound treatments in the depicted experiments were applied at the beginning of the day. When treatments were performed in the afternoon or evening, no growth effect was observed (data not shown), probably because of the fact that the leaves were practically not growing at this time at all. Even when wounding was performed in the early morning, total leaf area growth was not decreased strongly (Fig. 9b). Only for the first 24 h period immediately following wound treatment, slight effects were visible with RGR of water- and spit-treated plants ranging about 10% below that of control plants (19.2 and 19.5% d−1 versus 21.4% d−1, in water- and spit-treated plants versus control plants, respectively). Treated leaves showed a rapid initial decrease of RGR, with more pronounced diminution of growth in spit- versus water-treated leaves (Fig. 9c). Approximately 5–6 h after treatment, RGR of water- and spit-treated leaves was comparable again and did not differ strongly from RGR of control leaves anymore. Systemically affected leaves (Fig. 9d) showed an even weaker, but noticeable response, which also lasted for about 5–6 h. Leaf growth experiments have also been performed under laboratory conditions at lower light intensity. There, no detectable growth reduction following wound treatments was observed (data not shown).

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Figure 9. Leaf growth of Nicotiana attenuata after wounding and the application of 20 µL water (H2O) or oral secretions of Manduca sexta (experiment 7, spit). Treatments were performed at 0600 h. (a) Time series of relative growth rate (RGR) throughout 24 h (mean values ± SD, n = 3). (b) RGR of total leaf area after wounding and the application of H2O and spit (mean values ± SD, n = 3). (c) Time series of RGR of wounded leaf after the application of H2O and spit. (d) Time series of RGR of systemically affected leaf after wounding and application of spit.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The seedling system

The results demonstrate clearly that N. attenuata shows inducible defence in very early developmental stages. The application of MeJA induced strongly increased whole plant nicotine concentration, hypocotyl trichome length and trichome density within 4 d. It has been pointed out that PIs are not inducible in seedlings of N. attenuata, but only in later developmental stages (van Dam et al. 2001), indicating that not the entire suite of defence mechanisms is active in seedlings and is hence constrained by plant ontogeny. This fact is also supported by the finding that in Nicotiana sylvestris, nicotine induction after wounding only occurs during the rosette stage, but not during the elongation or flowering stages (Ohnmeiss & Baldwin 2000).

The induced increase of hypocotyl trichome length and density may play an important role for repelling herbivores from seedlings as here, the hypocotyl still represents a relatively high fraction of the plant tissue. Hitherto, increasing trichome densities have only been reported on a larger timescale for leaves emerging after herbivore attack (Agrawal 1999; Traw & Dawson 2002; Traw & Bergelson 2003). Leaves that are already differentiated by the time of herbivore attack are not capable to strengthen their physical defence systems as this can only be performed in developing and non-mature tissues (Mayers & Bazely 1991; Nagata et al. 1999). Clearly, the hypocotyl epidermis cells are still able to differentiate into trichomes, as this tissue is displaying secondary growth. Hence, hypocotyl trichome density may represent a powerful monitoring trait for non-invasive detection of defence induction in similar experiments.

The dynamics of nicotine and trichome induction were different for the two treatments investigated (Fig. 3). While the ‘pure’ wounding treatment (application of H2O) induced both nicotine and trichome formation rapidly, the spit treatment also led to a rapid increase of nicotine content, but trichome density was only increased 3 d after wounding. In addition to the root growth data, which will be discussed later, this may be an indication for the transiently reduced growth potential of the seedling upon herbivoral attack. If the application of spit specifically leads to a transient depletion of metabolites required for growth processes, a delayed elongation of trichomes would be a logical consequence.

It has to be pointed out that all experiments have been conducted at very low light intensity to allow high-resolution root growth monitoring. It is conceivable that at low light intensity, both growth and defence may be carbon limited, and hence it might be problematical to draw conclusions for a trade-off between growth and defence. It is well documented that seedling root growth is suboptimal under the light intensity reached in this cultivation system (Nagel et al. 2006). Yet, the fact that nicotine concentrations in greenhouse-grown seedlings and growth room-cultivated seedlings were comparable rather leads to the speculation that defence mechanisms are realized in a relatively ‘normal’ manner despite the limited availability of photosynthates. This may in turn be realized by the simple fact that the plant body itself is much smaller under low light conditions, and a low amount of nicotine or other defence-related substances consequently results in concentrations similar to those found in high-light-grown plants. A detailed analysis of the relation between light intensity and defence induction would thus surely be interesting in future studies.

Inhibition of root growth by application of MeJA

JA and MeJA inhibit root growth when applied directly to roots; however, growth-promoting effects at low concentrations are also reported in literature (Staswick et al. 1992; Tung et al. 1996; Creelman & Mullet 1997; Toro et al. 2003; Uppalapati et al. 2005). In this study, MeJA was applied to leaves and root growth inhibition was observed subsequently. In contrast to application of spit, no recovering of root growth was observed during the experiment, suggesting that MeJA was constantly diffusing from lanolin paste into the leaf and was thereby maintaining growth inhibition. Using radioactively labelled JA, Zhang & Baldwin (1997) demonstrated a rapid transport of JA from leaves to roots via the phloem in N. sylvestris. They suggest that wound-induced JA from leaves accounts for the systemic increase of JA in the roots peaking approximately 180 min after a herbivore attack and mediating de novo synthesis of nicotine. Moreover, Thorpe et al. (2007) reported a rapid transport of 11C-labelled MeJA from leaves to roots in Nicotiana tabacum within 60 min.

Growth reaction after wounding treatments

An immediate root growth response was observed for the wounding treatments. The first rapid decrease clearly points to a hydraulic response of the plant as loosing water or turgor pressure is immediately reducing root growth (Nagel et al. 2006). Apart from the hydraulic response, there is a strong response towards the spit of Manduca larvae, which coincides with the time frame of about 2–3 h that was reported for the systemic increase of JA transported from shoot to root. Baldwin et al. (1997) reported that JA pools in roots are increased systemically (3.5-fold) within 180 min following mechanical wounding. This increase in JA pools of roots could account for the reduction in root growth. The 10-fold higher JA burst following herbivory of M. sexta larvae or the application of their spit to wounds (Kahl et al. 2000) could be responsible for a more pronounced inhibition of root growth. Seventeen hours after the spit treatment, root growth of spit- and H2O-treated plants was comparable again and had even reached the same intensity as that of control plants. Hence, it is conceivable that JA pools or ethylene concentrations in seedling roots have decreased by this time again. However, the ethylene burst elicited by Manduca or their spit may mediate a reduction of root growth as well.

Recently, Schwachtje et al. (2006) reported that a β-subunit of an SNF1-related kinase, GAL83, which is induced via herbivore specific elicitors, regulates root–shoot partitioning of carbohydrates after herbivore attack independently of JA signalling. This change in source–sink relations could in itself act as a signal, as changes in carbohydrate concentrations are known to affect gene regulation (Koch 1996; Smeekens 1998). However, an increase in carbohydrate import to roots would rather result in an increase of root growth as reported by (Nagel et al. 2006), suggesting a down-regulation of root growth via wound signalling by ethylene or JA.

Decreased import of carbohydrates due to destruction of a relevant fraction of photosynthetic tissue can lead to decreased root growth (Aguirrezabal et al. 1994; Nagel et al. 2006). Hence, the permanent destruction of 13% of the source leaf tissue during the wounding treatment may be responsible for the slow recovery of root growth throughout several days (Fig. 5) and for lower growth of H2O- and spit-treated plants compared to control plants in the morning after the treatment day (Fig. 7).

The strongest difference concerning the distribution of REGR between H2O and spit treatment was initially observed about 1 mm behind the root tip in the beginning of the central elongation zone. Here, meristematic activity has practically ceased, which is indicated by increase of REGR. This means that spit-induced growth reduction might mainly be caused by reduction of growth during an early stage of cell expansion. Eight hours later, cells from this region have been displaced towards the end of the growth zone, where now strongest differences are observed. Hence, spit might mostly affect a cell file in an early stage of cell expansion, and the root zone recovers as new cells that are formed in the meristematic part of the root growth zone at the very tip are being displaced into the elongation zone.

Unfortunately, it was impossible to perform leaf and root growth monitoring in response to simulated herbivore attack in comparable stages of plant development. Nevertheless, the reaction of leaf growth was monitored in a later stage of development to gain some insight whether growth reactions might be restricted to roots or whether they might be more pronounced in the aboveground plant part, where herbivore attacks occur and where hence a growth reduction would intuitively seem more meaningful. The results show clearly that the leaf rosette, at least in this developmental stage, shows a weaker reaction than the primary root. If there is any reduction at all towards a single wounding event, it is also of transient nature and seems to prevail for a shorter time than observed in the root. Yet, it has to be pointed out that because of the overall low leaf growth rate during large parts of the diel cycle and because of the high variability among individuals, leaf growth results should be interpreted carefully.

In summary, our results obtained on seedling plants in laboratory conditions clearly indicate that a single herbivore attack at a leaf, which is sensed via the presence of FACs, leads to an immediate growth response in the root. The growth reduction is transient and is regulated by wound-induced JA transported from leaves to roots. Following herbivore attack, the plant diverges its metabolism towards the production of defence substances and structures within hours, leaving a significant imprint in a reduction of growth. The results of Schwachtje et al. (2006) show that in a more realistic situation under higher light intensity, allocation of sugars to roots is increased upon herbivoral attack, leading not to stronger growth, but to increased C storage there. Overall, the picture emerges that not the leaf attacked aboveground, but the belowground root system turns down its spatial expansion and strengthens its function as a ‘safe retreat’ for carbon to produce reproductive growth eventually.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

We would like to thank Andrés Chavarría-Krauser, Arnaud Lanoue, Hinrich Lühring, Ursula Röse, Kerstin Nagel and Gunnar Henkes for assistance. We are grateful for the provision of FACs by Ian T. Baldwin, and we acknowledge stimulating discussions with all members of the Virtual Institute for Biotic Interactions (ViBi) especially with Ian T. Baldwin, and Stefan Scheu. This work was financially supported by the Helmholtz Association of German Research Centres.

REFERENCES

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  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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