Jasmonic acid signalling and herbivore resistance traits constrain regrowth after herbivore attack in Nicotiana attenuata


Ian T. Baldwin. Fax: +49 3641 571102; e-mail: baldwin@ice.mpg.de


Because traits conferring resistance on herbivores can reduce fitness-associated traits, trade-offs may occur between tolerance and resistance responses. We examined these trade-offs in genotypes of Nicotiana attenuata that were transformed to silence trypsin proteinase inhibitor (TPI) production (AS-Natpi), an antiherbivore defense associated with (14%) reductions in seed production, and the jasmonate signal cascade that elicits these defenses (AS-Nalox3), by measuring stalk and axillary branch growth and seed production after two defoliation regimes and Manduca sexta larval attack to bottom or middle and top stalk leaves. Larval attack and defoliation at middle and top leaves depressed seed production and increased axillary branching more than at bottom leaves. AS-Nalox3 and AS-Natpi plants produced significantly longer (two- to fourfold) branches than did wild-type (WT) plants, results that are consistent with resource-based trade-offs between resistance and regrowth. Methyl jasmonate (MeJA) treatment of AS-Nalox3 plants restored WT branch growth, suggesting that jasmonic acid (JA) signalling suppresses regrowth and contributes to apical dominance. These results are consistent with the existence of JA- and resource-mediated trade-offs between regrowth and herbivore resistance traits.


Because many herbivores can overcome plant resistance traits, such as secondary compounds, plants have evolved the ability to tolerate a certain level of defoliation, and their ability to survive and reproduce after damage can differ markedly among species (Rosenthal & Kotanen 1994; Strauss & Agrawal 1999; Haukioja & Koricheva 2001; Strauss & Zangerl 2002). Tolerance, the ability of plants to regrow and/or reproduce after herbivory (Strauss & Agrawal 1999), can be influenced by extrinsic factors such as the availability of soil nutrients and the type of herbivory (Rosenthal & Kotanen 1994; Wise & Abrahamson 2005), and has been considered an alternative strategy to defense responses for plants to cope with attack from herbivores (van der Meijden, Wijn & Verkaar 1988). The identification of the mechanisms responsible for tolerance would facilitate a substantially more rigorous analysis of potential trade-offs between these major categories of evolutionary responses to herbivore attack (Baldwin 1999; Tiffin 2000).

Strong evidence indicates that there is a trade-off between apical dominance and branching (Sadras 2000). Removal of the apical meristems in Ipomopsis aggregata releases lateral meristems that would otherwise not be released until later in the growing season; these new branches produce as many seeds, if not more, than would have been produced by undamaged plants (Paige & Whitman 1987; Paige 1992; Paige 1999; Juenger & Bergelson 2000). In some species, leaf defoliation rather than decapitation can also induce regrowth (Topperwein 1993), and some species can experience 100% of leaf area loss without any concomitant effect on seed set (Wilson et al. 2003), suggesting that stem leaves have an inhibitory influence on axillary branching (Turnbull 2005). Because the way in which allocation patterns shift in response to damage is under hormonal control, regrowth may depend partly on hormonal control of meristem release (Stowe et al. 2000).

Dormant buds remain inactive because of apical dominance, which is a result of auxin-mediated hormonal suppression and resource availability (McSteen & Leyser 2005). After damage to the shoot apex, a major site of auxin biosynthesis, the growth of dormant axillary buds increases dramatically, similar to what also commonly occurs in the auxin-insensitive mutants (axr-1–12 Arabidopsis mutants) (Thimann & Skoog 1933; Stirnberg, Chatfield & Leyser 1999; Leyser 2003). Interestingly, a new study of the pea shows that RAMOSUS1 (RMS1) gene expression is up-regulated in nodal tissue in response to apical auxin addition and down-regulated by removing the endogenous auxin source, suggesting that the transcriptional up-regulation of RMS1 is a branch-inhibiting signal (Foo et al. 2005). By definition, hormones are merely signals which allow an organism to maximize its fitness by adjusting physiological processes to extant environmental conditions (Pugliucci 2001), and the hormonal signals that mediate tolerance responses are likely to interact with those that mediate defense responses.

Induced defenses allow organisms to forgo the costs of defense until the defenses are needed, and jasmonic acid (JA) plays a central role in mediating induced defense responses to herbivore attack in most plant species (Baldwin 1998; Halitschke & Baldwin 2005). JA not only strongly up-regulates the expression of many defense genes, but it also down-regulates the expression of many growth-related genes (Creelman & Mullet 1997; Hermsmeier, Schittko & Baldwin 2001; Halitschke & Baldwin 2003) and hence, it is likely involved in the reallocation of resources after damage. Herbivore damage to cultivated tobacco and tomato increases JA and reduces auxin levels, and down-regulates indoleacetic acid (IAA)-responsive genes in Arabidopsis (Cheong et al. 2002; Schmelz et al. 2003). In addition, exogenous IAA treatments inhibit wound-induced JA levels in Nicotiana sylvestris, suggesting crosstalk between JA and auxin (Baldwin et al. 1997; Cheong et al. 2002; Schmelz et al. 2003). The interaction of JA and auxin signalling may play a role not only in the expression of herbivore resistance traits, but also in the tolerance of plants to herbivore attack by regulating axillary branching.

Nicotiana attenuata Torr. ex S. Wats., a postfire annual inhabiting the Great Basin Desert, has a number of well-described herbivore-induced direct and indirect defenses that are mediated by JA signalling (Baldwin 2001; Halitschke & Baldwin 2005). Herbivore attack increases JA, which in turn increases plant resistance by increasing the accumulation of nicotine and trypsin proteinase inhibitors (TPIs) that reduce the performance of herbivores (Baldwin 2001; Halitschke & Baldwin 2003; Kessler, Halitschke & Baldwin 2004; Zavala et al. 2004b). However, constitutive and inducible TPI expression in N. attenuata is costly when plants are not attacked, reducing seed capsule production and plant growth (Zavala et al. 2004a), a result consistent with the large resource-based costs of TPI production. Because N. attenuata’s fire-chasing behaviour causes it to grow in soils whose fertility is highly variable (Lynds & Baldwin 1998) and because axillary branching is resource dependent in many species (Turnbull 2005), we propose that either increasing the below-ground resources level or down-regulating resistant traits in N. attenuata will increase the plant’s tolerance to defoliation by increasing regrowth and seed capsule production.

Here, we examine whether below-ground resources and plant resistance levels influence regrowth and seed capsule production after simulated and real herbivore attack. To determine if trade-offs between regrowth and resistance exist, we used a transformed N. attenuata genotype in which endogenous TPI production was down-regulated by antisense expression of the plant’s Natpi (AS-Natpi), which codes for a resistance trait that is effective against a variety of different herbivores (Glawe et al. 2003; Zavala & Baldwin 2004; Zavala et al. 2004b). We also used a transformed N. attenuata genotype in which JA signalling was silenced by antisense expression of Nalox3 (AS-Nalox3), which codes for the lipoxygenase that supplies hydroperoxides for JA biosynthesis (Halitschke & Baldwin 2003). These plants are severely impaired in their resistance to a variety of herbivores because of their impaired abilities to elicit either direct or indirect defenses after herbivore attack (Halitschke & Baldwin 2003; Kessler et al. 2004). All transformants were of the same wild-type (WT) genotype and thereby represent valuable tools with which to compare regrowth among plants that differ only in the expression of genes that control resistance (Tatar 2000). We compared the regrowth and seed production of WT, AS-Nalox3 and As-Natpi N. attenuata genotypes that were grown in either 1 or 5 L pots and were subjected to two different defoliation regimes (bottom or middle and top stalk leaves), each of which mimicked the pattern of leaf removal from two native lepidopteran insect herbivores: Manduca sexta larvae, which commonly defoliate leaves at the middle and top stalk leaves, or Spodotera larvae, which defoliate basal rosette leaves (Baldwin 1998; Kessler & Baldwin 2001). In a separate experiment, we used M. sexta larvae to defoliate leaves to determine if the regrowth responses to our clipping treatment were similar to those elicited by a native herbivore. In addition, we complemented the JA-deficient AS-Nalox3 genotype with methyl jasmonate (MeJA) treatments in an attempt to restore the regrowth pattern of WT plants.


Plant genotypes and growth

Seeds collected from Utah (Baldwin 1998) and inbred 10–11 generations were used as the WT genotype and were transformed by an Agrobacterium-mediated transformation procedure with pNATPI1 and pNATLOX3, which contained fragments of N. attenuata’s TPI and lipoxygenase 3 genes, respectively, in antisense orientations (AS), as described in Halitschke et al. (2003) and Zavala et al. (2004a). Southern gel blot analysis confirmed that all T3 lines were single-copy independent transformants and have been fully characterized (Halitschke et al. 2003; Zavala et al. 2004a). Flow cytometric analysis confirmed that all plants were diploid (Bubner et al. 2006). Analysis of the phenotypes demonstrated that gene silencing was effective: the AS-Natpi plants expressed only 39% of the constitutive and 52% of the MeJA-elicited TPI activity of the WT plants, and the AS-Nalox3 plants were severely impaired in JA signalling, and as well as in the production of the direct and indirect defenses elicited by JA. Seeds were germinated in diluted liquid smoke solutions as described in Baldwin, Staszak-Kozinski & Davidson (1994), and seedlings were transplanted in the pots in a glasshouse under the conditions described in Zavala et al. (2004a) with 1000–1300 µmol m−2 s−1 photosynthetic photon flux density (PPFD) supplied by 450 W Na-vapor high-intensity discharge (HID) bulbs.

Defoliation and MeJA treatments, TPI, regrowth and fitness estimates

Nicotiana attenuata typically grows as a rosette for 41 d after germination and bolts to produce a monopodial reproductive axis; on this axis, branches are produced from the axillary meristems above stem leaves (Fig. 1). The initiation of branches coincides with the onset of reproductive development, and the shoot apical meristem completely converts to the production of a determinate inflorescence. Because branching is a resource-dependent response, and traits conferring resistance on herbivores can reduce fitness-associated traits, we used transformed N. attenuata genotypes in which resistance to a variety of herbivores is severely impaired. One hundred and forty plants of each of the three genotypes: antisense lipoxygenase (line number: A300, AS-Nalox3), antisense proteinase inhibitor (line number A315, AS-Natpi) and WT were germinated on agar and after 25 d, transplanted to either 1 or 5 L pots containing 95% peat and 5% clay. The amounts of nutrients used in our experiments were (in mg L−1) N, 70; P, 80; and K, 90 at 5.5–6.0 pH. Thirty-six of the most uniform-sized plants from each genotype and pot size were selected for experimentation in which approximately one quarter of the total leaf area was clipped from plants or attacked by M. sexta larvae (Fig. 1).

Figure 1.

(a) Depiction of nodal location (top, middle and bottom) of leaves removed from defoliation treatments of Nicotiana attenuata plants and location of axillary branches at the first seven stalk nodes and stalk length, parameters measured as proxies of regrowth capacity 7 d after the defoliation treatments. (b) Defoliation treatments and Manduca sexta attack. Plants were undamaged (control) or had leaves removed at either bottom (bottom) or middle and top (middle + top) of the plant in one cutting 30 d after transplanting. Clipping treatments removed comparable amounts of leaf mass and area. Two M. sexta neonates were placed either at the bottom (bottom) or middle and top (middle + top) leaves of the plants in the M. sexta attack treatment.

We measured the constitutive levels of TPIs in leaves at the S1 position 48 d after transplanting to determine whether growth in different pot sizes influenced resistance traits. Protein concentrations and TPI activity were expressed as nmol mg−1 as described in van Dam et al. (2001).

Spodoptera and Manduca are two of the most abundant lepidopteran herbivore genera found on N. attenuata in their native habitat in the Great Basin Desert, and their larvae tend to attack leaves located at either the bottom or middle and top of the reproductive stalk. To simulate these two commonly found patterns of damage, we clipped the stem leaves (S) at the bottom (basal rosette leaves) or middle and top of the stalk [S1–7; leaf position defined in Zavala & Baldwin (2004)] of 12 plants of each genotype during the elongation stage of growth (48 d after transplanting; Fig. 1). The leaves were removed by cutting the petiole with a sharp scalpel, a procedure that does not elicit significant increases in either TPI or nicotine contents and allowed us to separate the effects of leaf removal from the effects of induced resistance (van Dam & Baldwin 2001; van Dam et al. 2001). The two damage regimes removed the same amounts of leaf material (P > 0.3; Supplementary Table S1). To compare regrowth and lifetime reproductive performance among genotypes, we recorded the following from each plant: (1) branch length from S1 to S7 nodes 7 d after clipping and (2) plant height and number of seed capsules 76 d after transplanting. The daily watering was stopped 15 d prior to mimic the length of a typical growing season in the plant’s natural environment. The number of capsules per plant reflects the lifetime reproductive output (seeds) in N. attenuata under natural or glasshouse conditions (Baldwin 1998; Baldwin et al. 1998).

To compare the regrowth responses to clipping and caterpillar attack, two M. sexta neonates were placed on 24 plants of each genotype on a leaf growing at the rosette level (bottom) or S5 (middle and top) and were allowed to move and feed freely for 7 d. Manduca sexta larvae normally do not change feeding location between leaves during the first two instars and only move to adjacent leaves after day 8, when they tend to attack leaves at higher stalk nodes within the plant (Zavala & Baldwin 2004). Furthermore, the damage produced by the caterpillars in the first 7 d of feeding was localized to the leaves on which the caterpillars were placed. Caterpillar attack not only defoliates the plant, but it also elicits TPI production in all stem leaves (Zavala & Baldwin 2004). Eggs of Manduca sexta L. (Lepidoptera: Sphingidae) were obtained from Carolina Biological Supply Company (Burlington, NC, USA) and placed in plastic containers (200 mL) on a moist tissue. The containers were kept in climate chambers at 28 °C and 65% relative humidity under a 16:8 h light:dark photoperiod until the eggs hatched. To estimate whether growth on plants in the two pot sizes or on the different leaf positions of the different genotypes influenced herbivore performance, we measured the mass of larvae after 7 d of feeding.

To determine whether the dramatic response in axillary branch regrowth observed in clipped AS-Nalox3 plants could be attributed to the JA deficiency of the AS-Nalox3 plants, we treated clipped plants of all genotypes with applications of 150 µg of MeJA (Sigma, St Louis, MO, USA; in 20 µL of lanolin paste) to the clipped stem leaves and then measured branch length from S1 to S7 nodes and plant height 10 d after MeJA treatment.

Statistical analysis

Data were analysed with Stat View, version 5.0 (SAS 1998). The TPI, stalk length, branch length and seed capsule number values were analysed by analysis of variances (anovas) followed by Fisher’s protected least significant different (LSD) post hoc comparisons in all experiments. All proportions were arcsine square root transformed before statistical analysis to correct non-normality.


Performance of undamaged genotypes

Constitutive TPI levels in the transformed and untransformed genotypes were used as an index of plant resistance to insect attack, particularly by M. sexta larvae (Zavala & Baldwin 2004; Zavala et al. 2004b). The lowest TPI level was found in the AS-Natpi genotype (F5,66 = 20.374, P < 0.0001) followed by the AS-Nalox3 plants (P < 0.0001), and the pot size in which genotypes were grown did not influence the TPI levels of WT (P = 0.2), AS-Natpi (P = 0.4) and AS-Nalox3 (P = 0.3) genotypes (Fig. 2a).

Figure 2.

(a) Constitutive trypsin proteinase inhibitor (TPI) activity [± 1 standard error of the mean (SEM)] in leaves growing at node S1, (b) cumulative branch length (± 1 SEM) 7 d after natural branching initiation, (c) stalk length (± 1 SEM) 76 d after transplanting and (d) lifetime seed capsule number (± 1 SEM) 76 d after transplanting of untransformed wild-type (WT) Nicotiana attenuata plants of the Utah genotype (WT) and two homozygous T3 independently transformed lines of the Utah genotype that had been transformed with a construct containing either the TPI (AS-Natpi) or lipoxygenase 3 (AS-Nalox3) genes in an antisense orientation (AS) to silence the expression of the endogenous genes. The plants were grown in either 1 or 5 L pots to vary the available below-ground resources. Symbols above columns indicate levels of significant differences between WT and AS genotypes (*, P < 0.05; **, P < 0.001; ***, P < 0.0001).

Down-regulation of resistance traits by transformation increased stalk length, cumulative and average branch length, and seed capsule production in the transformed genotypes with higher levels in plants grown in 5 L pots compared with 1 L pots (F2,33-1-L-STALKLENGTH = 32.377, F2,33-5L-STALKLENGTH = 257.923, F5,498-1L-5L-BRANCHING = 175.957, F2,33-1L-CAPSULES = 14.893, F2,33-5L-CAPSULES = 18.046, P < 0.0001; Fig. 2 and Supplementary Fig. S1). Interestingly, seed capsule production and growth parameters were higher in AS-Nalox3 than in AS-Natpi genotypes. While AS-Nalox3 genotypes grown in either 1 or 5 L pots had the tallest stalks (P < 0.0001, 1.26- and 1.71-fold), the longest axillary branches (P < 0.0001, 2.3- and 4.4-fold) and the greatest capsule production (P < 0.0001, 1.22- and 1.26-fold), plants of the WT genotype had the lowest growth and capsule production (Fig. 2).

Fitness and regrowth consequences of clipping

The clipping treatment affected seed capsule production and growth parameters in AS genotypes more than in the WT genotype. Interestingly, while clipping stem leaves at the bottom of the plant reduced stalk lengths in both WT (5% in 1 L; P = 0.29 and 2% in 5 L; P = 0.65) and AS-Natpi (4% in 1 L; P = 0.38 and 14% in 5 L; P < 0.0001) plants, bottom clipping slightly increased stalk lengths (2.8% in 1 L; P = 0.3 and 5% in 5 L; P = 0.03) in AS-Nalox3 plants (Fig. 3a). When stem leaves at the middle and top of the plant were clipped, the stalk length was reduced in all genotypes (WT: 12% in 1 L; P = 0.004 and 2% in 5 L; P = 0.01; AS-Natpi: 13% in 1 L; P = 0.01 and 9% in 5 L; P < 0.001; and AS-Nalox3: 7% in 1 L; P < 0.001 and 2% in 5 L; P = 0.6) (Fig. 2a). In summary, defoliation at the middle and top of the plant decreased stalk lengths more than defoliation at the bottom did, with the smallest decreases in the AS-Nalox3 genotype.

Figure 3.

(a) Stalk length [± 1 standard error of the mean (SEM)] 46 d after defoliation (76 d after transplanting), (b) cumulative branch length (± 1 SEM) 7 d after defoliation and natural branching initiation and (c) lifetime seed capsule number (± 1 SEM) 46 d after defoliation in wild type (WT), AS-Natpi and AS-Nalox3 genotypes grown in either 1 or 5 L pots and subjected to two different defoliation treatments in which the stem leaves at the bottom of the plant were clipped (bottom) or stem leaves at the middle and top of the plant (middle + top) were removed or left undamaged (control). Symbols above columns indicate levels of significant differences between control and defoliation treatments (*, P < 0.05; **, P < 0.001; ***, P < 0.0001).

While both clipping treatments increased branch length in WT and AS-Natpi genotypes and to a larger degree in plants grown in 5 L rather than 1 L pots, clipping reduced branch length in the AS-Nalox3 genotype grown in either 1 or 5 L pots. Clipping treatments increased branch length in AS-Natpi genotypes grown either in 1 L (39%, bottom; 76%, middle and top) or 5 L (69%, bottom; 79%, middle and top) pots (F5,498-1L-5L = 64.409, P < 0.0001), but in WT genotypes, clipping increased branch length only in plants grown in 1 L pots (27%, bottom; 49%, middle and top; P = 0.001; P < 0.0001; Fig. 3b and Supplementary Fig. S1). Although branch length in AS-Nalox3 was reduced (26 and 25% in 1 and 5 L pots, respectively) by clipping bottom stem leaves (F5,498-1L-5L = 60.158, P < 0.0001), both AS-Natpi and AS-Nalox3 had longer branches than did WT genotypes grown in either 1 or 5 L pots after stem leaves were clipped (F5,498-1L-5L-MIDDLE+TOP = 92.373, F5,498-1L-5L-BOTTOM = 70.474, P < 0.0001; Fig. 3b and Supplementary Fig. S1). These results demonstrate that after clipping, the growth of axillary branches is higher in genotypes with low resistance levels than in the WT genotype, and these differences are larger when plants are grown in 5 L rather than 1 L pots.

Although the impact of defoliation treatments on seed capsule production was higher on genotypes with low resistance levels than on those with high resistance levels, AS-Nalox3 produced more seed capsules than did WT plants even after defoliation (P < 0.0001; Fig. 3c). The reduction in seed capsule number after bottom stem defoliation was higher in AS-Natpi (43% in 1 L, P < 0.0001; 53% in 5 L, P < 0.0001) and AS-Nalox3 (22% in 1 L, P < 0.0001; 21% in 5 L, P < 0.0001) than in WT (17% in 1 L, P = 0.0005; 8% in 5 L, P = 0.01) plants grown in either 1 or 5 L pots, respectively (Fig. 3c). Defoliation at the middle and top of the plant had a more negative result on seed capsule production than did defoliation at the bottom of the plant. Defoliation at the middle and top of the plant reduced seed capsule production in WT plants (20% in 1 L, P < 0.0001; 18% in 5 L, P < 0.0001), AS-Natpi genotypes (19% in 1 L, P = 0.003; 0% in 5 L, P = 0.7) and AS-Nalox3 genotypes (14% in 1 L, P < 0.0001; 19% in 5 L, P < 0.0001) compared with defoliation at the bottom of plants grown in either 1 or 5 L pots, respectively (Fig. 3c).

In summary, defoliation at the middle and top of the plant had a more negative effect on a plant’s fitness correlates (stalk length and seed capsule production) and increased branch length more than did defoliation at the bottom of the plant. Genotypes with low resistance levels (AS-Natpi and AS-Nalox3) had larger reductions in seed capsule production and longer branches after clipping than did WT plants. Interestingly, while defoliation at the bottom of the plant increased branch length and decreased stalk length in AS-Natpi and WT genotypes, the AS-Nalox3 genotype had the opposite response, increasing stalk length and decreasing branch length after defoliation.

Fitness consequences of M. sexta attack

To compare the differences between clipping and caterpillar attack on fitness correlates, two M. sexta neonates were placed on each genotype on a leaf growing at rosette level (bottom) or node S5 (middle and top) and allowed to feed for 7 d. Caterpillar attack decreased seed capsule production in all genotypes to a greater degree in low-resistance genotypes (AS-Natpi and AS-Nalox3) than in the high-resistance genotype (WT). Seed capsule production after caterpillar attack at the bottom of the plant was lower in AS-Natpi (49% in 1 L and 49% in 5 L) and AS-Nalox3 (47% in 1 L and 23% in 5 L) than in WT (15% in 1 L and 17% in 5 L) genotypes grown in either 1 or 5 L pots (F2,33-1L = 21.906, P < 0.0001; F2,33-5L = 12.025, P = 0.0001; Fig. 4a). Neonates placed at the middle and top of the plant increased the negative effect of attack on fitness, with lower reductions in relative capsule number in WT (24% in 1 L and 20% in 5 L) than in AS-Natpi (66% in 1 L and 55% in 5 L) and AS-Nalox3 (54% in 1 L and 33% in 5 L) genotypes grown in either 1 or 5 L pots (F2,33-1L = 27.490, P < 0.0001; F2,33-5L = 24.909, P < 0.0001; Fig. 4a).

Figure 4.

(a) Lifetime seed capsule number [± 1 standard error of the mean (SEM)] of wild type (WT), AS-Natpi and AS-Nalox3 genotypes grown in either 1 or 5 L pots after Manduca sexta attack. Two neonates were placed on each genotype on a leaf growing at rosette level (bottom) or at S5 node (middle + top) and allowed to move and feed freely for 7 d, and unattacked (control) plants of each genotype. (b) Larval mass (± 1 SEM) of M. sexta larvae 7 d after hatching that fed either at the bottom (bottom) or middle and top (middle + top) of WT, AS-Natpi and AS-Nalox3 genotypes grown in either 1 or 5 L pots. Symbols above columns indicate levels of significant differences between either control and defoliation treatments or bottom and middle + top treatments (*, P < 0.05; **, P < 0.001; ***, P < 0.0001).

Larval mass reflected the resistance levels of genotypes used in our experiments. The lower the resistance levels of the genotype, the larger the weight gain of the larvae (Figs 1b & 4b). Caterpillars that fed at the middle and top of the plant and on plants grown in 5 L pots were heavier than those that fed at the bottom of the plant and on plants grown in 1 L pots (F2,69 = 23.555, P < 0.0001; F2,69 = 4.261, P = 0.01; Fig. 4b). In summary, caterpillar attack had a more negative impact on seed capsule number, and caterpillars attained larger masses when they attacked leaves at the middle and top of the plant and fed on genotypes with low resistance levels. Hence, the regrowth responses after caterpillar attack were similar to those elicited by clipping.

Effects of treating AS-NalOX3 genotypes with MeJA on regrowth

To determine whether jasmonate signalling is involved in regrowth and elongation after damage, we applied MeJA to the stem leaves of transformed and untransformed genotypes (AS-Nalox3, AS-Natpi and WT) to restore jasmonate signalling in AS-Nalox3 plants (Halitschke & Baldwin 2003). Interestingly, while MeJA elicitation reduced stalk length in all genotypes (F1,18-WT = 86.571, P < 0.0001; F1,18-AS-pi = 17.325, P = 0.0006; F1,18-AS-lox = 53.348, P < 0.0001; Fig. 5), MeJA treatment only reduced the cumulative branch length of AS-Nalox3 genotypes. MeJA treatment restored the branch length of AS-Nalox3 plants to the length of the branch of WT plants (F1,138 = 31.056, P < 0.0001), and no effect was found in either WT or AS-Natpi (F1,138-WT = 0.238, P = 0.6; F1,138-AS-pi = 0.089, P = 0.7) genotypes, suggesting that JA is involved in branching (Fig. 5).

Figure 5.

(a) Stalk length [± 1 standard error of the mean (SEM)] and (b) cumulative branch length (± 1 SEM) of AS-Nalox3, AS-Natpi and wild-type (WT) genotypes 10 d after natural branching initiation; 150 µg of methyl jasmonate (MeJA) was applied to the stem leaves in a lanolin paste or treated only with a lanolin paste (control). Symbols above columns indicate levels of significant differences between control and MeJA treatment (*, P < 0.05; **, P < 0.001; ***, P < 0.0001).


We demonstrated that when herbivore resistance is down-regulated in N. attenuata, fitness-associated traits and vegetative growth are increased; moreover, this response is amplified when the amount of below-ground resources is increased. Seed capsule production in the undamaged AS-Nalox3 genotype was 1.22- and 1.26-fold larger; stalk height was 1.26- and 1.71-fold taller, and axillary branches were 2.3- and 4.4-fold longer than in the WT genotype, suggesting that the allocation to resistance traits diverts resources from fitness and vegetative growth (Fig. 2). Because JA mediates resistance traits that are elicited by herbivore attack, surprisingly, the JA-deficient genotype (AS-Nalox3) outperformed the other genotypes when plants were undamaged and grown in the sheltered environment of the glasshouse. Neither TPI levels (Fig. 2a) nor herbivore performance measures (Fig. 4b) revealed that JA-deficient plants had invested less in herbivore defenses than the TPI-deficient plants, and yet they consistently outperformed the TPI-deficient genotype when plants were undamaged (Fig. 2) and had similar performance measures when damaged (Fig. 3) or attacked (Fig. 4). Silencing TPI expression increased seed capsule production and vegetative growth, results which are consistent with previous studies (Zavala et al. 2004a), but increases in growth performance were greater in AS-Nalox3 than in AS-Natpi genotypes. These results suggest that JA signalling inhibits axillary branch growth and its associated fitness traits.

JA up-regulates the expression of many constitutively expressed defense genes, but also down-regulates the expression of many growth-related genes (Creelman & Mullet 1997; Halitschke & Baldwin 2003), and the silencing of JA signalling may relieve this down-regulation of growth. Alternatively, glasshouse-grown plants may be challenged by environmental stresses (e.g. high root temperatures) or biotic stresses (elicitation by soil microbes) that may also be JA-mediated (Creelman & Mullet 1997), and the increased growth performance may be due to the suppression of responses to these unmeasured stresses in the JA-deficient genotype. Regardless of the reasons for the increase in growth of AS-Nalox3 plants, JA clearly influences the dominance of the apical meristem over the growth of axillary meristems, and this dominance is also influenced by defoliation.

Defoliation increased the growth of axillary branches while decreasing stalk height in WT and TPI-silenced plants. Defoliation in JA-deficient plants, which had already caused considerable growth of axillary branches in undamaged plants, did not increase branch growth (Fig. 3). Interestingly, damage to bottom leaves increased stalk height but decreased branch growth in AS-Nalox3 plants grown in 5 L pots, suggesting that JA mediates the interplay of stalk and branch growth. Treatment of JA-deficient plants with MeJA to the axillary leaves reduced branch growth to levels commonly found in WT plants (Fig. 5), confirming the role of JA in apical dominance. Similar MeJA treatments have been shown to restore herbivore resistance in the same JA-deficient lines (Halitschke & Baldwin 2003). Interestingly, MeJA treatment of the WT or TPI-deficient line did not influence branch growth (Fig. 5). Because branching is regulated by IAA (Thimann & Skoog 1933) and the IAA- and JA-proteasome pathways are directly connected through AXR1 (Tiryaki & Staswick 2002), these results are consistent with previous studies suggesting antagonistic relationships between JA and IAA signals. It has been suggested that auxin might have a role in regulating JA levels in Arabidopsis (Tiryaki & Staswick 2002). IAA applications to wounds inhibit the wound-induced increase in JA in N. sylvestris, and wounding in Arabidopsis negatively regulated IAA-responsive genes (Baldwin et al. 1997; Cheong et al. 2002). Mechanical damage elicited increases in JA and decreases in IAA levels within 6 h in cultivated tobacco (Schmelz et al. 2003). However, the crosstalk between JA and IAA may be mediated by downstream traits. For example, JA is necessary for TPI elicitation, and the increase in branching in both AS-Nalox3 and AS-Natpi genotypes may be due to TPI levels in both genotypes. Plant TPIs may play physiological roles as regulators of endogenous proteases (Koiwa, Bressan & Hasegawa 1997), and TPIs may inhibit enzymes that support the processes that result in branching.

Hormonal and resource-mediated responses are physiologically intertwined. Our results are consistent with the existence of trade-offs between the expression of resistance traits and vegetative growth, and between branching and plant elongation (Sadras & Fitt 1997; Jong de & van der Meijden 2000). These trade-offs became more pronounced when plants were grown with larger amounts of below-ground resources. Branching patterns are commonly resource-dependent (Turnbull 2005), and deficiencies in a combination of different soil nutrients are known to dramatically influence branching (Jenkins & Mahmood 2003). Hormonal signals are thought to adjust plant physiology to optimize their Darwinian fitness under different environmental conditions.

Silencing JA signalling increased the plants’ Darwinian fitness (lifetime seed production) by 24% when they were not damaged (Fig. 2) but did not dramatically improve seed production in damaged plants compared with WT plants suffering the same damage regimes (Figs 3 & 4). It has been suggested that intermediate regrowth levels following defoliation may benefit plant fitness (Tiffin 2000). In this study, the large increases in branch length after defoliation did not increase lifetime seed capsule production (Fig. 3), suggesting a trade-off between branch growth and seed capsule production. Genotypes that invest more in resistance traits may have fewer resources for regrowth and vice versa (van der Meijden et al. 1988), and regrowth may incur fitness costs because meristems activated after damage compete with developing flowers and fruits for water, nutrients and carbohydrates (Mabry & Wayne 1997). However, trade-offs between apical dominance and tolerance have been identified in some species (Sadras 2000).

Down-regulation of resistance increased apical dominance and decreased tolerance to defoliation resulting from both clipping and larval attack in AS-Nalox3 and AS-Natpi genotypes (Figs 2–4), suggesting that the resistance traits down-regulated in these genotypes influence seed production under the experimental conditions. Strong evidence indicates that some degree of apical dominance optimizes resistance to herbivory; however, up to certain point, further increases in apical dominance could increase competitive ability at the cost of reduced resistance to herbivory (Sadras 2000). For example, in Zea, constitutive resistance is positively correlated with tolerance, and similar results have been found in many crop varieties (Stowe et al. 2000). However, the environmental dependence of the tolerance measures cannot be overemphasized. In the experiments reported here, we mimicked the length of a typical growing season for N. attenuata in south-western Utah by stopping watering 62 d after transplanting. If the growing period had been extended for plants with large investments in axillary branches, the negative relationship between seed production and branch growth may well have been reversed. Performance in complex ecological environments depends heavily on an exquisite sense of timing (Oesterheld & McNaughton 1991), and the negative relationship between axillary branching and lifetime seed production in JA-silenced plants should be verified by experimentation in the plant’s native habitat.

In conclusion, our study demonstrates that axillary branching after herbivore attack is inhibited by the production of resistance traits and/or by the signal cascades that mediate the resistance traits. JA signalling allows plants to adjust their phenotypes to different environmental conditions by directly regulating the expression of resistance traits and axillary branch growth; in addition, it influences branch growth by freeing up resources that would otherwise have been used for the production of resistance traits.


We thank the Max Planck Gesellschaft for financial support, M. Lim for invaluable assistance in plant transformation, T. Krügel and A. Weber for help in growing the plants, and C. Kuhlemeier for his comments about the project.