Journal of Chemical Ecology, Vol. 21, No. 7, 1995
PLASTICITY IN ALLOCATION OF NICOTINE TO REPRODUCTIVE PARTS IN Nicotiana attenuata
I A N T. B A L D W I N *
and M I C H A E L
J. K A R B
Department of Biological Sciences SUNY, University at Buffalo Buffalo. New York 14260 (Received February 14, 1995: accepted March 10~ 1995)
Abstract--Although little is known about the patterns of chemical defense allocation in reproductive tissues, optimal defense theory predicts a high constitutive allocation due to the tissues' high fitness value. To examine this prediction, we quantified the short- and long-term changes in the nicotine pools of reproductive tissues in response to both floral and leaf damage. Recently opened flowers (stage 5 capsules) do not alter their nicotine pools within a day in response to herbivory by Manduca sexta larvae or mechanical damage to the corolla. Similarly, leaf damage during both vegetative and reproductive growth does not influence the nicotine pools of the first three stage-5 capsules produced. However, the nicotine pools of capsules produced later in reproductive growth were significantly larger (1.2- to 1.9-fold) on plants with leaf damage. These differences in floral nicotine pools were a result of both increases in nicotine pools of capsules on damaged plants and decreases in the nicotine pools of capsules on undamaged plants during reproductive growth. Leaf damage did not affect the rate of capsule maturation or the mass of stage-5 capsules at any time during reproductive growth. An allometric analysis of nicotine pools and biomass of reproductive parts in all stages of development from damaged and undamaged plants demonstrates that damaged plants allocated a significantly larger quantity of nicotine to reproductive parts in all stages of development than did undamaged plants. Given that nicotine is thought to be synthesized in the roots and transported to leaves and reproductive parts, nicotine could be allocated to reproductive parts in proportion to the number of developing capsules on a plant. We excised the first 27 stage-5 capsules on plants with and without leaf damage, with the expectation that plants with fewer capsules would allocate a larger amount of nicotine to the remaining capsules. In contrast to the prediction of this passive allocation model, floral excision did not affect nicotine pools on plants with or without leaf damage. These results demonstrate that the allocation of nicotine to reproductive parts is more strongly influenced by damage to vegetative rather than reproductive tissues. Reproductive parts are constitutively defended *To whom correspondence should be addressed. 897 0098-0331/95/0700-0897507.50/0 © 1995 Plenum PublishingCorporation
898
BALDWINANDKAR8 over the short term, but the set points for defense allocation are apparently increased by damage to vegetative tissues during reproductivegrowth. The decrease in allocation of nicotine to reproductive parts in undamaged plants during reproductivegrowth suggests an optimizationof resource allocationas plants realize their potential fitness. Key Words--Nicotine. defensive allometry, Nicotiana attenuata, flowers, reproductive parts, optimal defense theory. INTRODUCTION
Leaves are functionally plastic in the face of herbivory: after attack by herbivores, they change their allocation of resources to both their primary function, carbon gain, and to chemicals used in defense (Welter, 1989; Baldwin, 1993). Few studies have examined the coordination of herbivory-induced changes in defense allocation and photosynthetic capacity (Baldwin and Ohnmeiss, 1994: Zangerl, 1986, Zangerl and Bazzaz, 1992), but the pattern that has emerged is consistent with the predictions of optimal defense (OD) theory (Rhoades, 1979, 1983; McKey, 1974, 1979). OD theory holds that plant tissues contributing more to plant fitness and those more likely to encounter herbivore attack should be allocated higher levels of resources for defense than other plant tissues. For example, the increase in photosynthetic capacity induced by damage and the induced increase in nicotine concentrations are highly coordinated in the leaves of Nicotiana sylvestris, and the coordination changes with nitrogen supply rates (Baldwin and Ohnmeiss, 1994). In contrast to leaves, reproductive tissues are decidedly more determinate in growth form, and for these tissues, OD theory predicts a high constitutive allocation of defense metabotites, given the importance of reproductive parts to the realization of a plant's fitness. OD theory also predicts that the chemical defenses present in immature fleshy fruits should be neutralized in order to increase the attractiveness of mature fruit to dispersers. The latter prediction has received substantially more attention than the former; the changes in palatability that accompany ripening are legend and well described. For example, as a tomato ripens, glycoatkaloids are degraded (Heftmann and Schwimmer, 1972) and the production of proteinase inhibitors (Pearce et al., 1988) ceases, resulting in fruits that are less well defended than their immature counterparts, Much less is known about the allocation of defenses to the reproductive parts of plants that do not produce fleshy fruits, and wild parsnip (Pastinaca sativa) is one of the better studied plants in this regard. The concentrations of furanocoumarins in fruits are not affected by floral damage in either damaged flowers or in flowers produced after damage (Nitao, 1988). This lack of variation in floral furanocoumarin concentrations contrasts with the dramatic increases in leaf furanocoumarin concentrations that are induced locally by damage (Zangerl, 1990).
PLASTICITY OF FLORAL DEFENSE
899
Hence, despite wild parsnip's ability to compensate for floral damage by producing more secondary umbels (Hendrix and Trapp, 1981), its allocation of defense appears to be unchanged in response to damage. Here we examine whether the allocation of nicotine to reproductive parts is similarly unaffected by damage in a species, Nicotiana attenuata, in which leaf damage is known to increase the concentrations of nicotine both locally in damaged leaves and systemically throughout the plant (Baldwin and Ohnmeiss, 1993). However, first we need to consider how best to quantify changes in defense allocation in growing tissues. These issues of quantification are particularly germane to the analysis of the predictions of OD theory, which weighs the costs of producing secondary metabolites against the benefits of resistance resulting from the use of the secondary metabolite. For growing plants, costs can be envisioned as the diversion of resources from growth because reproductive output is frequently size-dependent (Samson and Werk, 1986). Hence, rigorous tests of the predictions of OD theory require secondary metabolite production to be quantified in the context of growth. Changes in allocation of secondary metabolites in growing tissues can be described by an allometric analysis: the metabolite pool in the plant or plant part is regressed against the metabolite-free biomass of the plant or plant part from a series of harvests taken at many stages during development. If the metabolite pool reflects metabolite production (e.g., there is little or no metabolite turnover), then, borrowing the terminology from the literature on heterochronic growth (McNamara, 1986), metabolite production can be envisioned as having onset and offset times. The slope of this relationship describes how a plant partitions resources to defense during growth. Changes in allocation to defense and growth could therefore be quantified as changes in slopes and onset and offset times in the allometric relationship, and allometric relationships found in undamaged plants can serve as a null model against which damage-induced plasticity can be compared. Ohnmeiss and Baldwin (1994) used an allometric analysis to describe the changes in nicotine pools of damaged and undamaged N. sylvestris plants during vegetative growth. Although damage did not significantly affect plant growth, it did increase the accumulation of nicotine for approximately 10 days after damage; consequently, the slope of allometric relationship increased. The slopes of plants harvested more than 10 days after damage were the same as those of undamaged plants. The larger intercept of this allometric relationship reflected the earlier period of increased production (Figure 1). We can conclude that leaf damage causes vegetatively growing plants to move from one allometric relationship to another. In this study we examined the plasticity in nicotine allocation to reproductive parts in N. attenuata Torr. ex Watson. This plant is a herbaceous annual native to North America found in dry washes and recently disturbed or burned areas throughout the Great Basin desert. It produces a central racemose or nar-
900
B A L D W I N AND KARB
"•2.0 L5 ~y =
0.5 0.0 -0,5
0.0
0.5
1.0
In Nicotine-free B i o m a s s (g)
Fro. 1. An allometric analysis of nicotine pools in vegetatively growing N. sylvestris plants that were either undamaged (open symbols) or damaged once (closed symbols). Damaged plants increased their accumulation rates for the first 10 days after the damage event (three harvests) but then returned to the same nicotine accumulation rate as undamaged plants (data from Ohnmeiss and Baldwin, 1994).
rowly peniculate indeterminate inflorescence with self-compatible perfect flowers that mature into green capsules that contain 10-300 small (120-160 p.g/seed) wind- and water-dispersed seeds (Goodspeed, 1954). The calyx is covered by many trichomes and contains approximately 80% of the nicotine of the entire reproductive part at all developmental stages. A large proportion of the nicotine in the calyx is located in the trichomes, and a single large trichome (of which there may be 27 per capsule) contains between 1.3- and 3.6 p,g of nicotine (I. Baldwin, M. Karb, E. Schmelz, N. Blenk and M. Euler, unpublished data). In contrast to the calyx, seeds contain very little nicotine. We found a range of 0-1.2 ng nicotine/seed from seeds collected from pods at different stages of development and grown under a variety of conditions. Hence, the amount of nicotine found in all the seeds of a pod represents a maximum of only 18% of nicotine found in a single large trichome. We ask the following two questions in two experiments: (1) Does the amount of nicotine in a mature flower increase rapidly in response to floral damage, as has been demonstrated for leaves? (2) Does damage to leaves influence the quantity of nicotine allocated to reproductive parts? These questions examine the prediction from OD theory that reproductive parts will be constitutively defended and examine the plasticity in defense allocation both over the lifetime of a flower and the lifetime of the plant. Few studies have examined the effect of damage to vegetative tissues on the defensive allocation in reproductive tissues despite the clear physiological reliance of reproductive tissues on the resources acquired during vegetative growth. The changes in the allocation over a plant's lifetime deserve attention because most of the nicotine in a reproductive capsule is stored in trichomes on the calyx. Trichomes are cell types that are active early in capsule development (Gershenzon and Croteau, 1991), and damage-induced changes may be constrained to the capsules initiated
PLASTICITY OF FLORAL DEFENSE
901
after damage. Additionally, because nicotine is synthesized in the roots and transported in the shoot in all nicotine-producing Nicotiana species studied to date (Baldwin, 1989), we examined the possibility that the allocation of nicotine to reproductive parts may occur simply as a function of the number of capsules on a plant and the amount of nicotine produced by the roots.
METHODS AND MATERIALS
Plant Growth. N. attenuata seeds were soaked in an aqueous extract of wood smoke in 9.8 mM KNO3 in order to stimulate germination (Baldwin et al., 1994b). Seeds were from the third generation of glasshouse-grown plants grown from seed collected from plants in 1988 on the DI Ranch in southwestern Utah (T40S R19W section 9). Approximately one week after germination, seedlings were transferred to Jiffypots (Jiffy Products Ltd., Canada), which were subsequently planted into 4- and 0.75 liter plastic pots containing Cornell mix A potting soil (Boodley and Sheldrake, 1977) for plants used in experiments 1 and 2, respectively. All plants were grown in a glasshouse and received supplemental lighting from 400-W high-pressure sodium vapor lamps for 14 hr/day. Ertraction and Quantification o f Nicotine. Reproductive parts were cut from the pedicel at the base of the calyx with a razor blade, weighed, frozen at - 4 0 ° C , and extracted in alkaloid extraction solution (40% methanol in 0.1% HC1) for a minimum of 2.5 days (the time necessary for complete nicotine extraction from frozen flower samples). The corollas of reproductive parts in developmental stage 5 (Table 1) were analyzed separately from the rest of the flower and are reported on in a companion paper (Euler and Baldwin, unpublished data); here we report the nicotine pools of the entire reproductive part. Twenty-four capsules from each developmental stage from one plant were
TABLE 1. CLASSIFICATION OF DEVELOPMENT IN Nicotiana attenuata CAPSULES Stage
Description
Corolla completelywithin calyx Corolla greenish and beginningto emerge from calyx Corolla greenish and fully elongated Corolla white, fully elongated but closed Corolla open Corolla senesced and pistil swollen
902
BALDWIN AND KARB
weighed, dried at 45-50°C, and reweighed for the calculation of stage-specific percentage dry mass conversion factors. Nicotine was separated by HPLC (Baldwin, 1988), quantified with external standard curves run every 50 samples, and expressed as micrograms of nicotine per capsule. Experiment 1. This experiment examined the effect of mechanical damage and herbivory to the corolla on capsule nicotine pools. Each afternoon we chose capsules in stage 4 (Table 1) on five plants that would open their corolla tube that evening (and enter stage 5) and haphazardly assigned them to the following three treatment groups on each plant: undamaged, razor damage, and caterpillar damage. Capsules in the razor-damaged treatment received one incision half the length of the corolla tube. One second- or third-instar Manduca sexta was placed on the corolla of each capsule in the caterpillar herbivory treatment. Caterpillars remained on the corolla until they had initiated feeding at least once before being removed. Caterpillars that did not feed within the first 30 min were replaced with others until the corolla was damaged. Capsules were damaged in the afternoon and harvested 26-28 hr later. Capsules that aborted before this time were not included in the analysis. Undamaged capsules aborted 7 % of the time (3 of 46); 23% of the razor-damaged capsules aborted (11 of 48); and 36% of the caterpillar-damaged capsules aborted (19 to 53). Experiment 2. This experiment examined the changes in nicotine pools in reproductive parts induced by damage to leaves both over the course of capsule ontogeny and over the reproductive phase of growth for the plant. We harvested capsules at the same stage in development (stage 5) at specified intervals of capsule production on each plant (capsules 1-3, 14-16, and 27-29) in order to track the developmental stage of each plant. Moreover, we included an additional pair of treatments in which the first 26 maturing capsules were excised and predicted that if nicotine is allocated to reproductive parts passively as a function of the total amount of nicotine produced by the vegetative tissues, the nicotine pools of capsules 27-29 of these plants would be larger than those from treatments that did not have their capsules excised. Forty plants in the early stages of stalk elongation were randomly assigned to the four following treatment groups: (i) both leaf damage and capsule excision, (2) capsule excision only, (3) leaf damage only, and (4) neither leaf damage nor capsule excision. Leaves were damaged every day throughout the period of stalk elongation and flowering. One row of evenly distributed pattern-wheel (Dritz, Spartanburg, South Carolina) damage was made across the length of the lamina on either the left or right side of the midrib on every other leaf of plants in the leaf-damage treatment groups. On average, pattern-wheel damage resulted in 4.5 1-mm 2 holes/cm of leaf. Holes were produced by crushing leaf tissue between the blunt spokes of the pattern wheel and a plastic card, and resulted in undetectable leaf dry mass loss (Ohnmeiss and Baldwin, 1994). Pattern-wheel damage was applied to all fully expanded leaves except the senescing leaves.
903
PLASTICITY OF FLORAL DEFENSE
Corolla tubes of stage-5 capsules open in the evening and remain open for two to three nights. Plants were inspected every evening between 8:30 and 11:30 PM for capsules with recently opened corrola tubes. The number of stage-5 capsules was recorded and the first 26 stage-5 capsules produced on plants in the flower excision treatments were removed. The first three and the fourteenth to sixteenth stage-5 capsules produced on the two nonexcision treatments were harvested for nicotine quantification. The twenty-seventh, twenty-eighth, and twenty-ninth stage-5 capsules of plants in all four treatments were harvested for nicotine analysis. An allometric analysis of nicotine contents in reproductive parts in all stages of development was conducted on the first five plants to produce 29 stage-5 capsules in treatments 3 and 4 (e.g., the two nonexcision treatments). Five capsules from each of six ontogenetic stages, for a total of 30 capsules per plant, were harvested for nicotine quantification. Statistical Analysis. Two-way ANOVAs with damage and plant as the main effects were performed on micrograms of nicotine per capsule and gram capsule mass data of experiment 1. Two-way ANOVAs with damage and excision as the main effects were performed on the third harvest of stage-5 capsules (numbered 27-29) of experiment 2. A repeated-measures one-way ANOVA with leaf damage as the main effect was performed on the three harvests of stage-5 capsules from the two nonexcision treatments of Experiment 2. The In-transformed amounts of nicotine per capsule were analyzed with an analysis of covariance with In-transformed amounts of nicotine-free biomass per capsule as the covariate and with individual regressions on the data from each plant, A test of homogeneity of slopes (parallelism) was conducted to determine whether the slopes of these relationships differed between plants with and without leaf damage. Data analysis was performed with the MGLH module of the SYSTAT statistical package (Evanston, Illinois). RESULTS
Experiment 1. The amount of nicotine in stage-5 capsules with caterpillar (37.4 _+ 3.4 ~tg nicotine/capsule) or mechanical damage (36.3 + 3.5/~g nicotine/capsule) to their corollas did not differ from that found in undamaged capsules (37.9 + 3.1 /zg nicotine/capsule) as determined by two-way ANOVAs (damage treatment F2,98 = 0.061; P = 0.94). Significant differences between plants were found (F4.98 = 2.66; P = 0.037), and the damage × plant interaction term was not significant (F8,98 = 0.16; P = 0,99). No significant differences were found in any of the flower biomass data (Ps > 0.40; two-way ANOVA). Experiment 2. Neither the leaf damage treatment nor the floral excision treatment significantly influenced the biomass of any of the stage-5 capsules
904
BALDWIN AND KARB
from any of the harvests as d e t e r m i n e d by the o n e - w a y repeated-measures A N O V A or the two-way A N O V A on the t w e n t y - s e v e n t h to twenty-ninth stage5 capsules produced on each plant (Table 2; Figure 2). Similarly, the floral excision treatment did not significantly affect the a m o u n t o f nicotine in the twenty-seventh to twenty-ninth stage-5 capsules produced on plants as determined by a two-way A N O V A (Table 2; Figure 2). L e a f d a m a g e had no significant effect on the a m o u n t o f nicotine found in the first three stage-5 capsules (F). ~7 = 1.15; P = 0.30; o n e - w a y A N O V A ; Figure 2) produced on each plant; however, leaf d a m a g e dramatically increased the a m o u n t of nicotine in capsules produced later during reproductive growth as d e t e r m i n e d by the repeated-measures one-way A N O V A and the two-way A N O V A (Table 2; Figure 3). By the third harvest (capsules n u m b e r e d 2 7 - 2 9 ) , stage-5 capsules on plants with leaf damage had nicotine pools that were three times those produced by u n d a m a g e d plants at the same stage o f d e v e l o p m e n t . This difference in nicotine pools is due in part to a significant decrease in nicotine o f capsules produced on u n d a m a g e d
TABLE 2. Two-WAY ANOVAs ON NICOTINE AND BIOMASSPOOLS OF STAGE-5 CAPSULES ON THIRD HARVEST (CAPSULES 27-29) FROM ALL FOUR TREATMENT GROUPS (FIGURE 2) AND ONE-WAY REPEATED-MEASURES ANOVAs BETWEEN-SUBJECTS EFFECTS ON NICOTINE AND BIOMASS POOLS OF STAGE-5 CAPSULES FROM ALL THREE HARVESTS FROM DAMAGED AND UNDAMAGED PLANT TREATMENTS (FIGURE 2). MS
F
P
13700.2 29.5 28.6 317.9
43.094 0.093 0.090
0.000 0.763 0.766
2.444 0.814 0.007
0.127 0.374 0.936
33
0.835 × 10-5 0.278 x 10-'~ 0.221 × 1 0 - 7 0.342 × 10-5
! 17
8524.3 582.0
14.6
0.0013
1 17
0.3799 × 10-5 0.4458 × 10-5
df
Third Harvest Nicotine (v.g/capsule) Leaf damage treatment (D) Floral excission treatment (E) D*E
Error Dry mass (rag/capsule) Leaf damage treatment (D) Floral excission treatment (E) D*E
Error Harvests 1-3 Nicotine (~g/capsule) Leaf damage treatment Error Dry mass (mg/capsule) Leaf damage treatment Error
1 I 1
33 1 I 1
0.8521
0.369
PLASTICITY
OF
FLORAL
DEFENSE
905
}'°I ~ '°l 0 (i
I
, ~
! io
,
i
,
L~
~n
25
i
g
Capsule Number FIG. 2. Mean (_+SEM) capsule (a) nicotine and (b) biomass at three stages during reproductive growth of l0 undamaged (open circles) plants and nine plants with leaves damaged during vegetative and reproductive growth (solid circles). Samples are of stage5 capsules (see Table 1) numbered 1-3, 14-16, and 27-29 on all plants where capsule 1 was the first capsule to reach stage 5. The nicotine and biomass pools of stage-5 capsules numbered 27-29 from an additional nine undamaged (open squares) and nine plants with leaves damaged during vegetative and reproductive growth (solid squares) are also depicted. These plants are from the floral excision treatment which had their first 1-26 stage-5 capsules excised.
plants (F].27 > 4.56; P < 0.042; one-way ANOVA contrasts between the first and second and first and third harvests). Neither leaf damage nor flower excision affected the plants" phenologies; the rates of flower production as determined by the time to produce 3, 14 or 27 stage-5 capsules did not differ among the treatment groups (F~.33 < 2.62; P > 0.11; two-way ANOVAs). Reproductive parts at all stages in development from plants with and without leaf damage used in the allometric analysis had significantly (FI.286 = 180.33; P < 0.00001; one-way ANOVA on In micrograms of nicotine) more nicotine than those from undamaged plants (Figure 3). Biomass values were not significantly different (FI.286 = 0.013; P = 0.91; one-way ANOVA on capsule In dry mass). The relationship between nicotine pools and capsule biomass as determined by a test of homogeneity of slopes (parallelism) did not differ sig-
906
BALDWIN AND KARB
oo
--=
o
oo
o
o o
o
In Nico6ne-Free Biomass (g/capsule)
FIG. 3. Natural log transformed nicotine pools plotted against the In-transformed nicotine-free biomass of five capsules from each of the six developmental stages described in Table 1, Capsules were sampled from five u n d a m a g e d (open circles) plants and five plants with leaves damaged during vegetative and reproductive growth (solid circles). All plants were sampled after producing 30 stage-5 capsules and hence were at the same stage in their reproductive phase as those plants in the third harvest of Figure 1, See Table 3 for the regression statistics for each individual plant and the text for the A N C O V A analysis,
TABLE 3. ESTIMATES ( _ S E M ) OF REGRESSION COEFFICIENTS (R 2, y INTERCEPT, AND SLOPE) FOR ALLOMETRIC REGRESSIONS (FIGURE 3) FOR EACH PLANTa R2
y-intercept
Slope
Undamaged plants 1
2 3 4 5 Damaged plants 1 2 3 4 5
0.116 0.575 0.5t5 0.423 0.382
4.212 5.288 5.498 5.693 5.875
± 0.753 ± 0,391 ::t: 0.474 ± 0.622 + 0.806
0.285 0A52 0.499 0.536 0.647
± ± ± ± ±
0,148 0,076 0,093 0.120 0.156
0.282 0.797 0.664 0.840 0.505
5.707 8,154 6.917 6.678 5.694
+ 0.654 ± 0,358 2:0.369 + 0.294 ± 0.414
0.410 0,719 0,546 0.644 0.422
± 0.126 ± 0.070 5:0.075 ± 0.057 ± 0.081
~Natural log-transformed nicotine pools in five capsules for each of the six developmental stages described in Table 1 were regressed against In-transformed nicotine-free dry biomass for each plant. Plants were either undamaged or had their leaves damaged throughout the reproductive phase of growth,
907
PLASTICITY OF FLORAL DEFENSE
nificantly (Fi.28 s = 0.115; P = 0.735) between damaged and undamaged plants. Because the assumption of homogeneity was not violated, an analysis of covariance (ANCOVA) with In nicotine as the dependent variable and In nicotinefree biomass as the covariate was performed, revealing that undamaged and damaged plants differed significantly (FI,289 = 72.3; P < 0.001). Within-plant regressions revealed the same pattern as the ANCOVA (Table 3). The y-intercept values were significantly higher for damaged plants (F~.8 = 5.93; P = 0.041; one-way ANOVA on In-transformed values), while the slopes of the individual plant regressions were not significantly different (F~,8 = 0.58; P = 0.47; oneway ANOVA on In-transformed values). In addition, the relationships between In-transformed nicotine pools and biomass tended to have less scatter with damaged plants than with undamaged plants (mean R 2 = 0.618 and 0.402 for damaged and undamaged plants, respectively; Table 3). DISCUSSION
The results of these experiments revealed some of the "'rules" that govern how nicotine is allocated to reproductive parts, First, nicotine is not passively allocated to reproductive parts as a function of the amount of nicotine produced in roots and the number of capsules on a plant. The nicotine content of stage5 capsules on plants that had the vast majority of their maturing capsules excised did not differ from those which did not have capsules excised (Figure 2). The excised stage-5 capsules contained between 20 and 60/zg of nicotine, depending on whether they developed on plants with or without leaf damage. Had these capsules remained on the plant through maturity, they would have accumulated 60-160 ~g of nicotine, respectively. Hence, the excision treatment should have doubled or tripled the nicotine pools or stage-5 capsules if the passive allocation model was correct. This deduction assumes that nicotine accumulation reflects nicotine production in N. attenuata as has been demonstrated in N. sylvestrius (Baldwin et al., 1994a) and that nicotine was not stored in other plant parts for allocation to capsules later in reproductive growth. Second, the allocation of nicotine to a capsule is not influenced by herbivory or mechanical damage to the corolla (experiment 1) or to the removal of other reproductive parts (experiment 2). Third, in contrast to damage to reproductive parts, damage to leaves dramatically increases the allocation of nicotine. Fourth, the allocation of nicotine to reproductive parts follows an allometric relationship that has less scatter in damaged plants, as indicated by the higher R z of the individual regressions (Table 3), and has a higher intercept. The effect of leaf damage on the nicotinebiomass allometry appears similar to that found in vegetatively growing N. sylvestrius plants (Figure 1). The long-term effect is to move plants from one allometric relationship to another with similar slopes but different intercepts.
908
BALDWIN AND KARB
The N. s v l v e s t r i s data set clearly showed that damaged plants moved from one allometric relationship to another by briefly increasing their rate of nicotine production during growth and, hence, increasing the slope of the relationship. A similar mechanism could be operating in N. a t t e n u a t a reproductive parts, although leaf damage may have initiated the production of capsules with higher nicotine accumulation set points at the beginning of capsule growth. Fifth, leaf damage does not appear to alter the allometric set points for nicotine accumulation for the first reproductive parts produced by a plant, and these apparent set points appear to decline during the later stages of reproductive growth in undamaged plants. These patterns of nicotine allocation underscore the important regulating influence that vegetative tissues have on the defense of reproductive tissues. Many of these patterns are broadly consistent with the predictions of OD theory. First, defense is constitutively allocated to individual reproductive parts as a function of capsule mass. OD theory predicts that defensive plasticity should be reserved for those tissues that are functionally plastic, such as leaves that are capable of physiologically rejuvenilizing and tissues whose contribution to plant fitness vary over ontogeny. Second, defense allocation to the first capsules produced on an undamaged plant appear to be higher than those produced later (Figure 2). For plants growing in unpredictable environments, the first offspring produced represent a larger proportion of realized fitness than those produced later and hence would be predicted to receive a larger defensive allocation. Moreover, if inducible defense allocation provides a means of conserving resources that could be otherwise used for offspring production, the inducible allocation of defense to offspring produced later during reproductive growth, as plants realize their potential fitness, is also consistent with the cost-benefit paradigm of OD theory. Whether or not plants that produce more defense in herbivore-free environments consequently produce fewer offspring of lower quality remains to be demonstrated, Acknowledgments--This research is supported by National Science Foundation grants BSR9157258, BSR-9118452 and a generous equipment grant from the Hewlett-PackardUniversityof Grants Program. We thank T. Ohnmeiss, C. Olney. E. Schmelz, N. Blenk, and M. Euler for expert technical assistance;E. Wheelerfor editorial assistance;and two reviewersfor improvingan earlier draft of this manuscript.
REFERENCES BALDWIN, I.T. 1988. Damaged-inducedalkaloids in tobacco: Pot-bound plants are not inducible. J. Chem. Ecol. 4:1113-1120. BALDWIN,I.T. 1969. Mechanismof damage-inducedalkaloid production in wild tobacco. J. Chem. Ecol. t5:1611-1680. BALDWIN,I.T. f993. Chemical changes rapidly induced by folivory, pp. 1-23, in E.A. Bemays
(ed.). Insect-Plant Interactions, Vol. 5. CRC Press, Boca Raton, Florida.
PLASTICITY OF FLORAL DEFENSE
909
BALDWIN, I.T., and OHNMEISS,T.E, 1993. Alkaloidal responses to damage in Nicotiana native to North America. J. Chem. EcoL 19:1143-1153. BALDWIN, I.T., and OHNMEISS,T.E. 1994, Coordination of photosynthetic and alkaloidal responses to leaf damage in uninducible and inducible Nicotiana sylvestris. Ecology 75:1003-1014. BALDWIN, I.T., KARB, M.J,, and OHNMEISS,T.E. 1994. Allocation of ~N from nitrate to nicotine: Production and turnover of an induced defense, Ecology. 75:1703-1713. BALDWIN, I.T.. STASCAK-KOZINSKI,L., and DAVIDSON,R. 1994b. Up in smoke: I. Smoke-derived germination cues for the post fire annual Nicotiana attenuata Ton" ex Watson. J. Chem. EcoL 20:2345-237 I. BOODLEY,J,W., and SHELDRAKE,R,J. 1977, Comell peat-lite mixes for commercial plant growing. Comell Information Bulletin 43. Ithaca, New York. GERSHENZON,J., and CROTEAU, R. 1991. Terpenoids, pp, 165-219, in G.A. Rosenthal and M.R, Berenbaum. Herbivores: Their Interactions with Secondary Plant Metabolites. Academic Press, San Diego. GOODSPEED, T H . 1954. The Genus Nicotiana. Chronica Botanica Co., Waltham, Massachusetts. HEFTMANN, E., and SCHW~MMER, S. 1972. Degradation of tomatine to 3B-hydroxy-5O-pregn-16enone by ripe tomatoes. Phytochemistn' I 1:2783-2787. HENDRIX, S.D., and TRAPP, E.J. 1981. Plant-herbivore interactions: Insect-induced changes in host plant sex expression and fecundity. Oecologia 49:119-122. McKEY, D. 1974~ Adaptive patterns in alkaloid physiology, Am. Nat. 108:305-320, MCKEY, D. t979. The distribution of secondary compounds within plants, pp. 55-133, in G.A. Rosenthal and D.H. Janzen (eds.I. Herbivores: Their Interactions with Secondary Plant Metabolites. Academic Press, New York. MCNAMARA, K. 1986. A guide to the nonmenclature of heterochrony. J. Paleontol. 60:4-13, NITAO, J.K. 1988. Artificial defloration and furanocoumarin induction in Pastinaca sativa (Umbelliferae/. J. Chem. Ecol. 14:1515-1522. OrtNMEtSS, T.E., and BALDWtN, I.T. 1994. The allometry of nitrogen allocation to growth and an inducible defense under nitrogen-limited growth. Ecology 75:995-1002. PEARCE, G., RYAN, C.A., and LIL/EGREN, D. 1988. Proteinase inhibitors I and II in frail of wild tomato species: Transient components to a mechanism for defense and seed dispersal. Planta 175:527-531. RHOADES, D.F. 1979. Evolution of plant chemical defense against herbivores, pp. 4-54, in G.A. Rosenthal and D.H. Janzen (eds.). Herbivores: Their Interactions with Secondary Plant Metabolites. Academic Press, New York. RI-IOADES, D.F. 1983. Herbivore population dynamics and plant chemistry, pp. 155-220, in R.F. Denno and M. McClure (eds.). Variable Plants and Herbivores in Natural and Managed Systems. Academic Press, New York. SAMSON, D.A., and WERK, K.S. 1986. Size-dependent effects in the analysis of reproductive effort in plants. Am. Nat. 127:667-680. WELTE~, S.C. 1989. Arthropod impact on plant gas exchange, pp. 135-150, in E.A. Bemays ted.). Insect-Plant Interactions. CRC Press, Boca Raton, Florida. ZANGERL, A.R. 1986. Leaf value and optimal defense in Pastinaca sativa L. (Umbelliferae). Am. MidL Nat. 116:432-437. ZANGEP,L, A.R. 1990. Furanocoumarin induction in wild parsnip: Evidence for an induced defense against herbivores. Ecology 71 : 1926-1932. ZANGERL, A.R., and BAZZAZ, F.A. 1992. Theory and pattern in plant defense allocation, pp. 36339l, in R.S. Fritz and E . L Simms (eds.). Plant Resistance to Herbivores and Pathogens: Ecology, Evolution, and Genetics. University of Chicago Press, Chicago.
PLASTICITY IN ALLOCATION OF NICOTINE TO REPRODUCTIVE PARTS IN Nicotiana attenuata
I A N T. B A L D W I N *
and M I C H A E L
J. K A R B
Department of Biological Sciences SUNY, University at Buffalo Buffalo. New York 14260 (Received February 14, 1995: accepted March 10~ 1995)
Abstract--Although little is known about the patterns of chemical defense allocation in reproductive tissues, optimal defense theory predicts a high constitutive allocation due to the tissues' high fitness value. To examine this prediction, we quantified the short- and long-term changes in the nicotine pools of reproductive tissues in response to both floral and leaf damage. Recently opened flowers (stage 5 capsules) do not alter their nicotine pools within a day in response to herbivory by Manduca sexta larvae or mechanical damage to the corolla. Similarly, leaf damage during both vegetative and reproductive growth does not influence the nicotine pools of the first three stage-5 capsules produced. However, the nicotine pools of capsules produced later in reproductive growth were significantly larger (1.2- to 1.9-fold) on plants with leaf damage. These differences in floral nicotine pools were a result of both increases in nicotine pools of capsules on damaged plants and decreases in the nicotine pools of capsules on undamaged plants during reproductive growth. Leaf damage did not affect the rate of capsule maturation or the mass of stage-5 capsules at any time during reproductive growth. An allometric analysis of nicotine pools and biomass of reproductive parts in all stages of development from damaged and undamaged plants demonstrates that damaged plants allocated a significantly larger quantity of nicotine to reproductive parts in all stages of development than did undamaged plants. Given that nicotine is thought to be synthesized in the roots and transported to leaves and reproductive parts, nicotine could be allocated to reproductive parts in proportion to the number of developing capsules on a plant. We excised the first 27 stage-5 capsules on plants with and without leaf damage, with the expectation that plants with fewer capsules would allocate a larger amount of nicotine to the remaining capsules. In contrast to the prediction of this passive allocation model, floral excision did not affect nicotine pools on plants with or without leaf damage. These results demonstrate that the allocation of nicotine to reproductive parts is more strongly influenced by damage to vegetative rather than reproductive tissues. Reproductive parts are constitutively defended *To whom correspondence should be addressed. 897 0098-0331/95/0700-0897507.50/0 © 1995 Plenum PublishingCorporation
898
BALDWINANDKAR8 over the short term, but the set points for defense allocation are apparently increased by damage to vegetative tissues during reproductivegrowth. The decrease in allocation of nicotine to reproductive parts in undamaged plants during reproductivegrowth suggests an optimizationof resource allocationas plants realize their potential fitness. Key Words--Nicotine. defensive allometry, Nicotiana attenuata, flowers, reproductive parts, optimal defense theory. INTRODUCTION
Leaves are functionally plastic in the face of herbivory: after attack by herbivores, they change their allocation of resources to both their primary function, carbon gain, and to chemicals used in defense (Welter, 1989; Baldwin, 1993). Few studies have examined the coordination of herbivory-induced changes in defense allocation and photosynthetic capacity (Baldwin and Ohnmeiss, 1994: Zangerl, 1986, Zangerl and Bazzaz, 1992), but the pattern that has emerged is consistent with the predictions of optimal defense (OD) theory (Rhoades, 1979, 1983; McKey, 1974, 1979). OD theory holds that plant tissues contributing more to plant fitness and those more likely to encounter herbivore attack should be allocated higher levels of resources for defense than other plant tissues. For example, the increase in photosynthetic capacity induced by damage and the induced increase in nicotine concentrations are highly coordinated in the leaves of Nicotiana sylvestris, and the coordination changes with nitrogen supply rates (Baldwin and Ohnmeiss, 1994). In contrast to leaves, reproductive tissues are decidedly more determinate in growth form, and for these tissues, OD theory predicts a high constitutive allocation of defense metabotites, given the importance of reproductive parts to the realization of a plant's fitness. OD theory also predicts that the chemical defenses present in immature fleshy fruits should be neutralized in order to increase the attractiveness of mature fruit to dispersers. The latter prediction has received substantially more attention than the former; the changes in palatability that accompany ripening are legend and well described. For example, as a tomato ripens, glycoatkaloids are degraded (Heftmann and Schwimmer, 1972) and the production of proteinase inhibitors (Pearce et al., 1988) ceases, resulting in fruits that are less well defended than their immature counterparts, Much less is known about the allocation of defenses to the reproductive parts of plants that do not produce fleshy fruits, and wild parsnip (Pastinaca sativa) is one of the better studied plants in this regard. The concentrations of furanocoumarins in fruits are not affected by floral damage in either damaged flowers or in flowers produced after damage (Nitao, 1988). This lack of variation in floral furanocoumarin concentrations contrasts with the dramatic increases in leaf furanocoumarin concentrations that are induced locally by damage (Zangerl, 1990).
PLASTICITY OF FLORAL DEFENSE
899
Hence, despite wild parsnip's ability to compensate for floral damage by producing more secondary umbels (Hendrix and Trapp, 1981), its allocation of defense appears to be unchanged in response to damage. Here we examine whether the allocation of nicotine to reproductive parts is similarly unaffected by damage in a species, Nicotiana attenuata, in which leaf damage is known to increase the concentrations of nicotine both locally in damaged leaves and systemically throughout the plant (Baldwin and Ohnmeiss, 1993). However, first we need to consider how best to quantify changes in defense allocation in growing tissues. These issues of quantification are particularly germane to the analysis of the predictions of OD theory, which weighs the costs of producing secondary metabolites against the benefits of resistance resulting from the use of the secondary metabolite. For growing plants, costs can be envisioned as the diversion of resources from growth because reproductive output is frequently size-dependent (Samson and Werk, 1986). Hence, rigorous tests of the predictions of OD theory require secondary metabolite production to be quantified in the context of growth. Changes in allocation of secondary metabolites in growing tissues can be described by an allometric analysis: the metabolite pool in the plant or plant part is regressed against the metabolite-free biomass of the plant or plant part from a series of harvests taken at many stages during development. If the metabolite pool reflects metabolite production (e.g., there is little or no metabolite turnover), then, borrowing the terminology from the literature on heterochronic growth (McNamara, 1986), metabolite production can be envisioned as having onset and offset times. The slope of this relationship describes how a plant partitions resources to defense during growth. Changes in allocation to defense and growth could therefore be quantified as changes in slopes and onset and offset times in the allometric relationship, and allometric relationships found in undamaged plants can serve as a null model against which damage-induced plasticity can be compared. Ohnmeiss and Baldwin (1994) used an allometric analysis to describe the changes in nicotine pools of damaged and undamaged N. sylvestris plants during vegetative growth. Although damage did not significantly affect plant growth, it did increase the accumulation of nicotine for approximately 10 days after damage; consequently, the slope of allometric relationship increased. The slopes of plants harvested more than 10 days after damage were the same as those of undamaged plants. The larger intercept of this allometric relationship reflected the earlier period of increased production (Figure 1). We can conclude that leaf damage causes vegetatively growing plants to move from one allometric relationship to another. In this study we examined the plasticity in nicotine allocation to reproductive parts in N. attenuata Torr. ex Watson. This plant is a herbaceous annual native to North America found in dry washes and recently disturbed or burned areas throughout the Great Basin desert. It produces a central racemose or nar-
900
B A L D W I N AND KARB
"•2.0 L5 ~y =
0.5 0.0 -0,5
0.0
0.5
1.0
In Nicotine-free B i o m a s s (g)
Fro. 1. An allometric analysis of nicotine pools in vegetatively growing N. sylvestris plants that were either undamaged (open symbols) or damaged once (closed symbols). Damaged plants increased their accumulation rates for the first 10 days after the damage event (three harvests) but then returned to the same nicotine accumulation rate as undamaged plants (data from Ohnmeiss and Baldwin, 1994).
rowly peniculate indeterminate inflorescence with self-compatible perfect flowers that mature into green capsules that contain 10-300 small (120-160 p.g/seed) wind- and water-dispersed seeds (Goodspeed, 1954). The calyx is covered by many trichomes and contains approximately 80% of the nicotine of the entire reproductive part at all developmental stages. A large proportion of the nicotine in the calyx is located in the trichomes, and a single large trichome (of which there may be 27 per capsule) contains between 1.3- and 3.6 p,g of nicotine (I. Baldwin, M. Karb, E. Schmelz, N. Blenk and M. Euler, unpublished data). In contrast to the calyx, seeds contain very little nicotine. We found a range of 0-1.2 ng nicotine/seed from seeds collected from pods at different stages of development and grown under a variety of conditions. Hence, the amount of nicotine found in all the seeds of a pod represents a maximum of only 18% of nicotine found in a single large trichome. We ask the following two questions in two experiments: (1) Does the amount of nicotine in a mature flower increase rapidly in response to floral damage, as has been demonstrated for leaves? (2) Does damage to leaves influence the quantity of nicotine allocated to reproductive parts? These questions examine the prediction from OD theory that reproductive parts will be constitutively defended and examine the plasticity in defense allocation both over the lifetime of a flower and the lifetime of the plant. Few studies have examined the effect of damage to vegetative tissues on the defensive allocation in reproductive tissues despite the clear physiological reliance of reproductive tissues on the resources acquired during vegetative growth. The changes in the allocation over a plant's lifetime deserve attention because most of the nicotine in a reproductive capsule is stored in trichomes on the calyx. Trichomes are cell types that are active early in capsule development (Gershenzon and Croteau, 1991), and damage-induced changes may be constrained to the capsules initiated
PLASTICITY OF FLORAL DEFENSE
901
after damage. Additionally, because nicotine is synthesized in the roots and transported in the shoot in all nicotine-producing Nicotiana species studied to date (Baldwin, 1989), we examined the possibility that the allocation of nicotine to reproductive parts may occur simply as a function of the number of capsules on a plant and the amount of nicotine produced by the roots.
METHODS AND MATERIALS
Plant Growth. N. attenuata seeds were soaked in an aqueous extract of wood smoke in 9.8 mM KNO3 in order to stimulate germination (Baldwin et al., 1994b). Seeds were from the third generation of glasshouse-grown plants grown from seed collected from plants in 1988 on the DI Ranch in southwestern Utah (T40S R19W section 9). Approximately one week after germination, seedlings were transferred to Jiffypots (Jiffy Products Ltd., Canada), which were subsequently planted into 4- and 0.75 liter plastic pots containing Cornell mix A potting soil (Boodley and Sheldrake, 1977) for plants used in experiments 1 and 2, respectively. All plants were grown in a glasshouse and received supplemental lighting from 400-W high-pressure sodium vapor lamps for 14 hr/day. Ertraction and Quantification o f Nicotine. Reproductive parts were cut from the pedicel at the base of the calyx with a razor blade, weighed, frozen at - 4 0 ° C , and extracted in alkaloid extraction solution (40% methanol in 0.1% HC1) for a minimum of 2.5 days (the time necessary for complete nicotine extraction from frozen flower samples). The corollas of reproductive parts in developmental stage 5 (Table 1) were analyzed separately from the rest of the flower and are reported on in a companion paper (Euler and Baldwin, unpublished data); here we report the nicotine pools of the entire reproductive part. Twenty-four capsules from each developmental stage from one plant were
TABLE 1. CLASSIFICATION OF DEVELOPMENT IN Nicotiana attenuata CAPSULES Stage
Description
Corolla completelywithin calyx Corolla greenish and beginningto emerge from calyx Corolla greenish and fully elongated Corolla white, fully elongated but closed Corolla open Corolla senesced and pistil swollen
902
BALDWIN AND KARB
weighed, dried at 45-50°C, and reweighed for the calculation of stage-specific percentage dry mass conversion factors. Nicotine was separated by HPLC (Baldwin, 1988), quantified with external standard curves run every 50 samples, and expressed as micrograms of nicotine per capsule. Experiment 1. This experiment examined the effect of mechanical damage and herbivory to the corolla on capsule nicotine pools. Each afternoon we chose capsules in stage 4 (Table 1) on five plants that would open their corolla tube that evening (and enter stage 5) and haphazardly assigned them to the following three treatment groups on each plant: undamaged, razor damage, and caterpillar damage. Capsules in the razor-damaged treatment received one incision half the length of the corolla tube. One second- or third-instar Manduca sexta was placed on the corolla of each capsule in the caterpillar herbivory treatment. Caterpillars remained on the corolla until they had initiated feeding at least once before being removed. Caterpillars that did not feed within the first 30 min were replaced with others until the corolla was damaged. Capsules were damaged in the afternoon and harvested 26-28 hr later. Capsules that aborted before this time were not included in the analysis. Undamaged capsules aborted 7 % of the time (3 of 46); 23% of the razor-damaged capsules aborted (11 of 48); and 36% of the caterpillar-damaged capsules aborted (19 to 53). Experiment 2. This experiment examined the changes in nicotine pools in reproductive parts induced by damage to leaves both over the course of capsule ontogeny and over the reproductive phase of growth for the plant. We harvested capsules at the same stage in development (stage 5) at specified intervals of capsule production on each plant (capsules 1-3, 14-16, and 27-29) in order to track the developmental stage of each plant. Moreover, we included an additional pair of treatments in which the first 26 maturing capsules were excised and predicted that if nicotine is allocated to reproductive parts passively as a function of the total amount of nicotine produced by the vegetative tissues, the nicotine pools of capsules 27-29 of these plants would be larger than those from treatments that did not have their capsules excised. Forty plants in the early stages of stalk elongation were randomly assigned to the four following treatment groups: (i) both leaf damage and capsule excision, (2) capsule excision only, (3) leaf damage only, and (4) neither leaf damage nor capsule excision. Leaves were damaged every day throughout the period of stalk elongation and flowering. One row of evenly distributed pattern-wheel (Dritz, Spartanburg, South Carolina) damage was made across the length of the lamina on either the left or right side of the midrib on every other leaf of plants in the leaf-damage treatment groups. On average, pattern-wheel damage resulted in 4.5 1-mm 2 holes/cm of leaf. Holes were produced by crushing leaf tissue between the blunt spokes of the pattern wheel and a plastic card, and resulted in undetectable leaf dry mass loss (Ohnmeiss and Baldwin, 1994). Pattern-wheel damage was applied to all fully expanded leaves except the senescing leaves.
903
PLASTICITY OF FLORAL DEFENSE
Corolla tubes of stage-5 capsules open in the evening and remain open for two to three nights. Plants were inspected every evening between 8:30 and 11:30 PM for capsules with recently opened corrola tubes. The number of stage-5 capsules was recorded and the first 26 stage-5 capsules produced on plants in the flower excision treatments were removed. The first three and the fourteenth to sixteenth stage-5 capsules produced on the two nonexcision treatments were harvested for nicotine quantification. The twenty-seventh, twenty-eighth, and twenty-ninth stage-5 capsules of plants in all four treatments were harvested for nicotine analysis. An allometric analysis of nicotine contents in reproductive parts in all stages of development was conducted on the first five plants to produce 29 stage-5 capsules in treatments 3 and 4 (e.g., the two nonexcision treatments). Five capsules from each of six ontogenetic stages, for a total of 30 capsules per plant, were harvested for nicotine quantification. Statistical Analysis. Two-way ANOVAs with damage and plant as the main effects were performed on micrograms of nicotine per capsule and gram capsule mass data of experiment 1. Two-way ANOVAs with damage and excision as the main effects were performed on the third harvest of stage-5 capsules (numbered 27-29) of experiment 2. A repeated-measures one-way ANOVA with leaf damage as the main effect was performed on the three harvests of stage-5 capsules from the two nonexcision treatments of Experiment 2. The In-transformed amounts of nicotine per capsule were analyzed with an analysis of covariance with In-transformed amounts of nicotine-free biomass per capsule as the covariate and with individual regressions on the data from each plant, A test of homogeneity of slopes (parallelism) was conducted to determine whether the slopes of these relationships differed between plants with and without leaf damage. Data analysis was performed with the MGLH module of the SYSTAT statistical package (Evanston, Illinois). RESULTS
Experiment 1. The amount of nicotine in stage-5 capsules with caterpillar (37.4 _+ 3.4 ~tg nicotine/capsule) or mechanical damage (36.3 + 3.5/~g nicotine/capsule) to their corollas did not differ from that found in undamaged capsules (37.9 + 3.1 /zg nicotine/capsule) as determined by two-way ANOVAs (damage treatment F2,98 = 0.061; P = 0.94). Significant differences between plants were found (F4.98 = 2.66; P = 0.037), and the damage × plant interaction term was not significant (F8,98 = 0.16; P = 0,99). No significant differences were found in any of the flower biomass data (Ps > 0.40; two-way ANOVA). Experiment 2. Neither the leaf damage treatment nor the floral excision treatment significantly influenced the biomass of any of the stage-5 capsules
904
BALDWIN AND KARB
from any of the harvests as d e t e r m i n e d by the o n e - w a y repeated-measures A N O V A or the two-way A N O V A on the t w e n t y - s e v e n t h to twenty-ninth stage5 capsules produced on each plant (Table 2; Figure 2). Similarly, the floral excision treatment did not significantly affect the a m o u n t o f nicotine in the twenty-seventh to twenty-ninth stage-5 capsules produced on plants as determined by a two-way A N O V A (Table 2; Figure 2). L e a f d a m a g e had no significant effect on the a m o u n t o f nicotine found in the first three stage-5 capsules (F). ~7 = 1.15; P = 0.30; o n e - w a y A N O V A ; Figure 2) produced on each plant; however, leaf d a m a g e dramatically increased the a m o u n t of nicotine in capsules produced later during reproductive growth as d e t e r m i n e d by the repeated-measures one-way A N O V A and the two-way A N O V A (Table 2; Figure 3). By the third harvest (capsules n u m b e r e d 2 7 - 2 9 ) , stage-5 capsules on plants with leaf damage had nicotine pools that were three times those produced by u n d a m a g e d plants at the same stage o f d e v e l o p m e n t . This difference in nicotine pools is due in part to a significant decrease in nicotine o f capsules produced on u n d a m a g e d
TABLE 2. Two-WAY ANOVAs ON NICOTINE AND BIOMASSPOOLS OF STAGE-5 CAPSULES ON THIRD HARVEST (CAPSULES 27-29) FROM ALL FOUR TREATMENT GROUPS (FIGURE 2) AND ONE-WAY REPEATED-MEASURES ANOVAs BETWEEN-SUBJECTS EFFECTS ON NICOTINE AND BIOMASS POOLS OF STAGE-5 CAPSULES FROM ALL THREE HARVESTS FROM DAMAGED AND UNDAMAGED PLANT TREATMENTS (FIGURE 2). MS
F
P
13700.2 29.5 28.6 317.9
43.094 0.093 0.090
0.000 0.763 0.766
2.444 0.814 0.007
0.127 0.374 0.936
33
0.835 × 10-5 0.278 x 10-'~ 0.221 × 1 0 - 7 0.342 × 10-5
! 17
8524.3 582.0
14.6
0.0013
1 17
0.3799 × 10-5 0.4458 × 10-5
df
Third Harvest Nicotine (v.g/capsule) Leaf damage treatment (D) Floral excission treatment (E) D*E
Error Dry mass (rag/capsule) Leaf damage treatment (D) Floral excission treatment (E) D*E
Error Harvests 1-3 Nicotine (~g/capsule) Leaf damage treatment Error Dry mass (mg/capsule) Leaf damage treatment Error
1 I 1
33 1 I 1
0.8521
0.369
PLASTICITY
OF
FLORAL
DEFENSE
905
}'°I ~ '°l 0 (i
I
, ~
! io
,
i
,
L~
~n
25
i
g
Capsule Number FIG. 2. Mean (_+SEM) capsule (a) nicotine and (b) biomass at three stages during reproductive growth of l0 undamaged (open circles) plants and nine plants with leaves damaged during vegetative and reproductive growth (solid circles). Samples are of stage5 capsules (see Table 1) numbered 1-3, 14-16, and 27-29 on all plants where capsule 1 was the first capsule to reach stage 5. The nicotine and biomass pools of stage-5 capsules numbered 27-29 from an additional nine undamaged (open squares) and nine plants with leaves damaged during vegetative and reproductive growth (solid squares) are also depicted. These plants are from the floral excision treatment which had their first 1-26 stage-5 capsules excised.
plants (F].27 > 4.56; P < 0.042; one-way ANOVA contrasts between the first and second and first and third harvests). Neither leaf damage nor flower excision affected the plants" phenologies; the rates of flower production as determined by the time to produce 3, 14 or 27 stage-5 capsules did not differ among the treatment groups (F~.33 < 2.62; P > 0.11; two-way ANOVAs). Reproductive parts at all stages in development from plants with and without leaf damage used in the allometric analysis had significantly (FI.286 = 180.33; P < 0.00001; one-way ANOVA on In micrograms of nicotine) more nicotine than those from undamaged plants (Figure 3). Biomass values were not significantly different (FI.286 = 0.013; P = 0.91; one-way ANOVA on capsule In dry mass). The relationship between nicotine pools and capsule biomass as determined by a test of homogeneity of slopes (parallelism) did not differ sig-
906
BALDWIN AND KARB
oo
--=
o
oo
o
o o
o
In Nico6ne-Free Biomass (g/capsule)
FIG. 3. Natural log transformed nicotine pools plotted against the In-transformed nicotine-free biomass of five capsules from each of the six developmental stages described in Table 1, Capsules were sampled from five u n d a m a g e d (open circles) plants and five plants with leaves damaged during vegetative and reproductive growth (solid circles). All plants were sampled after producing 30 stage-5 capsules and hence were at the same stage in their reproductive phase as those plants in the third harvest of Figure 1, See Table 3 for the regression statistics for each individual plant and the text for the A N C O V A analysis,
TABLE 3. ESTIMATES ( _ S E M ) OF REGRESSION COEFFICIENTS (R 2, y INTERCEPT, AND SLOPE) FOR ALLOMETRIC REGRESSIONS (FIGURE 3) FOR EACH PLANTa R2
y-intercept
Slope
Undamaged plants 1
2 3 4 5 Damaged plants 1 2 3 4 5
0.116 0.575 0.5t5 0.423 0.382
4.212 5.288 5.498 5.693 5.875
± 0.753 ± 0,391 ::t: 0.474 ± 0.622 + 0.806
0.285 0A52 0.499 0.536 0.647
± ± ± ± ±
0,148 0,076 0,093 0.120 0.156
0.282 0.797 0.664 0.840 0.505
5.707 8,154 6.917 6.678 5.694
+ 0.654 ± 0,358 2:0.369 + 0.294 ± 0.414
0.410 0,719 0,546 0.644 0.422
± 0.126 ± 0.070 5:0.075 ± 0.057 ± 0.081
~Natural log-transformed nicotine pools in five capsules for each of the six developmental stages described in Table 1 were regressed against In-transformed nicotine-free dry biomass for each plant. Plants were either undamaged or had their leaves damaged throughout the reproductive phase of growth,
907
PLASTICITY OF FLORAL DEFENSE
nificantly (Fi.28 s = 0.115; P = 0.735) between damaged and undamaged plants. Because the assumption of homogeneity was not violated, an analysis of covariance (ANCOVA) with In nicotine as the dependent variable and In nicotinefree biomass as the covariate was performed, revealing that undamaged and damaged plants differed significantly (FI,289 = 72.3; P < 0.001). Within-plant regressions revealed the same pattern as the ANCOVA (Table 3). The y-intercept values were significantly higher for damaged plants (F~.8 = 5.93; P = 0.041; one-way ANOVA on In-transformed values), while the slopes of the individual plant regressions were not significantly different (F~,8 = 0.58; P = 0.47; oneway ANOVA on In-transformed values). In addition, the relationships between In-transformed nicotine pools and biomass tended to have less scatter with damaged plants than with undamaged plants (mean R 2 = 0.618 and 0.402 for damaged and undamaged plants, respectively; Table 3). DISCUSSION
The results of these experiments revealed some of the "'rules" that govern how nicotine is allocated to reproductive parts, First, nicotine is not passively allocated to reproductive parts as a function of the amount of nicotine produced in roots and the number of capsules on a plant. The nicotine content of stage5 capsules on plants that had the vast majority of their maturing capsules excised did not differ from those which did not have capsules excised (Figure 2). The excised stage-5 capsules contained between 20 and 60/zg of nicotine, depending on whether they developed on plants with or without leaf damage. Had these capsules remained on the plant through maturity, they would have accumulated 60-160 ~g of nicotine, respectively. Hence, the excision treatment should have doubled or tripled the nicotine pools or stage-5 capsules if the passive allocation model was correct. This deduction assumes that nicotine accumulation reflects nicotine production in N. attenuata as has been demonstrated in N. sylvestrius (Baldwin et al., 1994a) and that nicotine was not stored in other plant parts for allocation to capsules later in reproductive growth. Second, the allocation of nicotine to a capsule is not influenced by herbivory or mechanical damage to the corolla (experiment 1) or to the removal of other reproductive parts (experiment 2). Third, in contrast to damage to reproductive parts, damage to leaves dramatically increases the allocation of nicotine. Fourth, the allocation of nicotine to reproductive parts follows an allometric relationship that has less scatter in damaged plants, as indicated by the higher R z of the individual regressions (Table 3), and has a higher intercept. The effect of leaf damage on the nicotinebiomass allometry appears similar to that found in vegetatively growing N. sylvestrius plants (Figure 1). The long-term effect is to move plants from one allometric relationship to another with similar slopes but different intercepts.
908
BALDWIN AND KARB
The N. s v l v e s t r i s data set clearly showed that damaged plants moved from one allometric relationship to another by briefly increasing their rate of nicotine production during growth and, hence, increasing the slope of the relationship. A similar mechanism could be operating in N. a t t e n u a t a reproductive parts, although leaf damage may have initiated the production of capsules with higher nicotine accumulation set points at the beginning of capsule growth. Fifth, leaf damage does not appear to alter the allometric set points for nicotine accumulation for the first reproductive parts produced by a plant, and these apparent set points appear to decline during the later stages of reproductive growth in undamaged plants. These patterns of nicotine allocation underscore the important regulating influence that vegetative tissues have on the defense of reproductive tissues. Many of these patterns are broadly consistent with the predictions of OD theory. First, defense is constitutively allocated to individual reproductive parts as a function of capsule mass. OD theory predicts that defensive plasticity should be reserved for those tissues that are functionally plastic, such as leaves that are capable of physiologically rejuvenilizing and tissues whose contribution to plant fitness vary over ontogeny. Second, defense allocation to the first capsules produced on an undamaged plant appear to be higher than those produced later (Figure 2). For plants growing in unpredictable environments, the first offspring produced represent a larger proportion of realized fitness than those produced later and hence would be predicted to receive a larger defensive allocation. Moreover, if inducible defense allocation provides a means of conserving resources that could be otherwise used for offspring production, the inducible allocation of defense to offspring produced later during reproductive growth, as plants realize their potential fitness, is also consistent with the cost-benefit paradigm of OD theory. Whether or not plants that produce more defense in herbivore-free environments consequently produce fewer offspring of lower quality remains to be demonstrated, Acknowledgments--This research is supported by National Science Foundation grants BSR9157258, BSR-9118452 and a generous equipment grant from the Hewlett-PackardUniversityof Grants Program. We thank T. Ohnmeiss, C. Olney. E. Schmelz, N. Blenk, and M. Euler for expert technical assistance;E. Wheelerfor editorial assistance;and two reviewersfor improvingan earlier draft of this manuscript.
REFERENCES BALDWIN, I.T. 1988. Damaged-inducedalkaloids in tobacco: Pot-bound plants are not inducible. J. Chem. Ecol. 4:1113-1120. BALDWIN,I.T. 1969. Mechanismof damage-inducedalkaloid production in wild tobacco. J. Chem. Ecol. t5:1611-1680. BALDWIN,I.T. f993. Chemical changes rapidly induced by folivory, pp. 1-23, in E.A. Bemays
(ed.). Insect-Plant Interactions, Vol. 5. CRC Press, Boca Raton, Florida.
PLASTICITY OF FLORAL DEFENSE
909
BALDWIN, I.T., and OHNMEISS,T.E, 1993. Alkaloidal responses to damage in Nicotiana native to North America. J. Chem. EcoL 19:1143-1153. BALDWIN, I.T., and OHNMEISS,T.E. 1994, Coordination of photosynthetic and alkaloidal responses to leaf damage in uninducible and inducible Nicotiana sylvestris. Ecology 75:1003-1014. BALDWIN, I.T., KARB, M.J,, and OHNMEISS,T.E. 1994. Allocation of ~N from nitrate to nicotine: Production and turnover of an induced defense, Ecology. 75:1703-1713. BALDWIN, I.T.. STASCAK-KOZINSKI,L., and DAVIDSON,R. 1994b. Up in smoke: I. Smoke-derived germination cues for the post fire annual Nicotiana attenuata Ton" ex Watson. J. Chem. EcoL 20:2345-237 I. BOODLEY,J,W., and SHELDRAKE,R,J. 1977, Comell peat-lite mixes for commercial plant growing. Comell Information Bulletin 43. Ithaca, New York. GERSHENZON,J., and CROTEAU, R. 1991. Terpenoids, pp, 165-219, in G.A. Rosenthal and M.R, Berenbaum. Herbivores: Their Interactions with Secondary Plant Metabolites. Academic Press, San Diego. GOODSPEED, T H . 1954. The Genus Nicotiana. Chronica Botanica Co., Waltham, Massachusetts. HEFTMANN, E., and SCHW~MMER, S. 1972. Degradation of tomatine to 3B-hydroxy-5O-pregn-16enone by ripe tomatoes. Phytochemistn' I 1:2783-2787. HENDRIX, S.D., and TRAPP, E.J. 1981. Plant-herbivore interactions: Insect-induced changes in host plant sex expression and fecundity. Oecologia 49:119-122. McKEY, D. 1974~ Adaptive patterns in alkaloid physiology, Am. Nat. 108:305-320, MCKEY, D. t979. The distribution of secondary compounds within plants, pp. 55-133, in G.A. Rosenthal and D.H. Janzen (eds.I. Herbivores: Their Interactions with Secondary Plant Metabolites. Academic Press, New York. MCNAMARA, K. 1986. A guide to the nonmenclature of heterochrony. J. Paleontol. 60:4-13, NITAO, J.K. 1988. Artificial defloration and furanocoumarin induction in Pastinaca sativa (Umbelliferae/. J. Chem. Ecol. 14:1515-1522. OrtNMEtSS, T.E., and BALDWtN, I.T. 1994. The allometry of nitrogen allocation to growth and an inducible defense under nitrogen-limited growth. Ecology 75:995-1002. PEARCE, G., RYAN, C.A., and LIL/EGREN, D. 1988. Proteinase inhibitors I and II in frail of wild tomato species: Transient components to a mechanism for defense and seed dispersal. Planta 175:527-531. RHOADES, D.F. 1979. Evolution of plant chemical defense against herbivores, pp. 4-54, in G.A. Rosenthal and D.H. Janzen (eds.). Herbivores: Their Interactions with Secondary Plant Metabolites. Academic Press, New York. RI-IOADES, D.F. 1983. Herbivore population dynamics and plant chemistry, pp. 155-220, in R.F. Denno and M. McClure (eds.). Variable Plants and Herbivores in Natural and Managed Systems. Academic Press, New York. SAMSON, D.A., and WERK, K.S. 1986. Size-dependent effects in the analysis of reproductive effort in plants. Am. Nat. 127:667-680. WELTE~, S.C. 1989. Arthropod impact on plant gas exchange, pp. 135-150, in E.A. Bemays ted.). Insect-Plant Interactions. CRC Press, Boca Raton, Florida. ZANGERL, A.R. 1986. Leaf value and optimal defense in Pastinaca sativa L. (Umbelliferae). Am. MidL Nat. 116:432-437. ZANGEP,L, A.R. 1990. Furanocoumarin induction in wild parsnip: Evidence for an induced defense against herbivores. Ecology 71 : 1926-1932. ZANGERL, A.R., and BAZZAZ, F.A. 1992. Theory and pattern in plant defense allocation, pp. 36339l, in R.S. Fritz and E . L Simms (eds.). Plant Resistance to Herbivores and Pathogens: Ecology, Evolution, and Genetics. University of Chicago Press, Chicago.
