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Defense signaling among interconnected ramets of a rhizomatous clonal plant, induced by jasmonic-acid application
Acta Oecologica 37 (2011) 355e360
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Original article
Defense signaling among interconnected ramets of a rhizomatous clonal plant, induced by jasmonic-acid application Jin-Song Chen a, Ning-Fei Lei b, Qing Liu a, * a b
Chengdu Institute of Biology, CAS, Chengdu 610041, China Chengdu University of Technology, Chengdu 610059, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2011 Accepted 2 April 2011
Resource sharing between ramets of clonal plants is a well-known phenomenon that allows stoloniferous and rhizomatous species to internally transport water, mineral nutrients and carbohydrates from sites of high supply to sites of high demand. Moreover, vascular ramet connections are likely to provide an excellent means to share substances other than resources, such as defense signals. In a greenhouse experiment, the rhizomatous sedge Carex alrofusca, consisting of integrated ramets of different ages, was used to study the transmission of defense signals through belowground rhizome connections in response to local spray with jasmonic-acid. A feeding preference test with the caterpillar Gynaephora rnenyuanensis was employed to assess benefits of rhizome connections on defense signaling. Young ramets were more responsive to jasmonic-acid treatment than middle-aged or old ramets. Condensed tannin content in the foliage of young ramets showed a significant increase and soluble carbohydrate and nitrogen content showed marginally significant decreases in the 1 mM jasmonic-acid treatment but not in control and/or 0.0001 mM jasmonic-acid treatments. The caterpillar G. rnenyuanensis preferentially grazed young ramets. After a localized spray of 1 mM jasmonic-acid, the leaf area of young ramets consumed by herbivores was greatly reduced. We propose that defense signals may be transmitted through physical connections (stolon or rhizome) among interconnected ramets of clonal plants. Induced resistance to herbivory may selectively enhance the protection of more vulnerable and valuable plant tissues and confer a significant benefit to clonal plants by a modular risk-spreading strategy, equalizing ontogenetic differences of unevenly-aged ramets in chemical defense compounds and nutritional properties of tissue. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: C. alrofusca G. rnenyuanensis Feeding preference Chemical defense compound Nutritional properties of tissue
1. Introduction Clonal plants produce new shoots at the nodes of horizontal stems (stolons or rhizomes). This type of clonal growth forms networks of physically interconnected, genetically identical, and functionally autonomous offspring individuals, called ramets. The internal transport of substances such as water, photo-assimilates and nutrients can be realized through physical connections among ramets. This is termed clonal integration or resource sharing. Clonal integration has been extensively investigated, and numerous studies have provided clear evidence of its ecological implications, such as allowing post-natal care for clonal offspring, acting as an efficient vehicle for resource extraction from heterogeneous environments, and provisioning internal support to damaged or stressed ramets (Pitelka and Ashmun, 1985; Alpert, 1991; Evans, 1992; Marshall and Price, 1997; Magori et al., 2003). * Corresponding author. Fax: þ86 28 85222753. E-mail address: [email protected] (Q. Liu). 1146-609X/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.actao.2011.04.002
It has been suggested that clonal plant networks may be used to share non-resource substances as well as water, carbohydrates and mineral nutrients (Cook,1978; Pitelka and Ashmun,1985). Evidence on the integration of the non-resource substances among interconnected ramets has recently been gathered from studies on a stoloniferous clonal plant Trifolium repens (Gómez and Stuefer, 2006; Gómez et al., 2007, 2008). These studies indicated that defense signaling could allow undamaged members of a clonal fragment to prepare themselves for impending herbivore attacks by up-regulating the expression of chemical and other defense traits. However, more studies are necessary to provide insight into the mechanisms, dynamics, and implications of clonal integration beyond resource sharing. Jasmonic-acid (JA) is found in many species and has been identified as an endogenous regulator of wound-induced chemistry in plants and a signal molecule in responses of plants to herbivory (Thaler et al., 1996; Karban and Baldwin, 1997; Thaler, 1999). When plants are attacked by herbivores, jasmonic-acid production increases in plant tissues (Creelman et al., 1992; Wasternack and Parthier, 1997), activating the natural defensive response of plants
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and thus enhancing resistance to attack (Cohen et al., 1993; Thaler et al., 1996). Treatment with jasmonic-acid may simulate the same defensive responses of plants to herbivory, leading to local and systemic induction of various classes of defensive compounds, such as alkaloids, phenolics, flavonoids and various terpenoids (Wasternack and Parthier, 1997). For example, Thaler et al. (1996), Cipollini and Redman (1999), and Arnold and Schultz (2002) showed that jasmonic-acid treatment induced the activity of the oxidative enzymes, peroxidase, and polyphenol oxidase, and condensed tannin contents, thus enhancing the resistance of these plants to herbivory. Furthermore, treating plants with jasmonicacid provides a convenient way to disassociate chemical defense and leaf loss, because jasmonic-acid causes the induction of several well documented defensive pathways without the removal of leaf tissue (Baldwin, 1998). Condensed tannins (CT) are secondary phenolic metabolites of plants and are involved in plantepathogen (Brownlee et al., 1990; Edwards, 1992; Heil et al., 2002) and planteherbivore (Bernays, 1981; Min et al., 2003; Forkner et al., 2004) interactions. They are widespread in nature, occurring in a range of herbaceous legumes (Jones et al., 1976; Terrill et al., 1992) and tree leaves (Kumar and Vaithiyanathan, 1990). Condensed tannins have been reported to play a significant role in the plant defense system against a wide variety of fungi, bacteria and insects (Feeny, 1968; Scalbert, 1991). The perennial rhizomatous clonal plant Carex alrofusca can produce condensed tannins, which have been shown to confer resistance to leaf-chewing insects such as the grassland caterpillar Gynaephora rnenyuanensis (Guo et al., 2004; Yan, 2006). Most studies on induced defenses have focused on the production of chemical defense compounds (Karban and Baldwin, 1997). However, animal grazing may alter the quality of hosteplant foliage, by reducing foliar nitrogen and carbohydrate concentrations. The nutritional properties of tissue may affect the feeding preferences and/or performance of herbivores so that they should be regarded in the context of inducible defense strategies (Jones and Roberts, 1991; Jensen, 1993; Redak and Capinera, 1994; Brathen et al., 2004; Gómez et al., 2008). A greenhouse experiment was set up using the rhizomatous clonal sedge C. alrofusca to examine the following two questions: (1) Does jasmonic-acid application induce defense signaling through physical connections among interconnected ramets? Because chemical defense compounds and the nutritional properties of tissue are regarded in the context of inducible defense strategies, we expect that localized spraying of jasmonic-acid may incur a significant change in condensed tannins, soluble carbohydrates, and nitrogen content in untreated ramets of the same clonal fragment. (2) Does defense signaling among interconnected ramets confer significant benefits to clonal plants? Defense signaling could allow untreated ramets of the same clonal fragment to improve their resistance for impending herbivore attacks by changing their chemical defense compounds and the nutritional properties of their tissue. Therefore, we expect that after localized spraying of jasmonic-acid, the proportion of consumed leaf areas in untreated ramets of the same clonal fragment may significantly decrease.
2. Materials and methods 2.1. Plant and herbivore C. alrofusca is a perennial, 20e30 cm tall rhizomatous herb distributed mainly in alpine meadows with altitudes ranging from
2600 to 5000 m a.s.l in western China (Liu and Lu, 1999). After expanding horizontally, the terminal bud of the rhizome grows upward to form the shoot, thereby forming extensive clonal fragments. In June 2006, six original clonal fragments, from sites at least 1000 m apart, were collected in Zoige County, Sichuan, China. Clonal fragments from the six sites are likely different genotypes due to the distance separating the collection sites. They were then vegetatively propagated in a greenhouse located in the Maoxian Mountain Ecosystem Research Station, Chinese Academy of Science (location 103 540 E, 31420 N, altitude1826 m a.s.l). The plants were watered as needed and fertilized with nutrient solution (0.083 mol l1 CO(NH2)2, 0.022 mol l1 KH2PO4) approximately every two weeks. G. rnenyuanensis is a grassland caterpillar with a broad range of hosts including Festuca ovina, Elymus nutans, and C. alrofusca in the alpine meadow of the TibeteQinghai plateau in China (Yan et al., 1995). In April 2007, first instar caterpillars were obtained from Qinghai University, Xining China. The caterpillars were reared using the methods described in Yan et al. (2005). 2.2. Experimental design In May 2007, three clonal fragments were collected from each of the six original genotypes and transported to a temperaturecontrolled greenhouse at Chengdu University of Technology, Chengdu, China. The greenhouse had a mean temperature of 23 5 C and natural light, supplemented with a sodium halide lamp as needed. These 18 clonal fragments were planted into individual wooden trays (100 20 20 cm) at least two weeks before the start of the induction treatment. The wooden trays were divided into sectors by a thin plastic wall. The rhizomes connecting ramets passed through notches in the wall that were caulked with a DAP acrylic sealant so that the interconnected ramets were grown in separate water-tight compartments within a single tray and all rhizome connections were maintained throughout the experiment. Each clonal fragment consisted of six successive ramets. The sixth and oldest ramet (as indicated by its relative proximity to the mother ramet) was subjected to one of the following three treatments: (1) control; (2) spray with a solution of 0.0001 mM jasmonic-acid; and (3) spray with a solution of 1 mM jasmonicacid. The jasmonic-acid dosage applied was based on a previous report (Dèlano-Frier et al., 2004) and on preliminary laboratory results. The fifth, third and first ramets were referred as old, middle-aged, and young ramets respectively. The control was placed between the two treatments to allow planteplant volatile signals (Fig. 1). Each of the six original plants and their descendants was subjected to all treatments. Given its low solubility in water, jasmonic-acid (Sigma Chemical Co.) was first dissolved in a small amount (about 5 ml) of ethanol, made up to two different concentrations of 0.0001 mM and 1 mM with distilled water. Triton-X 100 detergent (0.125% v/v) was added to facilitate penetration of leaf cuticles. Control solutions were identical but lacked jasmonic-acid. We sprayed 1 ml of control or jasmonic-acid solution evenly onto two fully unfolded leaves of the sixth ramet. Neighboring ramets were shielded from spray with a piece of plastic. Then, the target leaves of the sixth ramet were sealed in a transparent plastic bag until dry, approximately 40 min. Five days after the jasmonic-acid treatment, a feeding preference test in the untreated young, middle-aged and old ramets of the same clonal fragments was used to investigate the effects of defense signaling on consumption by the herbivore G. rnenyuanensis. Feeding preference tests were conducted among leaves of young, middle-aged and old ramets of induced and control plants, respectively (Fig. 1). The leaves of the untreated young, middle-aged and old ramets of the same clonal fragment were put
feeding preference test
Control/Jas monic-acid
6
5
4
3
2
1
Fig. 1. Schematic representation of the experimental design to test defense signaling through rhizomes among interconnected ramets of the clonal plant C. alrofusca. The clonal fragments consist of six successive ramets. The first ramet is the youngest and the sixth ramet is the oldest. The first, third and fifth ramets of same clonal fragment were referred to as young, middle-age and old ramets respectively. After the induction treatment on the sixth ramets, a feeding preference test with a fourth instar caterpillar G. rnenyuanensis was conducted on young, middle-aged, and old ramets.
into a petri dish and a fourth instar caterpillar was placed in the middle of the leaves, which is similar to the method described by Gómez et al. (2008). The caterpillar was allowed to feed for a fixed amount of time (24 h). Total leaf area and remaining leaf area were measured at the start and end of the experiment using a portable leaf area meter. The proportion of consumed leaf area was calculated as:
total leaf area remaining leaf area 100%: total leaf area Owing to small size of C. alrofusca leaves, a single clonal fragment was too small to use for all measurements. Therefore the experimental procedure was repeated four times; once for analysis of foliage chemical defense compounds, once for soluble carbohydrate content, once for nitrogen content and the fourth for the herbivory preference of G. rnenyuanensis. 2.3. Chemical analysis and measurement After jasmonic-acid treatment, leaves of old, middle-aged, and young ramets were harvested. We used about 2 g from each to investigate condensed tannin content by vanillineHCL assay (Wu and Guo, 2000) and another 1 g to measure soluble carbohydrates and nitrogen content using the anthrone reagent colorimetric method and the Kjeldahl nitrogen determination method (Dong, 1997). 2.4. Statistics analysis One-way repeated-measures ANOVA was used, where the repeated (within-subject) factor was ramet age and the fixed between-subject factor was jasmonic-acid induction. Means were
Condensed tannin content in the foliage (mg.g-1DM)
J.-S. Chen et al. / Acta Oecologica 37 (2011) 355e360
357
8
a
a
6
a
a
a
a
a
b 4
b
2
0
Control
1mM
0.0001mM
Fig. 2. Condensed tannins content (SE) in the foliage after the induction treatment was conducted for 5 days. , , , represent young, middle-age and old ramets. The bars with the same lowercase letters are not significantly different at p ¼ 0.05.
compared with SeNeK multiple contrast. All statistical analyses were conducted with SPSS 10.0. 3. Results Condensed tannin content in the foliage was significantly affected by jasmonic-acid induction, ramet age and their interaction (Table 1). Young ramets contained significantly less condensed tannins than did middle-aged or old ramets in control and 0.0001 mM jasmonic-acid treatments (Fig. 2) but in the 1 mM jasmonic-acid treatment the foliage of young ramets contained significantly more condensed tannins than that of young ramets from either control or 0.0001 mM jasmonic-acid treatments (Fig. 2). Jasmonic-acid did not significantly affect soluble carbohydrate or nitrogen content in the foliage of young, middle-aged and old ramets (Table 1). Nonetheless there was a tendency for soluble carbohydrate content of in the foliage of young ramets to be greater in control and 0.0001 mM jasmonic-acid treatments than in all other combinations (0.05 p 0.06; Fig. 3). Ramet age, jasmonic-acid induction and their interaction had a significant effect on the amount of leaf area consumed by the caterpillar (Table 1). The results of the feeding preference test showed that G. rnenyuanensis larvae consistently preferred young ramets over middle-aged and old ramets of C. alrofusca in control and 0.0001 mM jasmonic-acid treatments (Fig. 5). On average, young ramets incurred almost 50% higher damage than middleaged and old ramets. However, in the 1 mM jasmonic-acid treatment this preference for young leaves was not evident and caterpillars consumed significantly less leaf material of young ramets than in control and 0.0001 mM jasmonic-acid treatments (Fig. 5). Jasmonic-acid induction had a very strong effect on the damage inflicted on young ramets.
Table 1 Repeated-measure ANOVA for effects of jasmonic-acid induction on condensed tannins, soluble carbohydrates and nitrogen contents in the foliage as well as the proportion of consumed leaf area among interconnected young-age, middle-age and old-age ramet. Source
Foliage condensed tannins content Foliage soluble carbohydrates content Foliage nitrogen content Ratio of consumed leaf area
Between-subject effects Jasmonic-acid induction Error
df
F
df
F
df
F
df
F
2 15
4.57*
2 15
2.86ns
2 15
3.01ns
2 15
3.96*
23.19** 3.76*
2 4 30
3.16ns 1.94ns
2 4 30
2.41ns 1.63ns
2 4 30
10.67** 5.43**
Within-subject effects Ramet age 2 Ramet age jasmonic-acid induction 4 Error 30 Significance level:
ns
P > 0.05 *P < 0.05 **P < 0.01.
J.-S. Chen et al. / Acta Oecologica 37 (2011) 355e360 14 12
a
4
a a a
a
a
a
a
a
10 8 6 4 2 0
Control
0.0001mM
Nitrogen content in the foliage (%)
Soluble carbohydrate content in the foliage (%)
358
a
a
a a
a
a
a
2
0
1mM
a
a
Control
0.0001mM
1mM
Fig. 3. Soluble carbohydrates content (SE) in the foliage after the induction treatment was conducted for 5 days. , , represent young, middle-age and old ramets. The bars with the same lowercase letters are not significantly different at p ¼ 0.05.
Fig. 4. Nitrogen content (SE) in the foliage after the induction treatment was conrepresent young, middle-age and old ramets. The bars with ducted for 5 days. , , the same lowercase letters are not significantly different at p ¼ 0.05.
4. Discussion
treatment in young ramets than in middle-aged or old ramets (Fig. 2). The amounts of soluble carbohydrates and nitrogen in the foliage of young ramets were marginally lower in 1 mM jasmonicacid treatment than in control and/or 0.0001 mM jasmonic-acid treatments (0.05 p 0.06; Figs. 3 and 4). Therefore, it is suggested that the effects of jasmonic-acid induction on chemical defense compounds and nutritional properties of the tissue in the clonal plant C. alrofusca may be dependent on the ontogenetic stage of the ramets. The optimal defense theory predicts that plant tissues with a high contribution to fitness and a high risk of attack should be better protected than other plant tissues. Young ramets are primarily responsible for future vegetative growth and reproductive success of clonal plant individuals. Therefore, young ramets of clonal plants are the most valuable tissues for growth and fitness and should hence be especially well protected (Mckey, 1974). C. alrofusca can share defense signals through belowground rhizome connections and its young ramets are more responsive than middle-age or old ramets, which selectively enhances the protection of more vulnerable and valuable plant tissues and confers a significant benefit to clonal plants by employing a modular risk-spreading strategy, equalizing ontogenetic ramet differences in chemical defense compounds and nutritional properties of tissue (Gómez et al., 2008). The experiment provides concrete evidence on specific mechanism of induced defense resistance against generalist herbivore in clonal plants. In alpine meadow, seedling recruitment is infrequent, and vegetative propagation is the prevalent form for reproduction in rhizomatous sedges C. alrofusca (Zhao et al., 2007). 50
Proportion of consumed leaf area (%)
Our study provides clear evidence that belowground rhizome connections among interconnected ramets can serve as pathways for the transmission of induced defense signals in physiologically integrated clonal plant species. Therefore, clonal integration may not only allow for an efficient transport of resources such as water, mineral nutrients and carbohydrates in the system, but can also operate as an information sharing system among members of clonal fragments. In our experiment, interconnected ramets were grown in separate water-tight compartments within a single tray and the plants assigned to control and induction treatments were spatially arranged without physical barriers between them, which ensured that the induction effect among interconnected ramets was not caused by volatile info-chemicals or root exudates (Gómez and Stuefer, 2006). Physiological features of young plants may be different from mature plants and they often invest a lot of energy in the development of biomass (Cipollini and Redman, 1999). Due to different chemical defense compounds and nutritional properties of tissue in the foliage among various ontogenetic stages, young ramets in the clonal plant C. alrofusca might be more palatable to herbivores and incurred more herbivory damage than middle-age or old ramets (Fig. 5). A similar effect was reported for T. repens, in which phenolic profiles and foliage quality measured as foliage carbohydrate and nitrogen contents were different between young and old ramets (Gómez et al., 2008). It is frequently observed that young plants or tissues are more responsive to elicitors of defense than older plants or tissues, although the inverse can also be true (Karban and Myers, 1989; Faeth, 1991). For example, the ability of wounding to induce proteinase inhibitor activity in tomato decreases with plant age (Wolfson and Murdock, 1990). The mRNA of polyphenol oxidase F is induced more strongly by wounding or jasmonic-acid in young leaves of tomato than in older leaves on the same plant (Thipyapong and Steffens, 1997). Cipollini and Redman (1999) investigated the quantitative effect of plant age on the induction of defense by jasmonic-acid, indicating that constitutive polyphenol oxidase activity did not change with age in tomato plants and was more strongly induced by jasmonic-acid in young plants. Furthermore, changes in plant foliage quality after defoliation have often been attributed to defoliation-induced increases in plant defensive compounds (Wratten et al., 1984; Tallamy and Raupp, 1991), generalized plant-stress responses to insect damage (Wagner and Evans, 1985; Myers, 1988), or both (Bryant et al., 1983; Tuomi et al., 1984). Chemical defense compounds in the foliage such as condensed tannins are more responsive to jasmonic-acid
a
a
40
b 30
b
b b
20
b
b
b
10
0
Control
0.0001mM
1mM
Fig. 5. Proportion of consumed leaf area (SE) in a feeding preference test with a fourth instar caterpillar G. rnenyuanensis after the induction treatment was conrepresent young, middle-age and old ramets. The bars with ducted for 5 days. , , the same lowercase letters are not significantly different at p ¼ 0.05.
J.-S. Chen et al. / Acta Oecologica 37 (2011) 355e360
Defense signaling within clonal plant network may be very important for survival and growth of C. alrofusca in the harsh habitat when suffering from caterpillar G. rnenyuanensis herbivory. Clonal integration can confer clonal plant networks with considerable benefits by allowing for fast, specific and efficient defense signaling among interconnected ramets. The defense signaling may also lead to substantial costs if clonal plant network members become induced without being threatened by herbivores (Gómez and Stuefer, 2006). The ramets of C. alrofusca are connected by belowground rhizomes, which may reduce the probability of subsequent herbivore attacks (Stuefer et al., 2004). Thus, defense signaling can be costly. Alternatively, defense signaling through belowground connection (i.e. rhizome) may be beneficial if it minimizes eavesdropping by other plant competitors. A major caveat is that the 1 mM jasmonic-acid concentration may be not biologically relevant. To get more insight into the ecological and evolutionary implications of defense signaling in clonal plants, additional experiments using defense signaling induced by caterpillar herbivory will be needed. Acknowledgments This research was supported by National Science Fund of China (30870389) and by Knowledge Innovation Project of The Chinese Academy of Sciences (KZCX2-XB2-02). We would also like to thank Anne Bjorkman at the University of British Columbia for her assistance with English language and grammatical editing of the manuscript. References Alpert, P., 1991. Nitrogen sharing among ramets increases clonal growth in Fragaria chiloensis. Ecology 72, 69e80. Arnold, T.M., Schultz, J.C., 2002. Induced sink strength as a prerequisite for induced tannin biosynthesis in developing leaves of Populus. Oecologia 130, 585e593. Baldwin, I.T., 1998. Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences 95, 8113e8118. Bernays, E.A., 1981. Plant tannins and insect herbivores e an appraisal. Ecological Entomology 6, 353e360. Brathen, K.A., Agrell, J., Berteaux, D., Jonsdottir, I.S., 2004. Intraclonal variation in defence substances and palatability: a study on Carex and lemmings. Oikos 105, 461e470. Brownlee, H.E., McEuen, A.R., Hedger, J., Scott, I.M., 1990. Antifungal effects of cocoa tannin on the witches broom pathogen Crinipellis perniciosa. Physiological and Molecular Plant Pathology 36, 39e48. Bryant, J.P., Chapin, F.S., Klein, D.R., 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357e368. Cipollini, D.F., Redman, A.M., 1999. Age-dependent effects of jasmonic acid treatment and wind exposure on foliar oxidase activity and insect resistance in tomato. Journal of Chemical Ecology 25, 271e281. Cohen, Y., Gisi, U., Niderman, T., 1993. Local and systemic protection against Phytophthora infestans induced in potato and tomato plants by jasmonic acid and jasmonic methyl ester. Phytopathology 83, 1054e1062. Cook, R.E., 1978. Asexual reproduction: a further consideration. The American Naturalist 113, 769e772. Creelman, R.A., Tierney, M.L., Mullet, J.E., 1992. Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression. Proceedings of the National Academy of Sciences 89, 4938e4941. Dèlano-Frier, J.P., Martìnez-Gallardo, N.A., Martìnez-de la vega, O., SalasAraiza, M.D., et al., 2004. The effect of exogeneous jasmonic-acid on induced resistance and productivity in amaranth (Amaranthus hypochondriacus) is influenced by environmental conditions. Journal of Chemical Ecology 30 (5), 1001e1034. Dong, M., 1997. Observation and Analysis of Terrestrial Biocommunities. Standards Press of China, Beijing. Edwards, P.J., 1992. Resistance and defence: the role of secondary plant substances. In: Ayres, P.G. (Ed.), Pests and Pathogens: Plant Responses to Foliar Attack. BIOS Scientific Publishers, Oxford, pp. 69e84. Evans, J.P., 1992. The effect of local resource availability and clonal integration on ramet functional morphology in Hydrocotyle bonariensis. Oecologia 89, 265e276. Faeth, S.H., 1991. Variable induced responses: direct and indirect effects on oak herbivores. In: Tallamy, D.W., Raupp, M.J. (Eds.), Phytochemical Induction by Herbivores. John Wiley, New York, pp. 293e323,.
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Oecologia 61, 208e210. Wagner, M.R., Evans, P.D., 1985. Defoliation increases nutritional quality and allelochemics of pine seedlings. Oecologia 67, 235e237. Wasternack, C., Parthier, B., 1997. Jasmonate-signalled plant gene expression. Trends in Plant Science 2, 302e307. Wolfson, J.L., Murdock, L.L., 1990. Growth of Manduca sexta on wounded tomato plants: role of induced proteinase inhibitors. Entomologia Experimentalis et Applicata 54, 257e264. Wratten, S.D., Edwards, P.J., Dunn, I., 1984. Wound-induced changes in the palatability of Betula pubescens and B. pendula. Oecologia 61, 372e375.
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Wu, Y.Q., Guo, Y.Y., 2000. Determination of tannin in cotton plant. Chinese Journal of Applied Ecology 11 (2), 243e245. Yan, L., Liu, Z.K., Mei, J.R., Lan, J.H., 1995. Feed selection and utilization of grassland caterpillar in the field cage condition. Acta Agrestia Sinica 3 (4), 257e268. Yan, L., Jiang, X.L., Wang, G., 2005. Characterization of larvae development in Gynaephora menyuanensis. Acta Prataculturae Sinica 14 (2), 116e120.
Yan, L., 2006. Studies of taxonomy, geographic distribution in Gynaephora genus and life-history strategies on Gynaephora menyuanensis. PhD thesis. Lanzhou University. Zhao, J.Z., Liu, W., Zhou, H.K., et al., 2007. Influence of simulated warming effect on growth characteristic of Carex alrofusca. Journal of Gansu Agricultural University 42 (2), 84e90.
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Original article
Defense signaling among interconnected ramets of a rhizomatous clonal plant, induced by jasmonic-acid application Jin-Song Chen a, Ning-Fei Lei b, Qing Liu a, * a b
Chengdu Institute of Biology, CAS, Chengdu 610041, China Chengdu University of Technology, Chengdu 610059, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 January 2011 Accepted 2 April 2011
Resource sharing between ramets of clonal plants is a well-known phenomenon that allows stoloniferous and rhizomatous species to internally transport water, mineral nutrients and carbohydrates from sites of high supply to sites of high demand. Moreover, vascular ramet connections are likely to provide an excellent means to share substances other than resources, such as defense signals. In a greenhouse experiment, the rhizomatous sedge Carex alrofusca, consisting of integrated ramets of different ages, was used to study the transmission of defense signals through belowground rhizome connections in response to local spray with jasmonic-acid. A feeding preference test with the caterpillar Gynaephora rnenyuanensis was employed to assess benefits of rhizome connections on defense signaling. Young ramets were more responsive to jasmonic-acid treatment than middle-aged or old ramets. Condensed tannin content in the foliage of young ramets showed a significant increase and soluble carbohydrate and nitrogen content showed marginally significant decreases in the 1 mM jasmonic-acid treatment but not in control and/or 0.0001 mM jasmonic-acid treatments. The caterpillar G. rnenyuanensis preferentially grazed young ramets. After a localized spray of 1 mM jasmonic-acid, the leaf area of young ramets consumed by herbivores was greatly reduced. We propose that defense signals may be transmitted through physical connections (stolon or rhizome) among interconnected ramets of clonal plants. Induced resistance to herbivory may selectively enhance the protection of more vulnerable and valuable plant tissues and confer a significant benefit to clonal plants by a modular risk-spreading strategy, equalizing ontogenetic differences of unevenly-aged ramets in chemical defense compounds and nutritional properties of tissue. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: C. alrofusca G. rnenyuanensis Feeding preference Chemical defense compound Nutritional properties of tissue
1. Introduction Clonal plants produce new shoots at the nodes of horizontal stems (stolons or rhizomes). This type of clonal growth forms networks of physically interconnected, genetically identical, and functionally autonomous offspring individuals, called ramets. The internal transport of substances such as water, photo-assimilates and nutrients can be realized through physical connections among ramets. This is termed clonal integration or resource sharing. Clonal integration has been extensively investigated, and numerous studies have provided clear evidence of its ecological implications, such as allowing post-natal care for clonal offspring, acting as an efficient vehicle for resource extraction from heterogeneous environments, and provisioning internal support to damaged or stressed ramets (Pitelka and Ashmun, 1985; Alpert, 1991; Evans, 1992; Marshall and Price, 1997; Magori et al., 2003). * Corresponding author. Fax: þ86 28 85222753. E-mail address: [email protected] (Q. Liu). 1146-609X/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.actao.2011.04.002
It has been suggested that clonal plant networks may be used to share non-resource substances as well as water, carbohydrates and mineral nutrients (Cook,1978; Pitelka and Ashmun,1985). Evidence on the integration of the non-resource substances among interconnected ramets has recently been gathered from studies on a stoloniferous clonal plant Trifolium repens (Gómez and Stuefer, 2006; Gómez et al., 2007, 2008). These studies indicated that defense signaling could allow undamaged members of a clonal fragment to prepare themselves for impending herbivore attacks by up-regulating the expression of chemical and other defense traits. However, more studies are necessary to provide insight into the mechanisms, dynamics, and implications of clonal integration beyond resource sharing. Jasmonic-acid (JA) is found in many species and has been identified as an endogenous regulator of wound-induced chemistry in plants and a signal molecule in responses of plants to herbivory (Thaler et al., 1996; Karban and Baldwin, 1997; Thaler, 1999). When plants are attacked by herbivores, jasmonic-acid production increases in plant tissues (Creelman et al., 1992; Wasternack and Parthier, 1997), activating the natural defensive response of plants
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and thus enhancing resistance to attack (Cohen et al., 1993; Thaler et al., 1996). Treatment with jasmonic-acid may simulate the same defensive responses of plants to herbivory, leading to local and systemic induction of various classes of defensive compounds, such as alkaloids, phenolics, flavonoids and various terpenoids (Wasternack and Parthier, 1997). For example, Thaler et al. (1996), Cipollini and Redman (1999), and Arnold and Schultz (2002) showed that jasmonic-acid treatment induced the activity of the oxidative enzymes, peroxidase, and polyphenol oxidase, and condensed tannin contents, thus enhancing the resistance of these plants to herbivory. Furthermore, treating plants with jasmonicacid provides a convenient way to disassociate chemical defense and leaf loss, because jasmonic-acid causes the induction of several well documented defensive pathways without the removal of leaf tissue (Baldwin, 1998). Condensed tannins (CT) are secondary phenolic metabolites of plants and are involved in plantepathogen (Brownlee et al., 1990; Edwards, 1992; Heil et al., 2002) and planteherbivore (Bernays, 1981; Min et al., 2003; Forkner et al., 2004) interactions. They are widespread in nature, occurring in a range of herbaceous legumes (Jones et al., 1976; Terrill et al., 1992) and tree leaves (Kumar and Vaithiyanathan, 1990). Condensed tannins have been reported to play a significant role in the plant defense system against a wide variety of fungi, bacteria and insects (Feeny, 1968; Scalbert, 1991). The perennial rhizomatous clonal plant Carex alrofusca can produce condensed tannins, which have been shown to confer resistance to leaf-chewing insects such as the grassland caterpillar Gynaephora rnenyuanensis (Guo et al., 2004; Yan, 2006). Most studies on induced defenses have focused on the production of chemical defense compounds (Karban and Baldwin, 1997). However, animal grazing may alter the quality of hosteplant foliage, by reducing foliar nitrogen and carbohydrate concentrations. The nutritional properties of tissue may affect the feeding preferences and/or performance of herbivores so that they should be regarded in the context of inducible defense strategies (Jones and Roberts, 1991; Jensen, 1993; Redak and Capinera, 1994; Brathen et al., 2004; Gómez et al., 2008). A greenhouse experiment was set up using the rhizomatous clonal sedge C. alrofusca to examine the following two questions: (1) Does jasmonic-acid application induce defense signaling through physical connections among interconnected ramets? Because chemical defense compounds and the nutritional properties of tissue are regarded in the context of inducible defense strategies, we expect that localized spraying of jasmonic-acid may incur a significant change in condensed tannins, soluble carbohydrates, and nitrogen content in untreated ramets of the same clonal fragment. (2) Does defense signaling among interconnected ramets confer significant benefits to clonal plants? Defense signaling could allow untreated ramets of the same clonal fragment to improve their resistance for impending herbivore attacks by changing their chemical defense compounds and the nutritional properties of their tissue. Therefore, we expect that after localized spraying of jasmonic-acid, the proportion of consumed leaf areas in untreated ramets of the same clonal fragment may significantly decrease.
2. Materials and methods 2.1. Plant and herbivore C. alrofusca is a perennial, 20e30 cm tall rhizomatous herb distributed mainly in alpine meadows with altitudes ranging from
2600 to 5000 m a.s.l in western China (Liu and Lu, 1999). After expanding horizontally, the terminal bud of the rhizome grows upward to form the shoot, thereby forming extensive clonal fragments. In June 2006, six original clonal fragments, from sites at least 1000 m apart, were collected in Zoige County, Sichuan, China. Clonal fragments from the six sites are likely different genotypes due to the distance separating the collection sites. They were then vegetatively propagated in a greenhouse located in the Maoxian Mountain Ecosystem Research Station, Chinese Academy of Science (location 103 540 E, 31420 N, altitude1826 m a.s.l). The plants were watered as needed and fertilized with nutrient solution (0.083 mol l1 CO(NH2)2, 0.022 mol l1 KH2PO4) approximately every two weeks. G. rnenyuanensis is a grassland caterpillar with a broad range of hosts including Festuca ovina, Elymus nutans, and C. alrofusca in the alpine meadow of the TibeteQinghai plateau in China (Yan et al., 1995). In April 2007, first instar caterpillars were obtained from Qinghai University, Xining China. The caterpillars were reared using the methods described in Yan et al. (2005). 2.2. Experimental design In May 2007, three clonal fragments were collected from each of the six original genotypes and transported to a temperaturecontrolled greenhouse at Chengdu University of Technology, Chengdu, China. The greenhouse had a mean temperature of 23 5 C and natural light, supplemented with a sodium halide lamp as needed. These 18 clonal fragments were planted into individual wooden trays (100 20 20 cm) at least two weeks before the start of the induction treatment. The wooden trays were divided into sectors by a thin plastic wall. The rhizomes connecting ramets passed through notches in the wall that were caulked with a DAP acrylic sealant so that the interconnected ramets were grown in separate water-tight compartments within a single tray and all rhizome connections were maintained throughout the experiment. Each clonal fragment consisted of six successive ramets. The sixth and oldest ramet (as indicated by its relative proximity to the mother ramet) was subjected to one of the following three treatments: (1) control; (2) spray with a solution of 0.0001 mM jasmonic-acid; and (3) spray with a solution of 1 mM jasmonicacid. The jasmonic-acid dosage applied was based on a previous report (Dèlano-Frier et al., 2004) and on preliminary laboratory results. The fifth, third and first ramets were referred as old, middle-aged, and young ramets respectively. The control was placed between the two treatments to allow planteplant volatile signals (Fig. 1). Each of the six original plants and their descendants was subjected to all treatments. Given its low solubility in water, jasmonic-acid (Sigma Chemical Co.) was first dissolved in a small amount (about 5 ml) of ethanol, made up to two different concentrations of 0.0001 mM and 1 mM with distilled water. Triton-X 100 detergent (0.125% v/v) was added to facilitate penetration of leaf cuticles. Control solutions were identical but lacked jasmonic-acid. We sprayed 1 ml of control or jasmonic-acid solution evenly onto two fully unfolded leaves of the sixth ramet. Neighboring ramets were shielded from spray with a piece of plastic. Then, the target leaves of the sixth ramet were sealed in a transparent plastic bag until dry, approximately 40 min. Five days after the jasmonic-acid treatment, a feeding preference test in the untreated young, middle-aged and old ramets of the same clonal fragments was used to investigate the effects of defense signaling on consumption by the herbivore G. rnenyuanensis. Feeding preference tests were conducted among leaves of young, middle-aged and old ramets of induced and control plants, respectively (Fig. 1). The leaves of the untreated young, middle-aged and old ramets of the same clonal fragment were put
feeding preference test
Control/Jas monic-acid
6
5
4
3
2
1
Fig. 1. Schematic representation of the experimental design to test defense signaling through rhizomes among interconnected ramets of the clonal plant C. alrofusca. The clonal fragments consist of six successive ramets. The first ramet is the youngest and the sixth ramet is the oldest. The first, third and fifth ramets of same clonal fragment were referred to as young, middle-age and old ramets respectively. After the induction treatment on the sixth ramets, a feeding preference test with a fourth instar caterpillar G. rnenyuanensis was conducted on young, middle-aged, and old ramets.
into a petri dish and a fourth instar caterpillar was placed in the middle of the leaves, which is similar to the method described by Gómez et al. (2008). The caterpillar was allowed to feed for a fixed amount of time (24 h). Total leaf area and remaining leaf area were measured at the start and end of the experiment using a portable leaf area meter. The proportion of consumed leaf area was calculated as:
total leaf area remaining leaf area 100%: total leaf area Owing to small size of C. alrofusca leaves, a single clonal fragment was too small to use for all measurements. Therefore the experimental procedure was repeated four times; once for analysis of foliage chemical defense compounds, once for soluble carbohydrate content, once for nitrogen content and the fourth for the herbivory preference of G. rnenyuanensis. 2.3. Chemical analysis and measurement After jasmonic-acid treatment, leaves of old, middle-aged, and young ramets were harvested. We used about 2 g from each to investigate condensed tannin content by vanillineHCL assay (Wu and Guo, 2000) and another 1 g to measure soluble carbohydrates and nitrogen content using the anthrone reagent colorimetric method and the Kjeldahl nitrogen determination method (Dong, 1997). 2.4. Statistics analysis One-way repeated-measures ANOVA was used, where the repeated (within-subject) factor was ramet age and the fixed between-subject factor was jasmonic-acid induction. Means were
Condensed tannin content in the foliage (mg.g-1DM)
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357
8
a
a
6
a
a
a
a
a
b 4
b
2
0
Control
1mM
0.0001mM
Fig. 2. Condensed tannins content (SE) in the foliage after the induction treatment was conducted for 5 days. , , , represent young, middle-age and old ramets. The bars with the same lowercase letters are not significantly different at p ¼ 0.05.
compared with SeNeK multiple contrast. All statistical analyses were conducted with SPSS 10.0. 3. Results Condensed tannin content in the foliage was significantly affected by jasmonic-acid induction, ramet age and their interaction (Table 1). Young ramets contained significantly less condensed tannins than did middle-aged or old ramets in control and 0.0001 mM jasmonic-acid treatments (Fig. 2) but in the 1 mM jasmonic-acid treatment the foliage of young ramets contained significantly more condensed tannins than that of young ramets from either control or 0.0001 mM jasmonic-acid treatments (Fig. 2). Jasmonic-acid did not significantly affect soluble carbohydrate or nitrogen content in the foliage of young, middle-aged and old ramets (Table 1). Nonetheless there was a tendency for soluble carbohydrate content of in the foliage of young ramets to be greater in control and 0.0001 mM jasmonic-acid treatments than in all other combinations (0.05 p 0.06; Fig. 3). Ramet age, jasmonic-acid induction and their interaction had a significant effect on the amount of leaf area consumed by the caterpillar (Table 1). The results of the feeding preference test showed that G. rnenyuanensis larvae consistently preferred young ramets over middle-aged and old ramets of C. alrofusca in control and 0.0001 mM jasmonic-acid treatments (Fig. 5). On average, young ramets incurred almost 50% higher damage than middleaged and old ramets. However, in the 1 mM jasmonic-acid treatment this preference for young leaves was not evident and caterpillars consumed significantly less leaf material of young ramets than in control and 0.0001 mM jasmonic-acid treatments (Fig. 5). Jasmonic-acid induction had a very strong effect on the damage inflicted on young ramets.
Table 1 Repeated-measure ANOVA for effects of jasmonic-acid induction on condensed tannins, soluble carbohydrates and nitrogen contents in the foliage as well as the proportion of consumed leaf area among interconnected young-age, middle-age and old-age ramet. Source
Foliage condensed tannins content Foliage soluble carbohydrates content Foliage nitrogen content Ratio of consumed leaf area
Between-subject effects Jasmonic-acid induction Error
df
F
df
F
df
F
df
F
2 15
4.57*
2 15
2.86ns
2 15
3.01ns
2 15
3.96*
23.19** 3.76*
2 4 30
3.16ns 1.94ns
2 4 30
2.41ns 1.63ns
2 4 30
10.67** 5.43**
Within-subject effects Ramet age 2 Ramet age jasmonic-acid induction 4 Error 30 Significance level:
ns
P > 0.05 *P < 0.05 **P < 0.01.
J.-S. Chen et al. / Acta Oecologica 37 (2011) 355e360 14 12
a
4
a a a
a
a
a
a
a
10 8 6 4 2 0
Control
0.0001mM
Nitrogen content in the foliage (%)
Soluble carbohydrate content in the foliage (%)
358
a
a
a a
a
a
a
2
0
1mM
a
a
Control
0.0001mM
1mM
Fig. 3. Soluble carbohydrates content (SE) in the foliage after the induction treatment was conducted for 5 days. , , represent young, middle-age and old ramets. The bars with the same lowercase letters are not significantly different at p ¼ 0.05.
Fig. 4. Nitrogen content (SE) in the foliage after the induction treatment was conrepresent young, middle-age and old ramets. The bars with ducted for 5 days. , , the same lowercase letters are not significantly different at p ¼ 0.05.
4. Discussion
treatment in young ramets than in middle-aged or old ramets (Fig. 2). The amounts of soluble carbohydrates and nitrogen in the foliage of young ramets were marginally lower in 1 mM jasmonicacid treatment than in control and/or 0.0001 mM jasmonic-acid treatments (0.05 p 0.06; Figs. 3 and 4). Therefore, it is suggested that the effects of jasmonic-acid induction on chemical defense compounds and nutritional properties of the tissue in the clonal plant C. alrofusca may be dependent on the ontogenetic stage of the ramets. The optimal defense theory predicts that plant tissues with a high contribution to fitness and a high risk of attack should be better protected than other plant tissues. Young ramets are primarily responsible for future vegetative growth and reproductive success of clonal plant individuals. Therefore, young ramets of clonal plants are the most valuable tissues for growth and fitness and should hence be especially well protected (Mckey, 1974). C. alrofusca can share defense signals through belowground rhizome connections and its young ramets are more responsive than middle-age or old ramets, which selectively enhances the protection of more vulnerable and valuable plant tissues and confers a significant benefit to clonal plants by employing a modular risk-spreading strategy, equalizing ontogenetic ramet differences in chemical defense compounds and nutritional properties of tissue (Gómez et al., 2008). The experiment provides concrete evidence on specific mechanism of induced defense resistance against generalist herbivore in clonal plants. In alpine meadow, seedling recruitment is infrequent, and vegetative propagation is the prevalent form for reproduction in rhizomatous sedges C. alrofusca (Zhao et al., 2007). 50
Proportion of consumed leaf area (%)
Our study provides clear evidence that belowground rhizome connections among interconnected ramets can serve as pathways for the transmission of induced defense signals in physiologically integrated clonal plant species. Therefore, clonal integration may not only allow for an efficient transport of resources such as water, mineral nutrients and carbohydrates in the system, but can also operate as an information sharing system among members of clonal fragments. In our experiment, interconnected ramets were grown in separate water-tight compartments within a single tray and the plants assigned to control and induction treatments were spatially arranged without physical barriers between them, which ensured that the induction effect among interconnected ramets was not caused by volatile info-chemicals or root exudates (Gómez and Stuefer, 2006). Physiological features of young plants may be different from mature plants and they often invest a lot of energy in the development of biomass (Cipollini and Redman, 1999). Due to different chemical defense compounds and nutritional properties of tissue in the foliage among various ontogenetic stages, young ramets in the clonal plant C. alrofusca might be more palatable to herbivores and incurred more herbivory damage than middle-age or old ramets (Fig. 5). A similar effect was reported for T. repens, in which phenolic profiles and foliage quality measured as foliage carbohydrate and nitrogen contents were different between young and old ramets (Gómez et al., 2008). It is frequently observed that young plants or tissues are more responsive to elicitors of defense than older plants or tissues, although the inverse can also be true (Karban and Myers, 1989; Faeth, 1991). For example, the ability of wounding to induce proteinase inhibitor activity in tomato decreases with plant age (Wolfson and Murdock, 1990). The mRNA of polyphenol oxidase F is induced more strongly by wounding or jasmonic-acid in young leaves of tomato than in older leaves on the same plant (Thipyapong and Steffens, 1997). Cipollini and Redman (1999) investigated the quantitative effect of plant age on the induction of defense by jasmonic-acid, indicating that constitutive polyphenol oxidase activity did not change with age in tomato plants and was more strongly induced by jasmonic-acid in young plants. Furthermore, changes in plant foliage quality after defoliation have often been attributed to defoliation-induced increases in plant defensive compounds (Wratten et al., 1984; Tallamy and Raupp, 1991), generalized plant-stress responses to insect damage (Wagner and Evans, 1985; Myers, 1988), or both (Bryant et al., 1983; Tuomi et al., 1984). Chemical defense compounds in the foliage such as condensed tannins are more responsive to jasmonic-acid
a
a
40
b 30
b
b b
20
b
b
b
10
0
Control
0.0001mM
1mM
Fig. 5. Proportion of consumed leaf area (SE) in a feeding preference test with a fourth instar caterpillar G. rnenyuanensis after the induction treatment was conrepresent young, middle-age and old ramets. The bars with ducted for 5 days. , , the same lowercase letters are not significantly different at p ¼ 0.05.
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Defense signaling within clonal plant network may be very important for survival and growth of C. alrofusca in the harsh habitat when suffering from caterpillar G. rnenyuanensis herbivory. Clonal integration can confer clonal plant networks with considerable benefits by allowing for fast, specific and efficient defense signaling among interconnected ramets. The defense signaling may also lead to substantial costs if clonal plant network members become induced without being threatened by herbivores (Gómez and Stuefer, 2006). The ramets of C. alrofusca are connected by belowground rhizomes, which may reduce the probability of subsequent herbivore attacks (Stuefer et al., 2004). Thus, defense signaling can be costly. Alternatively, defense signaling through belowground connection (i.e. rhizome) may be beneficial if it minimizes eavesdropping by other plant competitors. A major caveat is that the 1 mM jasmonic-acid concentration may be not biologically relevant. To get more insight into the ecological and evolutionary implications of defense signaling in clonal plants, additional experiments using defense signaling induced by caterpillar herbivory will be needed. Acknowledgments This research was supported by National Science Fund of China (30870389) and by Knowledge Innovation Project of The Chinese Academy of Sciences (KZCX2-XB2-02). We would also like to thank Anne Bjorkman at the University of British Columbia for her assistance with English language and grammatical editing of the manuscript. References Alpert, P., 1991. Nitrogen sharing among ramets increases clonal growth in Fragaria chiloensis. Ecology 72, 69e80. Arnold, T.M., Schultz, J.C., 2002. Induced sink strength as a prerequisite for induced tannin biosynthesis in developing leaves of Populus. Oecologia 130, 585e593. Baldwin, I.T., 1998. Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences 95, 8113e8118. Bernays, E.A., 1981. Plant tannins and insect herbivores e an appraisal. Ecological Entomology 6, 353e360. Brathen, K.A., Agrell, J., Berteaux, D., Jonsdottir, I.S., 2004. Intraclonal variation in defence substances and palatability: a study on Carex and lemmings. Oikos 105, 461e470. Brownlee, H.E., McEuen, A.R., Hedger, J., Scott, I.M., 1990. Antifungal effects of cocoa tannin on the witches broom pathogen Crinipellis perniciosa. Physiological and Molecular Plant Pathology 36, 39e48. Bryant, J.P., Chapin, F.S., Klein, D.R., 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357e368. Cipollini, D.F., Redman, A.M., 1999. Age-dependent effects of jasmonic acid treatment and wind exposure on foliar oxidase activity and insect resistance in tomato. Journal of Chemical Ecology 25, 271e281. Cohen, Y., Gisi, U., Niderman, T., 1993. Local and systemic protection against Phytophthora infestans induced in potato and tomato plants by jasmonic acid and jasmonic methyl ester. Phytopathology 83, 1054e1062. Cook, R.E., 1978. Asexual reproduction: a further consideration. The American Naturalist 113, 769e772. Creelman, R.A., Tierney, M.L., Mullet, J.E., 1992. Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression. Proceedings of the National Academy of Sciences 89, 4938e4941. Dèlano-Frier, J.P., Martìnez-Gallardo, N.A., Martìnez-de la vega, O., SalasAraiza, M.D., et al., 2004. The effect of exogeneous jasmonic-acid on induced resistance and productivity in amaranth (Amaranthus hypochondriacus) is influenced by environmental conditions. Journal of Chemical Ecology 30 (5), 1001e1034. Dong, M., 1997. Observation and Analysis of Terrestrial Biocommunities. Standards Press of China, Beijing. Edwards, P.J., 1992. Resistance and defence: the role of secondary plant substances. In: Ayres, P.G. (Ed.), Pests and Pathogens: Plant Responses to Foliar Attack. BIOS Scientific Publishers, Oxford, pp. 69e84. Evans, J.P., 1992. The effect of local resource availability and clonal integration on ramet functional morphology in Hydrocotyle bonariensis. Oecologia 89, 265e276. Faeth, S.H., 1991. Variable induced responses: direct and indirect effects on oak herbivores. In: Tallamy, D.W., Raupp, M.J. (Eds.), Phytochemical Induction by Herbivores. John Wiley, New York, pp. 293e323,.
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