J Chem Ecol (2014) 40:21–30 DOI 10.1007/s10886-013-0374-0

Reserves Accumulated in Non-Photosynthetic Organs during the Previous Growing Season Drive Plant Defenses and Growth in Aspen in the Subsequent Growing Season Ahmed Najar & Simon M. Landhäusser & Justin G. A. Whitehill & Pierluigi Bonello & Nadir Erbilgin

Received: 10 June 2013 / Revised: 26 November 2013 / Accepted: 3 December 2013 / Published online: 24 December 2013 # Springer Science+Business Media New York 2013

Abstract Plants store non-structural carbohydrates (NSC), nitrogen (N), as well as other macro and micronutrients, in their stems and roots; the role of these stored reserves in plant growth and defense under herbivory pressure is poorly understood, particularly in trees. Trembling aspen (Populus tremuloides) seedlings with different NSC and N reserves accumulated during the previous growing season were generated in the greenhouse. Based on NSC and N contents, seedlings were assigned to one of three reserve statuses: Low N– Low NSC, High N–Medium NSC, or High N–High NSC. In the subsequent growing season, half of the seedlings in each reserve status was subjected to defoliation by forest tent caterpillar (Malacosoma disstria) while the other half was left untreated. Following defoliation, the effect of reserves was measured on foliar chemistry (N, NSC) and caterpillar performance (larval development). Due to their importance in herbivore feeding, we also quantified concentrations of phenolic glycoside compounds in foliage. Seedlings in Low N-Low NSC reserve status contained higher amounts of induced phenolic glycosides, grew little, and supported fewer caterpillars. In contrast, aspen seedlings in High N-Medium or High NSC reserve statuses contained lower amounts of induced phenolic glycosides, grew faster, and some of the caterpillars which fed on these seedlings developed up to their fourth instar. Furthermore, multiple regression analysis indicated that A. Najar : S. M. Landhäusser : N. Erbilgin (*) Department of Renewable Resources, University of Alberta, 442 Earth Sciences Building, Edmonton, AB, Canada e-mail: [email protected] J. G. A. Whitehill : P. Bonello Department of Plant Pathology, The Ohio State University, Columbus, OH, USA J. G. A. Whitehill Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

foliar phenolic glycoside concentration was related to reserve chemistry (NSC, N). Overall, these results demonstrate that reserves accumulated during the previous growing season can influence tree defense and growth in the subsequent growing season. Additionally, our study concluded that the NSC/N ratio of reserves in the previous growing season represents a better measure of resources available for use in defense and growth than the foliar NSC/N ratios. Keywords Aspen . Constitutive and induced defenses . Non-structural carbohydrates . Nitrogen . Phenolic glycosides

Introduction Trees deploy a combination of anatomical (e.g., polyphenolic parenchyma cells, or traumatic resin ducts) and biochemical (e.g., phenolic glycosides) defense mechanisms against herbivores and pathogens. These defenses can be constitutive or induced (Eyles et al. 2010; Franceschi et al. 2005). Constitutive defenses are always present in a tree to discourage attackers, while induced responses are triggered by tissue damage and can limit further injury from attacking organisms (Bonello et al. 2006; Eyles et al. 2010). However, the development of these defense responses is metabolically costly for plants and requires resources in the form of carbohydrates and nutrients (Frost et al. 2008). In woody plants, non-structural carbohydrates, such as starch and soluble sugars, as well as amino acids, other macro or micronutrients are stored in non-photosynthetic organs, such as branches, stems, and roots. Evidence emerging from studies that focus on functions of reserves in plant physiological processes, such as growth and defense, has suggested that reserves can be critical for plant survival when plants are under severe carbon stress resulting from limitations in photosynthesis (e.g., due to drought) or destruction of

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photosynthetic organs (e.g., by defoliation) (Dunn et al. 1990; Landhäusser and Lieffers 2012; McDowell et al. 2008; Sala et al. 2012; Sampedro et al. 2011). For instance, after repeated severe defoliations by herbivores, the basic functions of a plant and the renewal of the foliage are supported by the reserves accumulated in the non-photosynthetic organs (Babst et al. 2005, 2008; Donaldson et al. 2006). If these reserves are depleted or low before new photosynthetic organs are fully functional, plant growth and survivorship can be compromised even under resource-rich environments (Landhäusser and Lieffers 2002; Landhäusser et al. 2012a; Sprugel 2002; Stevens et al. 2008; Zhao et al. 2008). In light of these studies, it is critical to investigate the role of plant reserves in explaining plant growth and defense under herbivory pressure. Such investigations are particularly lacking in long-lived trees that have large storage organs (stems and roots), and are exposed to multiple and chronic biotic stresses throughout their lifetime (Goodsman et al. 2013; Landhäusser and Lieffers 2002, 2012; Sala et al. 2012). In this study, we tested whether the interaction between trembling aspen (Populus tremuloides Michx.) and its major insect defoliator, the forest tent caterpillar (Malacosoma disstria Hübner) (Lepidoptera: Lasiocampidae) is mediated by reserves accumulated in aspen’s non-photosynthetic organs during the previous growing season. Aspen is a prominent tree species in the boreal forests of North America, similarly to its sister species, Populus tremula L., the European aspen, which has similar ecological functions in Europe. Aspen has been a focal species in studies exploring patterns of variation in secondary metabolites and growth with respect to growing conditions and resource availability (Babst et al. 2005, 2008; Bryant et al. 1987; Donaldson et al. 2006; Donaldson and Lindroth 2007; Osier and Lindroth 2001). Likewise, studies in P. tremula have established relationships between plant secondary compounds and insect herbivores (Bernhardsson et al. 2013; Jansen et al. 2009). However, these studies generally focused on foliar chemistry and its effects on insect performance or investigated interactions between current sources and sinks, and largely did not account for reserves accumulated in non-photosynthetic organs during the previous growing seasons. More recently, Stevens et al. (2008) provided strong evidence that biomass accumulation in previous growing seasons can influence both herbivory and plant responses to herbivore damage, suggesting that biomass accumulation – and its content – are important features of subsequent plant-herbivore interactions. Therefore, the objective of this study was to investigate whether reserves such as nitrogen (N) or non-structural carbohydrates (NSC) accumulated in non-photosynthetic organs during the previous growing season can mediate chemistry of aspen foliage, particularly NSC, N, phenolic glycosides, and herbivore performance in the subsequent growing season.

J Chem Ecol (2014) 40:21–30

Methods and Materials Seedling Generation and Treatment Applications We generated aspen seedlings with different N and NSC reserve accumulations in their stems and roots (hereafter referred to as reserves) using the methods described in Landhäusser et al. (2012a) and Schott et al. (2013) (Table 1). Briefly, we used seeds collected near Edmonton (Alberta, CA) (53°32′N 113°30′W) and sowed them on 29-May-2009 into eight styroblocks (Beaverplastic, Alberta) with 66 cavities (cavity size: 5 cm dia×15 cm depth; 220 ml vol.). The planting substrate used was ten parts peat, two parts perlite, and one part clay particles. Greenhouse conditions were 18:6 h L:D photoperiod and 60 % relative humidity at 24 °C during the course of seedling growth. Germination of seeds occurred within 2 d and germinants were misted with water during the first 2 wk. On 14-June2009, a single fertilization took place using N-P-K (10-52-10) with chelated micronutrients (Plant Prod Co. ON, Canada) at 1 g L−1 concentration. We used fertilizers with high P concentration to facilitate early establishment of seedlings. From 28-June-2009 to 12-July-2009 we fertilized all aspen seedlings twice with a more balanced fertilizer, N-P-K (15-30-15) with chelated micronutrients at 1 g L−1concentration (Plant Prod Co.). In each of two fertilization treatments, a fertilizer (N-P-K: 10-52-10 or 15-30-15) was mixed with 6 L of water, and equally distributed among seedlings (66) in each styroblock. On 15-July-2009, we moved four of eight styroblocks of seedlings outside the greenhouse, while the remaining four stayed inside. Seedlings inside the greenhouse experienced lower light levels (40–50 % less) in addition to different Table 1 Various treatment combinations produced aspen seedlings with different nitrogen (N) and non-structural carbohydrate (NSC) reserves in their stems and roots Fertilization regimesa

Shoot growth inhibitor

Light levels

N and NSC status

High Low High Low High Low High Low

Applied Applied Absent Absent Applied Applied Absent Absent

Low Low Low Low High High High High

High N-Medium NSC Low N-Low NSC High N-Medium NSC Low N-Low NSC High N-High NSC Low N-Low NSC High N-High NSC Low N-Low NSC

a

Seedlings were grown either inside (low light) or outside (high light) the greenhouse and were subjected to either low or high fertilization treatments and either treated with shoot growth inhibitor or not. N=8 for of each of eight treatment combinations (location (2)×fertilization (2)×shoot growth inhibitor (2))

J Chem Ecol (2014) 40:21–30

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temperature, wind, and ambient moisture conditions than did seedlings outdoors. These additional factors were not evaluated in the current study. We assigned two of the inside and two of the outside styroblocks to either low (0.2 g L−1) or high (2 g L−1) fertilization (N-P-K: 15-30-15) treatments, applied at their respective concentrations once a week for 4 wk until August 10, as described above. One week after the fertilizer treatments started, we treated seedlings in one styroblock of each treatment (location×fertilization) combination once with a shoot growth inhibitor (active ingredient: Paclobutrazol; Bonzi®, Plant Growth Regulator, Syngenta Crop Protection Canada, Inc. ON, Canada). This treatment procedure is described in more detail by Landhäusser et al. (2012b). On 16-Aug-2009, we moved the seedlings inside the greenhouse to the outside to go through the natural hardening and dormancy process. When seedlings were dormant by midNovember, we took ten seedlings from each of eight treatment combinations (location (2)×fertilization (2)×shoot growth inhibitor (2)) (total 80 seedlings, N=10/treatment combination) to evaluate seedling characteristics as described below. The seedlings remaining in styroblocks were packed in plastic bags, put in wax-coated cardboard boxes, and stored at −4 °C until 12-Apr-2010. Chemical and Growth Characteristics of Dormant Seedling We measured dormant seedlings for shoot height, root collar diameter, root volume, total dry weight of root and shoot, and root-to-shoot ratio. Shoot height was determined by measuring the length of a shoot from the root collar to the tip of the terminal bud. Root volume was measured using the water displacement method (Harrington et al. 1994) after removal of planting medium from the roots. Following these measurements, the roots and shoots were oven dried at 70 °C for 72 h. Dry weight was recorded for each shoot and root system, and root-to-shoot ratios were calculated. We ground dried roots and shoots in a Wiley Mill (Thomas Scientific Wiley Laboratory Mill, NJ, U.S.A.). Plant material was sieved through 40 mesh (0.4 mm) and pooled to obtain a Table 2 Chemical (content) and growth characteristics of aspen seedlings used in the greenhouse study with different nitrogen (N) and non-structure carbohydrate (NSC) reserves

a

Means with the same letter within a row are not significantly different at α=0.05. N=32 for Low N-Low NSC, and 16 for each of the other two reserve statuses

Characteristics of aspen seedlings during dormancy

composite sample of non-photosynthetic tissues per plant. Using this pool, NSC was determined following the method described by Chow and Landhäusser (2004). Briefly, sugars were extracted three times from ground tissues using hot 85 % ethanol, and then analyzed colorimetrically using phenolsulphuric acid at 490 nm. Following sugar extraction, the starch in the remaining residue was digested with α-amylase (ICN 190151, from Bacillus lichenformis) and amyloglucosidase (Sigma A3514, from Aspergillus niger), and glucose equivalents were determined colorimetrically with peroxidase-glucose oxidase-o-dianisidine (Sigma Glucose Diagnostic Kit 510A). Non-structural carbohydrates were the sum of water soluble sugars and starch. We summed individual sugar and starch compounds but we used them jointly in our statistical analyses as sugars and starches can be transferred from one state to the other. Furthermore, from an herbivore perspective, sum of both would provide a better reflection of what herbivores are feeding on during defoliation. Nitrogen was determined using the Kjeldahl method (Bremner and Mulvaney 1982). Micro (Mn, Fe, Zn, Al, Cu, Pb) and macro (P, K, Ca, Mg, S) nutrients were measured using the microwave digestion / ICP OES method [EPA 3051, Instrumentation: Microwave=CEM MARS Express; ICP OES=Spectro Ciros, (Kalra and Maynard 1991)]. We grouped seedlings in terms of their similarity in N and NSC contents across the eight treatment combinations using linear discriminant analysis (LDA) (Table 2). LDA incorporated vertical and radial growth, including total dry weight of stem and roots, and chemical (NSC, N, micro and macronutrients) characteristics and converged the seedlings into three reserve statuses: Low N-Low NSC, High N-Medium NSC, or High N-High NSC (Fig. 1; Table 2). Seedlings in Low-N were characterized by low N, low NSC, high NSC/N ratio, low micro (Al) and macro (Ca, K, P) nutrients, slow growth, and had the smallest root volume, root collar diameter, and total dry weight. Seedlings in High-N were characterized by high N, low NSC/N ratio, high micro (Mn, Fe, Pb, Cu, Zn) and macro (Mg, S) nutrients, and faster growth. Seedlings in High-

Seedling reserve status (Mean±Standard Error)a Low N–Low NSC

High N–Medium NSC

High N–High NSC

N (mg) NSC (mg) NSC/N ratio Shoot height (cm) Root volume (cm3)

12.2±0.4c 640.7±21.2 c 53.0±1.3 a 25.0±1.2 c 3.9±0.4 b

38.1±2.4 b 1527.8±120.0 b 39.5±1.6 b 37.7±2.4 b 11.7±1.3 a

46.8±2.2 a 2018.4±150.0 a 43.5±2.6 b 57.3±2.1 a 9.5±1.0 a

Root collar diameter (mm) Total dry weight (g) Root:shoot ratio

3.5±0.1 b 1.9±0.1 b 2.6±0.2 a

5.6±0.2 a 5.5±0.5 a 2.7±0.3 a

5.3±0.2 a 5.6±0.4 a 1.1±0.2 b

F

P

253.77 91.26 17.08 65.35 22.38

Reserves accumulated in non-photosynthetic organs during the previous growing season drive plant defenses and growth in aspen in the subsequent growing season.

Plants store non-structural carbohydrates (NSC), nitrogen (N), as well as other macro and micronutrients, in their stems and roots; the role of these ...
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