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• Here, we examine the influence of source-to-sink carbohydrate (CHO) flow on the development of constitutive and inducible levels of phenylpropenoids in hybrid poplar (Populus nigra × P. deltoides) foliage to determine if secondary metabolic processes in plant modules can be inhibited in a predictable manner by events such as herbivory and the development of new leaves and reproductive structures, which alter the path of phloem-borne resources.
• Phenylpropenoid concentrations were determined for developing foliage after CHO flow, measured as the translocation of 13C from labeled sources was manipulated.
• Phenylpropenoid metabolism in both unwounded and induced sink leaves was directly and positively linked to rates of CHO import. Alterations in rates of translocation yielded different results, depending on how CHO import was affected: the removal of competing sinks rapidly and dramatically increased leaf phenolic contents, whereas phenolic levels (and their inducibility) tended to be reduced when import was interrupted.
• High and inducible sink strength in developing poplar leaves provides resources for phenolic biosynthesis and, as a result, restrictions or re-directions of CHOs affect the foliar quality. Sink strength and the vascular architecture of plants, which confer upon them a modular nature, can determine the direction and magnitude of defense responses in trees.
The ability of plants to respond to herbivores is constrained by the flow of signals and resources among modules as determined by vascular architecture, or phyllotaxy. For example, signaling molecules may travel via direct vascular connections to cue the systemic induction of proteinase inhibitors and related genes in orthostichous leaves of poplar, tomato, and tobacco (Davis et al., 1991; Shulaev et al., 1995; Orians et al., 2000; Schittko & Baldwin, 2003) and the distribution of defensive substances within individual leaves can be attributed to phyllotaxies (Rhodes et al., 1999; Orians et al., 2000). In addition, carbohydrates, translocated from source tissues to orthostichous sinks can provide needed resources for the production of chemical defenses in developing leaves (Kleiner et al., 1999; Arnold & Schultz, 2002). They are presumed to serve as a source of additional energy for wounded tissues (Truernit et al., 1996; Roitsch et al., 2003), and may, themselves, serve as signaling molecules (Rolland et al., 2002). Tuomi et al. (1988, Tuomi et al., 1991) argued that the flow of carbohydrates within a module, and not within plants as a whole, determines the production of carbon-based defenses in plant foliage. Taken together, these studies suggest that plants respond to herbivory on a modular, not whole-plant basis.
Haukioja and coworkers proposed that the modular nature of plants can explain why herbivory elicits responses from plants that seem erratic and often unpredictable (Haukioja, 1990; Honkanen et al., 1994). For example, they observed that mountain birch often develops induced resistance in response to caterpillar grazing, while induced susceptibility develops when buds and shoots are removed by moose. They suggested that this could be attributed to preferential herbivory of either source or sink tissues, which would, in turn, affect the resource flow within orthostichous modules (Haukioja, 1990; Tuomi et al., 1990; Honkanen et al., 1994). This hypothesis was tested experimentally in Scots pine, by removing a number of source and/or sink tissues from a single branch and, thus, manipulating the sink-source balance and the availability of carbohydrates to leaves in the coming season. Changes in the levels of reducing sugars and phenolic substances in new leaves were best explained by the changes in source–sink relations (Honkanen et al., 1994; Honkanen & Haukioja, 1994). From these results, Haukioja and coworkers proposed a model of plant defense, which suggests that these ‘induced defenses’ reflect the ability of meristematic tissues within a module to compete for resources that could in turn be allocated to growth or defense (Haukioja, 1990; Honkanen et al., 1994). In other words, ‘induced’ responses may be passive shifts in resource allocation, arising because herbivores remove source and sink units unequally.
Recently we observed that woody plants may also respond to herbivory by actively modifying sink-source relationships at a rapid pace. We demonstrated that grazing by gypsy moth caterpillars as well as treatment with jasmonic acid (JA) elicited enhanced sink strength in the developing leaves of hybrid poplar saplings (Arnold & Schultz, 2002). These leaves, including those that lost < 25% of their surface area to grazers, imported 3–4 times as much 13C-labeled carbohydrates from orthostichous source leaves compared with the untreated controls, and this 13C was used in the production of condensed tannins. This ‘induced sink strength’ co-occurred with elevated activities of cell wall invertase (CWI, EC 188.8.131.52), an enzyme that is ionically bound to plant cell walls and facilitates phloem unloading at sink tissues (Sturm, 1999).
Elevated CWI activities are familiar wound responses in herbaceous species such as tomato (Ohyama et al., 1998), carrot (Sturm & Chrispeels, 1990), Chenopodium rubrum (Ehnel & Roitsch, 1997), and pea (Zhang et al., 1996), among others. In herbs, CWI activities and gene expression are known to be affected by all classes of plant hormones, linked to the development of floral organs, associated with the expression of other defense-related genes (e.g. phenylalanine ammonia lyase), and to be involved in antimicrobial responses (Roitsch et al., 2003). Together, these studies strongly suggest that inducible CWI activities are components of defense responses, although we are unaware of any study that has directly tested the hypothetical link between sink strength and the ultimate production of chemical defenses in herbaceous plants.
We used hybrid poplar saplings as a model system in which to examine the effect of both increased and decreased CHO flow on constitutive and inducible levels of polyphenols in a woody plant, where translocation and the production of phenylpropenoids are of great importance. The results indicate that first constitutive polyphenol production and even so-called ‘local’ wound-responses often depend on long-distance carbohydrate transport; second any alteration in rates of translocation between source and sink modules is likely to affect the polyphenol concentrations of leaves; and third these responses can operate rapidly, within the timeframe of herbivore grazing.
Materials and Methods
The importance of carbohydrate import for the development of induced defense responses in Populus foliage was evaluated. In preliminary experiments, we identified the precise developmental stages during which induced responses (elevated CWI and polyphenol synthesis) were observed in leaves when elicitation was general throughout the canopy (all leaves elicited). In a larger experiment, we then disrupted carbohydrate flow to those leaves previously observed to be inducible and examined the impact of interrupting CHO flow on the ability of responding leaves to increase polyphenol production under these circumstances.
Hybrid poplar saplings (Populus nigra × P. deltoides; clone OP-367, Segal Ranch, WA, USA) were grown from cuttings as described by Arnold & Schultz (2002). Plant development was assessed by the plastochron index method. Specifically, each sapling leaf was assigned a leaf plastochron index (LPI) number, beginning with the most recently emerged ‘index’ leaf (≥ 3 cm), which was designated LPI0. This method allowed sapling leaves to be classified as either rapidly expanding sink leaves (LPI0– LPI4), leaves are making the sink-to_source transition (LPI5– LPI7), or fully expanded source leaves (≥ LPI8).
Preliminary experiments To identify the precise developmental stage at which ‘inducibility’ is lost we conducted a pair of preliminary experiments. In each experiment hybrid poplar saplings were treated with either 2 mm jasmonic acid (JA) or a control solution over their entire canopies every other day for 13 d and subsequently a single leaf (either LPI1, 3, 5, 7, or 9) was sampled from each individual. The presence or absence of induced tannin production and elevated activities of cell wall invertase were evaluated in replicated experiments. The results of these preliminary experiments were critical to the design of the main experiment.
Main experiment To test the hypothesis that source-to-sink carbohydrate flow is required for induced tannin biosynthesis, we disrupted the flow of sugars to sink leaves and measured changes in the strength of the induced response. To accomplish this, 250 hybrid poplar saplings were cultivated in a glasshouse at the Pennsylvania State University in April and May of 2002 (Arnold & Schultz, 2002). Supplemental lighting from 400 W sodium vapor lamps served to minimize fluctuations in daily irradiance levels and, hence plant development, by maintaining consistent average daily irradiances of 800–1200 µmol m−2 s−1 for 15 h d−1. The experiment began when the saplings were > 30 cm tall and had at least 29 leaves (i.e. were of LPI29).
We focused on two specific sink leaves that exhibited both induced tannin biosynthesis and induced CWI activities in the preliminary experiments. At the beginning of the experiment, the LPI1 and LPI2 sink leaves of each plant were banded and measured. These banded leaves developed rapidly during the course of the experiment so that by the conclusion of the 5-d experiment they were LPI3, 4, or 5 leaves, depending on the type of treatments they received. For clarity, these banded leaves are henceforth referred to as ‘A’ (apical) and ‘B’ (basal) sink leaves. The experiment examined the response of leaves during this critical phase and ended before banded leaves became mature and unresponsive. Each individual plant was randomly assigned to either the control group, or one of three treatment groups wherein the flow of carbohydrates to the banded sink leaves was to be altered.
Four treatments were established: first, in the first treatment group 46 saplings were subjected to steam girdling around the stems just below the banded sink leaves, effectively disrupting phloem-mediated import from potential source tissues below; second in the second group, 46 saplings were steam girdled at the soil line, in an attempt to affect import to induced sink leaves indirectly, by halting translocation between the above- and below-ground portions of the plants; third in the third treatment group, 30 saplings had all source leaves (LPI3 and below) removed at the petiole, thus eliminating source leaves, but not stems, roots, or the original cutting, as a potential source of carbohydrates; fourth finally, 46 plants were randomly allocated to the control group.
Steam girdling of stems was conducted using a clothes steamer (Proctor-Silex, Washington, NC, USA) with modified 8-mm nozzle. Brief, 3-second steam ‘bursts’ aimed at stems from four directions caused them to blacken within 24 h; however, stems maintained physical strength and plants remained healthy well beyond the duration of the experiment.
For each treatment or control group, half of the B leaves (at the LPI2 stage) were immediately treated with 5 mm JA as described previously by Arnold & Schultz (2002). A leaves were not directly treated. Jasmonic acid (Sigma Chemical, item no. J-2500, St Louis, MO, USA) was solubilized in 10% EtOH(aq) with 0.125% (w/v) Triton X-100 added to increase the penetration of JA through leaf cuticles. A high concentration of JA was used to ensure a clear response during the short window of time when the induced response could be detected, that is before A and B sink leaves would become unresponsive. A leaves from control plants were sprayed with the appropriate control solution. Treatments occurred once at noon and plants were allowed 48 h to respond.
The effectiveness of steam girdling was assessed by quantifying the translocation of 13C-labeled substrates between orthostichous source and sink leaves, as described previously (Arnold & Schultz, 2002). A subset of seven plants from each group were randomly selected for the 13C -tracer experiments (plants in the ‘source leaf – removal’ group were not labeled since they no longer possessed any source leaves that could be exposed to 13C). For each labeled sapling a single source leaf on each plant was exposed to 13CO2 rich air for 90 min and the mass of 13C imported by an orthostichous sink (leaf B) leaf after 24 h was measured. For labeling, sink leaves were enclosed in 500 ml chambers and the ambient CO2 was evacuated by purging the chamber with CO2-free air. A concentration of c. 370 ppm 13CO2 within each chamber was generated by reacting 38.8 mg NaH13CO2 with 200 µl lactic acid. The δ13C signatures of both source and sink leaves were determined as described in Arnold & Schultz (2002). The absence of 13CO2 leaks was confirmed by examining the δ13C signature of unlabeled plants, which were interspersed among the experimental saplings.
Methods common to all experiments
Invertase assay The activity of cell wall invertase (CWI) in select leaves was quantified colorimetrically as µmole sucrose cleaved per gram of tissue WM min−1 using the methods of Arnold & Schultz (2002).
Phenolic assays We focused on condensed tannins (proanthocyanins) as wound-induced defenses since their biosynthesis has been well characterized and is known to be elicited by wounding and JA (Richard et al., 2000) and because we had previously established that induced tannin production co-occurs with elevated sink strength in hybrid poplar foliage (Arnold & Schultz, 2002). Condensed tannin levels were measured as extracted proanthocyanins by the acid-butanol method (Hagerman & Butler, 1989). In addition, the concentrations of total reactive phenolics present in leaf extracts were determined using the Folin-Denis assay (Appel et al., 2001), which predominately measures phenolic glycoside levels in this clone of poplar (T. Schaeffer, pers. comm.). In both cases, standard curves were developed using standards purified from hybrid poplar (clone OP-367) foliage.
Induced tannin levels were observed in young, but not mature leaves (Fig. 1a). A two-way anova revealed a significant effect of both JA and leaf age. There was a significant interaction between these factors, indicating that the response to JA was dependent on the developmental status of individual leaves. LPI1 leaves exhibited the strongest induced tannin levels. LPI3 leaves also responded. However, leaves of LPI5 and older did not exhibit a detectable JA-induced increase in condensed tannins.
Leaf age also affected constitutive CWI activities, as well as the response of the enzyme to JA exposure (Fig. 1c). In untreated trees, mean CWI activities decreased with age from 4.3 to 2.0 µm sucrose cleaved g−1 tissue min−1. Enzyme activities were elevated in treated trees, but only in young leaves. Specifically, leaves of LPI1, 3, 5 exhibited JA-induced enzyme activities, whereas mature leaves (LPI7, 9) did not.
In both of the preliminary experiments, the growth rates of LPI1, 3, 5, 8 leaves decreased significantly in response to JA, whether those leaves exhibited other induced responses or not (Fig. 1b,c). Together, these results demonstrate that the induced responses are detectable in developing sink leaves (LPI1–5) of treated trees. We focused on these inducible sink leaves, and on the source tissues that supply carbohydrates to induced sinks, in the primary experiment.
13C translocation was disrupted LPI8 source leaves assimilated 13CO2 during the 1.5 h incubation period. The resulting leaf δ13C values ranged from +594 to +1250, compared with −28.16 for foliage on unlabeled plants. No leaks of 13CO2 were detected. Rates of 13C translocation, from labeled source leaves to orthostichous B leaves, were induced by JA treatment. Exposure to JA resulted in a threefold increase in 13C import within 24 h (Fig. 2). These results are consistent with the findings of Arnold & Schultz (2002) and indicate that local wound-responses can alter plant-wide patterns of carbohydrate translocation. However, induced sink strength was observed in saplings only where source–sink dynamics were not disrupted (Fig. 2). Steam girdling stems between source and sink effectively disrupted the flow of 13C-labeled substrates, in both JA-exposed and control trees. Saplings girdled at the soil line, however, retained the ability to transport carbohydrates among above ground tissues and, accordingly, we observed active 13C transport between source and sink leaves (Fig. 2).
Growth rates were reduced During the 5-d experiment the B leaves progressed from the LPI2 stage to the LPI3, 4, or 5 stage, depending on different rates of growth for leaves in each treatment group. Stem girdling (both types) and the removal of source leaves reduced the growth of focal sink leaves as well as the emergence of new leaves (Fig. 3). Sink leaf growth rates were reduced c. 20–30% and rates of new leaf emergence were reduced c. 15–68% in all three treatment groups, wherever CHO transport was disrupted. By contrast, there was no detectable effect of a single exposure to JA on the growth of focal sink leaves or on the emergence of new leaves on treated saplings during the 5-d experiment. Measurable differences in leaf growth rates usually require a longer period of time to emerge; for example, reduced growth was detected in the preliminary experiments after 13 d of repeated JA exposure (data not shown).
Source–sink balance affects constitutive phenolic levels Disruption of carbohydrate flow between above- and below-ground tissues affected the concentration of total phenolics and condensed tannins in sink leaves, whether saplings were exposed to JA or not. Both A and B sink leaves exhibited dramatically increased phenolic contents when saplings were girdled at the soil line (Figs 4 and 5). In these treatments, condensed tannin concentrations were increased threefold over all other treatment groups. The response could be clearly observed as a dramatic change in leaf coloration during the course of the experiment (Fig. 6). We presume that this treatment eliminated the flow of carbohydrates to roots, thus removing a competing sink and increasing the resources available for phenolic metabolism in sink leaves. Correspondingly, the elimination of contributing source tissues, by source leaf removal or stem girdling, resulted in lowered levels of total phenolics and condensed tannins in the sink leaves of some, but not all, treatment groups (Figs 4 and 5).
Source–sink balance affects induced responses In saplings where carbohydrate flow was not disrupted, JA exposure clearly resulted in induced phenolic concentrations in sink leaves A and B after 48 h (Fig. 4). JA-treatments affected the condensed tannin levels of A sink leaves (P = 0.018), but not of B leaves (P = 0.845) (Fig. 5). However, there was no induction of either total phenolic contents or condensed tannins where carbohydrate import was interrupted. In fact, some treatments resulted in JA-exposed sink leaves with lower phenol or tannin levels, on average, compared with controls (Figs 4 and 5). Induction did not occur in leaves that could not import resources from source tissues below.
Interestingly, the A leaves, located just above the treated B sink leaves seemed to exhibit stronger induced responses, on average when translocation below the B leaves was disrupted (Figs 4 and 5). For example, concentrations of total phenolic and/or tannins were inducible in the A sink leaves (but not the B leaves) of plants for which translocation was not disrupted and also in plants that were girdled at the soil line or had all source leaves removed by clipping. The only treatment that eliminated the response of the A leaves was girdling high on the stems, just below the focal sink leaves (Fig. 4). Apparently, the age of these leaves or presence of a single subtending leaf helps to maintain the induced response when source supply is disrupted.
The phenylpropenoid metabolism of young hybrid poplar foliage is linked to rates of CHO import. Both constitutive and inducible phenolic contents of the youngest leaves were affected by disruptions of translocation, including the removal of orthostichous sources, the elimination of competing sinks and the generation of ‘induced sink strength’. A consistent positive correlation between rates of CHO import and phenolic metabolism was evident. For example, when import to sink leaves was increased, phenolic contents also increased, and reductions in CHO flow tended to decrease phenolic levels and/or limit their inducibility. These responses were relatively rapid, occurring within a 5-d period, and could not be uncoupled in our experiments. The results support the view that induced sink strength serves to provide resources for defense-responses in woody plants like poplar (Arnold & Schultz, 2002), as has been proposed for herbaceous species (Truernit et al., 1996; Roitsch et al., 2003).
Constitutive polyphenol levels
In our study, manipulations of CHO flow affected sink leaf phenolic levels within 5 d. Dramatic increases of 300–400% were observed in sinks with greater access to CHOs (e.g. when the below ground tissues, presumed to be acting as competing sink organs, were disconnected; Figs 4 and 5) while decreases occurred in some cases where CHO flow to the sink leaves themselves was impeded. This is consistent with our previous finding, that the removal of source leaves reduced total phenolic concentrations of sink leaves by 16% within 24 h (Arnold & Schultz, 2002). Both types of change are consistent with the observation that imported CHOs are used for the synthesis of condensed tannins (Arnold & Schultz, 2002) and other phenolics such as phenolic glycosides (Kleiner et al., 1999).
Our findings offer some support for the argument that resource availability within plant modules may predict foliar polyphenolic concentrations, even though theories of plant defense founded on this principle generally do a poor job of predicting variations in levels of carbon-based compounds when applied to whole plants (Tuomi et al., 1984, 1990; Haukioja, 1990). We demonstrated covariation of carbon availability and leaf phenolic contents within an orthostichous module, like those observed in Pinus sylvestris (Honkanen et al., 1999), even though our environmental conditions remained unchanged. Other similarities include the presence of wound-induced phenolic contents and the development of ‘growth-defense tradeoffs’ between leaf growth rates and the production of phenolics that we observed for Populus saplings (Fig. 1, Arnold & Schultz, 2002). Further, both data sets show how the pattern of herbivory can be more decisive than its extent (Haukioja, 1990; Honkanen et al., 1999).
There is one notable difference, however, between our observations for Populus and those of Haukioja and coworkers for P. sylvestris. While Honkanen et al. (1999) observed that the production of phenolics was negatively correlated with sink strength, our data strongly indicate that leaf phenolics are positively correlated with sink strength and the rate of CHO import in Populus. This could indicate that phenolic metabolism is regulated differently in these species. There are fundamental differences in carbohydrate translocation between woody angiosperms and gymnosperms, for example, Karban & Baldwin (1997) remarked that the development of induced C-based defenses in evergreens may be affected by their tendency to store carbon in leaves, compared with deciduous species that hold reserves in roots and stems. In any case, there is solid evidence that, in angiosperms, the positive correlation between CHO import/phenolic metabolism is common. For example, molecular studies have shown that sink strength, as measured by CWI activity, is coinduced along with the expression of key enzymes of phenolic metabolism, including phenylalanine ammonia lyase (Roitsch et al., 2003). In addition, many resource availability theories developed from studies of deciduous tree species also predict a positive correlation between carbon resources and phenolics. As a result, we suggest that the sink/source model be amended so that, when angiosperms are considered, increased CHO flow to developing foliage is predicted to increase phenolic metabolism.
In hybrid poplar, increased phenolic levels in young leaves occur as leaves mature, in response to various JA treatments, in response to grazing by gypsy moth larvae, and after mechanical damage (Fig. 4; Arnold & Schultz, 2002, and unpublished data). Developing leaves of many plant species often exhibit the strongest herbivore-induced responses (Karban & Baldwin, 1997) and for hybrid poplar the ‘inducible’ stage lasts approx. 6–8 d in trees when the entire canopy has been treated. During this time leaves are progressing from a plastrochron index of LPI1 to LPI5, and are characterized by CWI activities and general sink strengths, which are higher and more plastic than in mature leaves of treated or untreated trees (Fig. 1). After this period, leaves are mature sources, which have lower CWI activities and lack detectable induced responses (Fig. 1, Arnold & Schultz, 2002) under our experimental conditions (whole-canopy elicitation). Treated, responding leaves may or may not exhibit reduced growth rates depending on the frequency of treatment and duration of the elicitation period.
The link between polyphenol production and CHO import was observed during wound-responses, as well as during normal development. When CHO import was halted by phloem girdling or source leaf removal, increased phenolic synthesis could not be elicited with JA. In fact, treatments that limited CHO flow often led to the opposite result, that is significantly reduced phenolic contents in JA-treated leaves. The apical (A) sink leaves generally possessed stronger responses than the basal (B) sink leaves. A sink leaves either benefited from having a single subtending B sink leaf below, which may have served as a source of resources or signals, or may have retained their ‘inducibility’ because of their younger developmental state and, presumably, greater sink strength (Fig. 1). Again, our data indicate that sink strength and the availability of orthostichous sources determine the supply of carbohydrates for defenses responses.
These and other recent findings blur the distinction between so-called local and systemic defense responses, suggesting that even ‘local’ wound responses in developing foliage depend, at least to some degree, on long-distance carbohydrate transport from source tissues.
A revised sink/source model may be helpful in accounting for specific patterns of defense responses in nature. The central prediction of this model, that CHO flow from source to sink tissues is positively correlated with the production of phenylpropenoids in those sinks, is consistent with previous observations concerning first the induction of CWI gene transcripts, CWI enzyme activities, and the emergence of induced sink strength, second the role of vascular architecture in determining within-canopy patterns of leaf quality (Orians & Jones, 2001), and third the development of phenolic-rich galls, which are strong sinks (Larson & Whitham, 1997). Sink/source dynamics may also account for changes in the defense levels of developing seedlings, such as oak, that alternate the flow of CHOs between active shoot and root growth during development, and it may help to explain why induced defenses are often reduced or lost in reproductive plants. Our results support the view of Roitsch et al. (2003) that CWI is a pathogenesis-related protein (or more generally, defense-related) and provide strong evidence for an intimate link between the local production of phenolics and the long-distance transport of CHOs throughout plants.
This work was supported by NSF grants DEB −9974067 and IBN −0114565 to J.C. Schultz and IBN −0336717 and OCE −0336716 to T.M. Arnold, as well as a grants from the Department of Biology at Dickinson College and the College of Charleston. We thank Carolina Velez for assistance in cultivating the trees and Brain Rehill and Toni Schaeffer for useful discussions.