ORIGINAL RESEARCH published: 06 August 2021 doi: 10.3389/fpls.2021.715399 Reserve Accumulation Is Prioritized Over Growth Following Single or Combined Injuries in Three Common North American Urban Tree Species Jorge Andres Ramirez 1,2* , Valentina Vitali 2,3 , Jordi Martínez-Vilalta 4 , I. Tanya Handa 2,5† and Christian Messier 2,5,6† 1 Facultad de Ciencias Agrarias, Universidad del Cauca, Popayán, Colombia, 2 Centre d’Étude de la Forêt (CEF), Université du Québec à Montréal, Montreal, QC, Canada, 3 WSL Research Unit Forest Dynamics, Research Group Ecosystem-Ecology Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland, 4 CREAF, Universitat Autònoma de Barcelona, Bellaterra, Spain, 5 Faculté des Sciences, Département des Sciences Biologiques, Université du Québec à Montréal, Montreal, QC, Canada, 6 Institut des Sciences de la Foret Tempérée, Université du Québec en Outaouais, Ripon, QC, Canada Trees that grow in urban areas are confronted with a wide variety of stresses that undermine their long-term survival. These include mechanical damage to the crown, root reduction and stem injury, all of which remove significant parts of plant tissues. Edited by: Penny J. Tricker, The single or combined effects of these stresses generate a complex array of growth University of Adelaide, Australia and ecophysiological responses that are hard to predict. Here we evaluated the Reviewed by: effects of different individual and combined damage on the dynamics of non-structural Gary Johnson, carbohydrates (NSC, low weight sugars plus starch) concentration and new tissue University of Minnesota Twin Cities, United States growth (diameter increment) in young trees. We hypothesized that (i) tissue damage Alessio Collalti, will induce larger reductions in diameter growth than in NSC concentrations and National Research Council (CNR), Italy (ii) combinations of stress treatments that minimally alter the “functional equilibrium” *Correspondence: Jorge Andres Ramirez (e.g., similar reductions of leaf and root area) would have the least impact on NSC j.ramirez@unicauca.edu.co concentrations (although not on growth) helping to maintain tree health and integrity. † These authors share senior To test these hypotheses, we set up a manipulative field experiment with 10-year- authorship old trees of common urban species (Celtis occidentalis, Fraxinus pennsylvanica, and Specialty section: Tilia cordata). These trees were treated with a complete array of mechanical damage This article was submitted to combinations at different levels of intensity (i.e., three levels of defoliation and root Plant Abiotic Stress, reduction, and two levels of stem damage). We found that tree growth declined in a section of the journal Frontiers in Plant Science relation to the total amount of stress inflicted on the trees, i.e., when the combined Received: 26 May 2021 highest level of stress was applied, but NSC concentrations were either not affected or, Accepted: 19 July 2021 in some cases, increased with an increasing level of stress. We did not find a consistent Published: 06 August 2021 response in concentration of reserves in relation to the combined stress treatments. Citation: Ramirez JA, Vitali V, Therefore, trees appear to reach a new “functional equilibrium” that allows them to Martínez-Vilalta J, Handa IT and adjust their levels of carbohydrate reserves, especially in stems and roots, to meet their Messier C (2021) Reserve metabolic demand under stressful situations. Our results provide a unique insight into Accumulation Is Prioritized Over Growth Following Single or Combined the carbon economy of trees facing multiple urban stress conditions in order to better Injuries in Three Common North predict long-term tree performance and vitality. American Urban Tree Species. Front. Plant Sci. 12:715399. Keywords: non-structural carbohydrates, storage, growth, stress, urban forest, Celtis occidentalis, Fraxinus doi: 10.3389/fpls.2021.715399 pennsylvanica, Tilia cordata Frontiers in Plant Science | www.frontiersin.org 1 August 2021 | Volume 12 | Article 715399 Ramirez et al. Reserve Accumulation Following Multiple Stresses INTRODUCTION long-distance transport between the carbon sources (either NSC in pools or carbohydrates synthesized by leaves) and carbon Trees are among the most valuable components of urban green sinks (mainly respiratory metabolism, storage of NSC, and areas due to the wide range of environmental, social, cultural, tissue growth) (Lacointe, 2000; Minchin and Lacointe, 2005). and economic benefits they provide (Konijnendijk et al., 2005). Therefore, damage that results in tissue loss will affect the carbon Nevertheless, urban trees are often facing both biotic and abiotic allocation priorities. This response will depend both on the damage, which affect their health and integrity from canopy functional role of the organs involved and timing of the damage, to root level. Such damage includes defoliation by insects and as different organs may function as carbon sources or carbon wind-breakage, stem damage due to frost or injuries leading to sinks at different times (Li et al., 2002). loss of woody tissues and transport capacity (Sieghardt et al., Concurrent damage can cause urban tree health to decline 2005), as well as root damage due to road and house repair and and, in extreme cases, lead to mortality (Calfapietra et al., 2015). construction (North et al., 2017). Gray infrastructure often limits However, the physiological response of trees to a combination the growing space of trees, and when combined with compacted of stress-factors is generally unclear (Niinemets and Valladares, soils, water and atmospheric pollution, further exacerbates the 2008; Niinemets, 2010). The interaction of several stress-factors stress conditions caused by various damage (Konijnendijk and generates a unique response that may be more severe (negative Randrup, 2004; Tubby and Webber, 2010). Additionally, other interaction) or less severe (positive interaction) than the sum of damage such as girdling, or ring-barking, often occurs from their individual effects (Mittler, 2006; Niinemets, 2010). Here, bicycles chained to street trees, lawn mowers, weed trimmers and we evaluated the effects of different individual and combined human vandalism (Moore, 2013; Purcell, 2014). stresses on the dynamics of NSC reserves in tree saplings using Tissue loss changes the growth (the annual change in standing a fully-factorial manipulative experiment with three common biomass accumulated) and allocation patterns between above- North American urban tree species (Fraxinus pennsylvanica, and below-ground tree components, affecting the functional Celtis occidentalis, and Tilia cordata). Trees were subjected to balance of the tree (Quentin et al., 2012; Wiley et al., 2017; Dong increasing levels of commonly occurring urban damage: (1) three et al., 2019). The reduction in photosynthetic tissue leads to a levels of defoliation, (2) three levels of root reduction, and (3) decrease in carbon resources produced, and in photosynthates two levels of stem damage. These stress treatments were applied available for growth and reserve accumulation (Handa et al., individually and in combinations of two or three simultaneously 2005; Wiley et al., 2013; Atkinson et al., 2014; Deslauriers et al., at different intensities. These disturbance treatments offered the 2015). Severe defoliation may even cause root mortality by potential to act in a complementary way modifying primarily decreasing metabolic activity (e.g., water and nutrient uptake) the carbon sources (defoliation), sinks (root reduction), and (Snyder and Williams, 2003). Root reduction causes a deficiency transport capacity (stem damage). Hence, each treatment created of water and nutrients reducing photosynthetic metabolism an imbalance between resource production and reserve demand (Dong et al., 2016). Additionally, root reduction removes and availability (Farrar and Jones, 2000). sink-structures and reduces storage capacity (Landhäusser and Since plants require a minimum concentration of NSC stored Lieffers, 2003). The removal of the bark and cambium through to maintain their basic functioning (McDowell, 2011; Sala et al., stem damage influences the transport capacity between sources 2012; Martínez-Vilalta et al., 2016), we hypothesized that (i) and sinks thus hindering the refilling or mobilization of reserves tissue damage will induce larger reductions in growth than in (Högberg et al., 2001; Moore, 2013; Purcell, 2014; Mei et al., NSC concentrations, and (ii) combinations of stress treatments 2015). However, stem damage typically allows respiration as that minimally alter the “functional equilibrium” (e.g., similar water transport is carried out through the xylem. reductions of leaf and root area) will have the least impact on NSC New growth to restore the functional balance between above- concentrations (although not on tree growth) helping to maintain and below-ground tissues after stress may depend on the amount tree health and integrity. of carbohydrate reserves in storage pools, as the remobilization of reserves support metabolic functioning during stressful periods and can produce compensatory growth or additional reserve MATERIALS AND METHODS investment facilitating recovery after the stress (Chapin et al., 1990; Dietze et al., 2014; Ramirez et al., 2018). Carbohydrate Study Site reserves are mainly comprised of non-structural carbohydrates The study was conducted in the municipal nursery of the (NSC) that are formed from low weight sugars and starch (Hoch city of Montreal, province of Quebec, Canada. The site lies et al., 2003). Sugars are mobilized easily and used for short-term at 45◦ 3000 N, 73◦ 330 W (about 35 m of elevation). The mean metabolism, while starch is stored in a more recalcitrant form for annual precipitation is 978 mm (215 mm snow and 763 mm long-term use during and after periods of severe stress (Chapin rain). The mean annual temperature is 6.2◦ C and the mean et al., 1990; Dietze et al., 2014; Martínez-Vilalta et al., 2016). annual growing season temperature is 14.4◦ C, lasting generally Photoassimilates are allocated to growth or to reserves and from May to October. other physiological functions such as defense (Chapin et al., 1990; Dietze et al., 2014; Hoch, 2015). Thus, the concentration Study Species of NSC in tree tissue depends on the ability of components We studied three tree species that are among the most commonly to use plant-available resources (sink strength), and on the planted trees in the city of Montreal: Celtis occidentalis Linnaeus Frontiers in Plant Science | www.frontiersin.org 2 August 2021 | Volume 12 | Article 715399 Ramirez et al. Reserve Accumulation Following Multiple Stresses (Common Hackberry; native), Fraxinus pennsylvanica Marsh. per treatment given the availability of individuals. Thus, three (Green ash; native), and Tilia cordata Mill. (little-leaf linden; blocks were established in the case of the T. cordata, four introduced in America from Europe). These species have blocks for F. pennsylvanica and six blocks for C. occidentalis growth strategies and growth rates considered as moderate, (Supplementary Figure 1). intermediate and rapid, respectively, which could determine The experiment was set up as a fully factorial design with different responses of reserve allocation and growth under three levels of defoliation (DF) [severe (75%), moderate (37%) stress (Table 1). and control (0%)], three levels of root reduction (RR) [severe (75%), moderate (37%) and control (0%)], two levels of stem Growth Measurements damage (SD) [severe (50%) and control (0%)], and all the possible The trees were field-grown seedling-propagated in 2003 and combinations among these three treatments and intensities 2004 in the Montreal municipal nursery. The average diameter (Figure 1). The defoliation (DF) treatment consisted of removing at 40 cm above the ground and height of the trees at the leaves manually at the base of the petiole, removing 37% of beginning of the study (July 2012) were 53.5 (7.8) mm and leaves per branch in the moderate treatment, and 75% in the 3.1 (0.6) m for C. occidentalis, 51.8 (8.9) mm and 4.4 (0.6) m severe treatment (Figure 1). The root reduction (RR) treatment for Fraxinus pennsylvanica, and 63.5 (4.7) mm and 4.3 (0.3) m consisted of removing part of the root system outside a 30 cm for Tilia cordata (standard deviation in brackets). The diameter radius from the trunk using a tree spade machine (Figure 1). The was measured with a Mitutoyo Digimatic caliper (0.01 mm machine, with independent blades that encircled the tree, cut the accuracy) at 40 cm above ground level to avoid actively growing roots to a depth of 1.2 m, and removed 37% in the moderate branches. To increase accuracy, diameter was measured along treatment and 75% in the severe treatment. The stem damage two perpendicular axes at a height marked with a permanent (SD) treatment consisted of removing a 40 mm wide band around steel nail. Tree height was measured with a TruPulse 360 laser 50% of the stem circumference at 30 cm above the ground using with a resolution of 10 cm for linear lengths (Laser Technology, a bark blaster girdling tool that removed both the cambium and Inc., CO, United States). Trunk diameter (at 40 cm height) phloem (Figure 1). and total tree height were measured periodically between July 2012 and May 2015. Diameter was measured approximately Analysis of Carbohydrate Concentrations every 2 months during the growing season while the height In the spring and autumn of 2014, we measured the was measured annually. To allow for comparisons between the concentrations of NSC (low weight sugars plus starch) in roots, different tree species, growth measurements were normalized. stems, and branches of all 267 tree saplings. These measurements took place the growing season after the stress treatments were Stress Treatments applied in July 2013 to monitor tree reaction in terms of growth At the beginning of the study, trees were assigned randomly to reallocation and reserve utilization. The concentrations of NSC one of 18 treatment combinations (see details below). Treatments in leaves were only measured in the summer of 2014. Stem were first applied in July 2012 and repeated in July 2013, samples were taken with a 4.3 mm diameter increment borer at which corresponded to the month of maximum leaf area. The 130 cm from the ground for each individual. Root samples were stress treatments consisted of various gradients of defoliation, taken with a 4.3 mm increment borer from large surface roots root reduction, and stem damage. The total number of trees within 10 cm of the base of the stem. Stem and root samples in the experiment was 267 individuals (116 C. occidentalis, were comprised of phloem and xylem from all growth rings 86 F. pennsylvanica, and 65 T. cordata). Individuals of each including the pith but not the outer bark. Top branches were species were divided in blocks ensuring at least three trees cut with a tree trimmer so that the twenty leaves per tree were sampled from the entire canopy. Collected samples were placed in paper bags and refrigerated right after collection. Within 8 h of TABLE 1 | Functional characteristics of the tree species studied. collection samples were microwaved in the lab to stop enzymatic activity (Popp et al., 1996), oven dried, and ground using a ball Species/Trait Celtis Fraxinus Tilia cordata mill. Samples were analyzed for NSC concentration following occidentalis pennsylvanica the standard methods proposed by Hoch et al. (2002). Ground Foliar carbon (%) 41.0 46.5 47.0 plant material was dissolved for 30 min in distilled water. Starch Foliar nitrogen (%) 1.2 2.0 2.7 was broken down into glucose, and sucrose into glucose and Foliar carbon/nitrogen 33.9 23.4 18.0 fructose with clarase (Aspergillus oryzae, Enzyme Solutions Pty Specific leaf area 17.3 15.2 18.6 Ltd., Croydon South, VIC, Australia) incubation at 40◦ C for (mm2 mg−1 ) 15 h. Phosphoglucose-isomerase was added to the solution. The Photosynthetic capacity 6.0 13.7 15.3 total amount of glucose, which corresponded to total NSC, was (µmol CO2 m−2 s−1 ) quantified photometrically in a microplate photometer at 340 nm Wood density (mg mm−3 ) 0.7 0.6 0.4 (Thermo Fisher Scientific, Waltham, MA, United States) after Growth rate Moderate Intermediate Rapid the conversion of glucose to gluconate-6-phosphate (hexokinase; Shade tolerance* Intermediate Intermediate Tolerant Sigma-Aldrich, St. Louis, MO, United States). Subsequently, an Lifespan Moderate Short Moderate aliquot of the original extract was treated with invertase and *Data from Niinemets and Valladares (2006). phosphoglucose-isomerase (both Sigma-Aldrich) to determine Frontiers in Plant Science | www.frontiersin.org 3 August 2021 | Volume 12 | Article 715399 Ramirez et al. Reserve Accumulation Following Multiple Stresses FIGURE 1 | Schematic representation of the stress treatments applied to trees of F. pennsylvanica, C. occidentalis, and T. cordata: defoliation, root reduction, and stem damage. the amount of glucose, fructose, and sucrose with a glucose severe stress had a significant effect on the normalized growth test (see above). Starch was calculated as NSC minus soluble (75% DF or 75% RR), except for C. occidentalis and T. cordata sugars (soluble sugars = sucrose + fructose + glucose). where there was already a significant effect at low root reduction Pure starch and glucose, fructose, and sucrose solutions were intensities. Stem damage only significantly affected the diameter used as standards. Plant powder from peach leaves (Leco, growth of F. pennsylvanica. Interactions seemed mostly non- St. Joseph, MI, United States) was included to control the significant. Only the interaction between defoliation and root replicability of the extractions. NSC concentrations are reported reduction (DF:RR) reduced diameter growth of C. occidentalis as percentage of dry matter. and the interaction between defoliation and stem damage (DF:SD) reduced height growth on F. pennsylvanica (Figure 2, Statistical Analysis Supplementary Figures 2, 3, and Supplementary Table 1). To avoid autocorrelation of errors produced by continuous measures of diameter and height, and reserve concentrations Effects of Single Stress Treatments on (soluble sugars, starch, and NSC), linear mixed-effect models NSC Concentrations were used to evaluate the effect of the stress treatments (DF, In spring 2014, the first measurements of NSC in woody RR, and SD) and their interactions on growth and reserve tissues showed concentrations between 0.4 and 12.0% and at the concentrations in the different tissues (leaves, roots, stems, and second assessment in fall 2014 concentrations varied between branches) per species (Gregoire, 1987). Blocks were used as 1.1 and 11.7%, depending on tissues, species, and treatments random effect (Supplementary Figure 1). Model calculations (Supplementary Figures 4, 5). Concentrations of NSC in leaves were performed using the R package “lme4” (Bates et al., 2014). measured in summer 2014 varied between 3.3 and 12.7%, with The function difflsmeans in the “lmerTest” package (Kuznetsova highest concentrations in the control trees of C. occidentalis and et al., 2016) was used as a post hoc test to determine differences lowest in F. pennsylvanica (Supplementary Figure 6). Reserve in the least square means among treatment combinations. The concentrations were in most cases not significantly affected by relationship between concentration of reserves and growth was single treatments in neither spring nor autumn measurements assessed through the coefficient of determination (Pearson’s r). (Figure 3 and Supplementary Table 2). When the effects were All statistical analyses were performed with the software R (R significant, high intensity treatments tended to significantly Foundation for Statistical Computing, Vienna, Austria). increase reserve concentrations in stems and roots in both periods, but reduced their concentration in branches (Figure 3). Single treatments of root reduction and stem damage significantly RESULTS decreased the NSC concentrations in leaves of C. occidentalis only (Figure 3 and Supplementary Table 2). Effects of Stress on Tree Growth No mortality was recorded during the study period. All Effects of Combined Stress Treatments treatments had a negative impact on both diameter and on NSC Concentrations height normalized growth for all species, except for stem The number of significant interactions between stress treatments damage in F. pennsylvanica that caused a significant increase and reserve concentrations was extremely low (Figure 5 and in diameter growth (Figure 2, Supplementary Figures 2, 3, Supplementary Table 2). In spring 2014, severe defoliation and Supplementary Table 1). In most of the stress cases, only and stem damage interactions were significant and increased Frontiers in Plant Science | www.frontiersin.org 4 August 2021 | Volume 12 | Article 715399 Ramirez et al. Reserve Accumulation Following Multiple Stresses FIGURE 2 | Single effects of a gradient of defoliation, root reduction, and stem damage on diameter and height growth of Celtis occidentalis, Fraxinus pennsylvanica, and Tilia cordata. Error bars represent the standard error of the mean. Different letters represent significant mean differences between stress levels. FIGURE 3 | Single effects of a gradient of defoliation, root reduction, and stem damage on non-structural carbohydrates (NSC) concentrations in tissues of Celtis occidentalis, Fraxinus pennsylvanica, and Tilia cordata in the spring, summer and fall of 2014. Error bars represent the standard error of the mean. Different letters represent significant mean differences between stress levels. Frontiers in Plant Science | www.frontiersin.org 5 August 2021 | Volume 12 | Article 715399 Ramirez et al. Reserve Accumulation Following Multiple Stresses NSC concentrations in C. occidentalis stems. In Fraxinus results are quantitatively similar to the growth reduction reported pennsylvanica, moderate defoliation (37%) along with an increase for defoliation and root reduction treatments in other studies in stem damage decreased NSC concentrations in roots of (Quentin et al., 2011; Jacquet et al., 2012; Wiley, 2013; Dong F. pennsylvanica relative to severe defoliation (75%) with stem et al., 2016). This reduction in growth can be explained by the damage. In fall 2014, severe root reduction and stem damage had lower carbon uptake due to defoliation, and lower water and a significant effect and reduced NSC concentrations in roots. The nutrient uptake due to root reduction. Interestingly, although interaction of root reduction and stem damage had a significant we expected stem damage to induce a decrease in tree growth negative effect on NSC concentrations in leaves in summer 2014 due to the disruption of the transport system and consequent in both C. occidentalis and F. pennsylvanica. For T. cordata no reduction of photosynthate movement to the roots (Regier et al., significant interactions were found. 2010; Mei et al., 2015), we instead found a significant increase in tree diameter for F. pennsylvanica. The stem damage applied Relationship Between NSC to the trees was relatively mild as it only affected 50% of the Concentrations and Tree Growth circumference. It has been reported that most coniferous species are able to resist up to 25% basal girdling (Filip et al., 2007) while Higher NSC concentrations were consistently associated with some broadleaf species are able to resist more than 50% girdling lower diameter increments for all three species (Figure 5). (Holmes, 1984; Moore, 2013) or even 100% girdling damage such Specifically, we found significant negative correlations between as in white poplar (Glass, 2011). The increase presented in tree diameter increment and NSC concentrations in roots and stems diameter of F. pennsylvanica may be due to an accumulation of of C. occidentalis, roots of F. pennsylvanica, and roots, and carbohydrates above the wound zone (Moore, 2013) in reaction stems and branches of T. cordata. On the contrary, we did not to the injury as shown in Vitali et al. (2019). find any significant correlation between height increment and Most of the single defoliation stress treatments did not NSC concentrations in any tissues of the three species for both lead to a significant reduction in the concentrations of NSC treatment periods (data not shown). in the different tree tissues (Figure 3). In most cases, root reduction caused an increase in the NSC concentrations in the stem as well as the roots. However, in C. occidentalis DISCUSSION there was a reduction in the NSC concentrations in branches, Assessing the impact of different levels of defoliation, root especially after root reduction. Reduction of carbohydrate reduction, and stem damage separately and in combinations, reserves in tree organs has been shown to occur in the weeks we observed a consistent decline in tree diameter and height following stress caused by defoliation (Palacio et al., 2012; growth with increasing stress intensity; the sole exception was the Wiley et al., 2013; Atkinson et al., 2014), and stem girdling, positive effect on diameter growth of F. pennsylvanica following especially in the tissues that mobilize reserves to maintain stem damage (Figure 2 and Supplementary Table 1). However, physiological activity (Mei et al., 2015). Nevertheless, a higher the impacts of different levels of defoliation, root reduction and concentration of carbohydrates in stems and roots for all stem damage on carbohydrate reserves were (1) less consistent, three tree species 9 months after the stress treatments in our (2) did not follow a simple linear trend with increasing stress study may indicate a fast recovery for tree species following intensities, (3) were relatively mild, and (4) changed with each injuries to below- and above-ground tree organs, probably organ, species and combination of treatments. We found some indicating a good resilience of these tree species to these significant interactions among the different stress treatments that types of injuries. make any interpretation of single effects difficult. Nonetheless, in The increase in carbohydrate reserve concentrations in stems cases where some effects were observed, single stress treatments and roots after carbon-limiting conditions may indicate that the induced an increase in reserve concentrations in stem and roots, stem and roots are secure places to store carbohydrates, and thus and a decrease in branches. On the contrary, combined stress they can maintain the availability of resources for resprouting or treatment effects on reserve concentrations were more variable re-foliation after stress or under possible further stress episodes both among species and the tree organs evaluated. (Gibon et al., 2009; Wiley, 2013). This carbohydrate increase in these tissues may have been reached through compensatory mechanisms, such as increasing nitrogen concentrations and Effects of Single Stress Treatments on photosynthetic rates in the remaining foliage after defoliation Tree Growth and Reserve (Pinkard and Beadle, 1998; Vanderklein and Reich, 1999; Concentrations Handa et al., 2005; Eyles et al., 2009). Nevertheless, although As hypothesized, both diameter and height growth were it is expected that an increase in nitrogen concentrations negatively affected by the increasing level of stress intensity and photosynthetic rates leads to an increase in carbohydrate inflicted on the trees (Figure 2 and Supplementary Table 1). At concentrations (Li et al., 2016), unlike woody tissues, only the end of the study, differences in growth between control and a few stress treatments had a significant effect on reserve the most affected trees were between 13 and 19% for diameter concentrations in leaves, and those that had a significant effect and between 9 and 16% for height (Supplementary Figure 2). caused a reduction in the carbohydrate reserves (Figure 4), Although the growth responses to stress treatments are complex which suggests no evidence of photosynthetic up-regulation and and depend on the species and type of stress applied, our compensatory responses. Frontiers in Plant Science | www.frontiersin.org 6 August 2021 | Volume 12 | Article 715399 Ramirez et al. Reserve Accumulation Following Multiple Stresses FIGURE 4 | Significant interacting effects of defoliation, root reduction, and stem damage on non-structural carbohydrates (NSC) concentrations in tissues of Celtis occidentalis, Fraxinus pennsylvanica, and Tilia cordata in the spring and fall of 2014. Error bars represent the standard error of the mean. Different letters represent significant mean differences between stress levels. Effects of Multiple Stress Treatments on concentrations in leaves of C. occidentalis and F. pennsylvanica Tree Growth and Reserve (Figure 5), decreasing NSC in C. occidentalis and increasing reserves in F. pennsylvanica. A decrease in reserve concentrations Concentrations after stress treatments in C. occidentalis may indicate the lack of We hypothesized that combinations of stress treatments that alter compensatory photosynthesis reactions to recover carbon supply the “functional equilibrium” the least (e.g., similar reductions (McNaughton, 1983). A reduction in the carbon supply through of leaf and root area) would have the least impact on NSC photosynthesis may lead to a fast depletion of the reserve pools, concentrations; however, we did not find a consistent response. thus increasing the stress effect on the trees (Niinemets, 2010). We expected a positive interaction in reserve concentrations It should be noted that treatments were applied in summer in treatments that involved a combination of defoliation and 2012 and 2013, while the NSC concentration measurements were root reduction compared to single stress treatments. Yet, none performed the next growing season in spring and autumn of of the interactions between DF and RR on reserves were 2014 for woody tissues and in summer for leaves. Sampled leaves significant (Figure 4 and Supplementary Table 2). We also were less than 1 year old, branches were generally from the expected that a combination of tissue loss (by either defoliation current year and stems and roots were up to several years old. or root reduction) and stem damage would lead to a negative Thus, stem and roots sampled combine pre-treatment, treatment interaction (reduction of reserves concentration) because stem and post-treatment tissues. Since NSC reserves stored in stems damage limits the supply of reserves either to leaves (from roots) and roots serve as a long-term reservoir for responding to high or to roots (from new photosynthates). We did find several impact damage (Clark and Clark, 1991; Poorter et al., 2010; significant interactions between tissue loss and stem damage Clarke et al., 2013) and stay stable over time (Hartmann and but with different patterns. We found that severe defoliation Trumbore, 2016), this may be a possible explanation for the (75%) with stem damage (50%) increased reserve concentrations lack of a consistent relationship between stress treatments and in roots and stems of F. pennsylvanica and C. occidentalis, NSC concentrations in woody tissues. Also, since many models respectively (Figure 4). As suggested above, this may indicate were fitted simultaneously, some of the interactive effects may be the mobilization of reserves to more secure tissues under severe spurious due to multiple testing. stress conditions (Gibon et al., 2009; Wiley et al., 2013). However, moderate defoliation (37%) with stem damage (50%) reduced reserve concentrations in roots in F. pennsylvanica. Severe root Relationship Between Carbohydrate reduction (75%) and stem damage (50%) also caused a reduction Reserves and Tree Growth in reserve concentrations in roots of C. occidentalis, which may We found a negative relationship between NSC concentrations indicate that stem damage is limiting the supply of reserves from and diameter increment suggesting an overall conservative leaves to roots and thus, roots are spending their reserves in strategy to cope with maintenance respiration, tissue metabolism and/or increasing root production to compensate reconstruction or new tissue production following some for root loss and exploit new available soil nutrients and water form of stress (Körner, 2003, 2015; Muller et al., 2011; Figure 5). resources (Mei et al., 2015). Moderate root reduction (37%) This relationship was significant in the three woody tissues of and stem damage (50%) caused a contrasting effect in reserve T. cordata and persistent in roots of all three tree species. This Frontiers in Plant Science | www.frontiersin.org 7 August 2021 | Volume 12 | Article 715399 Ramirez et al. Reserve Accumulation Following Multiple Stresses FIGURE 5 | Relationships between non-structural carbohydrates concentrations in spring 2014 and diameter increment in Celtis occidentalis, Fraxinus pennsylvanica, and Tilia cordata. Model lines represent significant relationships between non-structural carbohydrates (NSC) concentrations and diameter increment. may indicate that trees of T. cordata showed higher response to (Sala et al., 2012; Wiley and Helliker, 2012). This behavior may stress than the other two species. Although T. cordata is a shade be even more marked at increased age (with higher biomass tolerant species, it presents characteristics of fast-growing species accumulation) and thus, the effect of age should be considered such as high foliar nitrogen, photosynthetic capacity, and lower for future analyses since urban forests are mainly composed wood density (Table 1). This may indicate a lower allocation by mature trees. of carbon to defense traits and thus a higher dependence on reserves than the other species to maintain a positive carbon balance. These results support the idea that under stress CONCLUSION conditions, fast-growing species respond with higher flexibility than slow-growing species (Atkinson et al., 2014). This study examined the single and combined effects of three Our results suggest that following a disturbance to some part frequent urban stresses (defoliation, root reduction, and stem of the tree, trees may mobilize accumulated stored NSC over the damage) on the growth and NSC reserve concentrations in short term to repair the damage and increase growth, but over young trees of three common species under field conditions. the medium to long term the strategy seems to replenish the Our results showed a consistent inverse relationship between reserve pool as quickly as possible to the detriment of tree growth. diameter growth and total NSC reserve in all three tree species, Increasing reserves under the conditions of lower carbon uptake indicating that trees prioritize reserve accumulation over growth imposed by the stress treatments is consistent with previous following injuries. Globally, trees tended to accumulate NSC in studies suggesting that allocation of carbon to reserves is an active roots and stems rather than branches 9–12 months following process that does not depend on the balance between carbon various combinations of stresses. Significant interactions were supply and demand for growth and metabolism; trees regulate found between the three stresses applied, indicating that some the levels of reserves at the expense of growth (Chapin et al., combinations of stresses while not showing simple additive 1990; Silpi et al., 2007; Sala et al., 2012; Wiley and Helliker, 2012). effects, could modify tree responses. Thus, moderate and mild Such behavior in carbohydrate reserves suggests that trees adjust stress caused by defoliation, root reduction and girdling events do their level of reserves to meet the new metabolic demands (Silpi not generally adversely affect plant physiology in terms of growth et al., 2007) because survival under stress conditions may require and reserves. However, trees under severe stress, especially under a higher availability of carbon for maintaining physiological conditions that alter the functional equilibrium, may drastically functions, such as metabolism, hydraulic integrity and osmotic reduce their growth rates and, depending on the severity, could exchange of the soluble sugars, instead of maintaining growth eventually die with large amounts of NSC stored in their tissues. Frontiers in Plant Science | www.frontiersin.org 8 August 2021 | Volume 12 | Article 715399 Ramirez et al. Reserve Accumulation Following Multiple Stresses DATA AVAILABILITY STATEMENT Foundation for Innovation infrastructure grant and by a scholarship from the Quebec research funds for nature The raw data supporting the conclusions of this article will be and technology. made available by the authors, without undue reservation. ACKNOWLEDGMENTS AUTHOR CONTRIBUTIONS We would like to thank David Andres Herrera, JR, IH, and CM contributed to the design of the research. JR Sergio Martinez, and Mathieu Messier for their carried out the fieldwork. JR performed laboratory analysis with fieldwork support. We would also like to thank Martin the support CM and IH. JR, VV, and JM-V wrote the manuscript Gaudet from the Montreal municipal nursery for in collaboration with all co-authors. All authors contributed to logistical support. the article and approved the submitted version. SUPPLEMENTARY MATERIAL FUNDING The Supplementary Material for this article can be found This research was supported by the NSERC/Hydro- online at: https://www.frontiersin.org/articles/10.3389/fpls.2021. Quebec research chair on tree growth control, a Canadian 715399/full#supplementary-material REFERENCES Gibon, Y., Pyl, E., Sulpice, R., Lunn, J. E., Höhne, M., Günther, M., et al. (2009). Adjustment of growth, starch turnover, protein content and central metabolism Atkinson, R. R. L., Burrell, M. M., Rose, K. E., Osborne, C. P., and Rees, M. (2014). to a decrease of the carbon supply when Arabidopsis is grown in very short The dynamics of recovery and growth: how defoliation affects stored resources. photoperiods. Plant Cell Environ. 32, 859–874. doi: 10.1111/j.1365-3040.2009. Proc. R. Soc. B 281:20133355. doi: 10.1098/rspb.2013.3355 01965.x Bates, D., Maechler, M., Bolker, B., and Walker, S. (2014). Lme4: Linear Mixed- Glass, W. (2011). Vegetation Management Guideline. 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Sample preservation for determination of organic compounds: open-access article distributed under the terms of the Creative Commons Attribution microwave versus freeze-drying. J. Exp. Bot. 47, 1469–1473. doi: 10.1093/jxb/ License (CC BY). The use, distribution or reproduction in other forums is permitted, 47.10.1469 provided the original author(s) and the copyright owner(s) are credited and that the Purcell, L. (2014). Mechanical Damage to Trees: Mowing and Maintenance original publication in this journal is cited, in accordance with accepted academic Equipment (FNR-492-W). West Lafayette, IN: Purdue University, Forestry and practice. No use, distribution or reproduction is permitted which does not comply Natural Resources. with these terms. Frontiers in Plant Science | www.frontiersin.org 10 August 2021 | Volume 12 | Article 715399