ORIGINAL RESEARCH published: 04 June 2019 doi: 10.3389/fpls.2019.00701 Plasticity of Fine-Root Traits Under Long-Term Irrigation of a Water-Limited Scots Pine Forest Ivano Brunner 1* , Claude Herzog 1 , Lucía Galiano 2,3,4 and Arthur Gessler 2 1 Forest Soils and Biogeochemistry, Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland, 2 Forest Dynamics, Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland, 3 Centre for Research on Ecology and Forestry Applications, Barcelona, Spain, 4 Department of Animal, Plant Biology and Ecology, Universitat Autònoma de Barcelona, Barcelona, Spain Trait-based approaches are increasingly used to investigate plant strategies for resource acquisition, growth, or competition between individual organisms or across species. However, the characterization of responses to environmental stimuli by fine-root systems of trees at the trait level is rather limited, particularly regarding the timing and degree of plasticity of the traits involved. These aspects become especially relevant under current Edited by: climate-driven shifts in environmental conditions. In the present study, we examined Boris Rewald, University of Natural Resources the responses of the fine roots of Scots pines to increased soil water availability from and Life Sciences, Vienna, Austria long-term irrigation starting in the year 2003. The Scots pine forest is situated in a Reviewed by: water-limited region in the central European Alps where increased tree mortality has Louise Comas, United States Department been observed over the last two decades. The fine-root traits investigated include root of Agriculture, United States system traits, root dynamic traits, architectural traits, and morphological traits. A first Jorge E. Mayer, survey of fine-root traits in 2005 using ingrowth cores did not reveal any trait-based Ag RD&IP Consult P/L, Australia responses resulting from the irrigation treatment over a three-year period. Fine-root *Correspondence: Ivano Brunner biomass, as periodically recorded by coring the topsoil from 2003 to 2016, showed ivano.brunner@wsl.ch a significant increase compared to the non-irrigated controls between three and nine years after the start of treatment. Overall, a maximum biomass increase due to the Specialty section: This article was submitted to irrigation treatment was recorded in 2016 with about 80% higher biomass compared to Functional Plant Ecology, controls. The analysis of fine-root traits revealed that irrigation significantly increased a section of the journal Frontiers in Plant Science biomass, length, and production, but did not alter morphological and architectural Received: 31 January 2019 traits, such as diameter, frequency of tips, specific root length (SRL), and root tissue Accepted: 13 May 2019 density (RTD). In contrast, clear significant differences were found for all traits except Published: 04 June 2019 for length when comparing the two root sampling methods, namely, ingrowth cores Citation: and soil coring. However, there were no interactions between the irrigation treatment Brunner I, Herzog C, Galiano L and Gessler A (2019) Plasticity and the sampling methods used and, therefore, the methods used did not affect the of Fine-Root Traits Under Long-Term documented patterns, just the actual measured trait values. Irrigation of a Water-Limited Scots Pine Forest. Front. Plant Sci. 10:701. Keywords: architectural traits, biomass, drought, dynamic traits, ingrowth cores, morphological traits, doi: 10.3389/fpls.2019.00701 production, soil coring Frontiers in Plant Science | www.frontiersin.org 1 June 2019 | Volume 10 | Article 701 Brunner et al. Plasticity of Fine-Root Traits INTRODUCTION and Mediterranean climates are characteristic landscape elements in dry and warm inner Alpine valleys in the Central Alps (Rigling Trait-based approaches are increasingly being used to et al., 2013). Increasing Scots pine mortality has been recorded investigate plant strategies for resource acquisition, growth, for several decades, with a dieback of up to 50% in particularly and competition, as well as for plant impacts on ecosystem water-limited stands in the Swiss Rhone Valley since 1995, as well processes (McCormack et al., 2017). However, most efforts have as in other valleys in the central Alps in Italy and Austria (Rebetez thus far focused on aboveground plant traits, while belowground and Dobbertin, 2004; Vacchiano et al., 2012). High mortality of traits and strategies still remain under-researched. To fill Scots pine on long-term monitoring sites in the Swiss Rhone this gap, a Fine-Root Ecology Database (FRED1 ) was created valley, with up to 20% of the trees in a stand dying in a given exclusively for fine-root traits and which now includes over year, was observed in 1999, 2004, and 2017. At the Pfynwald 70,000 observations (Iversen et al., 2017). site, cumulated mortality between 2003 (when the irrigation Ideally, the range of a trait’s response to environmental stimuli treatment started) and 2016 was about 16% in the control plots or plant-intrinsic factors should be known. Regrettably, this and about 9% in the irrigated plots. Although average annual knowledge is still limited for belowground traits (Shipley et al., precipitation has remained constant in recent decades, there is 2016). Part of the problem may stem from the inconsistency and evidence that climate warming has increased evaporation rates, inaccuracy of belowground measurements (Freschet et al., 2015; and that water has become the main factor limiting growth and McCormack et al., 2017). reducing stress resilience in trees (Rigling et al., 2013). It was The variability of a trait in response to environmental changes, therefore hypothesized that reducing stress from water limitation either across environmental gradients or in experimental using irrigation could improve tree vitality and reduce mortality. treatments, is called “phenotypic plasticity” (Callaway et al., Indeed, after 3–9 years of irrigation, trees showed increased leaf 2003; Iversen et al., 2017). The capacity of organisms to alter area and increased fine-root biomass (Dobbertin et al., 2010; their phenotype under changing conditions is widely recognized Herzog et al., 2014). Furthermore, irrigation was found to cause as an important mechanism to avoid migration or extinction significant shifts in plant community composition and increased (Valladares et al., 2014). The term phenotypic plasticity is vegetation cover (Herzog et al., 2014). In response to the doubling currently applied in a broad sense, and is used to describe the of precipitation over the decade-long experimental period, the phenotypic responses of organisms to environmental change, monthly mean volumetric water content in the top soil increased e.g., acclimation (equivalent to acclimatization) (Kelly et al., significantly, from 28% in the controls to 34% in the irrigated 2012). One aboveground example is the particular plasticity of plots (Hartmann et al., 2017). the leaves of tropical trees, which grow differently depending In the present study, our research question was to determine on light conditions. In the light, leaves are thicker, whereas whether the irrigation treatment (i.e., dry controls vs. irrigation) in the shade they tend to be thinner (Rozendaal et al., 2006). would affect the recorded fine-root traits. The second research Weemstra et al. (2017) and Zadworny et al. (2017) have recently question was to determine whether the method of recording shown that phenotypic plasticity occurs in tree roots, where roots (soil cores vs. ingrowth cores) under the irrigation physical and chemical soil parameters are the influencing factors treatment would influence the traits. It was the aim of the instead of light. study to investigate the fine-root traits of several trait categories Plasticity is understood as an adaptive mechanism that allows (McCormack et al., 2017), such as “root system,” “morphology,” plants to optimally respond to environmental heterogeneity “architecture,” and “root dynamics.” (Palacio-Lopez et al., 2015). The phenotypic shift can either be the result of a genetic differentiation of populations to become locally adapted, or of the phenotypic plasticity of individuals expressing MATERIALS AND METHODS the optimal phenotype for the corresponding environment via physiological and molecular regulatory mechanism (Palacio- Study Site and Experimental Setup Lopez et al., 2015). However, the ability of an organism to express The study site is located in the Pfynwald forest situated in the plasticity within a given trait is most likely mediated at the Rhone Valley in Switzerland (46◦ 180 N, 07◦ 370 E, 615 m.a.s.l.), molecular level (Nicotra et al., 2010). in a Scots pine forest containing trees of about 100 years of age, Here, we examine the response of Scots pine (Pinus sylvestris a stand density of about 730 stems ha−1 , and with occasional L.) fine-root traits to precipitation changes using a unique interspersed pubescent oak (Quercus pubescens Willd) (Brunner long-term irrigation experiment in a water-limited pine forest et al., 2009). The mean yearly precipitation of the nearby climate ecosystem in the central European Alps (Dobbertin et al., 2010; station Sion is 603 mm, with a mean annual temperature 10.2◦ C Hartmann et al., 2017). The irrigation experiment was established (1980–2010; MeteoSchweiz, 2018). Over the last three decades, in the Pfyn forest in 2003 to better understand how forest precipitation has dropped toward 90% of the long-term average ecosystems respond to water limitation (Dobbertin et al., 2010). and the temperature has risen by more than 1◦ C (1980–2010; The Pfyn forest represents the largest continuous forest of Scots MeteoSchweiz, 2018). For the long-term irrigation treatment, a pine in Switzerland, and is located in the dry Rhone Valley. Large- 1.23-ha study area containing about 1,100 tree individuals, was scale Scots pine forests in the transition zone between continental subdivided into eight plots of 25 × 40 m (1,000 m2 ) with 5 m buffer areas between and around each plot. The plots were aligned 1 http://roots.ornl.gov side by side along a channel fed by the Rhone River, from where Frontiers in Plant Science | www.frontiersin.org 2 June 2019 | Volume 10 | Article 701 Brunner et al. Plasticity of Fine-Root Traits water was taken to irrigate four randomly selected plots (hereafter A second series of ingrowth cores, using identical ingrowth referred to as “irrigated”). Four plots were left untreated as cores as before, was installed in April 2014 and harvested in control plots (hereafter “dry”). Irrigation started for the first time spring 2016. The ingrowth cores were installed in the same way in spring 2003. The irrigation system was activated on rainless as the first series, at a distance of about 0.5–1 m from the stems. nights during the vegetation period (May-October), doubling However, trees other than those from the first series were probed, the annual rainfall amount. Volumetric soil water content was and three ingrowth cores per tree instead of two were installed. In monitored hourly using time-domain reflectometry (Tektronix total, six trees were selected from the dry treatment and six trees 1502B cable tester, Beaverton, OR) at soil depth of 10 cm at from the irrigated treatment. four different locations in irrigated and dry plots. The mean In order to compare the datasets of the two series of ingrowth volumetric water content in the soil significantly increased from cores, the data were averaged per tree. If one of the ingrowth 28% in the dry plots to 34% in the irrigated plot (Herzog et al., cores per sample tree remained without roots, then only those 2014). In 2013, irrigation was stopped in about a third of the with roots were taken into consideration. area of each of the irrigated plots to track the return to a ‘dry’ forest. More details on the study site and experimental setup Fine-Root Scanning and Analyses are described in Brunner et al. (2009), Herzog et al. (2014), and The fine roots from the ingrowth campaign of 2005 and 2016, Hartmann et al. (2017), and data on long-term growth as well as and from the soil-coring sampling in 2016 were scanned for on relative leaf areas of trees from the irrigated and control plots morphological characteristics before drying and weighing. The are given by Schönbeck et al. (2018). scanned pictures were then analyzed using the WinRHIZO software package (version 4.1c, Regent Instruments Inc., Quebec, Soil Cores Canada) for morphological and architectural traits such as length, Fine roots (<2 mm in diameter) were sampled from healthy diameter, root volume, tips, and forks. sample trees with a soil-coring cylinder (diameter 4.5 cm) about 0.5–1 m away from the stems and down to the rocks of the subsoil to a depth of 8–12 cm. Two soil cores per tree were taken, and Fine-Root Traits three trees per plot were sampled before irrigation treatment The measured fine-root traits were root biomass density (g m−2 ), started and in April / May in 2003, 2004, 2005, 2012, 2014, root length density (m m−2 ), mean diameter (mm), tip frequency and 2016 (Brunner et al., 2009; Herzog et al., 2014). The same (n cm−1 ), and fork frequency (n cm−1 ). The calculated fine- trees were sampled throughout the duration of the experiment. root traits were specific root length (SRL; m g−1 ) and root tissue Overall, twelve trees in the dry- and nine trees in the irrigation- density (RTD; g cm−3 ). Biomass and length were calculated per treatment were included in the analysis. After sampling, the soil soil area and to a soil depth of 10 cm (topsoil), tips and forks to cores were packed in plastic bags, transported to the laboratory, the root length, SRL to the fine-root biomass, and RTD to the and stored at a low temperature (in a cold room at 4◦ C) until fine-root volume (Brunner et al., 2009). The fine-root turnover they were analyzed. The soil cores were then washed in a sieve, rate (year−1 ) was calculated by dividing annual production by the roots collected, and the fine roots of Scots pine sorted out standing biomass (Gill and Jackson, 2000; Brunner et al., 2013) by hand, dried at 60◦ C for 3 days, and weighed. In order to by using ingrowth core data for the annual production and soil- compare data over the 13-year period, the two samples per coring data for the standing biomass for the years 2005 and tree were averaged. Fine roots were calculated per cm3 of soil 2016. The method used to determine turnover rate, however, is and then balanced for 0–10 cm depth and per m2 to obtain only a rough estimate because of root pruning that occurs when comparable data. ingrowth cores are installed (Hendricks et al., 2006). The lifespan of the fine roots (year) is equal to the inverse value of the turnover Ingrowth Cores rate (Brunner et al., 2013). A first series of ingrowth cores (glass-fiber-netting cylinders 11 cm in height, 5 cm in diameter, with a 5 mm mesh size) Statistical Analyses were set in April 2003 with the ingrowth cores inserted into The statistical analyses performed were one- and two-way the holes where soil core samples had been taken previously analyses of variance (ANOVA), repeated-measures ANOVA, and for the fine-root biomass (Brunner et al., 2009). The ingrowth simple linear regression with ANOVA using StatView software cores were refilled with sieved topsoil from outside the plots. The (Version 5.0, SAS Institute, Cary, NC). Repeated-measures ingrowth cores were then harvested with a large soil corer 8.5 cm ANOVA was applied when samples were taken consecutively in diameter after 2 years in May 2005. After harvest, the ingrowth from the same trees at different times. The significance of cores were packed undisturbed in plastic bags, transported to the the differences between treatments was tested by pairwise laboratory, and stored at 4◦ C until they were analyzed several comparison of the sample means using Fisher’s protected days later. The ingrowth cores were then cut out with a knife, the least significant difference at P < 0.05. All data were tested core length of the samples recorded, the netting removed with for normal distributions using the Kolmogorov-Smirnov Test scissors, the soils sieved, and the fine roots rinsed with tap water. of Normality2 . The fine roots of the Scots pine were then sorted out by hand, and stored in tap water in a refrigerator until fine-root morphology and architecture was analyzed (Brunner et al., 2009). 2 https://www.socscistatistics.com/tests/kolmogorov/Default.aspx Frontiers in Plant Science | www.frontiersin.org 3 June 2019 | Volume 10 | Article 701 Brunner et al. Plasticity of Fine-Root Traits RESULTS Repeated measures ANOVA revealed that the biomass increase in the irrigation treatment was statistically significant Fine-Root Biomass over the 13-year period (Figure 1A). Considering the ratio of The fine-root biomass of the Scot pines, as recorded with soil biomass from the irrigated plots to dry plots, it is evident that over coring over more than a decade, changed considerably over the the years there has been a steady increase in the biomass ratio duration of the experiment (Figure 1A). Under the dry (control) with a high coefficient of determination R2 of 0.95 (Figure 1B). condition, biomass varied from 87 to 311 g m−2 in the first 10 cm We found no indication for a slowdown or reversal of this of the soil. In the first 2 years after the beginning of irrigation increase over time. treatment, no significant change in the fine-root biomass was measured. Only when fine-roots were recorded again after a Fine-Root Production and Turnover Rate gap of 7 years in 2012, did they show a significant difference, Comparing the 2005 fine-root production with that of 2016 with a biomass increase of approximately 50% in the irrigated reveals that values in general can be highly plastic, ranging compared to the dry plots (Figure 1B). This difference between from 33 to 66 g m−2 year−1 (Table 1). The data also show treatments increased steadily over the following years (2014, that production in 2005 was not significantly affected by the 2016) resulting in the fine-root biomass of irrigated plots being irrigation treatment. In contrast, in 2016, fine-root production more than 50% higher than in controls, with a maximum biomass significantly increased by a factor of 2. However, the turnover rate of 411 g m−2 in 2012. in 2016 was only slightly influenced by the irrigation treatment (Table 1) because the biomass recorded through soil coring had also greatly increased due to irrigation (compare also Figure 1A). The lifespan of the fine-roots, as calculated from the turnover rates, was from 1.4 to 1.6 year in 2005, whereas in 2016 it was found to range from 3.0 to 3.3 year (Table 1). Fine-Root Traits Analysis of ingrowth cores sampled in 2005 showed that the fine-root traits measured were not significantly influenced by the irrigation treatment after 2 years of growth (Table 2). However, when the traits were again measured in 2016 from ingrowth cores, significant increases were then recorded for the biomass, length, and frequency of forks, but not for the average diameter or frequency of root tips, SRL, or RTD (Table 2). The strongest effects due to irrigation were recorded for root biomass, which grew 1.9-fold, and for overall root length, which increased 1.8- fold. When the 2016 soil coring samples were evaluated, only root biomass and overall root length increased significantly, 1.6 and 1.8-fold, respectively. This is slightly lower compared to the ingrowth cores (Table 2). Overall, fine-root architectural traits, such as the frequency of tips and forks, and morphological traits, such as average diameter, SRL and RTD, were not significantly influenced by the irrigation treatment (with one exception, namely, the frequency of forks in ingrowth cores in 2016). However, traits varied considerably across survey years and the method applied. Specifically, average diameters ranged from 0.7 to 1.1 mm, root tips from 1.1 to 3.4 cm−1 , fork frequency from 1.1 to 5.9 cm−1 , that of SRL from 7.2 to 13.3 m g−1 , and that of RTD from 0.17 to 0.32 g cm−3 (Table 2). Soil Cores vs. Ingrowth Cores When fine-root traits from both ingrowth cores collected after two-year of growth and soil cores were sampled and analyzed in 2016, significant differences between irrigated and control FIGURE 1 | Fine-root biomass of the topsoil (0–10 cm) under dry and irrigated conditions of the Scots pine forest for the years 2003 to 2016 recorded by soil samples were obtained for biomass, length and forks, but not coring. (A) Course of biomass (±SE). Repeated measures ANOVA, significant for other fine-root traits (Table 3). A significant effect for the effects (P < 0.05) are shown in bold; One-way ANOVA, significant effects sampling method (ingrowth cores vs. soil cores) was observed for (P < 0.05) are indicated with ∗ . (B) Relationship between the biomass ratio almost all fine-root traits, except for root length (Table 3). The (irrigated/dry) and the sample years, a significant effect (P < 0.05) is shown in greatest significant differences were recorded for fine-root tips, bold. forks, average diameter, and SRL. Interactions between the two Frontiers in Plant Science | www.frontiersin.org 4 June 2019 | Volume 10 | Article 701 Brunner et al. Plasticity of Fine-Root Traits TABLE 1 | Biomass, production, turnover rate, and lifespan of fine roots in the years 2005 and 2016 of the Scots pine forest under dry and irrigated conditions. Fine-root traits Year 2005 Year 2016 Dry Irrigated P Dry Irrigated P Biomass (g m−2 ) 87.2 105.3 0.55 111.5 198.5 0.01 Production (g m−2 y−1 ) 54.9 53.2 0.89 33.8 66.1 0.02 Turnover (y−1 ) 0.73 0.61 – 0.30 0.33 – Lifespan (year) 1.38 1.64 – 3.31 3.00 – Biomass corresponds to standing biomass estimated by soil coring, production corresponds to annual production estimated by ingrowth cores, turnover corresponds to turnover rate (calculated as production divided by biomass), lifespan corresponds to root age and is the inverse of turnover. Turnover and lifespan were calculated per treatment. One-way ANOVA; significant effects (P < 0.05) are shown in bold. TABLE 2 | Comparison of fine-root traits of roots from ingrowth cores from 2005 and 2016 after 2 years of growth, and of roots from the soil-coring sample from 2016 of the Scots pine forest under dry and irrigated conditions. Fine-root traits Ingrowth cores (2005) Ingrowth cores (2016) Soil cores (2016) Dry Irrigated P Dry Irrigated P Dry Irrigated P Biomass (g m−2 ) 109.9 106.3 0.89 67.6 132.2 0.02 111.9 198.5 0.01 Length (m m−2 ) 38.9 59.9 0.14 87.3 157.0 0.01 100.2 155.4 0.049 Diameter (mm) 1.10 1.05 0.63 0.73 0.70 0.37 0.91 0.93 0.55 Tips (n cm−1 ) 1.12 1.10 0.81 3.21 3.43 0.26 2.39 2.40 0.85 Forks (n cm−1 ) 1.04 1.17 0.60 4.72 5.91 0.01 3.42 3.74 0.23 SRL (m g−1 ) 8.11 7.19 0.77 12.5 13.3 0.61 9.79 8.56 0.33 RTD (g cm−3 ) 0.32 0.37 0.64 0.20 0.22 0.19 0.17 0.18 0.50 One-way ANOVA; significant effects (P < 0.05) are indicated in bold. SRL, specific root length; RTD, root tissue density. TABLE 3 | Results of two-way ANOVA testing the effect of the irrigation treatments (dry vs. irrigated) and the fine-root sampling methods (ingrowth cores vs. soil cores) on fine-root traits in 2016. Fine-root traits Treatment Method Treatment × Method dF F P dF F P dF F P Biomass (g m−2 ) 1 11.9 0.002 1 6.36 0.02 1 0.25 0.62 Length (m m−2 ) 1 10.2 0.003 1 0.09 0.77 1 0.14 0.71 Diameter (mm) 1 0.12 0.73 1 67.5 <0.001 1 1.21 0.28 Tips (n cm−1 ) 1 1.82 0.19 1 110.6 <0.001 1 1.34 0.26 Forks (n cm−1 ) 1 12.1 0.002 1 63.4 <0.001 1 4.06 0.05 SRL (m g−1 ) 1 0.05 0.83 1 14.4 <0.001 1 1.06 0.31 RTD (g cm−3 ) 1 1.70 0.20 1 7.85 0.01 1 0.15 0.71 Significant effects (P < 0.05) are shown in bold. SRL, specific root length; RTD, root tissue density. factors, irrigation treatment and fine-root sampling method were, Although the exact time-point of a significant increase in fine- however, not detected (Table 3). root biomass was not recorded, root biomass acclimation had started before year nine as indicated by the slight but not significant irrigation effects in 2004 and 2005. It also appears DISCUSSION that the ratio of irrigated to dry treatment biomass was still increasing, rising from 1.5 in 2012 to 1.8 in 2016. This indicates Several attributes in the ecosystem of the experimentally irrigated that, on the one hand, a saturation of the fine-root biomass pine forest changed due to the prolonged treatment over more in the topsoil had not yet been reached, and, on the other than a decade. Herb and moss coverage increased significantly, hand, the density of fine-roots is strongly dependent on water as did crown cover (from 57 to 71%), tree biomass (from availability. Thus, water rather than nutrients (e.g., nitrogen) 6.8 to 7.9 kg m−2 ), and litter-fall (from 0.31 to 0.46 kg m−2 seems to be the primarily limiting factor for fine-root abundance year−1 ) (Herzog et al., 2014; Hartmann et al., 2017). The in this Scots pine forest. Long-term irrigation did not affect increase in fine-root biomass only became apparent 9 years the carbon-to-nitrogen ratio, although the fine-root biomass after the irrigation experiment began (Herzog et al., 2014). increased by approximately 50% (Herzog et al., 2014). Thus, Frontiers in Plant Science | www.frontiersin.org 5 June 2019 | Volume 10 | Article 701 Brunner et al. Plasticity of Fine-Root Traits higher water availability did not mobilize more nutrients from synthesis, which help to regulate water uptake and contribute the topsoil, which, in fact, could also promote fine-root growth to osmotic adjustment. Hydraulic conduits and suberin are (e.g., Yuan and Chen, 2012). An additional indication for the increasingly produced to better handle water transport and water water limitation is the decrease in the δ13 C-values in fine- loss, respectively (Fonti and Jansen, 2012; Barberon et al., 2016). roots from −26 to −27h (Herzog et al., 2014), and in trunk Morphological and architectural traits, in contrast, do not appear tree rings, from −22 to −24h (Timofeeva et al., 2017) in the to be strongly affected by water limitation. Weemstra et al. irrigation treatment. These lower values are most likely a result of (2017) investigated fine-root trait plasticity in European beech higher stomatal conductance, leading to higher leaf internal CO2 and Norway spruce in two contrasting soils (clay vs. sand) and concentrations and subsequent higher photosynthetic carbon observed that dynamic traits, such as biomass and length, were isotope fractionation (Farquhar et al., 1989). variable, but that morphological traits such as diameter, SRL and Only recently, Solly et al. (2018) unraveled the real age of RTD were not. It is indeed hypothesized that dynamic traits fine roots by counting annual rings, and discovered a mean age follow the resource economics spectrum (Grime, 1977; Bardgett (=lifespan) of fine roots of 1 to 2 year (including the fine roots of et al., 2014; Reich, 2014), and are thus influenced by soil resource the Scots pines in the Pfyn forest). Thus, the short lifespan of the availability (i.e., water and nutrients). This study has come to the fine roots explains the strong fluctuation between 2004 and 2005 same conclusion, that while biomass and length were found to be and between 2014 and 2016, when fine-root biomass was reduced positively affected by the irrigation treatment – and thus by soil by more than half. This decrease was most likely due to the heat water availability – diameter, SRL, and RTD remained unaffected. waves and summer droughts in 2003 and 2015, respectively (Ciais In general, roots can show considerable trait plasticity et al., 2005; Dietrich et al., 2018), which negatively affected fine- (Callaway et al., 2003). A recent meta-analysis of the effects root biomass build-up with a delay of one to two years, which is of precipitation changes on fine-root traits at a global scale when reduced storage reserves came to bear. concluded that the responses of fine-root biomass, production, The fine-root turnover rate, estimated by dividing annual decomposition, and morphology to precipitation can be either production by standing biomass, revealed turnover values of 0.30 positive or negative (Zhang et al., 2019). Joslin et al. (2000) to 0.73 year−1 . These values appear to be lower than the mean previously reviewed the dynamics of fine-root biomass in long- values (0.76–1.40 year−1 ) determined for Scots pine in European term stand-level irrigation experiments (2–4 year) with no forests (Brunner et al., 2013), and reach lower values than those conclusive results. Particularly with European beech, results were of other tree species (0.48 ± 0.52 year−1 ; Finer et al., 2011), ambiguous, as decreases in precipitation resulted in more fine- both estimated by the ingrowth method. Yuan and Chen (2010) root biomass in some cases (e.g., Hertel et al., 2013) and less in analyzed fine roots worldwide, obtaining a mean turnover rate others (e.g., Meier and Leuschner, 2008). Hommel et al. (2016) of 0.61 ± 0.17 year−1 for pines. While our values are still in the showed that the allocation of assimilates to roots in European range covered by these studies, Finer et al. (2011) stated that the beech and Norway maple (Acer platanoides) increased under ingrowth core method usually yields lower turnover values than mild, but strongly decreased under more intensive drought, those obtained by the sequential coring or minirhizotron method, suggesting that drought regimes may impact fine-root biomass most likely because of a shorter exposure (<2 year). Keeping in production. While there are indications that water sensing mind the turnover values of 0.5 to 1.0 year−1 obtained by Solly by the root cap might stimulate root growth along moisture et al. (2018), our values are in a similar range, with the exception gradients allowing plants to forage water (Eapen et al., 2005; of 2016, which was remarkably low at 0.3 year−1 . Dietrich, 2018) strong drought will impair root metabolic activity Comparing the two methods, soil coring and ingrowth (Chaves et al., 2003; Beck et al., 2007). Hagedorn et al. (2016) cores, has been done previously (e.g., Persson, 1983; Neill, showed that the allocation of assimilates to the roots under 1992; Makkonen and Helmisaari, 1999), but mainly to compare drought is driven by this root metabolic activity rather than dynamic fine-root traits and not to compare morphological by an impairment of phloem transport. In coordination with or architectural traits. Our data shows that the experimental impaired root activity and growth, supply with new carbon treatment in the Pfynwald forest had a similar effect across is ceased. Some studies with conifers have shown biomass methods, although absolute values significantly differed between increases (e.g., Gower et al., 1992; De Visser et al., 1994), methods for most traits. Hendricks et al. (2006) concluded that while others have not (e.g., Bredemeier et al., 1998). Bakker ingrowth core estimates are in general comparable with soil et al. (2009) also conducted a long-term irrigation experiment core estimates, although ingrowth cores may underestimate root on 13-year-old maritime pine (Pinus pinaster), and overall production in water-limited environments. In our study, we found no effects on fine-root length, SRL, or ramification after observed a similar trend regarding fine-root biomass, where 7 years of treatment. However, in the latter study, we must values obtained from ingrowth cores were about one-third of assume that non-irrigated control plants did not suffer from those from soil cores. a lack of water, because the mean annual precipitation of the Drought is known to affect both fine-root traits, such as study site is approximately 950 mm. This means that plants the dynamic traits of biomass and length, and physiological under both treatments, control and irrigation, were most likely and molecular traits (Gessler et al., 2005; Brunner et al., 2015; not water-limited. In contrast, Konopka and Lukac (2013), Volkmann et al., 2016; Polle et al., 2019). The production of conducting a rain-shelter experiment in a 90-year-old Norway the plant hormone abscisic acid (ABA) is strongly enhanced, spruce (Picea abies) stand, found that water shortage reduced influencing root growth, as well as aquaporins and proline fine-root biomass and increased necromass, which supports the Frontiers in Plant Science | www.frontiersin.org 6 June 2019 | Volume 10 | Article 701 Brunner et al. Plasticity of Fine-Root Traits hypothesis that biomass is expected to be affected only when fertility, fractals (=tip abundance) with soil moisture, and length water is limited. Gaul et al. (2008), in a rain-shelter experiment with soil carbon. with Norway spruce, showed that experimental drought did The use of single-pool and diameter-based classification not result in significant changes in fine-root biomass during of fine-roots, defined here as <2 mm diameter roots, has a six-week treatment period. We can assume here, however, limitations. Functional aspects of the fine roots can hardly that 6 weeks is too short a period to observe acclimation of be described with this classification system. To overcome tree root biomass. these limitations, McCormack et al. (2015) proposed an Investigating the fine roots of Scots pine across a alternative approach using an order-based classification that temperature and latitudinal gradient in Europe, Zadworny allows more standardized comparisons of root traits, and et al. (2017) observed that the root diameter of absorptive a functional classification that allows for the distinction roots (defined by the absence of a suberized cell layer) between absorption and transport functions. Absorptive fine was variable, with the diameter decreasing as the mean roots in their concept correspond to the most distal roots, annual temperature (MAT) increased (0.4 mm at −2◦ C; which are mainly involved in acquisition and uptake, while 0.15 mm at 8◦ C). In contrast, SRL and the carbon-to- transport fine roots in the branching hierarchy are higher nitrogen ratio of absorptive roots were not dependent on ordered and serve mainly structural and transport functions the MAT. The average root age of transport roots (seventh (McCormack et al., 2015). Such a new approach would to ninth root order), however, was variable with the MAT, allow for more consistent and accurate comparisons of root with root age decreasing with increasing MAT (4 year at traits as well as comparisons between functionally similar −2◦ C; 1.4 year at 8◦ C). Other studies with Scots pine, roots in the future. especially on precipitation gradients, are, unfortunately, lacking. Nevertheless, Zadworny et al. (2017) study provide insight into which Scots pine root traits are potentially plastic CONCLUSION and which are not. Zadworny et al. (2017) proposed that there are two major Our study demonstrates that the fine-root biomass of Scots strategic paths that plants use. Either plants produce thin roots pine in the topsoil of a water-limited site increases when water (with a high SRL), which grow quickly and are able to enhance is added to the soil. Other studies with broader purposes water and nutrient acquisition, or they produce thick roots (with (meta-analyses and reviews) have shown, in contrast, that low SRL and high RTD), which grow slowly and are able to the reactions of fine roots can be ambiguous and divergent, live longer, store resources, and are better protected against with increasing soil moisture leading either to an increase in environmental hazards. In fact, having larger root diameters may fine-root biomass, a decrease, or no reaction at all. In these be a more effective strategy to enhance resource acquisition when cases, the experimental design or the climatic gradient under the environment is nutrient-limited (Zadworny et al., 2017). study played a crucial role, and factors such as the length In our study, however, larger diameters and altered SRL or of the experiment, soil type, or method used to measure the RTD were not obvious due to the irrigation treatment. Even fine roots influenced the results. Even though we observed though we investigated these traits with two different methods in our study an increase in the fine-root biomass in the (soil coring and ingrowth cores), no significant shift in either topsoil, we did not record root acclimation at deeper soil direction was observed. horizons. Given that the Pfynwald is located on a former In a recent analysis of root traits, Liese et al. (2017) alluvial fan composed of large rocks, a change in fine-root demonstrated that the branching pattern of roots enables biomass at deeper levels is likely, although its measurement a plastic response to a changing environment. Species with is not feasible. Since we observed a positive change solely high branching intensity are able to rapidly and extensively in terms of fine-root biomass and length, and not in other proliferate into resource-rich patches. Consequently, fine-root morphological or architectural traits, such as diameter, SRL, branching, including tips and forks, and not necessarily the RTD, tips, or forks, we can assume that the majority of root diameter, reflects the environment. It should be noted, however, traits are not plastic. As Rewald et al. (2014) and Pierret et al. that the goal of that study was to investigate inter-species (2016) correctly pointed out, it is complicated to study whole and not intra-species variation. A better understanding could root systems, and intra-root variability occurs with respect to potentially be gained from the new global Fine-Root Ecology dynamic, morphological, and architectural traits. As observed Database FRED, introduced by Iversen et al. (2017). In their in our study, some traits undergo a change only after several review, the authors ask the question “How do root traits years of treatment, suggesting that more research on long- vary along environmental gradients?” and refer to the studies term experiments in natural forests is needed. To understand of Larson and Funk (2016) and Zadworny et al. (2016) in why certain fine-root traits do adjust to soil environment relation to phenotypic responses to environmental change, changes and some do not, requires “more attention to the although the former study did not investigate trees. Overall, it underlying drivers of fine-root mass and morphology, and to appears that soil factors have a strong effect on intra-species other mechanisms that are involved in soil resource uptake” plasticity, as Valverde-Barrantes et al. (2013) pointed out when (Weemstra et al., 2017). The same authors speculate that investigating 14 angiosperm tree species. Biomass, diameter, SRL, plasticity in fine-root biomass may be even more important and RTD, were found to be significantly correlated with soil than morphological plasticity. Further, it must be taken into Frontiers in Plant Science | www.frontiersin.org 7 June 2019 | Volume 10 | Article 701 Brunner et al. Plasticity of Fine-Root Traits consideration that the plasticity of certain fine-root traits might FUNDING be species-specific, or dependent on the evolutionary status (gymnosperms, angiosperms, monocots, dicots) or mycorrhizal CH was supported by the Swiss National Science Foundation status (ectomycorrhizal, arbuscular mycorrhizal, ericoid, non- (SNF), Grant number SNF 31003A_149507. LG was supported mycorrhizal) of the trees in question (Comas et al., 2014; by a postdoctoral fellowship from the Alexander von Humboldt- Valverde-Barrantes et al., 2017). In conclusion, more studies are Foundation (AvH). needed to investigate the main drivers and patterns of root- trait plasticity to finally link those to overall tree performance. Under future global change scenarios, it will be highly relevant ACKNOWLEDGMENTS to know which fine-root traits are susceptible to environmental changes, whether they be water, temperature, pH, or nutrients, The analysis was based on data from the long-term Pfynwald and which are not. forest irrigation experiment, which is part of the Swiss Long-term Forest Ecosystem Research Program LWF (www.lwf.ch). We are particularly grateful to Andreas Rigling and Marcus Schaub who AUTHOR CONTRIBUTIONS are responsible for the Pfynwald irrigation experiment. We are also grateful for the assistance of Peter Bleuler and Christian Hug, IB and CH dealt with the 2005 ingrowth cores and the soil who provided technical support for the experiment, as well as coring from 2003–2016 and analyzed the data with WinRHIZO the long-standing support received from HYDRO Exploitation Software. LG and AG dealt with the 2016 ingrowth cores. All SA in Sion. 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New conducted in the absence of any commercial or financial relationships that could Phytol. 212, 389–399. doi: 10.1111/nph.14048 be construed as a potential conflict of interest. Zadworny, M., McCormack, M. L., Zytkowiak, R., Karolewski, P., Mucha, J., and Oleksyn, J. (2017). Patterns of structural and defense investments in fine roots Copyright © 2019 Brunner, Herzog, Galiano and Gessler. This is an open-access of Scots pine (Pinus sylvestris L.) across a strong temperature and latitudinal article distributed under the terms of the Creative Commons Attribution License gradient in Europe. Global Change Biol. 23, 1218–1231. doi: 10.1111/gcb.13514 (CC BY). The use, distribution or reproduction in other forums is permitted, provided Zhang, X., Xing, Y., Yan, G., Han, S., and Wang, Q. (2019). Effects of precipitation the original author(s) and the copyright owner(s) are credited and that the original change on fine root morphology and dynamics at a global scale: a meta-analysis. publication in this journal is cited, in accordance with accepted academic practice. No Can. J. Soil Sci. 99, 1–11. doi: 10.1139/CJSS-2018-0114 use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 10 June 2019 | Volume 10 | Article 701