Soil microbes aid in the drought tolerance of plants, yet the extent to which a microbial community’s previous drought exposure can affect plant responses to contemporary drought is largely unknown. We used a greenhouse experiment to investigate whether microbes exposed to reduced moisture in the past affect the sensitivity of trees to future water-stress. We planted saplings of 3 species in field soils exposed to experimentally-induced drought or ambient moisture from adjacent forest plots, and then altered the watering regime of the saplings to induce contemporary drought. When trees were grown in ambient soils with no drought history, contemporary drought reduced C assimilation rates, stomatal conductance, and leaf water potential in all species. However, when Prunus virginiana were grown in soils with a drought history, they were buffered from the effects of contemporary drought, as physiological performance was mostly unchanged by water stress. P. virginiana grown in drought history soils also increased soluble sugars during contemporary drought to a lesser extent than those in soils with no drought history, suggesting the plants experienced less water stress. Sterilized soils confirmed the “soil drought history effect” likely resulted from drought-adapted microbes in soils exposed to drought previously. None of these effects were apparent in Liriodendron tulipifera and Quercus rubra, which reduced their physiological performance when water-stressed regardless of soil drought history. To the extent mature tree responses to environmental stress are similar, our results suggest that forest sensitivity to drought may depend, in part, on plant-microbial interactions shaped by past stress exposures.
Research Article
Soil microbial drought history affects tree physiology of select species
https://doi.org/10.21203/rs.3.rs-4902672/v1
This work is licensed under a CC BY 4.0 License
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anisohydry
drought sensitivity
gas exchange
isohydry
legacy effects
Predicted increases in the frequency and intensity of drought (Büntgen et al. 2021; Masson-Delmotte et al. 2022) have prompted interest in quantifying the drought sensitivity of forests, one of the largest carbon (C) sinks on earth (Pan et al. 2011). Most studies of tree drought tolerance have focused on hydraulic traits of the dominant species (Anderegg 2015; Anderegg et al. 2016; Choat et al. 2018), with limited consideration of how microbial interactions – often hidden from view – can affect tree water use. Microbial mediation of plant drought tolerance has been reported for numerous taxa (Lau and Lennon 2012; Kumar and Verma 2018; Allsup and Lankau 2019; Allsup et al. 2023), calling into question a plant-centric, trait-based perspective of drought sensitivity. If microbial mediation of drought tolerance is widespread, linking this information to other factors known to affect ecosystem sensitivity to drought (e.g., plant hydraulic traits, rooting depth, soil texture, etc.) (Anderegg et al. 2018; Kannenberg et al. 2019) will be critical for making robust predictions about climate change impacts on the land C sink.
Soil microbes can affect plant drought tolerance via hormonal signaling (Hussain et al. 2014; Cohen et al. 2015) or by increasing the availability of soil water (Duñabeitia et al. 2004, Pavithra and Yapa 2018), though little is known about the relative importance of each process. Microbes send hormonal signals to plants that influence their physiology and morphology, such as upregulating stomatal and root growth responses to water deficits (Kumar and Verma 2018). Some soil microbes excrete compounds that degrade ethylene precursors to release plants from drought-induced growth reductions as well as stimulate root growth (Akbari et al. 2007; Hussain et al. 2014). Additionally, certain soil microbes release the stress hormone abscisic acid to trigger stomatal closure thus relieving plant water stress (Cohen et al. 2015). In terms of increasing water availability, plant-associated microbes can increase plant access to and uptake of water or alter soil properties in ways that increase water retention. For instance, many plant taxa increase their associations with mycorrhizal fungi when exposed to water-stress given that fungi can extend hyphae into small soil pores to access water (Augé et al. 2001, Duñabeitia et al. 2004, Smith et al. 2010, Pavithra and Yapa 2018). Mycorrhizal fungi also increase water retention by binding soil particles together with hyphae (Augé et al. 2001). Likewise, roots promote microbes that excrete extracellular polymeric substances (EPS), which enhance the formation of water-retentive aggregates (Alami et al. 2000; Costa et al. 2018). While microbes have been shown to affect plant drought tolerance in myriad ways, we lack understanding of whether such dynamics are widespread or relatively rare, especially in long-lived woody plants (Phillips et al. 2016).
Given that plant-associated microbes have been shown to alter stomatal conductance (Forchetti et al. 2007; Salomon et al. 2014; Cohen et al. 2015; Tsukanova et al. 2017), an open question is whether microbes affect plant hydraulic strategies. Some plants close stomata at the first signs of drought (strict isohydry), some keep their stomata open during drought and make other physiological adjustments (strict anisohydry), and some adopt a strategy that is intermediate between the two extremes (Tardieu and Simonneau 1998). If plant-associated microbes secrete hormones (e.g., abscisic acid) in response to drought to trigger stomatal closure, the plant’s hydraulic strategy may shift (Tardieu and Simonneau 1998; Kannenberg and Phillips 2017; Tsukanova et al. 2017). Kannenberg and Phillips (2017) reported that Liriodendron tulipifera and Acer saccharum (but not Quercus rubra) saplings became more isohydric when exposed to a soil inoculum (as opposed to sterile control), indicating that microbial signals can influence plant drought tolerance. Moreover, if C assimilation is downregulated, other plant physiological responses can be anticipated (Tardieu and Simonneau 1998; McDowell 2011; Mencuccini et al. 2015).
Non-structural carbohydrates (NSCs), which are produced by plants in times when supply of C assimilates exceeds demand (Chapin et al. 1990; Kozlowski 1992; Dietze et al. 2014), are sensitive to drought-induced changes in C assimilation (O’Brien et al. 2014; Dickman et al. 2015; Tomasella et al. 2019). During times of drought, plants use NSC stores to maintain metabolism (McDowell 2011; Dietze et al. 2014) and osmotic regulation (O’Brien et al. 2014; Tomasella et al. 2019). Starches are often used to support plant metabolism when stressed, including during drought (McDowell 2011; Dietze et al. 2014; Martínez-Vilalta et al. 2016), whereas soluble sugars are used as metabolic substrates and osmoregulatory compounds to maintain turgor (Woodruff and Meinzer 2011; Sala et al. 2012; Martínez‐Vilalta et al. 2016). Thus, if a plant’s resident microbes cause it to become more isohydric, a cascade of C consequences may follow. Despite this, investigations of microbial effects on whole plant C balance are relatively rare (Naylor and Coleman-Derr 2018).
Plant species likely differ in their responsiveness to microbial signals, with possible consequences for plant drought tolerance. In a study where trees were inoculated with microbes with different water stress histories, Allsup et al. (2023) found that inoculating tree species with drought-stressed microbes can increase tree survival under contemporary drought conditions. However, the response was inconsistent among tree species. Trees that associated with arbuscular mycorrhizal benefitted from the drought-stressed inoculum (owing to greater diversity of arbuscular mycorrhizal fungi), whereas trees that associated with ectomycorrhizal fungi were insensitive to the historical conditions of their microbes and did not experience the microbial-mediated benefits under drought (Allsup et al. 2023). Thus, if plants have interspecific differences in their responsiveness to microbial influence, interspecific differences in whole-plant sensitivity to drought may occur.
Finally, the degree to which microbes affect plant drought tolerance may also depend on previous drought exposure (i.e., soil history) of the microbes. Microbes possess adaptations for dealing with drought, and it is well-established that microbial community composition is sensitive to water stress (Lau and Lennon 2012; Ochoa-Hueso et al. 2018; Munoz-Ucros et al. 2022; Evans et al. 2022). Certain species of microbes (e.g. Actinobacteria) tolerate drought (Bouskill et al. 2013; Wipf et al. 2021; Munoz-Ucros et al. 2022) owing to their ability produce thick cell walls (Ebrahimi-Zarandi et al. 2023) and their capacity to produce osmolytes (Bouskill et al. 2013). Thus, changes in microbial community composition owing to drought legacies could affect plant sensitivity to drought if such compositional shifts impact plant fitness. Whether drought-tolerant microbial species affect plant drought tolerance is incompletely understood.
To date, much of the research on microbial mediation of plant drought tolerance has focused on how microbes affect growth and survival during drought, with limited consideration of morphological, physiological, and chemical responses. Moreover, few studies have been carried out with trees and have examined the impact of microbes that experienced different historical water conditions. Here we use a controlled greenhouse experiment to uncover the effects of microbially-mediated drought history on trees' response to subsequent drought conditions. We hypothesized that trees exposed to drought-stressed microbes will be buffered from the effects of succeeding drought and will exhibit changes in their degree of isohydry. By understanding how microbial drought history affects tree physiology under subsequent droughts, we can better predict how trees will fare against the more frequent and intense droughts induced by climate change.
Site description for soil harvesting
We collected soil from two adjacent forest plots in Griffy Woods, Indiana, USA (39°11’N, 86°30’W). The site is a deciduous hardwood forest dominated primarily by Quercus rubra (red oak), Acer saccharum (sugar maple), and Liriodendron tulipifera (tulip poplar). One plot had been exposed to ambient levels of precipitation, and therefore the resident soil microbes experienced an ambient precipitation history; we designate this soil as the “control” soil history. The other plot has been exposed to a 55% reduction of throughfall using a 40m x 40m throughfall displacement design (Asbjornsen et al. 2018) for ~ 4.5 years. Hereafter we use the term “drought-stressed” to refer to the soil history in this plot, as the soil and microbes have been exposed to a 162% reduction in soil water potential. The soil at Griffy Woods is a silty-loam derived from sandstone or shale at a boundary of ultisols and alfisols.
Experimental design
We collected soil to 10cm depth from multiple locations from each of the forest plots and homogenized the soils to generate two soils: a “control” and a “drought-stressed” soil. While we only collected drought-stressed soils from a single site, the size of the plot (40m x 40m) and number of samplings gave us a representative sample, which is appropriate given our interest in analyzing the average effect of soil history (Cahill et al. 2017). In the greenhouse, we planted 1–2-year-old saplings of L. tulipifera (Cold Stream Farm), Q. rubra (Vallonia State Nursery), and Prunus virginiana (chokecherry; Cold Stream Farm) in each of the two soils. Liriodendron tulipifera is a water-demanding tree that adopts an isohydric hydraulic strategy (Roman et al. 2015; Yi et al. 2017) and associates with arbuscular mycorrhizal (AM) fungi. Quercus rubra is anisohydric and associates with ectomycorrhizal fungi (ECM) (Roman et al. 2015; Yi et al. 2017). Prunus virginiana associates with AM fungi (Bainard et al. 2011). While typically found in northern but not southern Indiana, a close relative (P. serontina; black cherry) is a component of Griffy Woods where the soil was collected. There have been no reports of P. virginiana hydraulic traits and thus its drought strategy is unknown.
When planting, we first placed a layer of sterilized sand in tall tree pots and then positioned the sapling in the pot. We placed the appropriate field soil completely around the root zone of the tree and added sterilized sand on top of the field soil (Fig. 1). We used this sand-soil-sand layering design to minimize the amount of field soil removed from the field site and accommodate for the large size of the tree pots compared to sapling size. We acclimated the plants for 3 weeks starting in May 2022, allowing them to leaf out before starting the watering treatments and measurements. We submitted the saplings to either a well-watered (weekly watering) or water-stressed (biweekly watering or “contemporary drought”) watering treatment. For each of the treatment combinations, we had 6 replicate trees (n = 72, 24 trees per species). In addition, we sterilized a subset of soils using an autoclave (1hr at 120℃) using the same experimental design (n = 3 pots per treatment combination). We took measurements at the conclusion of 10 weeks within +/- 3 hours of solar noon, ending in September 2022.
Physiological measurements
We measured photosynthetic assimilation (A) and stomatal conductance (gsw) using the LI-COR 6800 (LI-COR Inc., Lincoln, NE). We set the chamber conditions to a constant 600 µmol/s flow rate, 60% relative humidity, 420 µmol/mol CO2 concentration, 10,000 rpm fan speed, 29°C temperature, and 1500 µmol/m2/s light setpoint. We calculated intrinsic water use efficiency (iWUE) by dividing A by gsw. We measured spot leaf water potential (ΨLeaf) using a Model 610 Scholander-type pressure chamber (PMS Instrument Company, Corvallis, OR) at the midpoint of the experiment due to the destructive nature of the process. At the conclusion of the experiment, we determined the tree hydraulic strategy for each treatment to determine if soil history influenced tree hydraulic status. To determine hydraulic strategy, we measured ΨLeaf every other day for 2 weeks while the soil dried out to capture differences in ΨLeaf with changing soil moisture levels. We simultaneously measured soil volumetric water content (%) using a Hydrosense II meter (Campbell Scientific, Logan, UT) to determine volumetric water content. We then converted volumetric water content to soil water potential (ΨSoil) using a soil water retention curve that was created using soil psychrometers (WP4-C, Decagon Devices Inc., Pullman, WA, United States) on our sand-soil mix upon the completion of our experiment. We saved the leaves used for ΨLeaf measurements to later determine total leaf dry biomass.
Growth and biomass
Each week, we measured stem elongation and diameter. We measured the distance from the base of the branch to the tip to track stem elongation. In addition, we used calipers to measure stem diameter growth throughout the experiment at the base of the branch used for stem elongation. We calculated stem elongation and diameter relative growth rates (RGR) by subtracting the initial measure from the final measure then dividing the initial measure. After the conclusion of the experiment, we harvested the trees and separated them into stems, leaves, and roots. We wet-weighed the stems and leaves, dried them at 60°C for at least 48 hours, and dry-weighed them for biomass. For root biomass, we first subsampled the roots for morphological analyses, which is detailed in the subsequent section. We wet-weighed both the larger mass of roots and the subsample. We then dried the larger root mass at 60°C for at least 48 hours and dry-weighed the sample. We determined the linear relationship between wet weight and dry weight for each of the species, and from this relationship, we calculated the inferred dry weight of the root subsample and added this to the final dry biomass measurement.
Root morphology
Root subsamples collected after harvesting were scanned using an EPSON GT-20000 flatbed scanner (Epson, Nagano, Japan) at 600 dpi. We took particular care to spread the roots out for more accurate structural analyses. We analyzed root morphology and architecture using the software Rhizovision and following the protocols described in Seethepalli and York (2020). We investigated if there was a difference in the average branching intensity (BI), diameter (D), and specific root length (SRL) among treatments.
Nitrogen concentration and non-structural carbohydrates
For N concentration and NSC analyses, we ground the leaf samples using a SPEX 2010 GenoGrinder (SPEX® Sample Prep, Metuchen, NJ, USA), and we ground the stem and root samples using a Thomas Scientific-Wiley Mini-Mill (Thomas Scientific, Swedesboro, NJ, USA). We measured two types of NSCs, soluble sugar and soluble starch, from each of the tissue types (stem, leaf, and root) using an extraction protocol adapted from Chow and Landhausser (2004). We extracted the soluble sugars in a liquid phase using a mixture of methanol, chloroform and water, while starches were precipitated in the form of a starch pellet. We depolymerized the starch pellet using diluted sulfuric acid in a 90°C water bath for 30 minutes. We then took the resulting soluble starches and sugars and added concentrated sulfuric acid and 2% phenol. In a dark room, we allowed the resulting yellow color to develop for 10 minutes after mixing. We used a UV-1700 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) to determine the colorimetric concentration of soluble sugars and starches in the solution at a wavelength of 490nm. We converted spectrophotometric data to NSC concentrations (% dry mass of the plant tissue) using 1:1:1 D-glucose:D-fructose:D-galactose for the standard curve and the total dry mass of the tissue (Kannenberg and Phillips 2020). To determine the N concentrations, we analyzed the ground material using the Elemental Combustion System 4010 (Costech Analytical Technologies, Valencia, CA, USA).
Statistical analyses
We performed all statistical analyses and created all visualizations using R (RStudio Team 2020). We employed nested, factorial ANOVAs to compare differences across treatments for each species. We also computed Hedges’ g effect size and the 95% confidence intervals to reflect the difference the water-stressed treatment made on a measure within each of the soil history treatments. To calculate Hedges’ g, we used the package “effsize” (Torchiano 2016). For comparing hydraulic strategies, we employed ANCOVAs to compare slopes using the package “lsmeans” (Lenth 2016). Lastly, we ran a linear mixed model to investigate the effects of watering treatments, microbial history, and their interactions using the package “DHARMa” (Hartig 2017).
Physiological responses and strength of drought
While trees responded to elevated water stress by decreasing photosynthesis, conductance, and ΨLeaf (Table 1), exposure to soils with a history of drought stress influenced some species more than others (Table S1). Water-stressed saplings experienced a 192% lower ΨSoil compared to the well-watered saplings, decreasing from -0.336 MPa to -0.981 MPa (p<0.0001). In P. virginiana, exposure to drought-stressed soil buffered tree responses to contemporary drought (Fig. 2). For instance, photosynthesis was reduced by 82% in trees that experienced water stress (compared to well-watered controls) and were planted in soil with no drought history (Hedges’ g = -2.52, CI= [-4.20, -0.846], Fig. 2). In contrast, photosynthetic rates were unaffected by water stress when trees were planted in soils with a drought history (Hedges’ g = -0.0745, CI= [-1.26, 1.11], Fig. 2, Table 1). Importantly, when P. virginiana were grown in sterilized soils, with or without a history of drought, physiological responses were the marginally the same in contemporary drought as in the well-watered treatment (p=0.069; Table S2), suggesting that living microbes were likely responsible for the observed soil history treatment differences. In addition, P. virginiana planted in sterilized soils had lower photosynthetic assimilation when well-watered compared to those planted in unsterilized soils (p=0.043), indicating that microbes are also instrumental to plant physiology when under optimal moisture conditions.
For Q. rubra, both the drought-stressed (Hedges’ g = -2.30, CI= [-3.92, -0.692], Fig. 2) and control (Hedges’ g = -1.46, CI= [-2.80, -0.124], Fig. 2) soil histories did not buffer the tree’s photosynthetic assimilation under the water-stressed conditions. The L. tulipifera trees planted in the soil with a history of drought-stress (Hedges’ g = -3.32, CI= [-5.25, -1.39], Fig. 2) as well as those planted in the soil with a control moisture history (Hedges’ g = -3.68, CI= [-5.74, -1.65], Fig. 2) experienced reductions in photosynthetic assimilation with further water-stress.
The results for stomatal conductance mirrored the photosynthetic assimilation results. Prunus virginiana experienced an 86% decline in gsw when water-stressed and planted with control history soils (Hedges’ g = -2.99, CI= [-4.81, -1.17], Fig. 2), but there was no effect of subsequent water-stress on gsw when planted with drought-stressed soils (Hedges’ g = 0.0447, CI= [-1.14,1.23], Fig. 2). Quercus rubra and L. tulipifera experienced declines in gsw under water-stressed conditions when planted with control history soils (Q. rubra: Hedges’ g = -1.57, CI= [-2.93,-0.212], Fig. 2; L. tulipifera: Hedges’ g = -2.63, CI= [-4.34, -0.925, Fig. 2) as well as drought-stressed soils (Q. rubra: Hedges’ g = -2.69, CI= [-4.41, -0.963], Fig. 2; L. tulipifera: Hedges’ g = -3.70, CI= [-5.76, -1.65], Fig. 2).
Prunus virginiana experienced only a 42% decline in ΨLeaf under water-stress when planted with drought-stressed soils (Hedges’ g = -1.67, CI= [-3.05, -0.289, Fig. 2) compared to a decrease of 110% when P. virginiana was planted with control history soils (Hedges’ g = -3.86, CI= [-5.97, -1.75], Fig. 2). Quercus rubra did not decrease its ΨLeaf when water-stressed, regardless of soil history (drought-stressed microbial history: Hedges’ g = -0.713, CI= [-2.00, 0.578]; control soil history: Hedges’ g = -0.376, CI= [-1.57, 0.822], Fig. 2). Liriodendron tulipifera only decreased its ΨLeaf under water-stress when planted in control soil histories (Hedges’ g = -1.49, CI= [-2.90, -0.075], Fig. 2), while L. tulipifera planted in drought-stressed soils did not change their ΨLeaf (Hedges’ g = -0.668, CI= [-1.89, 0.552], Fig. 2).
Liriodendron tulipifera and P. virginiana saplings did not differ in their iWUE when exposed to water-stress under both soil histories and neither did Q. rubra saplings when planted in a control soil history (Fig. S1 and Table S3). However, when planted in soil with the drought-stressed history, Q. rubra water-stressed saplings had a higher iWUE compared to the well-watered saplings (Fig. S1 and Table S3), indicating a physiological response to the drought-stressed microbes.
Plant hydraulic strategy – the degree of isohydry versus anisohydry – was mostly unaffected by soil history. For instance, the slope of the line describing the relationship between ΨSoil and ΨLeaf was unaffected for P. virginiana (Fig. 3, Table S4, p=0.48) and Q. rubra saplings (Fig. 3, Table S4, p=0.43). Drought history soils pushed L. tulipifera trees to become more anisohydric, though only in the well-watered treatment (Fig. 3, Table S4, p=0.026).
Growth, biomass, and morphology
Watering regime and soil history had little effect on aboveground growth and root traits (Fig. S1 and Table S3). Watering treatment did not significantly alter stem elongation RGR and diameter RGR (Fig. S1 and Table S3). Root:shoot responses mirrored the patterns of RGR, as soil history did not influence root:shoot for any species (Fig. S1 and Table S3) and root:shoot in well-watered saplings were not different from water-stressed saplings (Fig. S1 and Table S3). Watering treatment did not significantly alter root BI, D, and SRL for any species, regardless of soil history (Fig. S1 and Table S3).
Nitrogen concentration and non-structural carbohydrates
Stem N concentrations did not differ between sapling watering treatments (Fig. S1, Table S3). For P virginiana, root N was 27% lower in the water-stressed saplings compared to the well-watered saplings when planted in soils containing drought-stressed soils, but no differences in root N among treatments for the other species was seen (Fig. S1, Table S3). Watering treatment did not influence leaf N in Q. rubra saplings. However, leaf N in P. virginiana was 15.2% lower in the water-stressed saplings when planted in drought-stressed soil history, and in L. tulipifera, leaf N was 27% higher in the water-stressed saplings compared to the well-watered saplings when planted in the control soil history (Fig. S1, Table S3).
NSC sugars were 562% higher in P. virginiana when water-stressed and planted in control history soils (Hedges’ g = 3.50, CI= [1.51, 5.49], Fig. 2), whereas those planted in soils with a history of drought increased by only 283% in response to water-stress (Hedges’ g = 1.33, CI= [0.0219, 2.65, Fig. 2). Quercus rubra had 81% less NSC sugars under water-stressed conditions when planted in soils with a history of drought-stress (Hedges’ g =-1.71, CI= [-3.25, -0.17], Fig. 2) compared to no difference in the well-watered treatment (Hedges’ g =-0.81, CI= [-2.20, 0.59], Fig. 2). Lastly, L tulipifera did not change stem concentrations of NSC sugars when water-stressed with both soil histories (Fig. 2, Table 1). There was no effect of watering treatment on NSC starches for all species regardless of soil history (Fig. 2, Table 1).
Discerning the role that soil microbes play in tree drought tolerance may lead to an improved understanding of how and why ecosystems differ in their sensitivity to water stress. In this study, we hypothesized that previously drought-stressed microbes will mediate the effects of succeeding drought stress in trees and will impact their degree of isohydry. We found partial support for our main hypothesis, as one species, P. virginiana, planted in soils with a history of drought-stress was buffered from the effects of subsequent drought, as demonstrated in their physiological responses (Fig. 2). Given that these patterns were not observed in sterilized soils (Table S2), we conclude that soil microbes (or microbially-derived residues) were likely responsible for the drought history effect. Moreover, given that NSC sugars in P. virginiana were less affected by water stress (i.e., increased less) when planted in soils with a drought history, our results support the interpretation that the soil history treatment buffered P. virginiana from drought stress (Fig. 2). We did not find support for our sub-hypothesis that soil history would influence a tree’s degree of isohydry. Collectively, our results indicate that in addition to affecting tree survivorship under contemporary drought (Allsup et al. 2023), soil drought history can influence a tree physiological responses to drought. Thus, plant-microbe interactions and previous exposure to drought may be important yet underappreciated modulators of forest responses to drought.
What factors might be responsible for the mediation of drought tolerance in P. virginiana? While a mechanistic understanding is beyond the scope of this study, microbes were likely responsible for increasing the drought tolerance of P. virginiana. When soils were sterilized – thereby negating any microbial effect – P. virginiana experienced the same A across all treatments (Table S2). This indicates that the soil history treatment was driven primarily by the characteristics of the microbial community as opposed to abiotic factors as the even P. virginiana that was well-watered were stressed by the absence of soil microbes. Further, P. virginiana planted in unsterilized soils had higher A when well-watered compared to those planted in sterilized soils (p = 0.043), adding to the argument that microbes are involved in determining plant physiological responses independent of water availability. A possible driver of microbial community change among the unsterilized treatments is drought-induced shifts in the quantity and quality of root exudates. Exudation rates and profiles often change in response to drought, especially in acquisitive, rapid C-assimilating species like P. virginiana (Williams and De Vries 2020). If changes in exudation stimulated microbes to produce more extracellular polymeric substances (EPS), ΨSoil would likely increase (Roberson and Firestone 1992; Alami et al. 2000; Costa et al. 2018). Although we did not measure EPS, P. virginiana pots that contained soils with a history of drought-stress had greater ΨSoil (Fig. S2), despite being irrigated with the same absolute amount of water as other pots within the water-stressed treatment. Changes in EPS would also have triggered the increases in A, gsw, and ΨLeaf in P. virginiana, which we observed.
Likewise, drought-induced alteration of specific microbial guilds like mycorrhizal fungi might be responsible for the observed drought history effects. P. virginiana associate with arbuscular mycorrhizal fungi (AMF) and drought-induced changes in the diversity or abundance of AMF have been linked to altered drought history effects (Allsup et al. 2023). Arbuscular mycorrhizal fungi can increase plant ΨLeaf by accessing water within microsites via small diameter, high surface area hyphae (Porcel and Ruiz-Lozano 2004), thereby mitigating the effects of chronic water stress. Additionally, AMF alter plant drought tolerance by selecting for rhizosphere microbes that minimize root desiccation and enhance soil water availability (Williams and De Vries 2020). While we did not measure the mycorrhizal colonization of P. virginiana, the greater ΨLeaf in soils with a history of drought-stress (Table 1) indicates that P. virginiana had increased access to water for leaf turgor, potentially as a result of mycorrhizal-assisted water uptake or mycorrhizal enhancement of water availability (Porcel and Ruiz-Lozano 2004).
Why then did soil history have little influence on physiological metrics in L. tulipifera and Q. rubra? Liriodendron tulipifera are strongly isohydric and close their stomata at the onset of drought (Kannenberg and Phillips 2017). As such, their soils may not dry out to the same degree, which would lessen the water stress experienced by microbes and limit microbial physiological adjustments (EPS) or activities (hyphal foraging). For instance, our biweekly watering treatment (to generate water stress) was unable to achieve especially negative water potentials in L. tulipifera pots (Fig. 3) since stomatal closure and reduced water uptake limited the water stress experienced by the plants. This may have precluded us from detecting microbial-induced shifts in isohydric-anisohydric behavior (Kannenberg and Phillips 2017). Moreover, L. tulipifera fine roots, which are relatively thick in diameter, tend to exude less C than many heterospecifics (Yin et al. 2014). Given that increases in exudation can stimulate microbial EPS production (Redmile-Gordon et al. 2015), low exudation rates in L. tulipifera may have limited EPS and its intending effects on water retention. The lack of microbial effects in Q. rubra is more puzzling. Q. rubra, which are more anisohydric than L. tulipifera (Fig. 3), experienced drier soils which presumably would have triggered strong microbial responses such as enhanced EPS production and mycorrhizal foraging. However, Q. rubra also associates with ectomycorrhizal fungi, which may have lesser effects on tree drought-tolerance (Allsup et al. 2023) owing to the high interspecific variability in fungal desiccation tolerance (Di Pietro et al. 2007). While P. virginiana appears to be nearly as anisohydric as Q. rubra (Fig. 3), rates of A in P. virginiana varied with soil water content, whereas Q. rubra tended to maintain its assimilation rate despite soil water content changes. Thus, the unique pairing of anisohydricity and photosynthetic sensitivity to water stress (McDowell et al. 2008) may have allowed us to detect (microbial-induced) changes in P. virginiana physiology but not in Q. rubra. Future work linking exudation rates and microbial-EPS in tree species with different hydraulic strategies should shed light on microbial mediation of tree drought tolerance.
Our NSC results partially align with our first hypothesis, as only P. virginiana NSCs indicated a physiological buffering when planted with drought-stressed soil microbes. P. virginiana trees increased NSC sugar concentrations under drought; however, those planted in soil with a control history had a much higher increase in NSCs than those planted in soil with a drought-stressed soil history. Since water-stress did not impact A and gsw of P. virginiana trees planted in drought-stressed soils, less osmolytes would be needed to maintain sap flow and turgor. In addition, there was an effect of water stress on Q. rubra NSC sugars in the drought-stressed soil history, most likely due to the slightly lower A and gsw under water stress, necessitating use of sugars for metabolism. On the other hand, there was no effect of water-stress on NSC concentrations in L. tulipifera regardless of soil history, an indication that its isohydric strategy led to physiological shut-down of the trees regardless of soil history. NSC starches were unchanged in all species, indicating that the water-stress treatment was not severe enough to trigger NSC sugar to starch conversion. These results mostly agree with Kannenberg and Phillips (2020) which found that NSC pools did not decrease for anisohydric or isohydric species, suggesting that hydraulic strategy may not be a robust predictor of NSC fluctuations (Kannenberg and Phillips 2020).
In our study, microbial effects on P. virginiana’s photosynthetic assimilation rates and NSCs did not coincide with aboveground growth, total biomass, or root morphology. In this way our growth results were inconsistent with Allsup and Lankau (2019), who found higher total seedling biomass under drought when seedlings were planted with microbes sourced from drier sites. Differences in the tree growth stage and the drought exposure of the inoculum may explain this paradox. The Allsup and Lankau (2019) study utilized young seedlings that were raised from seed as opposed to the 1–2-year-old saplings used in this study. Seedlings are often more sensitive to experimental drought than saplings (Cavender-Bares and Bazzaz 2000), and our short-term sapling experiment may have precluded us from detecting effects on growth due to their lower drought-sensitivity. In a similar study to Allsup and Lankau (2019), Allsup et al. (2023) found increased survivorship of seedlings under drought when planted in soils sourced from more arid sites. While we used soil with microbes sourced from a 4.5 year drought experiment, Allsup and Lankau (2019) and Allsup et al. (2023) inoculated their trees from a natural precipitation gradient where microbial drought-tolerance may have evolved over the long-term. Whether experimentally-induced droughts affect microbial communities to the same degree as moisture changes across a precipitation gradient is not well-known, though recent work indicates microbial resistance to (and recovery from) experimentally-imposed drought may be independent from a microbial community’s precipitation history (Leizeaga et al. 2021). Likewise, gas exchange responses to drought can be asymmetric to growth responses (Kannenberg et al. 2022), indicating that how trees experience drought may depend on the scale of inference. Nevertheless, whether soil history is affecting gas exchange (as shown in this study) or growth and survival (as shown in the studies of Allsup and colleagues), there is emerging evidence that plant-microbe interactions and drought legacies have the potential to shape ecosystem sensitivity to drought.
Our study is one of the first to show that soil history influences physiological drought responses of trees differently based on species identity, which builds on the growth and survivorship findings in Allsup et al. (2023). Studies solely considering how plant traits determine tree drought tolerance may therefore be missing a biotic factor: soil microbes and the histories that shape them. Microbially-mediated drought tolerance plays less of a role at the center of a range where microbes may already be well-adapted to the fluctuating environmental conditions. Rather, trees at the trailing end of its range limit that are experiencing the extreme of that species’ niche may rely more on their soil microbial communities. Thus, soil history may play a more of a role in predicting tree drought tolerance at this range limit. Without microbial mediation of drought tolerance, there could be large implications for the function and carbon storage of certain species of trees as evidenced by this experiment’s results. Overall, this study highlights that microbes that are experiencing current climate change-induced reductions in precipitation may buffer select species’ physiological stresses to future droughts, which can help us predict how ecosystems will fare in the face of climatic selective pressures.
Acknowledgements
We would like to thank Kimberly Novick, Mallory Barnes, and Michael Benson for supplying and helping with the analytical equipment. We would also like to thank Daniel Beverly for his data analysis advice. Special thanks to Elizabeth Huenupi, Adam Weiler, Amy Herendeen, Madison Berger, Mary Huynh, Damien Sparks, Zoe Worman, and Morgan Familo for their help with taking measurements and processing samples.
Funding – Funding for this grant came from the Department of Energy Environmental System Science Program (Award# DE-SC0021980). Additional funding came from the Indiana University Research and Teaching Preserve, through a grant awarded to NMS.
Conflicts of interest/Competing interests – The authors declare that they have no conflict of interest.
Ethics approval – Not applicable
Consent to participate – Not applicable
Consent for publication – Not applicable
Availability of data and material – The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability – Code available on request from the authors.
Authors' contributions - NMS and RPP conceived and designed the experiments. NMS performed the experiments, processed the samples, and analyzed the data. NMS and RPP wrote and edited the manuscript.
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Table 1 Means ± standard error of A (photosynthetic assimilation), gsw (stomatal conductance), ΨLeaf (leaf water potential), NSC sugar, and NSC starch for each of the treatment combinations and species. Superscript letters represent significant differences among treatments within a species for a given measurement.
