A multi-omic survey of black cottonwood tissues highlights coordinated transcriptomic and metabolomic mechanisms for plant adaptation to phosphorus deficiency.

Introduction: Phosphorus (P) deficiency in plants creates a variety of metabolic perturbations that decrease photosynthesis and growth. Phosphorus deficiency is especially challenging for the production of bioenergy feedstock plantation species, such as poplars (Populus spp.), where fertilization may not be practically or economically feasible. While the phenotypic effects of P deficiency are well known, the molecular mechanisms underlying whole-plant and tissue-specific responses to P deficiency, and in particular the responses of commercially valuable hardwoods, are less studied. Methods: We used a multi-tissue and multi-omics approach using transcriptomic, proteomic, and metabolomic analyses of the leaves and roots of black cottonwood (Populus trichocarpa) seedlings grown under P-deficient (5 µM P) and replete (100 µM P) conditions to assess this knowledge gap and to identify potential gene targets for selection for P efficiency. Results: In comparison to seedlings grown at 100 µM P, P-deficient seedlings exhibited reduced dry biomass, altered chlorophyll fluorescence, and reduced tissue P concentrations. In line with these observations, growth, C metabolism, and photosynthesis pathways were downregulated in the transcriptome of the P-deficient plants. Additionally, we found evidence of strong lipid remodeling in the leaves. Metabolomic data showed that the roots of P-deficient plants had a greater relative abundance of phosphate ion, which may reflect extensive degradation of P-rich metabolites in plants exposed to long-term P-deficiency. With the notable exception of the KEGG pathway for Starch and Sucrose Metabolism (map00500), the responses of the transcriptome and the metabolome to P deficiency were consistent with one another. No significant changes in the proteome were detected in response to P deficiency. Discussion and conclusion: Collectively, our multi-omic and multi-tissue approach enabled the identification of important metabolic and regulatory pathways regulated across tissues at the molecular level that will be important avenues to further evaluate for P efficiency. These included stress-mediating systems associated with reactive oxygen species maintenance, lipid remodeling within tissues, and systems involved in P scavenging from the rhizosphere.

Plant-microbial responses to reduced precipitation depend on tree species in a temperate forest.

Given that global change is predicted to increase the frequency and severity of drought in temperate forests, it is critical to understand the degree to which plant belowground responses cascade through the soil system to drive ecosystem responses to water stress. While most research has focused on plant and microbial responses independently of each other, a gap in our understanding lies in the integrated response of plant-microbial interactions to water stress. We investigated the extent to which divergent belowground responses to reduced precipitation between sugar maple trees (Acer saccharum) versus oak trees (Oak spp.) may influence microbial activity via throughfall exclusion in the field. Evidence that oak trees send carbon belowground to prime microbial activity more than maples under ambient conditions and in response to water stress suggests there is the potential for corresponding impacts of reduced precipitation on microbial activity. As such, we tested the hypothesis that differences in belowground C allocation between oaks and maples would stimulate microbial activity in the oak treatment soils and reduce microbial activity in in the sugar maple treatment soils compared to their respective controls. We found that the treatment led to declines in N mineralization, soil respiration, and oxidative enzyme activity in the sugar maple treatment plot. These declines may be due to sugar maple trees reducing root C transfers to the soil. By contrast, the reduced precipitation treatment enhanced soil respiration, as well as rates of N mineralization and peroxidase activity in the oak rhizosphere. This enhanced activity suggests that oak roots provided optimal rhizosphere conditions during water stress to prime microbial activity to support net primary production. With future changes in precipitation predicted for forests in the Eastern US, we show that the strength of plant-microbial interactions drives the degree to which reduced precipitation impacts soil C and nutrient cycling.

Fast-decaying plant litter enhances soil carbon in temperate forests but not through microbial physiological traits.

Conceptual and empirical advances in soil biogeochemistry have challenged long-held assumptions about the role of soil micro-organisms in soil organic carbon (SOC) dynamics; yet, rigorous tests of emerging concepts remain sparse. Recent hypotheses suggest that microbial necromass production links plant inputs to SOC accumulation, with high-quality (i.e., rapidly decomposing) plant litter promoting microbial carbon use efficiency, growth, and turnover leading to more mineral stabilization of necromass. We test this hypothesis experimentally and with observations across six eastern US forests, using stable isotopes to measure microbial traits and SOC dynamics. Here we show, in both studies, that microbial growth, efficiency, and turnover are negatively (not positively) related to mineral-associated SOC. In the experiment, stimulation of microbial growth by high-quality litter enhances SOC decomposition, offsetting the positive effect of litter quality on SOC stabilization. We suggest that microbial necromass production is not the primary driver of SOC persistence in temperate forests. Factors such as microbial necromass origin, alternative SOC formation pathways, priming effects, and soil abiotic properties can strongly decouple microbial growth, efficiency, and turnover from mineral-associated SOC.

Altered plant carbon partitioning enhanced forest ecosystem carbon storage after 25 years of nitrogen additions.

Decades of atmospheric nitrogen (N) deposition in the northeastern USA have enhanced this globally important forest carbon (C) sink by relieving N limitation. While many N fertilization experiments found increased forest C storage, the mechanisms driving this response at the ecosystem scale remain uncertain. Following the optimal allocation theory, augmented N availability may reduce belowground C investment by trees to roots and soil symbionts. To test this prediction and its implications on soil biogeochemistry, we constructed C and N budgets for a long-term, whole-watershed N fertilization study at the Fernow Experimental Forest, WV, USA. Nitrogen fertilization increased C storage by shifting C partitioning away from belowground components and towards aboveground woody biomass production. Fertilization also reduced the C cost of N acquisition, allowing for greater C sequestration in vegetation. Despite equal fine litter inputs, the C and N stocks and C : N ratio of the upper mineral soil were greater in the fertilized watershed, likely due to reduced decomposition of plant litter. By combining aboveground and belowground data at the watershed scale, this study demonstrates how plant C allocation responses to N additions may result in greater C storage in both vegetation and soil.

Root-derived inputs are major contributors to soil carbon in temperate forests, but vary by mycorrhizal type.

Roots promote the formation of slow-cycling soil carbon (C), yet we have a limited understanding of the magnitude and controls on this flux. We hypothesised arbuscular mycorrhizal (AM)- and ectomycorrhizal (ECM)-associated trees would exhibit differences in root-derived C accumulation in the soil, and that much of this C would be transferred into mineral-associated pools. We installed δ13 C-enriched ingrowth cores across mycorrhizal gradients in six Eastern U.S. forests (n = 54 plots). Overall, root-derived C was 54% greater in AM versus ECM-dominated plots. This resulted in nearly twice as much root-derived C in putatively slow-cycling mineral-associated pools in AM compared to ECM plots. Given that our estimates of root-derived inputs were often equal to or greater than leaf litter inputs, our results suggest that variation in root-derived soil C accumulation due to tree mycorrhizal dominance may be a key control of soil C dynamics in forests.

Differences in microbial community response to nitrogen fertilization result in unique enzyme shifts between arbuscular and ectomycorrhizal-dominated soils.

While the effect of nitrogen (N) deposition on belowground carbon (C) cycling varies, emerging evidence shows that forest soils dominated by trees that associate with ectomycorrhizal fungi (ECM) store more C than soils dominated by trees that associate with arbuscular mycorrhizae (AM) with increasing N deposition. We hypothesized that this is due to unique nutrient cycling responses to N between AM and ECM-dominated soils. ECM trees primarily obtain N through fungal mining of soil organic matter subsidized by root-C. As such, we expected the largest N-induced responses of C and N cycling to occur in ECM rhizospheres and be driven by fungi. Conversely, as AM trees rely on bacterial scavengers in bulk soils to cycle N, we predicted the largest AM responses to be driven by shifts in bacteria and occur in bulk soils. To test this hypothesis, we measured microbial community composition, metatranscriptome profiles, and extracellular enzyme activity in bulk, rhizosphere, and organic horizon (OH) soils in AM and ECM-dominated soils at Bear Brook Watershed in Maine, USA. After 27 years of N fertilization, fungal community composition shifted across ECM soils, but bacterial communities shifted across AM soils. These shifts were mirrored by enhanced C relative to N mining enzyme activities in both mycorrhizal types, but this occurred in different soil fractions. In ECM stands these shifts occurred in rhizosphere soils, but in AM stands they occurred in bulk soils. Additionally, ECM OH soils exhibited the opposite response with declines in C relative to N mining. As rhizosphere soils account for only a small portion of total soil volume relative to bulk soils, coupled with declines in C to N enzyme activity in ECM OH soils, we posit that this may partly explain why ECM soils store more C than AM soils as N inputs increase.

Interactions among plants, bacteria, and fungi reduce extracellular enzyme activities under long-term N fertilization.

Atmospheric nitrogen (N) deposition has enhanced soil carbon (C) stocks in temperate forests. Most research has posited that these soil C gains are driven primarily by shifts in fungal community composition with elevated N leading to declines in lignin degrading Basidiomycetes. Recent research, however, suggests that plants and soil microbes are dynamically intertwined, whereby plants send C subsidies to rhizosphere microbes to enhance enzyme production and the mobilization of N. Thus, under elevated N, trees may reduce belowground C allocation leading to cascading impacts on the ability of microbes to degrade soil organic matter through a shift in microbial species and/or a change in plant-microbe interactions. The objective of this study was to determine the extent to which couplings among plant, fungal, and bacterial responses to N fertilization alter the activity of enzymes that are the primary agents of soil decomposition. We measured fungal and bacterial community composition, root-microbial interactions, and extracellular enzyme activity in the rhizosphere, bulk, and organic horizon of soils sampled from a long-term (>25 years), whole-watershed, N fertilization experiment at the Fernow Experimental Forest in West Virginia, USA. We observed significant declines in plant C investment to fine root biomass (24.7%), root morphology, and arbuscular mycorrhizal (AM) colonization (55.9%). Moreover, we found that declines in extracellular enzyme activity were significantly correlated with a shift in bacterial community composition, but not fungal community composition. This bacterial community shift was also correlated with reduced AM fungal colonization indicating that declines in plant investment belowground drive the response of bacterial community structure and function to N fertilization. Collectively, we find that enzyme activity responses to N fertilization are not solely driven by fungi, but instead reflect a whole ecosystem response, whereby declines in the strength of belowground C investment to gain N cascade through the soil environment.

Feedbacks between plant N demand and rhizosphere priming depend on type of mycorrhizal association.

Ecosystem carbon (C) balance is hypothesised to be sensitive to the mycorrhizal strategies that plants use to acquire nutrients. To test this idea, we coupled an optimality-based plant nitrogen (N) acquisition model with a microbe-focused soil organic matter (SOM) model. The model accurately predicted rhizosphere processes and C-N dynamics across a gradient of stands varying in their relative abundance of arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) trees. When mycorrhizal dominance was switched - ECM trees dominating plots previously occupied by AM trees, and vice versa - legacy effects were apparent, with consequences for both C and N stocks in soil. Under elevated productivity, ECM trees enhanced decomposition more than AM trees via microbial priming of unprotected SOM. Collectively, our results show that ecosystem responses to global change may hinge on the balance between rhizosphere priming and SOM protection, and highlight the importance of dynamically linking plants and microbes in terrestrial biosphere models.

Dominant mycorrhizal association of trees alters carbon and nutrient cycling by selecting for microbial groups with distinct enzyme function.

While it is well established that plants associating with arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi cycle carbon (C) and nutrients in distinct ways, we have a limited understanding of whether varying abundance of ECM and AM plants in a stand can provide integrative proxies for key biogeochemical processes. We explored linkages between the relative abundance of AM and ECM trees and microbial functioning in three hardwood forests in southern Indiana, USA. Across each site's 'mycorrhizal gradient', we measured fungal biomass, fungal : bacterial (F : B) ratios, extracellular enzyme activities, soil carbon : nitrogen ratio, and soil pH over a growing season. We show that the percentage of AM or ECM trees in a plot promotes microbial communities that both reflect and determine the C to nutrient balance in soil. Soils dominated by ECM trees had higher F : B ratios and more standing fungal biomass than AM stands. Enzyme stoichiometry in ECM soils shifted to higher investment in extracellular enzymes needed for nitrogen and phosphorus acquisition than in C-acquisition enzymes, relative to AM soils. Our results suggest that knowledge of mycorrhizal dominance at the stand or landscape scale may provide a unifying framework for linking plant and microbial community dynamics, and predicting their effects on ecological function.

Tree-mycorrhizal associations detected remotely from canopy spectral properties.

A central challenge in global ecology is the identification of key functional processes in ecosystems that scale, but do not require, data for individual species across landscapes. Given that nearly all tree species form symbiotic relationships with one of two types of mycorrhizal fungi - arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi - and that AM- and ECM-dominated forests often have distinct nutrient economies, the detection and mapping of mycorrhizae over large areas could provide valuable insights about fundamental ecosystem processes such as nutrient cycling, species interactions, and overall forest productivity. We explored remotely sensed tree canopy spectral properties to detect underlying mycorrhizal association across a gradient of AM- and ECM-dominated forest plots. Statistical mining of reflectance and reflectance derivatives across moderate/high-resolution Landsat data revealed distinctly unique phenological signals that differentiated AM and ECM associations. This approach was trained and validated against measurements of tree species and mycorrhizal association across ~130 000 trees throughout the temperate United States. We were able to predict 77% of the variation in mycorrhizal association distribution within the forest plots (P < 0.001). The implications for this work move us toward mapping mycorrhizal association globally and advancing our understanding of biogeochemical cycling and other ecosystem processes.

Carbon cost of plant nitrogen acquisition: global carbon cycle impact from an improved plant nitrogen cycle in the Community Land Model.

Plants typically expend a significant portion of their available carbon (C) on nutrient acquisition - C that could otherwise support growth. However, given that most global terrestrial biosphere models (TBMs) do not include the C cost of nutrient acquisition, these models fail to represent current and future constraints to the land C sink. Here, we integrated a plant productivity-optimized nutrient acquisition model - the Fixation and Uptake of Nitrogen Model - into one of the most widely used TBMs, the Community Land Model. Global plant nitrogen (N) uptake is dynamically simulated in the coupled model based on the C costs of N acquisition from mycorrhizal roots, nonmycorrhizal roots, N-fixing microbes, and retranslocation (from senescing leaves). We find that at the global scale, plants spend 2.4 Pg C yr(-1) to acquire 1.0 Pg N yr(-1) , and that the C cost of N acquisition leads to a downregulation of global net primary production (NPP) by 13%. Mycorrhizal uptake represented the dominant pathway by which N is acquired, accounting for ~66% of the N uptake by plants. Notably, roots associating with arbuscular mycorrhizal (AM) fungi - generally considered for their role in phosphorus (P) acquisition - are estimated to be the primary source of global plant N uptake owing to the dominance of AM-associated plants in mid- and low-latitude biomes. Overall, our coupled model improves the representations of NPP downregulation globally and generates spatially explicit patterns of belowground C allocation, soil N uptake, and N retranslocation at the global scale. Such model improvements are critical for predicting how plant responses to altered N availability (owing to N deposition, rising atmospheric CO2 , and warming temperatures) may impact the land C sink.

Mycorrhizal type determines the magnitude and direction of root-induced changes in decomposition in a temperate forest.

Although it is increasingly being recognized that roots play a key role in soil carbon (C) dynamics, the magnitude and direction of these effects are unknown. Roots can accelerate soil C losses by provisioning microbes with energy to decompose organic matter or impede soil C losses by enhancing microbial competition for nutrients. We experimentally reduced belowground C supply to soils via tree girdling, and contrasted responses in control and girdled plots for three consecutive growing seasons. We hypothesized that decreases in belowground C supply would have stronger effects in plots dominated by ectomycorrhizal (ECM) trees rather than arbuscular mycorrhizal (AM) trees. In ECM-dominated plots, girdling decreased the activity of enzymes that break down soil organic matter (SOM) by c. 40%, indicating that, in control plots, C supply from ECM roots primes microbial decomposition. In AM-dominated plots, girdling had little effect on SOM-degrading enzymes, but increased the decomposition of AM leaf litter by c. 43%, suggesting that, in control plots, AM roots may intensify microbial competition for nutrients. Our findings indicate that root-induced changes in soil processes depend on forest composition, and that shifts in the distribution of AM and ECM trees owing to climate change may determine soil C gains and losses.

Rhizosphere processes are quantitatively important components of terrestrial carbon and nutrient cycles.

While there is an emerging view that roots and their associated microbes actively alter resource availability and soil organic matter (SOM) decomposition, the ecosystem consequences of such rhizosphere effects have rarely been quantified. Using a meta-analysis, we show that multiple indices of microbially mediated C and nitrogen (N) cycling, including SOM decomposition, are significantly enhanced in the rhizospheres of diverse vegetation types. Then, using a numerical model that combines rhizosphere effect sizes with fine root morphology and depth distributions, we show that root-accelerated mineralization and priming can account for up to one-third of the total C and N mineralized in temperate forest soils. Finally, using a stoichiometrically constrained microbial decomposition model, we show that these effects can be induced by relatively modest fluxes of root-derived C, on the order of 4% and 6% of gross and net primary production, respectively. Collectively, our results indicate that rhizosphere processes are a widespread, quantitatively important driver of SOM decomposition and nutrient release at the ecosystem scale, with potential consequences for global C stocks and vegetation feedbacks to climate.

The rhizosphere and hyphosphere differ in their impacts on carbon and nitrogen cycling in forests exposed to elevated CO2.

While multiple experiments have demonstrated that trees exposed to elevated CO2 can stimulate microbes to release nutrients from soil organic matter, the importance of root- versus mycorrhizal-induced changes in soil processes are presently unknown. We analyzed the contribution of roots and mycorrhizal activities to carbon (C) and nitrogen (N) turnover in a loblolly pine (Pinus taeda) forest exposed to elevated CO2 by measuring extracellular enzyme activities at soil microsites accessed via root windows. Specifically, we quantified enzyme activity from soil adjacent to root tips (rhizosphere), soil adjacent to hyphal tips (hyphosphere), and bulk soil. During the peak growing season, CO2 enrichment induced a greater increase of N-releasing enzymes in the rhizosphere (215% increase) than in the hyphosphere (36% increase), but a greater increase of recalcitrant C-degrading enzymes in the hyphosphere (118%) than in the rhizosphere (19%). Nitrogen fertilization influenced the magnitude of CO2 effects on enzyme activities in the rhizosphere only. At the ecosystem scale, the rhizosphere accounted for c. 50% and 40% of the total activity of N- and C-releasing enzymes, respectively. Collectively, our results suggest that root exudates may contribute more to accelerated N cycling under elevated CO2 at this site, while mycorrhizal fungi may contribute more to soil C degradation.

Chronic water stress reduces tree growth and the carbon sink of deciduous hardwood forests.

Predicted decreases in water availability across the temperate forest biome have the potential to offset gains in carbon (C) uptake from phenology trends, rising atmospheric CO2 , and nitrogen deposition. While it is well established that severe droughts reduce the C sink of forests by inducing tree mortality, the impacts of mild but chronic water stress on forest phenology and physiology are largely unknown. We quantified the C consequences of chronic water stress using a 13-year record of tree growth (n = 200 trees), soil moisture, and ecosystem C balance at the Morgan-Monroe State Forest (MMSF) in Indiana, and a regional 11-year record of tree growth (n > 300 000 trees) and water availability for the 20 most dominant deciduous broadleaf tree species across the eastern and midwestern USA. We show that despite ~26 more days of C assimilation by trees at the MMSF, increasing water stress decreased the number of days of wood production by ~42 days over the same period, reducing the annual accrual of C in woody biomass by 41%. Across the deciduous forest region, water stress induced similar declines in tree growth, particularly for water-demanding 'mesophytic' tree species. Given the current replacement of water-stress adapted 'xerophytic' tree species by mesophytic tree species, we estimate that chronic water stress has the potential to decrease the C sink of deciduous forests by up to 17% (0.04 Pg C yr(-1) ) in the coming decades. This reduction in the C sink due to mesophication and chronic water stress is equivalent to an additional 1-3 days of global C emissions from fossil fuel burning each year. Collectively, our results indicate that regional declines in water availability may offset the growth-enhancing effects of other global changes and reduce the extent to which forests ameliorate climate warming.

An improved approach for remotely sensing water stress impacts on forest C uptake.

Given that forests represent the primary terrestrial sink for atmospheric CO2 , projections of future carbon (C) storage hinge on forest responses to climate variation. Models of gross primary production (GPP) responses to water stress are commonly based on remotely sensed changes in canopy 'greenness' (e.g., normalized difference vegetation index; NDVI). However, many forests have low spectral sensitivity to water stress (SSWS) - defined here as drought-induced decline in GPP without a change in greenness. Current satellite-derived estimates of GPP use a vapor pressure deficit (VPD) scalar to account for the low SWSS of forests, but fail to capture their responses to water stress. Our objectives were to characterize differences in SSWS among forested and nonforested ecosystems, and to develop an improved framework for predicting the impacts of water stress on GPP in forests with low SSWS. First, we paired two independent drought indices with NDVI data for the conterminous US from 2000 to 2011, and examined the relationship between water stress and NDVI. We found that forests had lower SSWS than nonforests regardless of drought index or duration. We then compared satellite-derived estimates of GPP with eddy-covariance observations of GPP in two deciduous broadleaf forests with low SSWS: the Missouri Ozark (MO) and Morgan Monroe State Forest (MMSF) AmeriFlux sites. Model estimates of GPP that used VPD scalars were poorly correlated with observations of GPP at MO (r(2) = 0.09) and MMSF (r(2) = 0.38). When we included the NDVI responses to water stress of adjacent ecosystems with high SSWS into a model based solely on temperature and greenness, we substantially improved predictions of GPP at MO (r(2) = 0.83) and for a severe drought year at the MMSF (r(2) = 0.82). Collectively, our results suggest that large-scale estimates of GPP that capture variation in SSWS among ecosystems could improve predictions of C uptake by forests under drought.

Substrate supply, fine roots, and temperature control proteolytic enzyme activity in temperate forest soils.

Temperature and substrate availability constrain the activity of the extracellular enzymes that decompose and release nutrients from soil organic matter (SOM). Proteolytic enzymes are the primary class of enzymes involved in the depolymerization of nitrogen (N) from proteinaceous components of SOM, and their activity affects the rate of N cycling in forest soils. The objectives of this study were to determine whether and how temperature and substrate availability affect the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ectomycorrhizal or arbuscular mycorrhizal fungi. Temperature limitation of proteolytic enzyme activity was observed only early in the growing season when soil temperatures in the field were near 4 degrees C. Substrate limitation to proteolytic activity persisted well into the growing season. Ligninolytic enzyme activity was higher in soils dominated by ectomycorrhizal associated tree species. In contrast, the activity of proteolytic and chitinolytic enzymes did not differ, but there were differences between mycorrhizal association in the control of roots on enzyme activity. Roots of ectomycorrhizal species but not those of arbuscular mycorrhizal species exerted significant control over proteolytic, chitinolytic, and ligninolytic enzyme activity; the absence of ectomycorrhizal fine roots reduced the activity of all three enzymes. These results suggest that climate warming in the absence of increases in substrate availability may have a modest effect on soil-N cycling, and that global changes that alter belowground carbon allocation by trees are likely to have a larger effect on nitrogen cycling in stands dominated by ectomycorrhizal fungi.