Uncovering the dominant role of root metabolism in shaping rhizosphere metabolome under drought in tropical rainforest plants.

Plant-soil-microbe interactions are crucial for driving rhizosphere processes that contribute to metabolite turnover and nutrient cycling. With the increasing frequency and severity of water scarcity due to climate warming, understanding how plant-mediated processes, such as root exudation, influence soil organic matter turnover in the rhizosphere is essential. In this study, we used 16S rRNA gene amplicon sequencing, rhizosphere metabolomics, and position-specific 13C-pyruvate labeling to examine the effects of three different plant species (Piper auritum, Hibiscus rosa sinensis, and Clitoria fairchildiana) and their associated microbial communities on soil organic carbon turnover in the rhizosphere. Our findings indicate that in these tropical plants, the rhizosphere metabolome is primarily shaped by the response of roots to drought rather than direct shifts in the rhizosphere bacterial community composition. Specifically, the reduced exudation of plant roots had a notable effect on the metabolome of the rhizosphere of P. auritum, with less reliance on neighboring microbes. Contrary to P. auritum, H. rosa sinensis and C. fairchildiana experienced changes in their exudate composition during drought, causing alterations to the bacterial communities in the rhizosphere. This, in turn, had a collective impact on the rhizosphere's metabolome. Furthermore, the exclusion of phylogenetically distant microbes from the rhizosphere led to shifts in its metabolome. Additionally, C. fairchildiana appeared to be associated with only a subset of symbiotic bacteria under drought conditions. These results indicate that plant species-specific microbial interactions systematically change with the root metabolome. As roots respond to drought, their associated microbial communities adapt, potentially reinforcing the drought tolerance strategies of plant roots. These findings have significant implications for maintaining plant health and preference during drought stress and improving plant performance under climate change.

Drought re-routes soil microbial carbon metabolism towards emission of volatile metabolites in an artificial tropical rainforest.

Drought impacts on microbial activity can alter soil carbon fate and lead to the loss of stored carbon to the atmosphere as CO2 and volatile organic compounds (VOCs). Here we examined drought impacts on carbon allocation by soil microbes in the Biosphere 2 artificial tropical rainforest by tracking 13C from position-specific 13C-pyruvate into CO2 and VOCs in parallel with multi-omics. During drought, efflux of 13C-enriched acetate, acetone and C4H6O2 (diacetyl) increased. These changes represent increased production and buildup of intermediate metabolites driven by decreased carbon cycling efficiency. Simultaneously,13C-CO2 efflux decreased, driven by a decrease in microbial activity. However, the microbial carbon allocation to energy gain relative to biosynthesis was unchanged, signifying maintained energy demand for biosynthesis of VOCs and other drought-stress-induced pathways. Overall, while carbon loss to the atmosphere via CO2 decreased during drought, carbon loss via efflux of VOCs increased, indicating microbially induced shifts in soil carbon fate.

Asthma-protective agents in dust from traditional farm environments.

BACKGROUND: Growing up on traditional European or US Amish dairy farms in close contact with cows and hay protects children against asthma, and airway administration of extracts from dust collected from cowsheds of those farms prevents allergic asthma in mice. OBJECTIVES: This study sought to begin identifying farm-derived asthma-protective agents. METHODS: Our work unfolded along 2 unbiased and independent but complementary discovery paths. Dust extracts (DEs) from protective and nonprotective farms (European and Amish cowsheds vs European sheep sheds) were analyzed by comparative nuclear magnetic resonance profiling and differential proteomics. Bioactivity-guided size fractionation focused on protective Amish cowshed DEs. Multiple in vitro and in vivo functional assays were used in both paths. Some of the proteins thus identified were characterized by in-solution and in-gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis enzymatic digestion/peptide mapping followed by liquid chromatography/mass spectrometry. The cargo carried by these proteins was analyzed by untargeted liquid chromatography-high-resolution mass spectrometry. RESULTS: Twelve carrier proteins of animal and plant origin, including the bovine lipocalins Bos d 2 and odorant binding protein, were enriched in DEs from protective European cowsheds. A potent asthma-protective fraction of Amish cowshed DEs (≈0.5% of the total carbon content of unfractionated extracts) contained 7 animal and plant proteins, including Bos d 2 and odorant binding protein loaded with fatty acid metabolites from plants, bacteria, and fungi. CONCLUSIONS: Animals and plants from traditional farms produce proteins that transport hydrophobic microbial and plant metabolites. When delivered to mucosal surfaces, these agents might regulate airway responses.

Automating methods for estimating metabolite volatility.

The volatility of metabolites can influence their biological roles and inform optimal methods for their detection. Yet, volatility information is not readily available for the large number of described metabolites, limiting the exploration of volatility as a fundamental trait of metabolites. Here, we adapted methods to estimate vapor pressure from the functional group composition of individual molecules (SIMPOL.1) to predict the gas-phase partitioning of compounds in different environments. We implemented these methods in a new open pipeline called volcalc that uses chemoinformatic tools to automate these volatility estimates for all metabolites in an extensive and continuously updated pathway database: the Kyoto Encyclopedia of Genes and Genomes (KEGG) that connects metabolites, organisms, and reactions. We first benchmark the automated pipeline against a manually curated data set and show that the same category of volatility (e.g., nonvolatile, low, moderate, high) is predicted for 93% of compounds. We then demonstrate how volcalc might be used to generate and test hypotheses about the role of volatility in biological systems and organisms. Specifically, we estimate that 3.4 and 26.6% of compounds in KEGG have high volatility depending on the environment (soil vs. clean atmosphere, respectively) and that a core set of volatiles is shared among all domains of life (30%) with the largest proportion of kingdom-specific volatiles identified in bacteria. With volcalc, we lay a foundation for uncovering the role of the volatilome using an approach that is easily integrated with other bioinformatic pipelines and can be continually refined to consider additional dimensions to volatility. The volcalc package is an accessible tool to help design and test hypotheses on volatile metabolites and their unique roles in biological systems.

Elucidating Drought-Tolerance Mechanisms in Plant Roots through 1H NMR Metabolomics in Parallel with MALDI-MS, and NanoSIMS Imaging Techniques.

As direct mediators between plants and soil, roots play an important role in metabolic responses to environmental stresses such as drought, yet these responses are vastly uncharacterized on a plant-specific level, especially for co-occurring species. Here, we aim to examine the effects of drought on root metabolic profiles and carbon allocation pathways of three tropical rainforest species by combining cutting-edge metabolomic and imaging technologies in an in situ position-specific 13C-pyruvate root-labeling experiment. Further, washed (rhizosphere-depleted) and unwashed roots were examined to test the impact of microbial presence on root metabolic pathways. Drought had a species-specific impact on the metabolic profiles and spatial distribution in Piper sp. and Hibiscus rosa sinensis roots, signifying different defense mechanisms; Piper sp. enhanced root structural defense via recalcitrant compounds including lignin, while H. rosa sinensis enhanced biochemical defense via secretion of antioxidants and fatty acids. In contrast, Clitoria fairchildiana, a legume tree, was not influenced as much by drought but rather by rhizosphere presence where carbohydrate storage was enhanced, indicating a close association with symbiotic microbes. This study demonstrates how multiple techniques can be combined to identify how plants cope with drought through different drought-tolerance strategies and the consequences of such changes on below-ground organic matter composition.

Ecosystem fluxes during drought and recovery in an experimental forest.

Severe droughts endanger ecosystem functioning worldwide. We investigated how drought affects carbon and water fluxes as well as soil-plant-atmosphere interactions by tracing 13CO2 and deep water 2H2O label pulses and volatile organic compounds (VOCs) in an enclosed experimental rainforest. Ecosystem dynamics were driven by different plant functional group responses to drought. Drought-sensitive canopy trees dominated total fluxes but also exhibited the strongest response to topsoil drying. Although all canopy-forming trees had access to deep water, these reserves were spared until late in the drought. Belowground carbon transport was slowed, yet allocation of fresh carbon to VOCs remained high. Atmospheric VOC composition reflected increasing stress responses and dynamic soil-plant-atmosphere interactions, potentially affecting atmospheric chemistry and climate feedbacks. These interactions and distinct functional group strategies thus modulate drought impacts and ecosystem susceptibility to climate change.

The role of innate immunity in asthma development and protection: Lessons from the environment.

Asthma, a complex, chronic disease characterized by airway inflammation, hyperresponsiveness and remodelling, affects over 300 million people worldwide. While the disease is typically associated with exaggerated allergen-induced type 2 immune responses, these responses are strongly influenced by environmental exposures that stimulate innate immune pathways capable of promoting or protecting from asthma. The dual role played by innate immunity in asthma pathogenesis offers multiple opportunities for both research and clinical interventions and is the subject of this review.

Effect of Re-acidification on Buffalo Grass Rhizosphere and Bulk Microbial Communities During Phytostabilization of Metalliferous Mine Tailings.

Phytostabilized highly acidic, pyritic mine tailings are susceptible to re-acidification over time despite initial addition of neutralizing amendments. Studies examining plant-associated microbial dynamics during re-acidification of phytostabilized regions are sparse. To address this, we characterized the rhizosphere and bulk bacterial communities of buffalo grass used in the phytostabilization of metalliferous, pyritic mine tailings undergoing re-acidification at the Iron King Mine and Humboldt Smelter Superfund Site in Dewey-Humboldt, AZ. Plant-associated substrates representing a broad pH range (2.35-7.76) were sampled to (1) compare the microbial diversity and community composition of rhizosphere and bulk compartments across a pH gradient, and (2) characterize how re-acidification affects the abundance and activity of the most abundant plant growth-promoting bacteria (PGPB; including N2-fixing) versus acid-generating bacteria (AGB; including Fe-cycling/S-oxidizing). Results indicated that a shift in microbial diversity and community composition occurred at around pH 4. At higher pH (>4) the species richness and community composition of the rhizosphere and bulk compartments were similar, and PGPB, such as Pseudomonas, Arthrobacter, Devosia, Phyllobacterium, Sinorhizobium, and Hyphomicrobium, were present and active in both compartments with minimal presence of AGB. In comparison, at lower pH (<4) the rhizosphere had a significantly higher number of species than the bulk (p < 0.05) and the compartments had significantly different community composition (unweighted UniFrac; PERMANOVA, p < 0.05). Whereas some PGPB persisted in the rhizosphere at lower pH, including Arthrobacter and Devosia, they were absent from the bulk. Meanwhile, AGB dominated in both compartments; the most abundant were the Fe-oxidizer Leptospirillum and Fe-reducers Acidibacter and Acidiphilium, and the most active was the Fe-reducer Aciditerrimonas. This predominance of AGB at lower pH, and even their minimal presence at higher pH, contributes to acidifying conditions and poses a significant threat to sustainable plant establishment. These findings have implications for phytostabilization field site management and suggest re-application of compost or an alternate buffering material may be required in regions susceptible to re-acidification to maintain a beneficial bacterial community conducive to long-term plant establishment.

Bacterial Rhizoplane Colonization Patterns of Buchloe dactyloides Growing in Metalliferous Mine Tailings Reflect Plant Status and Biogeochemical Conditions.

Plant establishment during phytostabilization of legacy mine tailings in semiarid regions is challenging due to low pH, low organic carbon, low nutrients, and high toxic metal(loid) concentrations. Plant-associated bacterial communities are particularly important under these harsh conditions because of their beneficial services to plants. We hypothesize that bacterial colonization profiles on rhizoplane surfaces reflect deterministic processes that are governed by plant health and the root environment. The aim of this study was to identify associations between bacterial colonization patterns on buffalo grass (Buchloe dactyloides) rhizoplanes and both plant status (leaf chlorophyll and plant cover) and substrate biogeochemistry (pH, electrical conductivity, total organic carbon, total nitrogen, and rhizosphere microbial community). Buffalo grass plants from mesocosm- and field-scale phytostabilization trials conducted with tailings from the Iron King Mine and Humboldt Smelter Superfund Site in Dewey-Humboldt, Arizona, were analyzed. These tailings are extremely acidic and have arsenic and lead concentrations of 2-4 g kg-1 substrate. Bacterial communities on rhizoplanes and in rhizosphere-associated substrate were characterized using fluorescence in situ hybridization and 16S rRNA gene amplicon sequencing, respectively. The results indicated that the metabolic status of rhizoplane bacterial colonizers is significantly related to plant health. Principal component analysis revealed that root-surface Alphaproteobacteria relative abundance was associated most strongly with substrate pH and Gammaproteobacteria relative abundance associated strongly with substrate pH and plant cover. These factors also affected the phylogenetic profiles of the associated rhizosphere communities. In summary, rhizoplane bacterial colonization patterns are plant specific and influenced by plant status and rhizosphere biogeochemical conditions.

Resolving colocalization of bacteria and metal(loid)s on plant root surfaces by combining fluorescence in situ hybridization (FISH) with multiple-energy micro-focused X-ray fluorescence (ME µXRF).

Metal(loid)-contamination of the environment due to anthropogenic activities is a global problem. Understanding the fate of contaminants requires elucidation of biotic and abiotic factors that influence metal(loid) speciation from molecular to field scales. Improved methods are needed to assess micro-scale processes, such as those occurring at biogeochemical interfaces between plant tissues, microbial cells, and metal(loid)s. Here we present an advanced method that combines fluorescence in situ hybridization (FISH) with synchrotron-based multiple-energy micro-focused X-ray fluorescence microprobe imaging (ME µXRF) to examine colocalization of bacteria and metal(loid)s on root surfaces of plants used to phytostabilize metalliferous mine tailings. Bacteria were visualized on a small root section using SytoBC nucleic acid stain and FISH probes targeting the domain Bacteria and a specific group (Alphaproteobacteria, Gammaproteobacteria, or Actinobacteria). The same root region was then analyzed for elemental distribution and metal(loid) speciation of As and Fe using ME µXRF. The FISH and ME µXRF images were aligned using ImageJ software to correlate microbiological and geochemical results. Results from quantitative analysis of colocalization show a significantly higher fraction of As colocalized with Fe-oxide plaques on the root surfaces (fraction of overlap 0.49±0.19) than to bacteria (0.072±0.052) (p<0.05). Of the bacteria that colocalized with metal(loid)s, Actinobacteria, known for their metal tolerance, had a higher correlation with both As and Fe than Alphaproteobacteria or Gammaproteobacteria. This method demonstrates how coupling these micro-techniques can expand our understanding of micro-scale interactions between roots, metal(loid)s and microbes, information that should lead to improved mechanistic models of metal(loid) speciation and fate.