Identifying causes and consequences of rhizosphere microbiome heritability.

Host genotype affects microbiome composition in many plants, but the mechanisms and implications of this phenomenon are understudied. New work in PLOS Biology illustrates how host genotype leads to differential gene expression and fitness in bacteria of the barley rhizosphere.

Microbe-dependent heterosis in maize.

Hybrids account for nearly all commercially planted varieties of maize and many other crop plants because crosses between inbred lines of these species produce first-generation [F1] offspring that greatly outperform their parents. The mechanisms underlying this phenomenon, called heterosis or hybrid vigor, are not well understood despite over a century of intensive research. The leading hypotheses-which focus on quantitative genetic mechanisms (dominance, overdominance, and epistasis) and molecular mechanisms (gene dosage and transcriptional regulation)-have been able to explain some but not all of the observed patterns of heterosis. Abiotic stressors are known to impact the expression of heterosis; however, the potential role of microbes in heterosis has largely been ignored. Here, we show that heterosis of root biomass and other traits in maize is strongly dependent on the belowground microbial environment. We found that, in some cases, inbred lines perform as well by these criteria as their F1 offspring under sterile conditions but that heterosis can be restored by inoculation with a simple community of seven bacterial strains. We observed the same pattern for seedlings inoculated with autoclaved versus live soil slurries in a growth chamber and for plants grown in steamed or fumigated versus untreated soil in the field. In a different field site, however, soil steaming increased rather than decreased heterosis, indicating that the direction of the effect depends on community composition, environment, or both. Together, our results demonstrate an ecological phenomenon whereby soil microbes differentially impact the early growth of inbred and hybrid maize.

Soil variation among natural habitats alters glucosinolate content in a wild perennial mustard.

Baseline levels of glucosinolates-important defensive phytochemicals in brassicaceous plants-are determined by both genotype and environment. However, the ecological causes of glucosinolate plasticity are not well characterized. Fertilization is known to alter glucosinolate content of Brassica crops, but the effect of naturally occurring soil variation on glucosinolate content of wild plants is unknown. Here, we conducted greenhouse experiments using Boechera stricta to ask (i) whether soil variation among natural habitats shapes leaf and root glucosinolate profiles; (ii) whether such changes are caused by abiotic soil properties, soil microbes, or both; and (iii) whether soil-induced glucosinolate plasticity is genetically variable. Total glucosinolate quantity differed up to 2-fold between soils from different natural habitats, while the relative amounts of different compounds were less responsive. This effect was due to physico-chemical soil properties rather than microbial communities. We detected modest genetic variation for glucosinolate plasticity in response to soil. In addition, glucosinolate composition, but not quantity, of field-grown plants could be accurately predicted from measurements from greenhouse-grown plants. In summary, soil alone is sufficient to cause plasticity of baseline glucosinolate levels in natural plant populations, which may have implications for the evolution of this important trait across complex landscapes.

The Boechera model system for evolutionary ecology.

Model systems in biology expand the research capacity of individuals and the community. Closely related to Arabidopsis, the genus Boechera has emerged as an important ecological model owing to the ability to integrate across molecular, functional, and eco-evolutionary approaches. Boechera species are broadly distributed in relatively undisturbed habitats predominantly in western North America and provide one of the few experimental systems for identification of ecologically important genes through genome-wide association studies and investigations of selection with plants in their native habitats. The ecologically, evolutionarily, and agriculturally important trait of apomixis (asexual reproduction via seeds) is common in the genus, and field experiments suggest that abiotic and biotic environments shape the evolution of sex. To date, population genetic studies have focused on the widespread species B. stricta, detailing population divergence and demographic history. Molecular and ecological studies show that balancing selection maintains genetic variation in ~10% of the genome, and ecological trade-offs contribute to complex trait variation for herbivore resistance, flowering phenology, and drought tolerance. Microbiome analyses have shown that host genotypes influence leaf and root microbiome composition, and the soil microbiome influences flowering phenology and natural selection. Furthermore, Boechera offers numerous opportunities for investigating biological responses to global change. In B. stricta, climate change has induced a shift of >2 weeks in the timing of first flowering since the 1970s, altered patterns of natural selection, generated maladaptation in previously locally-adapted populations, and disrupted life history trade-offs. Here we review resources and results for this eco-evolutionary model system and discuss future research directions.

Prioritizing host phenotype to understand microbiome heritability in plants.

Breeders and evolutionary geneticists have grappled with the complexity of the 'genotype-to-phenotype map' for decades. Now, recent studies highlight the relevance of this concept for understanding heritability of plant microbiomes. Because host phenotype is a more proximate cause of microbiome variation than host genotype, microbiome heritability varies across plant anatomy and development. Fine-scale variation of plant traits within organs suggests that the well-established concept of 'microbiome compartment' should be refined. Additionally, recent work shows that the balance of deterministic processes (including host genetic effects) vs stochastic processes also varies over time and space. Together, these findings suggest that re-centering plant phenotype - both as a predictor and a readout of microbiome function - will accelerate new insights into microbiome heritability.

Microbial effects on plant phenology and fitness.

Plant development and the timing of developmental events (phenology) are tightly coupled with plant fitness. A variety of internal and external factors determine the timing and fitness consequences of these life-history transitions. Microbes interact with plants throughout their life history and impact host phenology. This review summarizes current mechanistic and theoretical knowledge surrounding microbe-driven changes in plant phenology. Overall, there are examples of microbes impacting every phenological transition. While most studies have focused on flowering time, microbial effects remain important for host survival and fitness across all phenological phases. Microbe-mediated changes in nutrient acquisition and phytohormone signaling can release plants from stressful conditions and alter plant stress responses inducing shifts in developmental events. The frequency and direction of phenological effects appear to be partly determined by the lifestyle and the underlying nature of a plant-microbe interaction (i.e., mutualistic or pathogenic), in addition to the taxonomic group of the microbe (fungi vs. bacteria). Finally, we highlight biases, gaps in knowledge, and future directions. This biotic source of plasticity for plant adaptation will serve an important role in sustaining plant biodiversity and managing agriculture under the pressures of climate change.

Heterosis of leaf and rhizosphere microbiomes in field-grown maize.

Macroorganisms' genotypes shape their phenotypes, which in turn shape the habitat available to potential microbial symbionts. This influence of host genotype on microbiome composition has been demonstrated in many systems; however, most previous studies have either compared unrelated genotypes or delved into molecular mechanisms. As a result, it is currently unclear whether the heritability of host-associated microbiomes follows similar patterns to the heritability of other complex traits. We take a new approach to this question by comparing the microbiomes of diverse maize inbred lines and their F1 hybrid offspring, which we quantified in both rhizosphere and leaves of field-grown plants using 16S-v4 and ITS1 amplicon sequencing. We show that inbred lines and hybrids differ consistently in the composition of bacterial and fungal rhizosphere communities, as well as leaf-associated fungal communities. A wide range of microbiome features display heterosis within individual crosses, consistent with patterns for nonmicrobial maize phenotypes. For leaf microbiomes, these results were supported by the observation that broad-sense heritability in hybrids was substantially higher than narrow-sense heritability. Our results support our hypothesis that at least some heterotic host traits affect microbiome composition in maize.

Analysis of leaf microbiome composition of near-isogenic maize lines differing in broad-spectrum disease resistance.

Plant genotype strongly affects disease resistance, and also influences the composition of the leaf microbiome. However, these processes have not been studied and linked in the microevolutionary context of breeding for improved disease resistance. We hypothesised that broad-spectrum disease resistance alleles also affect colonisation by nonpathogenic symbionts. Quantitative trait loci (QTL) conferring resistance to multiple fungal pathogens were introgressed into a disease-susceptible maize inbred line. Bacterial and fungal leaf microbiomes of the resulting near-isogenic lines were compared with the microbiome of the disease-susceptible parent line at two time points in multiple fields. Introgression of QTL from disease-resistant lines strongly shifted the relative abundance of diverse fungal and bacterial taxa in both 3-wk-old and 7-wk-old plants. Nevertheless, the effects on overall community structure and diversity were minor and varied among fields and years. Contrary to our expectations, host genotype effects were not any stronger in fields with high disease pressure than in uninfected fields, and microbiome succession over time was similar in heavily infected and uninfected plants. These results show that introgressed QTL can greatly improve broad-spectrum disease resistance while having only limited and inconsistent pleiotropic effects on the leaf microbiome in maize.

Research priorities for harnessing plant microbiomes in sustainable agriculture.

Feeding a growing world population amidst climate change requires optimizing the reliability, resource use, and environmental impacts of food production. One way to assist in achieving these goals is to integrate beneficial plant microbiomes-i.e., those enhancing plant growth, nutrient use efficiency, abiotic stress tolerance, and disease resistance-into agricultural production. This integration will require a large-scale effort among academic researchers, industry researchers, and farmers to understand and manage plant-microbiome interactions in the context of modern agricultural systems. Here, we identify priorities for research in this area: (1) develop model host-microbiome systems for crop plants and non-crop plants with associated microbial culture collections and reference genomes, (2) define core microbiomes and metagenomes in these model systems, (3) elucidate the rules of synthetic, functionally programmable microbiome assembly, (4) determine functional mechanisms of plant-microbiome interactions, and (5) characterize and refine plant genotype-by-environment-by-microbiome-by-management interactions. Meeting these goals should accelerate our ability to design and implement effective agricultural microbiome manipulations and management strategies, which, in turn, will pay dividends for both the consumers and producers of the world food supply.

Adaptive evolution: evaluating empirical support for theoretical predictions.

Adaptive evolution is shaped by the interaction of population genetics, natural selection and underlying network and biochemical constraints. Variation created by mutation, the raw material for evolutionary change, is translated into phenotypes by flux through metabolic pathways and by the topography and dynamics of molecular networks. Finally, the retention of genetic variation and the efficacy of selection depend on population genetics and demographic history. Emergent high-throughput experimental methods and sequencing technologies allow us to gather more evidence and to move beyond the theory in different systems and populations. Here we review the extent to which recent evidence supports long-established theoretical principles of adaptation.

Long-term structural and biomass dynamics of virgin Tsuga canadensis-Pinus strobus forests after hurricane disturbance.

The development of old-growth forests in northeastern North America has largely been within the context of gap-scale disturbances given the rarity of stand-replacing disturbances. Using the 10-ha old-growth Harvard Tract and its associated 90-year history of measurements, including detailed surveys in 1989 and 2009, we document the long-term structural and biomass development of an old-growth Tsuga canadensis-Pinus strobus forest in southern New Hampshire, USA following a stand-replacing hurricane in 1938. Measurements of aboveground biomass pools were integrated with data from second- and old-growth T. canadensis forests to evaluate long-term patterns in biomass development following this disturbance. Ecosystem structure across the Tract prior to the hurricane exhibited a high degree of spatial heterogeneity with the greatest levels of live tree basal area (70-129 m2 /ha) on upper west-facing slopes where P. strobus was dominant and intermixed with T. canadensis. Live-tree biomass estimates for these stratified mixtures ranged from 159 to 503 Mg/ha at the localized, plot scale (100 m2 ) and averaged 367 Mg/ha across these portions of the landscape approaching the upper bounds for eastern forests. Live-tree biomass 71 years after the hurricane is more uniform and lower in magnitude, with T. canadensis currently the dominant overstory tree species throughout much of the landscape. Despite only one living P. strobus stem in the 2009 plots (and fewer than five stems known across the entire 10-ha area), the detrital legacy of this species is pronounced with localized accumulations of coarse woody debris exceeding 237.7-404.2 m3 /ha where this species once dominated the canopy. These patterns underscore the great sizes P. strobus attained in pre-European landscapes and its great decay resistance relative to its forest associates. Total aboveground biomass pools in this 71-year-old forest (255 Mg/ha) are comparable to those in modern old-growth ecosystems in the region that also lack abundant white pine. Results highlight the importance of disturbance legacies in affecting forest structural conditions over extended periods following stand-replacing events and underscore that post-disturbance salvage logging can alter ecosystem development for decades. Moreover, the dominant role of old-growth P. strobus in live and detrital biomass pools before and after the hurricane, respectively, demonstrate the disproportionate influence this species likely had on carbon storage at localized scales prior to the widespread, selective harvesting of large P. strobus across the region in the 18th and 19th centuries.

Natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative.

Plant phenology is known to depend on many different environmental variables, but soil microbial communities have rarely been acknowledged as possible drivers of flowering time. Here, we tested separately the effects of four naturally occurring soil microbiomes and their constituent soil chemistries on flowering phenology and reproductive fitness of Boechera stricta, a wild relative of Arabidopsis. Flowering time was sensitive to both microbes and the abiotic properties of different soils; varying soil microbiota also altered patterns of selection on flowering time. Thus, soil microbes potentially contribute to phenotypic plasticity of flowering time and to differential selection observed between habitats. We also describe a method to dissect the microbiome into single axes of variation that can help identify candidate organisms whose abundance in soil correlates with flowering time. This approach is broadly applicable to search for microbial community members that alter biological characteristics of interest.

Evaluation of ready-to-use freezer stocks of a synthetic microbial community for maize root colonization.

IMPORTANCE: Synthetic communities (SynComs) are an invaluable tool to characterize and model plant-microbe interactions. Multimember SynComs approximate intricate real-world interactions between plants and their microbiome, but the complexity and time required for their construction increase enormously for each additional member added to the SynCom. Therefore, researchers who study a diversity of microbiomes using SynComs are looking for ways to simplify the use of SynComs. In this manuscript, we evaluate the feasibility of creating ready-to-use freezer stocks of a well-studied seven-member SynCom for maize roots. The frozen ready-to-use SynCom stocks work according to the principle of "just add buffer and apply to sterilized seeds or seedlings" and thus can save time applied in multiple days of laborious growing and combining of multiple microorganisms. We show that ready-to-use SynCom stocks provide comparable results to those of freshly constructed SynComs and thus allow for significant time savings when working with SynComs.

Interactions with fungi vary among Tripsacum dactyloides genotypes from across a precipitation gradient.

Plant-associated microbes, specifically fungal endophytes, augment the ability of many grasses to adapt to extreme environmental conditions. Tripsacum dactyloides (Eastern gamagrass) is a perennial, drought-tolerant grass native to the tallgrass prairies of the central USA. The extent to which the microbiome of T. dactyloides contributes to its drought tolerance is unknown. Ninety-seven genotypes of T. dactyloides were collected from native populations across an east-west precipitation gradient in Kansas, Oklahoma and Texas, and then grown together in a common garden for over 20 years. Root and leaf samples were visually examined for fungal density. Because fungal endophytes confer drought-tolerant capabilities to their host plants, we expected to find higher densities of fungal endophytes in plants from western, drier regions, compared to plants from eastern, wetter regions. Results confirmed a negative correlation between endophyte densities in roots and precipitation at the genotype's original location (r = -0.21 P = 0.04). Our analyses reveal that the host genotype's origin along the precipitation gradient predicts the absolute abundance of symbionts in the root, but not the relative abundances of particular organisms or the overall community composition. Overall, these results demonstrate that genetic variation for plant-microbe interactions can reflect historical environment, and reinforce the importance of considering plant genotype in conservation and restoration work in tallgrass prairie ecosystems.

How hungry roots get their microbes.

Maize genes influence which species of bacteria are recruited from the soil, especially in the absence of nitrogen supplied by fertilizer.

Plant Genetics as a Tool for Manipulating Crop Microbiomes: Opportunities and Challenges.

Growing human population size and the ongoing climate crisis create an urgent need for new tools for sustainable agriculture. Because microbiomes have profound effects on host health, interest in methods of manipulating agricultural microbiomes is growing rapidly. Currently, the most common method of microbiome manipulation is inoculation of beneficial organisms or engineered communities; however, these methods have been met with limited success due to the difficulty of establishment in complex farm environments. Here we propose genetic manipulation of the host plant as another avenue through which microbiomes could be manipulated. We discuss how domestication and modern breeding have shaped crop microbiomes, as well as the potential for improving plant-microbiome interactions through conventional breeding or genetic engineering. We summarize the current state of knowledge on host genetic control of plant microbiomes, as well as the key challenges that remain.

Ecological factors influence balancing selection on leaf chemical profiles of a wildflower.

Balancing selection is frequently invoked as a mechanism that maintains variation within and across populations. However, there are few examples of balancing selection operating on loci underpinning complex traits, which frequently display high levels of variation. We investigated mechanisms that may maintain variation in a focal polymorphism-leaf chemical profiles of a perennial wildflower (Boechera stricta, Brassicaceae)-explicitly interrogating multiple ecological and genetic processes including spatial variation in selection, antagonistic pleiotropy and frequency-dependent selection. A suite of common garden and greenhouse experiments showed that the alleles underlying variation in chemical profile have contrasting fitness effects across environments, implicating two ecological drivers of selection on chemical profile: herbivory and drought. Phenotype-environment associations and molecular genetic analyses revealed additional evidence of past selection by these drivers. Together, these data are consistent with balancing selection on chemical profile, probably caused by pleiotropic effects of secondary chemical biosynthesis genes on herbivore defence and drought response.

Plasticity of plant defense and its evolutionary implications in wild populations of Boechera stricta.

Phenotypic plasticity is thought to impact evolutionary trajectories by shifting trait values in a direction that is either favored by natural selection ("adaptive" plasticity) or disfavored ("nonadaptive" plasticity). However, it is unclear how commonly each of these types of plasticity occurs in natural populations. To answer this question, we measured glucosinolate defensive chemistry and reproductive fitness in over 1500 individuals of the wild perennial mustard Boechera stricta, planted in four common gardens across central Idaho, United States. Glucosinolate profiles-including total glucosinolate concentration as well as the relative abundances and overall diversity of different compounds-were strongly plastic both among habitats and within habitats. Patterns of glucosinolate plasticity varied greatly among genotypes. Plasticity among sites was predicted to affect fitness in 27.1% of cases; more often than expected by chance, glucosinolate plasticity increased rather than decreased relative fitness. In contrast, we found no evidence for within-habitat selection on glucosinolate reaction norm slopes (i.e., plasticity along a continuous environmental gradient). Together, our results indicate that glucosinolate plasticity may improve the ability of B. stricta populations to persist after migration to new habitats.