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IntroductionPlants employ the Calvin-Benson cycle (CBC) to fix atmospheric CO₂for the production of biomass. The flux of carbon through the CBC is limited by the activity and selectivity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO). Alternative CO₂fixation pathways that do not use RuBisCO to fix CO₂have evolved in some anaerobic, autotrophic microorganisms. MethodsRather than modifying existing routes of carbon metabolism in plants, we have developed a synthetic carbon fixation cycle that does not exist in nature but is inspired by metabolisms of bacterial autotrophs. In this work, we build and characterize a condensed, reverse tricarboxylic acid (crTCA) cyclein vitroandin planta. ResultsWe demonstrate that a simple, synthetic cycle can be used to fix carbon in vitro under aerobic and mesophilic conditions and that these enzymes retain activity whenexpressed transientlyin planta. We then evaluate stable transgenic lines ofCamelina sativathat have both phenotypic and physiologic changes. TransgenicC. sativaare shorter than controls with increased rates of photosynthetic CO₂assimilation and changes in photorespiratory metabolism. DiscussionThis first iteration of a build-test-learn phase of the crTCA cycle provides promising evidence that this pathway can be used to increase photosynthetic capacity in plants. 

Studies of plant–microbe interactions using synthetic microbial communities (SynComs) often require the removal of seed-associated microbes by seed sterilization prior to inoculation to provide gnotobiotic growth conditions. Diverse seed sterilization protocols have been developed and have been used on different plant species with various amounts of validation. From these studies it has become clear that each plant species requires its own optimized sterilization protocol. It has, however, so far not been tested whether the same protocol works equally well for different varieties and seed sources of one plant species. We evaluated six seed sterilization protocols on two different varieties (Sugar Bun and B73) of maize. All unsterilized maize seeds showed fungal growth upon germination on filter paper, highlighting the need for a sterilization protocol. A short sterilization protocol with hypochlorite and ethanol was sufficient to prevent fungal growth on Sugar Bun germinants; however a longer protocol with heat treatment and germination in fungicide was needed to obtain clean B73 germinants. This difference may have arisen from the effect of either genotype or seed source. We then tested the protocol that performed best for B73 on three additional maize genotypes from four sources. Seed germination rates and fungal contamination levels varied widely by genotype and geographic source of seeds. Our study shows that consideration of both variety and seed source is important when optimizing sterilization protocols and highlights the importance of including seed source information in plant–microbe interaction studies that use sterilized seeds. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license . 

ABSTRACT Synthetic microbial communities (SynComs) are a valuable tool to study community assembly patterns, host–microbe interactions, and microbe–microbe interactions in a fully controllable setting. Constructing the SynCom inocula for plant–microbe experiments can be time-consuming and difficult because a large number of isolates with different medium requirements and growth rates are grown in parallel and mixed to appropriate titers. A potential workaround to assembling fresh SynCom inocula for every experiment could be to prepare and freeze SynComs on a large scale, creating ready-to-use inocula. The objective of this study was to compare the reproducibility, stability, and colonization ability of freshly prepared versus frozen SynCom inocula. We used a community of seven species known to colonize maize roots. The results from inoculation with the frozen SynCom were as consistent as those of standardizedde novoconstruction of fresh SynCom. Our results indicate that creating frozen SynCom inocula for repeated use in experiments not only saves time but could also improve cross-experiment reproducibility. Although this approach was only validated with one SynCom, it demonstrates a principle that can be tested for improving approaches in constructing other SynComs. IMPORTANCESynthetic 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. 

Abstract Arbuscular mycorrhizal symbiosis (AM) is a beneficial trait originating with the first land plants, which has subsequently been lost by species scattered throughout the radiation of plant diversity to the present day, including the modelArabidopsis thaliana. To explore if elements of this apparently beneficial trait are still present and could be reactivated we generatedArabidopsisplants expressing a constitutively active form ofInteracting Protein of DMI3, a key transcription factor that enables AM within the Common Symbiosis Pathway, which was lost fromArabidopsisalong with the AM host trait. We characterize the transcriptomic effect of expressingIPD3inArabidopsiswith and without exposure to the AM fungus (AMF)Rhizophagus irregularis, and compare these results to the AM modelLotus japonicusand itsipd3knockout mutantcyclops-4. Despite its long history as a non-AM species, restoringIPD3in the form of its constitutively active DNA-binding domain toArabidopsisaltered expression of specific gene networks. Surprisingly, the effect of expressingIPD3inArabidopsisand knocking it out inLotuswas strongest in plants not exposed to AMF, which is revealed to be due to changes inIPD3genotype causing a transcriptional state, which partially mimics AMF exposure in non-inoculated plants. Our results indicate that molecular connections to symbiosis machinery remain in place in this nonAM species, with implications for both basic science and the prospect of engineering this trait for agriculture. 

Abstract Bathymodioline mussels rely on thiotrophic and/or methanotrophic chemosynthetic symbionts for nutrition, yet, secondary heterotrophic symbionts are often present and play an unknown role in the fitness of the organism. The bathymodioline Idas mussels that thrive in gas seeps and on sunken wood in the Mediterranean Sea and the Atlantic Ocean, host at least six symbiont lineages that often co-occur. These lineages include the primary symbionts chemosynthetic methane- and sulfur-oxidizing gammaproteobacteria, and the secondary symbionts, Methylophagaceae, Nitrincolaceae and Flavobacteriaceae, whose physiology and metabolism are obscure. Little is known about if and how these symbionts interact or exchange metabolites. Here we curated metagenome-assembled genomes of Idas modiolaeformis symbionts and used genome-centered metatranscriptomics and metaproteomics to assess key symbiont functions. The Methylophagaceae symbiont is a methylotrophic autotroph, as it encoded and expressed the ribulose monophosphate and Calvin-Benson-Bassham cycle enzymes, particularly RuBisCO. The Nitrincolaceae ASP10-02a symbiont likely fuels its metabolism with nitrogen-rich macromolecules and may provide the holobiont with vitamin B12. The Urechidicola (Flavobacteriaceae) symbionts likely degrade glycans and may remove NO. Our findings indicate that these flexible associations allow for expanding the range of substrates and environmental niches, via new metabolic functions and handoffs. 

Metaproteomics is a powerful tool for the characterization of metabolism, physiology, and functional interactions in microbial communities, including plant-associated microbiota. However, the metaproteomic methods that have been used to study plant-associated microbiota are very laborious and require large amounts of plant tissue, hindering wider application of these methods. We optimized and evaluated different protein extraction methods for metaproteomics of plant-associated microbiota in two different plant species ( Arabidopsis and maize). Our main goal was to identify a method that would work with low amounts of input material (40 to 70 mg) and that would maximize the number of identified microbial proteins. We tested eight protocols, each comprising a different combination of physical lysis method, extraction buffer, and cell-enrichment method on roots from plants grown with synthetic microbial communities. We assessed the performance of the extraction protocols by liquid chromatography-tandem mass spectrometry–based metaproteomics and found that the optimal extraction method differed between the two species. For Arabidopsis roots, protein extraction by beating whole roots with small beads provided the greatest number of identified microbial proteins and improved the identification of proteins from gram-positive bacteria. For maize, vortexing root pieces in the presence of large glass beads yielded the greatest number of microbial proteins identified. Based on these data, we recommend the use of these two methods for metaproteomics with Arabidopsis and maize. Furthermore, detailed descriptions of the eight tested protocols will enable future optimization of protein extraction for metaproteomics in other dicot and monocot plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license . 

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 F₁offspring 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.