The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

Identification of genetic and environmental factors influencing aerial root traits that support biological nitrogen fixation in sorghum.

Plant breeding and genetics play a major role in the adaptation of plants to meet human needs. The current requirement to make agriculture more sustainable can be partly met by a greater reliance on biological nitrogen fixation (BNF) by symbiotic diazotrophic microorganisms that provide crop plants with ammonium. Select accessions of the cereal crop sorghum (Sorghum bicolor (L.) Moench) form mucilage-producing aerial roots that harbor nitrogen-fixing bacteria. Breeding programs aimed at developing sorghum varieties that support diazotrophs will benefit from a detailed understanding of the genetic and environmental factors contributing to aerial root formation. A genome-wide association study (GWAS) of the sorghum minicore, a collection of 242 landraces, and 30 accessions from the sorghum association panel (SAP) was conducted in Florida and Wisconsin and under two fertilizer treatments to identify loci associated with the number of nodes with aerial roots and aerial root diameter. Sequence variation in genes encoding transcription factors that control phytohormone signaling and root system architecture showed significant associations with these traits. In addition, the location had a significant effect on the phenotypes. Concurrently, we developed F2 populations from crosses between bioenergy sorghums and a landrace that produced extensive aerial roots to evaluate the mode of inheritance of the loci identified by the GWAS. Furthermore, the mucilage collected from aerial roots contained polysaccharides rich in galactose, arabinose, and fucose, whose composition displayed minimal variation among 10 genotypes and two fertilizer treatments. These combined results support the development of sorghums with the ability to acquire nitrogen via BNF.

Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes.

The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: Azotobacter vinelandii, Stutzerimonas stutzeri (Pseudomonas stutzeri), and a new isolate of Klebsiella variicola. All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. S. stutzeri and K. variicola showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.

A glimpse into the fungal metabolomic abyss: Novel network analysis reveals relationships between exogenous compounds and their outputs.

Fungal specialized metabolites are a major source of beneficial compounds that are routinely isolated, characterized, and manufactured as pharmaceuticals, agrochemical agents, and industrial chemicals. The production of these metabolites is encoded by biosynthetic gene clusters that are often silent under standard growth conditions. There are limited resources for characterizing the direct link between abiotic stimuli and metabolite production. Herein, we introduce a network analysis-based, data-driven algorithm comprising two routes to characterize the production of specialized fungal metabolites triggered by different exogenous compounds: the direct route and the auxiliary route. Both routes elucidate the influence of treatments on the production of specialized metabolites from experimental data. The direct route determines known and putative metabolites induced by treatments and provides additional insight over traditional comparison methods. The auxiliary route is specific for discovering unknown analytes, and further identification can be curated through online bioinformatic resources. We validated our algorithm by applying chitooligosaccharides and lipids at two different temperatures to the fungal pathogen Aspergillus fumigatus. After liquid chromatography-mass spectrometry quantification of significantly produced analytes, we used network centrality measures to rank the treatments' ability to elucidate these analytes and confirmed their identity through fragmentation patterns or in silico spiking with commercially available standards. Later, we examined the transcriptional regulation of these metabolites through real-time quantitative polymerase chain reaction. Our data-driven techniques can complement existing metabolomic network analysis by providing an approach to track the influence of any exogenous stimuli on metabolite production. Our experimental-based algorithm can overcome the bottlenecks in elucidating novel fungal compounds used in drug discovery.

Scripting a new dialogue between diazotrophs and crops.

Diazotrophs are bacteria and archaea that can reduce atmospheric dinitrogen (N2) into ammonium. Plant-diazotroph interactions have been explored for over a century as a nitrogen (N) source for crops to improve agricultural productivity and sustainability. This scientific quest has generated much information about the molecular mechanisms underlying the function, assembly, and regulation of nitrogenase, ammonium assimilation, and plant-diazotroph interactions. This review presents various approaches to manipulating N fixation activity, ammonium release by diazotrophs, and plant-diazotroph interactions. We discuss the research avenues explored in this area, propose potential future routes, emphasizing engineering at the metabolic level via biorthogonal signaling, and conclude by highlighting the importance of biocontrol measures and public acceptance.

A Dual Transcriptomic Approach Reveals Contrasting Patterns of Differential Gene Expression During Drought in Arbuscular Mycorrhizal Fungus and Carrot.

While arbuscular mycorrhizal (AM) fungi are known for providing host plants with improved drought tolerance, we know very little about the fungal response to drought in the context of the fungal-plant relationship. In this study, we evaluated the drought responses of the host and symbiont, using the fungus Rhizophagus irregularis with carrot (Daucus carota) as a plant model. Carrots inoculated with spores of R. irregularis DAOM 197198 were grown in a greenhouse. During taproot development, carrots were exposed to a 10-day water restriction. Compared with well-watered conditions, drought caused diminished photosynthetic activity and reduced plant growth in carrot with and without AM fungi. Droughted carrots had lower root colonization. For R. irregularis, 93% of 826 differentially expressed genes (DEGs) were upregulated during drought, including phosphate transporters, several predicted transport proteins of potassium, and the aquaporin RiAQPF2. In contrast, 78% of 2,486 DEGs in AM carrot were downregulated during drought, including the symbiosis-specific genes FatM, RAM2, and STR, which are implicated in lipid transfer from the host to the fungus and were upregulated exclusively in AM carrot during well-watered conditions. Overall, this study provides insight into the drought response of an AM fungus in relation to its host; the expression of genes related to symbiosis and nutrient exchange were downregulated in carrot but upregulated in the fungus. This study reveals that carrot and R. irregularis exhibit contrast in their regulation of gene expression during drought, with carrot reducing its apparent investment in symbiosis and the fungus increasing its apparent symbiotic efforts. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Running the numbers on plant synthetic biology solutions to global problems.

Synthetic biology and metabolic engineering promise to deliver sustainable solutions to global problems such as phasing out fossil fuels and replacing industrial nitrogen fixation. While this promise is real, scale matters, and so do knock-on effects of implementing solutions. Both scale and knock-on effects can be estimated by 'Fermi calculations' (aka 'back-of-envelope calculations') that use uncontroversial input data plus simple arithmetic to reach rough but reliable conclusions. Here, we illustrate how this is done and how informative it can be using two cases: oilcane (sugarcane engineered to accumulate triglycerides instead of sugar) as a source of bio-jet fuel, and nitrogen fixation by bacteria in mucilage secreted by maize aerial roots. We estimate that oilcane could meet no more than about 1% of today's U.S. jet fuel demand if grown on all current U.S. sugarcane land and that, if cane land were expanded to meet two-thirds of this demand, the fertilizer and refinery requirements would create a large carbon footprint. Conversely, we estimate that nitrogen fixation in aerial-root mucilage could replace up to 10% of the fertilizer nitrogen applied to U.S. maize, that 2% of plant carbon income used for growth would suffice to fuel the fixation, and that this extra carbon consumption would likely reduce grain yield only slightly.

Genetic mapping and prediction for novel lesion mimic in maize demonstrates quantitative effects from genetic background, environment and epistasis.

KEY MESSAGE: A novel locus was discovered on chromosome 7 associated with a lesion mimic in maize; this lesion mimic had a quantitative and heritable phenotype and was predicted better via subset genomic markers than whole genome markers across diverse environments. Lesion mimics are a phenotype of leaf micro-spotting in maize (Zea mays L.), which can be early signs of biotic or abiotic stresses. Dissecting its inheritance is helpful to understand how these loci behave across different genetic backgrounds. Here, 538 maize recombinant inbred lines (RILs) segregating for a novel lesion mimic were quantitatively phenotyped in Georgia, Texas, and Wisconsin. These RILs were derived from three bi-parental crosses using a tropical pollinator (Tx773) as the common parent crossed with three inbreds (LH195, LH82, and PB80). While this lesion mimic was heritable across three environments based on phenotypic ([Formula: see text] = 0.68) and genomic ([Formula: see text] = 0.91) data, transgressive segregation was observed. A genome-wide association study identified a single novel locus on chromosome 7 (at 70.6 Mb) also covered by a quantitative trait locus interval (69.3-71.0 Mb), explaining 11-15% of the variation, depending on the environment. One candidate gene identified in this region, Zm00001eb308070, is related to the abscisic acid pathway involving in cell death. Genomic predictions were applied to genome-wide markers (39,611 markers) contrasted with a marker subset (51 markers). Population structure explained more variation than environment in genomic prediction, but other substantial genetic background effects were additionally detected. Subset markers explained substantially less genetic variation (24.9%) for the lesion mimic than whole genome markers (55.4%) in the model, yet predicted the lesion mimic better (0.56-0.66 vs. 0.26-0.29). These results indicate this lesion mimic phenotype was less affected by environment than by epistasis and genetic background effects, which explain its transgressive segregation.

Carnivorous Nepenthes Pitchers with Less Acidic Fluid House Nitrogen-Fixing Bacteria.

Carnivorous pitcher plants are uniquely adapted to nitrogen limitation, using pitfall traps to acquire nutrients from insect prey. Pitcher plants in the genus Sarracenia may also use nitrogen fixed by bacteria inhabiting the aquatic microcosms of their pitchers. Here, we investigated whether species of a convergently evolved pitcher plant genus, Nepenthes, might also use bacterial nitrogen fixation as an alternative strategy for nitrogen capture. First, we constructed predicted metagenomes of pitcher organisms from three species of Singaporean Nepenthes using 16S rRNA sequence data and correlated predicted nifH abundances with metadata. Second, we used gene-specific primers to amplify and quantify the presence or absence of nifH directly from 102 environmental samples and identified potential diazotrophs with significant differential abundance in samples that also had positive nifH PCR tests. Third, we analyzed nifH in eight shotgun metagenomes from four additional Bornean Nepenthes species. Finally, we conducted an acetylene reduction assay using greenhouse-grown Nepenthes pitcher fluids to confirm nitrogen fixation is indeed possible within the pitcher habitat. Results show active acetylene reduction can occur in Nepenthes pitcher fluid. Variation in nifH from wild samples correlates with Nepenthes host species identity and pitcher fluid acidity. Nitrogen-fixing bacteria are associated with more neutral fluid pH, while endogenous Nepenthes digestive enzymes are most active at low fluid pH. We hypothesize Nepenthes species experience a trade-off in nitrogen acquisition; when fluids are acidic, nitrogen is primarily acquired via plant enzymatic degradation of insects, but when fluids are neutral, Nepenthes plants take up more nitrogen via bacterial nitrogen fixation. IMPORTANCE Plants use different strategies to obtain the nutrients that they need to grow. Some plants access their nitrogen directly from the soil, while others rely on microbes to access the nitrogen for them. Carnivorous pitcher plants generally trap and digest insect prey, using plant-derived enzymes to break down insect proteins and generate a large portion of the nitrogen that they subsequently absorb. In this study, we present results suggesting that bacteria living in the fluids formed by Nepenthes pitcher plants can fix nitrogen directly from the atmosphere, providing an alternative pathway for plants to access nitrogen. These nitrogen-fixing bacteria are only likely to be present when pitcher plant fluids are not strongly acidic. Interestingly, the plant's enzymes are known to be more active under strongly acidic conditions. We propose a potential trade-off where pitcher plants sometimes access nitrogen using their own enzymes to digest prey and at other times take advantage of bacterial nitrogen fixation.

The putative transporter MtUMAMIT14 participates in nodule formation in Medicago truncatula.

Transport systems are crucial in many plant processes, including plant-microbe interactions. Nodule formation and function in legumes involve the expression and regulation of multiple transport proteins, and many are still uncharacterized, particularly for nitrogen transport. Amino acids originating from the nitrogen-fixing process are an essential form of nitrogen for legumes. This work evaluates the role of MtN21 (henceforth MtUMAMIT14), a putative transport system from the MtN21/EamA-like/UMAMIT family, in nodule formation and nitrogen fixation in Medicago truncatula. To dissect this transporter's role, we assessed the expression of MtUMAMIT14 using GUS staining, localized the corresponding protein in M. truncatula root and tobacco leaf cells, and investigated two independent MtUMAMIT14 mutant lines. Our results indicate that MtUMAMIT14 is localized in endosomal structures and is expressed in both the infection zone and interzone of nodules. Comparison of mutant and wild-type M. truncatula indicates MtUMAMIT14, the expression of which is dependent on the presence of NIN, DNF1, and DNF2, plays a role in nodule formation and nitrogen-fixation. While the function of the transporter is still unclear, our results connect root nodule nitrogen fixation in legumes with the UMAMIT family.

Lipo-Chitooligosaccharides Induce Specialized Fungal Metabolite Profiles That Modulate Bacterial Growth.

Lipo-chitooligosaccharides (LCOs) are historically known for their role as microbial-derived signaling molecules that shape plant symbiosis with beneficial rhizobia or mycorrhizal fungi. Recent studies showing that LCOs are widespread across the fungal kingdom have raised questions about the ecological function of these compounds in organisms that do not form symbiotic relationships with plants. To elucidate the ecological function of these compounds, we investigate the metabolomic response of the ubiquitous human pathogen Aspergillus fumigatus to LCOs. Our metabolomics data revealed that exogenous application of various types of LCOs to A. fumigatus resulted in significant shifts in the fungal metabolic profile, with marked changes in the production of specialized metabolites known to mediate ecological interactions. Using network analyses, we identify specific types of LCOs with the most significant effect on the abundance of known metabolites. Extracts of several LCO-induced metabolic profiles significantly impact the growth rates of diverse bacterial species. These findings suggest that LCOs may play an important role in the competitive dynamics of non-plant-symbiotic fungi and bacteria. This study identifies specific metabolomic profiles induced by these ubiquitously produced chemicals and creates a foundation for future studies into the potential roles of LCOs as modulators of interkingdom competition. IMPORTANCE The activation of silent biosynthetic gene clusters (BGC) for the identification and characterization of novel fungal secondary metabolites is a perpetual motion in natural product discoveries. Here, we demonstrated that one of the best-studied symbiosis signaling compounds, lipo-chitooligosaccharides (LCOs), play a role in activating some of these BGCs, resulting in the production of known, putative, and unknown metabolites with biological activities. This collection of metabolites induced by LCOs differentially modulate bacterial growth, while the LCO standards do not convey the same effect. These findings create a paradigm shift showing that LCOs have a more prominent role outside of host recognition of symbiotic microbes. Importantly, our work demonstrates that fungi use LCOs to produce a variety of metabolites with biological activity, which can be a potential source of bio-stimulants, pesticides, or pharmaceuticals.

Temporal change in chromatin accessibility predicts regulators of nodulation in Medicago truncatula.

BACKGROUND: Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules. RESULTS: We profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis-regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia. CONCLUSIONS: Our transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.

Nitrogen fixation and mucilage production on maize aerial roots is controlled by aerial root development and border cell functions.

Exploring natural diversity for biological nitrogen fixation in maize and its progenitors is a promising approach to reducing our dependence on synthetic fertilizer and enhancing the sustainability of our cropping systems. We have shown previously that maize accessions from the Sierra Mixe can support a nitrogen-fixing community in the mucilage produced by their abundant aerial roots and obtain a significant fraction of their nitrogen from the air through these associations. In this study, we demonstrate that mucilage production depends on root cap and border cells sensing water, as observed in underground roots. The diameter of aerial roots correlates with the volume of mucilage produced and the nitrogenase activity supported by each root. Young aerial roots produce more mucilage than older ones, probably due to their root cap's integrity and their ability to produce border cells. Transcriptome analysis on aerial roots at two different growth stages before and after mucilage production confirmed the expression of genes involved in polysaccharide synthesis and degradation. Genes related to nitrogen uptake and assimilation were up-regulated upon water exposure. Altogether, our findings suggest that in addition to the number of nodes with aerial roots reported previously, the diameter of aerial roots and abundance of border cells, polysaccharide synthesis and degradation, and nitrogen uptake are critical factors to ensure efficient nitrogen fixation in maize aerial roots.

An LCO-responsive homolog of NODULE INCEPTION positively regulates lateral root formation in Populus sp.

The transcription factor NODULE INCEPTION (NIN) has been studied extensively for its multiple roles in root nodule symbiosis within plants of the nitrogen-fixing clade (NFC) that associate with soil bacteria, such as rhizobia and Frankia. However, NIN homologs are present in plants outside the NFC, suggesting a role in other developmental processes. Here, we show that the biofuel crop Populus sp., which is not part of the NFC, contains eight copies of NIN with diversified protein sequence and expression patterns. Lipo-chitooligosaccharides (LCOs) are produced by rhizobia and a wide range of fungi, including mycorrhizal ones, and act as symbiotic signals that promote lateral root formation. RNAseq analysis of Populus sp. treated with purified LCO showed induction of the PtNIN2 subfamily. Moreover, the expression of PtNIN2b correlated with the formation of lateral roots and was suppressed by cytokinin treatment. Constitutive expression of PtNIN2b overcame the inhibition of lateral root development by cytokinin under high nitrate conditions. Lateral root induction in response to LCOs likely represents an ancestral function of NIN retained and repurposed in nodulating plants, as we demonstrate that the role of NIN in LCO-induced root branching is conserved in both Populus sp. and legumes. We further established a visual marker of LCO perception in Populus sp. roots, the putative sulfotransferase PtSS1 that can be used to study symbiotic interactions with the bacterial and fungal symbionts of Populus sp.

Transcription Factors Controlling the Rhizobium-Legume Symbiosis: Integrating Infection, Organogenesis and the Abiotic Environment.

Legume roots engage in a symbiotic relationship with rhizobia, leading to the development of nitrogen-fixing nodules. Nodule development is a sophisticated process and is under the tight regulation of the plant. The symbiosis initiates with a signal exchange between the two partners, followed by the development of a new organ colonized by rhizobia. Over two decades of study have shed light on the transcriptional regulation of rhizobium-legume symbiosis. A large number of transcription factors (TFs) have been implicated in one or more stages of this symbiosis. Legumes must monitor nodule development amidst a dynamic physical environment. Some environmental factors are conducive to nodulation, whereas others are stressful. The modulation of rhizobium-legume symbiosis by the abiotic environment adds another layer of complexity and is also transcriptionally regulated. Several symbiotic TFs act as integrators between symbiosis and the response to the abiotic environment. In this review, we trace the role of various TFs involved in rhizobium-legume symbiosis along its developmental route and highlight the ones that also act as communicators between this symbiosis and the response to the abiotic environment. Finally, we discuss contemporary approaches to study TF-target interactions in plants and probe their potential utility in the field of rhizobium-legume symbiosis.

Genetic Determinants of Ammonium Excretion in nifL Mutants of Azotobacter vinelandii.

The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting rnf1 gene expression as an additional requirement for ammonium excretion. These ammonium-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. IMPORTANCE There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

Stress-associated developmental reprogramming in moss protonemata by synthetic activation of the common symbiosis pathway.

Symbioses between angiosperms and rhizobia or arbuscular mycorrhizal fungi are controlled through a conserved signaling pathway. Microbe-derived, chitin-based elicitors activate plant cell surface receptors and trigger nuclear calcium oscillations, which are decoded by a calcium/calmodulin-dependent protein kinase (CCaMK) and its target transcription factor interacting protein of DMI3 (IPD3). Genes encoding CCaMK and IPD3 have been lost in multiple non-mycorrhizal plant lineages yet retained among non-mycorrhizal mosses. Here, we demonstrated that the moss Physcomitrium is equipped with a bona fide CCaMK that can functionally complement a Medicago loss-of-function mutant. Conservation of regulatory phosphosites allowed us to generate predicted hyperactive forms of Physcomitrium CCaMK and IPD3. Overexpression of synthetically activated CCaMK or IPD3 in Physcomitrium led to abscisic acid (ABA) accumulation and ectopic development of brood cells, which are asexual propagules that facilitate escape from local abiotic stresses. We therefore propose a functional role for Physcomitrium CCaMK-IPD3 in stress-associated developmental reprogramming.

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade.

Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root-nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.