Molecular Mechanisms of Pseudomonas-Assisted Plant Nitrogen Uptake: Opportunities for Modern Agriculture.

Pseudomonas spp. make up 1.6% of the bacteria in the soil and are found throughout the world. More than 140 species of this genus have been identified, some beneficial to the plant. Several species in the family Pseudomonadaceae, including Azotobacter vinelandii AvOP, Pseudomonas stutzeri A1501, Pseudomonas stutzeri DSM4166, Pseudomonas szotifigens 6HT33bT, and Pseudomonas sp. strain K1 can fix nitrogen from the air. The genes required for these reactions are organized in a nitrogen fixation island, obtained via horizontal gene transfer from Klebsiella pneumoniae, Pseudomonas stutzeri, and Azotobacter vinelandii. Today, this island is conserved in Pseudomonas spp. from different geographical locations, which, in turn, have evolved to deal with different geo-climatic conditions. Here, we summarize the molecular mechanisms behind Pseudomonas-driven plant growth promotion, with particular focus on improving plant performance at limiting nitrogen (N) and improving plant N content. We describe Pseudomonas-plant interaction strategies in the soil, noting that the mechanisms of denitrification, ammonification, and secondary metabolite signaling are only marginally explored. Plant growth promotion is dependent on the abiotic conditions and differs at sufficient and deficient N. The molecular controls behind different plant responses are not fully elucidated. We suggest that superposition of transcriptome, proteome, and metabolome data and their integration with plant phenotype development through time will help fill these gaps. The aim of this review is to summarize the knowledge behind Pseudomonas-driven nitrogen fixation and to point to possible agricultural solutions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The molecular basis of zinc homeostasis in cereals.

Plants require zinc (Zn) as an essential cofactor for diverse molecular, cellular and physiological functions. Zn is crucial for crop yield, but is one of the most limiting micronutrients in soils. Grasses like rice, wheat, maize and barley are crucial sources of food and nutrients for humans. Zn deficiency in these species therefore not only reduces annual yield but also directly results in Zn malnutrition of more than two billion people in the world. There has been good progress in understanding Zn homeostasis and Zn deficiency mechanisms in plants. However, our current knowledge of monocots, including grasses, remains insufficient. In this review, we provide a summary of our knowledge of molecular Zn homeostasis mechanisms in monocots, with a focus on important cereal crops. We additionally highlight divergences in Zn homeostasis of monocots and the dicot model Arabidopsis thaliana, as well as important gaps in our knowledge that need to be addressed in future research on Zn homeostasis in cereal monocots.

N-dependent dynamics of root growth and nitrate and ammonium uptake are altered by the bacterium Herbaspirillum seropedicae in the cereal model Brachypodium distachyon.

Nitrogen (N) fixation in cereals by root-associated bacteria is a promising solution for reducing use of chemical N fertilizers in agriculture. However, plant and bacterial responses are unpredictable across environments. We hypothesized that cereal responses to N-fixing bacteria are dynamic, depending on N supply and time. To quantify the dynamics, a gnotobiotic, fabricated ecosystem (EcoFAB) was adapted to analyse N mass balance, to image shoot and root growth, and to measure gene expression of Brachypodium distachyon inoculated with the N-fixing bacterium Herbaspirillum seropedicae. Phenotyping throughput of EcoFAB-N was 25-30 plants h-1 with open software and imaging systems. Herbaspirillum seropedicae inoculation of B. distachyon shifted root and shoot growth, nitrate versus ammonium uptake, and gene expression with time; directions and magnitude depended on N availability. Primary roots were longer and root hairs shorter regardless of N, with stronger changes at low N. At higher N, H. seropedicae provided 11% of the total plant N that came from sources other than the seed or the nutrient solution. The time-resolved phenotypic and molecular data point to distinct modes of action: at 5 mM NH4NO3 the benefit appears through N fixation, while at 0.5 mM NH4NO3 the mechanism appears to be plant physiological, with H. seropedicae promoting uptake of N from the root medium.Future work could fine-tune plant and root-associated microorganisms to growth and nutrient dynamics.

Transcriptional regulation of ZIP genes is independent of local zinc status in Brachypodium shoots upon zinc deficiency and resupply.

The biological processes underlying zinc homeostasis are targets for genetic improvement of crops to counter human malnutrition. Detailed phenotyping, ionomic, RNA-Seq analyses and flux measurements with 67 Zn isotope revealed whole-plant molecular events underlying zinc homeostasis upon varying zinc supply and during zinc resupply to starved Brachypodium distachyon (Brachypodium) plants. Although both zinc deficiency and excess hindered Brachypodium growth, accumulation of biomass and micronutrients into roots and shoots differed depending on zinc supply. The zinc resupply dynamics involved 1,893 zinc-responsive genes. Multiple zinc-regulated transporter and iron-regulated transporter (IRT)-like protein (ZIP) transporter genes and dozens of other genes were rapidly and transiently down-regulated in early stages of zinc resupply, suggesting a transient zinc shock, sensed locally in roots. Notably, genes with identical regulation were observed in shoots without zinc accumulation, pointing to root-to-shoot signals mediating whole-plant responses to zinc resupply. Molecular events uncovered in the grass model Brachypodium are useful for the improvement of staple monocots.

Plant genome and transcriptome annotations: from misconceptions to simple solutions.

Next-generation sequencing has triggered an explosion of available genomic and transcriptomic resources in the plant sciences. Although genome and transcriptome sequencing has become orders of magnitudes cheaper and more efficient, often the functional annotation process is lagging behind. This might be hampered by the lack of a comprehensive enumeration of simple-to-use tools available to the plant researcher. In this comprehensive review, we present (i) typical ontologies to be used in the plant sciences, (ii) useful databases and resources used for functional annotation, (iii) what to expect from an annotated plant genome, (iv) an automated annotation pipeline and (v) a recipe and reference chart outlining typical steps used to annotate plant genomes/transcriptomes using publicly available resources.

Dynamics in plant roots and shoots minimize stress, save energy and maintain water and nutrient uptake.

Plants are inherently dynamic. Dynamics minimize stress while enabling plants to flexibly acquire resources. Three examples are presented for plants tolerating saline soil: transport of sodium chloride (NaCl), water and macronutrients is nonuniform along a branched root; water and NaCl redistribute between shoot and soil at night-time; and ATP for salt exclusion is much lower in thinner branch roots than main roots, quantified using a biophysical model and geometry from anatomy. Noninvasive phenotyping and precision agriculture technologies can be used together to harness plant dynamics, but analytical methods are needed. A plant advancing in time through a soil and atmosphere space is proposed as a framework for dynamic data and their relationship to crop improvement.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

Multilab EcoFAB study shows highly reproducible physiology and depletion of soil metabolites by a model grass.

There is a dynamic reciprocity between plants and their environment: soil physiochemical properties influence plant morphology and metabolism, and root morphology and exudates shape the environment surrounding roots. Here, we investigate the reproducibility of plant trait changes in response to three growth environments. We utilized fabricated ecosystem (EcoFAB) devices to grow the model grass Brachypodium distachyon in three distinct media across four laboratories: phosphate-sufficient and -deficient mineral media allowed assessment of the effects of phosphate starvation, and a complex, sterile soil extract represented a more natural environment with yet uncharacterized effects on plant growth and metabolism. Tissue weight and phosphate content, total root length, and root tissue and exudate metabolic profiles were consistent across laboratories and distinct between experimental treatments. Plants grown in soil extract were morphologically and metabolically distinct, with root hairs four times longer than with other growth conditions. Further, plants depleted half of the metabolites investigated from the soil extract. To interact with their environment, plants not only adapt morphology and release complex metabolite mixtures, but also selectively deplete a range of soil-derived metabolites. The EcoFABs utilized here generated high interlaboratory reproducibility, demonstrating their value in standardized investigations of plant traits.

Evolution of the Phosphoenolpyruvate Carboxylase Protein Kinase Family in C3 and C4 Flaveria spp.

The key enzyme for C4 photosynthesis, Phosphoenolpyruvate Carboxylase (PEPC), evolved from nonphotosynthetic PEPC found in C3 ancestors. In all plants, PEPC is phosphorylated by Phosphoenolpyruvate Carboxylase Protein Kinase (PPCK). However, differences in the phosphorylation pattern exist among plants with these photosynthetic types, and it is still not clear if they are due to interspecies differences or depend on photosynthetic type. The genus Flaveria contains closely related C3, C3-C4 intermediate, and C4 species, which are evolutionarily young and thus well suited for comparative analysis. To characterize the evolutionary differences in PPCK between plants with C3 and C4 photosynthesis, transcriptome libraries from nine Flaveria spp. were used, and a two-member PPCK family (PPCKA and PPCKB) was identified. Sequence analysis identified a number of C3- and C4-specific residues with various occurrences in the intermediates. Quantitative analysis of transcriptome data revealed that PPCKA and PPCKB exhibit inverse diel expression patterns and that C3 and C4 Flaveria spp. differ in the expression levels of these genes. PPCKA has maximal expression levels during the day, whereas PPCKB has maximal expression during the night. Phosphorylation patterns of PEPC varied among C3 and C4 Flaveria spp. too, with PEPC from the C4 species being predominantly phosphorylated throughout the day, while in the C3 species the phosphorylation level was maintained during the entire 24 h. Since C4 Flaveria spp. evolved from C3 ancestors, this work links the evolutionary changes in sequence, PPCK expression, and phosphorylation pattern to an evolutionary phase shift of kinase activity from a C3 to a C4 mode.

Precision, proteome coverage, and dynamic range of Arabidopsis proteome profiling using (15)N metabolic labeling and label-free approaches.

This study reports the comprehensive comparison of (15)N metabolic labeling and label free proteomic strategies for quantitation, with particular focus on plant proteomics. Our investigation of proteome coverage, dynamic range and quantitative precision for a wide range of mixing ratios and protein loadings aim to aid the investigators in the decision making process during experimental design. One of the main characteristics of the label free strategy is the applicability to all starting material, which is a limitation to the metabolic labeling. However, particularly at mixing ratios up to 10-fold the (15)N metabolic labeling proved to be more precise. Contrary to usual practice based on the results from this study, we suggest that nonequal mixing ratios in metabolic labeling could further increase the proteome coverage for quantitation. On the other hand, the label free strategy, in combination with low protein loading allows the extension of the dynamic range for quantitation and it is more precise at very high ratios, which could be important for certain types of experiments.

Seed coating with phages for sustainable plant biocontrol of plant pathogens and influence of the seed coat mucilage.

Pathogens resistant to classical control strategies pose a significant threat to crop yield, with seeds being a major transmission route. Bacteriophages, viruses targeting bacteria, offer an environmentally sustainable biocontrol solution. In this study, we isolated and characterized two novel phages, Athelas and Alfirin, which infect Pseudomonas syringae and Agrobacterium fabrum, respectively, and included the recently published Pfeifenkraut phage infecting Xanthomonas translucens. Using a simple immersion method, phages coated onto seeds successfully lysed bacteria post air-drying. The seed coat mucilage (SCM), a polysaccharide-polymer matrix exuded by seeds, plays a critical role in phage binding. Seeds with removed mucilage formed five to 10 times less lysis zones compared to those with mucilage. The podovirus Athelas showed the highest mucilage dependency. Phages from the Autographiviridae family also depended on mucilage for seed adhesion. Comparative analysis of Arabidopsis SCM mutants suggested the diffusible cellulose as a key component for phage binding. Long-term activity tests demonstrated high phage stability on seed surfaces and significantly increasing seedling survival rates in the presence of pathogens. Using non-virulent host strains enhanced phage presence on seeds but also has potential limitations. These findings highlight phage-based interventions as promising, sustainable strategies for combating pathogen resistance and improving crop yield.

Expanding the Phage Galaxy: Isolation and Characterization of Five Novel Streptomyces Siphoviruses Ankus, Byblos, DekoNeimoidia, Mandalore, and Naboo.

Background: Key features of the actinobacterial genus Streptomyces are multicellular, filamentous growth, and production of a broad portfolio of bioactive molecules. These characteristics appear to play an important role in phage-host interactions and are modulated by phages during infection. To accelerate research of such interactions and the investigation of novel immune systems in multicellular bacteria, phage isolation, sequencing, and characterization are needed. This is a prerequisite for establishing systematic collections that appropriately cover phage diversity for comparative analyses. Material & Methods: As part of a public outreach program within the priority program SPP 2330, involving local schools, we describe the isolation and characterization of five novel Streptomyces siphoviruses infecting S. griseus, S. venezuelae, and S. olivaceus. Results: All isolates are virulent members of two existing genera and, additionally, establish a new genus in the Stanwilliamsviridae family. In addition to an extensive set of tRNAs and proteins involved in phage replication, about 80% of phage genes encode hypothetical proteins, underlining the yet underexplored phage diversity and genomic dark matter still found in bacteriophages infecting actinobacteria. Conclusions: Taken together, phages Ankus, Byblos, DekoNeimoidia, Mandalore, and Naboo expand the phage diversity and contribute to ongoing research in the field of Streptomyces phage-host interactions.

Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana.

Here, we characterize a plastidial thioredoxin (TRX) isoform from Arabidopsis thaliana that defines a previously unknown branch of plastidial TRXs lying between x- and y-type TRXs and thus was named TRX z. An Arabidopsis knockout mutant of TRX z had a severe albino phenotype and was inhibited in chloroplast development. Quantitative real-time RT-PCR analysis of the mutant suggested that the expressions of genes that depend on a plastid-encoded RNA polymerase (PEP) were specifically decreased. Similar results were obtained upon virus-induced gene silencing (VIGS) of the TRX z ortholog in Nicotiana benthamiana. We found that two fructokinase-like proteins (FLN1 and FLN2), members of the pfkB-carbohydrate kinase family, were potential TRX z target proteins and identified conserved Cys residues mediating the FLN-TRX z interaction. VIGS in N. benthamiana and inducible RNA interference in Arabidopsis of FLNs also led to a repression of PEP-dependent gene transcription. Remarkably, recombinant FLNs displayed no detectable sugar-phosphorylating activity, and amino acid substitutions within the predicted active site imply that the FLNs have acquired a new function, which might be regulatory rather than metabolic. We were able to show that the FLN2 redox state changes in vivo during light/dark transitions and that this change is mediated by TRX z. Taken together, our data strongly suggest an important role for TRX z and both FLNs in the regulation of PEP-dependent transcription in chloroplasts.

Isolation of Novel Xanthomonas Phages Infecting the Plant Pathogens X. translucens and X. campestris.

The genus of Xanthomonas contains many well-known plant pathogens with the ability to infect some of the most important crop plants, thereby causing significant economic damage. Unfortunately, classical pest-control strategies are neither particularly efficient nor sustainable and we are, therefore, in demand of alternatives. Here, we present the isolation and characterization of seven novel phages infecting the plant-pathogenic species Xanthomonas translucens and Xanthomonas campestris. Transmission electron microscopy revealed that all phages show a siphovirion morphology. The analysis of genome sequences and plaque morphologies are in agreement with a lytic lifestyle of the phages making them suitable candidates for biocontrol. Moreover, three of the isolated phages form the new genus "Shirevirus". All seven phages belong to four distinct clusters underpinning their phylogenetic diversity. Altogether, this study presents the first characterized isolates for the plant pathogen X. translucens and expands the number of available phages for plant biocontrol.

Modulators or facilitators? Roles of lipids in plant root-microbe interactions.

Lipids have diverse functions in regulating the plasma membrane's cellular processes and signaling mediation. Plasma membrane lipids are also involved in the plant's complex interactions with the surrounding microorganisms, with which plants are in various forms of symbiosis. The roles of lipids influence the whole microbial colonization process, thus shaping the rhizomicrobiome. As chemical signals, lipids facilitate the stages of rhizospheric interactions - from plant root to microbe, microbe to microbe, and microbe to plant root - and modulate the plant's defense responses upon perception or contact with either beneficial or phytopathogenic microorganisms. Although studies have come a long way, further investigation is needed to discover more lipid species and elucidate novel lipid functions and profiles under various stages of plant root-microbe interactions.

Plant Growth Promotion and Heat Stress Amelioration in Arabidopsis Inoculated with Paraburkholderia phytofirmans PsJN Rhizobacteria Quantified with the GrowScreen-Agar II Phenotyping Platform.

High temperatures inhibit plant growth. A proposed strategy for improving plant productivity under elevated temperatures is the use of plant growth-promoting rhizobacteria (PGPR). While the effects of PGPR on plant shoots have been extensively explored, roots-particularly their spatial and temporal dynamics-have been hard to study, due to their below-ground nature. Here, we characterized the time- and tissue-specific morphological changes in bacterized plants using a novel non-invasive high-resolution plant phenotyping and imaging platform-GrowScreen-Agar II. The platform uses custom-made agar plates, which allow air exchange to occur with the agar medium and enable the shoot to grow outside the compartment. The platform provides light protection to the roots, the exposure of it to the shoots, and the non-invasive phenotyping of both organs. Arabidopsis thaliana, co-cultivated with Paraburkholderia phytofirmans PsJN at elevated and ambient temperatures, showed increased lengths, growth rates, and numbers of roots. However, the magnitude and direction of the growth promotion varied depending on root type, timing, and temperature. The root length and distribution per depth and according to time was also influenced by bacterization and the temperature. The shoot biomass increased at the later stages under ambient temperature in the bacterized plants. The study offers insights into the timing of the tissue-specific, PsJN-induced morphological changes and should facilitate future molecular and biochemical studies on plant-microbe-environment interactions.

The Metabolic Response of Brachypodium Roots to the Interaction with Beneficial Bacteria Is Affected by the Plant Nutritional Status.

The potential of plant growth promoting (PGP) bacteria in improving the performance of plants in suboptimal environments is increasingly acknowledged, but little information is available on the mechanisms underlying this interaction, particularly when plants are subjected to a combination of stresses. In this study, we investigated the effects of the inoculation with the PGP bacteria Azospirillum brasilense (Azospirillum) on the metabolism of the model cereal Brachypodium distachyon (Brachypodium) grown at low temperatures and supplied with insufficient phosphorus. Investigating polar metabolite and lipid fluctuations during early plant development, we found that the bacteria initially elicited a defense response in Brachypodium roots, while at later stages Azospirillum reduced the stress caused by phosphorus deficiency and improved root development of inoculated plants, particularly by stimulating the growth of branch roots. We propose that the interaction of the plant with Azospirillum was influenced by its nutritional status: bacteria were sensed as pathogens while plants were still phosphorus sufficient, but the interaction became increasingly beneficial for the plants as their phosphorus levels decreased. Our results provide new insights on the dynamics of the cereal-PGP bacteria interaction, and contribute to our understanding of the role of beneficial microorganisms in the growth of cereal crops in suboptimal environments.

MapMan4: A Refined Protein Classification and Annotation Framework Applicable to Multi-Omics Data Analysis.

Genome sequences from over 200 plant species have already been published, with this number expected to increase rapidly due to advances in sequencing technologies. Once a new genome has been assembled and the genes identified, the functional annotation of their putative translational products, proteins, using ontologies is of key importance as it places the sequencing data in a biological context. Furthermore, to keep pace with rapid production of genome sequences, this functional annotation process must be fully automated. Here we present a redesigned and significantly enhanced MapMan4 framework, together with a revised version of the associated online Mercator annotation tool. Compared with the original MapMan, the new ontology has been expanded almost threefold and enforces stricter assignment rules. This framework was then incorporated into Mercator4, which has been upgraded to reflect current knowledge across the land plant group, providing protein annotations for all embryophytes with a comparably high quality. The annotation process has been optimized to allow a plant genome to be annotated in a matter of minutes. The output results continue to be compatible with the established MapMan desktop application.

Monitoring of Plant Protein Post-translational Modifications Using Targeted Proteomics.

Protein post-translational modifications (PTMs) are among the fastest and earliest of plant responses to changes in the environment, making the mechanisms and dynamics of PTMs an important area of plant science. One of the most studied PTMs is protein phosphorylation. This review summarizes the use of targeted proteomics for the elucidation of the biological functioning of plant PTMs, and focuses primarily on phosphorylation. Since phosphorylated peptides have a low abundance, usually complex enrichment protocols are required for their research. Initial identification is usually performed with discovery phosphoproteomics, using high sensitivity mass spectrometers, where as many phosphopeptides are measured as possible. Once a PTM site is identified, biological characterization can be addressed with targeted proteomics. In targeted proteomics, Selected/Multiple Reaction Monitoring (S/MRM) is traditionally coupled to simple, standard protein digestion protocols, often omitting the enrichment step, and relying on triple-quadruple mass spectrometer. The use of synthetic peptides as internal standards allows accurate identification, avoiding cross-reactivity typical for some antibody based approaches. Importantly, internal standards allow absolute peptide quantitation, reported down to 0.1 femtomoles, also useful for determination of phospho-site occupancy. S/MRM is advantageous in situations where monitoring and diagnostics of peptide PTM status is needed for many samples, as it has faster sample processing times, higher throughput than other approaches, and excellent quantitation and reproducibility. Furthermore, the number of publicly available data-bases with plant PTM discovery data is growing, facilitating selection of modified peptides and design of targeted proteomics workflows. Recent instrument developments result in faster scanning times, inclusion of ion-trap instruments leading to parallel reaction monitoring- which further facilitates S/MRM experimental design. Finally, recent combination of data independent and data dependent spectra acquisition means that in addition to anticipated targeted data, spectra can now be queried for unanticipated information. The potential for future applications in plant biology is outlined.