Exploring Endophytic Bacteria from Artemisia spp. and Beneficial Traits on Pea Plants.

Endophytic microorganisms represent promising solutions to environmental challenges inherent in conventional agricultural practices. This study concentrates on the identification of endophytic bacteria isolated from the root, stem, and leaf tissues of four Artemisia plant species. Sixty-one strains were isolated and sequenced by 16S rDNA. Sequencing revealed diverse genera among the isolated bacteria from different Artemisia species, including Bacillus, Pseudomonas, Enterobacter, and Lysinibacillus. AR11 and VR24 obtained from the roots of A. absinthium and A. vulgaris demonstrated significant inhibition on Fusarium c.f. oxysporum mycelial growth. In addition, AR11, AR32, and CR25 exhibited significant activity in phosphatase solubilization, nitrogen fixation, and indole production, highlighting their potential to facilitate plant growth. A comparative analysis of Artemisia species showed that root isolates from A. absinthium, A. campestris, and A. vulgaris have beneficial properties for inhibiting pathogen growth and enhancing plant growth. AR11 with 100% similarity to Bacillus thuringiensis, could be considered a promising candidate for further investigation as microbial biofertilizers. This finding highlights their potential as environmentally friendly alternatives to chemical pesticides, thereby contributing to sustainable crop protection practices.

Adaptive Modifications in Plant Sulfur Metabolism over Evolutionary Time.

Sulfur (S) is an essential element for life on Earth. Plants are able to take up and utilize sulfate (SO42-), the most oxidized inorganic form of S compounds on Earth, through the reductive S assimilatory pathway that couples with the photosynthetic energy conversion. Organic S compounds are subsequently synthesized in plants and made accessible to animals, primarily as the amino acid methionine. Thus, the plant S metabolism clearly has nutritional importance in the global food chain. The S metabolites may be part of the redox regulation and drivers of essential metabolic pathways as cofactors and prosthetic groups, such as Fe-S centers, coenzyme A, thiamine, and lipoic acid. The evolution of the S metabolic pathways and enzymes reflects critical importance of the functional innovation and diversifications. Here we review the major evolutionary alterations that took place in the S metabolism across different scales and outline research directions that may take advantage of understanding the evolutionary adaptations.

The Arabidopsis SGN3/GSO1 receptor kinase integrates soil nitrogen status into shoot development.

The Casparian strip is a barrier in the endodermal cell walls of plants that allows the selective uptake of nutrients and water. In the model plant Arabidopsis thaliana, its development and establishment are under the control of a receptor-ligand mechanism termed the Schengen pathway. This pathway facilitates barrier formation and activates downstream compensatory responses in case of dysfunction. However, due to a very tight functional association with the Casparian strip, other potential signaling functions of the Schengen pathway remain obscure. In this work, we created a MYB36-dependent synthetic positive feedback loop that drives Casparian strip formation independently of Schengen-induced signaling. We evaluated this by subjecting plants in which the Schengen pathway has been uncoupled from barrier formation, as well as a number of established barrier-mutant plants, to agar-based and soil conditions that mimic agricultural settings. Under the latter conditions, the Schengen pathway is necessary for the establishment of nitrogen-deficiency responses in shoots. These data highlight Schengen signaling as an essential hub for the adaptive integration of signaling from the rhizosphere to aboveground tissues.

The fungus Acremonium alternatum enhances salt stress tolerance by regulating host redox homeostasis and phytohormone signaling.

While endophytic fungi offer promising avenues for bolstering plant resilience against abiotic stressors, the molecular mechanisms behind this biofortification remain largely unknown. This study employed a multifaceted approach, combining plant physiology, proteomic, metabolomic, and targeted hormonal analyses to illuminate the early response of Brassica napus to Acremonium alternatum during the nascent stages of their interaction. Notably, under optimal growth conditions, the initial reaction to fungus was relatively subtle, with no visible alterations in plant phenotype and only minor impacts on the proteome and metabolome. Interestingly, the identified proteins associated with the Acremonium response included TUDOR 1, Annexin D4, and a plastidic K+ efflux antiporter, hinting at potential processes that could counter abiotic stressors, particularly salt stress. Subsequent experiments validated this hypothesis, showcasing significantly enhanced growth in Acremonium-inoculated plants under salt stress. Molecular analyses revealed a profound impact on the plant's proteome, with over 50% of salt stress response proteins remaining unaffected in inoculated plants. Acremonium modulated ribosomal proteins, increased abundance of photosynthetic proteins, enhanced ROS metabolism, accumulation of V-ATPase, altered abundances of various metabolic enzymes, and possibly promoted abscisic acid signaling. Subsequent analyses validated the accumulation of this hormone and its enhanced signaling. Collectively, these findings indicate that Acremonium promotes salt tolerance by orchestrating abscisic acid signaling, priming the plant's antioxidant system, as evidenced by the accumulation of ROS-scavenging metabolites and alterations in ROS metabolism, leading to lowered ROS levels and enhanced photosynthesis. Additionally, it modulates ion sequestration through V-ATPase accumulation, potentially contributing to the observed decrease in chloride content.

Transcriptional and metabolic profiling of sulfur starvation response in two monocots.

BACKGROUND: Sulfur (S) is a mineral nutrient essential for plant growth and development, which is incorporated into diverse molecules fundamental for primary and secondary metabolism, plant defense, signaling, and maintaining cellular homeostasis. Although, S starvation response is well documented in the dicot model Arabidopsis thaliana, it is not clear if the same transcriptional networks control the response also in the monocots. RESULTS: We performed series of physiological, expression, and metabolite analyses in two model monocot species, one representing the C3 plants, Oryza sativa cv. kitaake, and second representing the C4 plants, Setaria viridis. Our comprehensive transcriptomic analysis revealed twice as many differentially expressed genes (DEGs) in S. viridis than in O. sativa under S-deficiency, consistent with a greater loss of sulfur and S-containing metabolites under these conditions. Surprisingly, most of the DEGs and enriched gene ontology terms were species-specific, with an intersect of only 58 common DEGs. The transcriptional networks were different in roots and shoots of both species, in particular no genes were down-regulated by S-deficiency in the roots of both species. CONCLUSIONS: Our analysis shows that S-deficiency seems to have different physiological consequences in the two monocot species and their nutrient homeostasis might be under distinct control mechanisms.

Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge.

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.

Traits linked to natural variation of sulfur content in Arabidopsis thaliana.

Sulfur (S) is an essential mineral nutrient for plant growth and development; it is important for primary and specialized plant metabolites that are crucial for biotic and abiotic interactions. Foliar S content varies up to 6-fold under a controlled environment, suggesting an adaptive value under certain natural environmental conditions. However, a major quantitative regulator of S content in Arabidopsis thaliana has not been identified yet, pointing to the existence of either additional genetic factors controlling sulfate/S content or of many minor quantitative regulators. Here, we use overlapping information of two separate ionomics studies to select groups of accessions with low, mid, and high foliar S content. We quantify series of metabolites, including anions (sulfate, phosphate, and nitrate), thiols (cysteine and glutathione), and seven glucosinolates, gene expression of 20 genes, sulfate uptake, and three biotic traits. Our results suggest that S content is tightly connected with sulfate uptake, the concentration of sulfate and phosphate anions, and glucosinolate and glutathione synthesis. Additionally, our results indicate that the growth of pathogenic bacteria is enhanced in the A. thaliana accessions containing higher S in their leaves, suggesting a complex regulation between S homeostasis, primary and secondary metabolism, and biotic pressures.

PlantACT! - how to tackle the climate crisis.

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels.

Shoot-root interaction in control of camalexin exudation in Arabidopsis.

Plants exude secondary metabolites from the roots to shape the composition and function of their microbiome. Many of these compounds are known for their anti-microbial activities and play a role in plant immunity, such as the indole-derived phytoalexin camalexin. Here we studied the dynamics of camalexin synthesis and exudation upon interaction of Arabidopsis thaliana with the plant growth promoting bacteria Pseudomonas sp. CH267 or the bacterial pathogen Burkholderia glumae PG1. We show that while camalexin accumulation and exudation is more rapidly but transiently induced upon interaction with the growth promoting bacteria, the pathogen induces higher and more stable camalexin levels. By combination of experiments with cut shoots and roots, and grafting of wild-type plants with mutants in camalexin synthesis, we showed that while camalexin can be produced and released by both organs, in intact plants exuded camalexin originates in the shoots. We also reveal that the root specific CYP71A27 protein specifically affects the outcome of the interaction with the plant growth promoting bacteria and that its transcript levels are controlled by a shoot derived signal. In conclusion, camalexin synthesis seems to be controlled on a whole plant level and is coordinated between the shoots and the roots.

Sulfate supplementation affects nutrient and photosynthetic status of Arabidopsis thaliana and Nicotiana tabacum differently under prolonged exposure to cadmium.

Hydroponic experiments were performed to examine the effect of prolonged sulfate limitation combined with cadmium (Cd) exposure in Arabidopsis thaliana and a potential Cd hyperaccumulator, Nicotiana tabacum. Low sulfate treatments (20 and 40 µM MgSO4) and Cd stress (4 µM CdCl2) showed adverse effects on morphology, photosynthetic and biochemical parameters and the nutritional status of both species. For example, Cd stress decreased NO3- root content under 20 µM MgSO4 to approximately 50% compared with respective controls. Interestingly, changes in many measured parameters, such as chlorophyll and carotenoid contents, the concentrations of anions, nutrients and Cd, induced by low sulfate supply, Cd exposure or a combination of both factors, were species-specific. Our data showed opposing effects of Cd exposure on Ca, Fe, Mn, Cu and Zn levels in roots of the studied plants. In A. thaliana, levels of glutathione, phytochelatins and glucosinolates demonstrated their distinct involvement in response to sub-optimal growth conditions and Cd stress. In shoot, the levels of phytochelatins and glucosinolates in the organic sulfur fraction were not dependent on sulfate supply under Cd stress. Altogether, our data showed both common and species-specific features of the complex plant response to prolonged sulfate deprivation and/or Cd exposure.

Unique features of regulation of sulfate assimilation in monocots.

Sulfate assimilation is an essential pathway of plant primary metabolism, regulated by the demand for reduced sulfur (S). The S-containing tripeptide glutathione (GSH) is the key signal for such regulation in Arabidopsis, but little is known about the conservation of these regulatory mechanisms beyond this model species. Using two model monocot species, C3 rice (Oryza sativa) and C4Setaria viridis, and feeding of cysteine or GSH, we aimed to find out how conserved are the regulatory mechanisms described for Arabidopsis in these species. We showed that while in principle the regulation is similar, there are many species-specific differences. For example, thiols supplied by the roots are translocated to the shoots in rice but remain in the roots of Setaria. Cysteine and GSH concentrations are highly correlated in Setaria, but not in rice. In both rice and Setaria, GSH seems to be the signal for demand-driven regulation of sulfate assimilation. Unexpectedly, we observed cysteine oxidation to sulfate in both species, a reaction that does not occur in Arabidopsis. This reaction is dependent on sulfite oxidase, but the enzyme(s) releasing sulfite from cysteine still need to be identified. Altogether our data reveal a number of unique features in the regulation of S metabolism in the monocot species and indicate the need for using multiple taxonomically distinct models to better understand the control of nutrient homeostasis, which is important for generating low-input crop varieties.

Integration of transcriptomic and metabolomic analyses provides insights into response mechanisms to nitrogen and phosphorus deficiencies in soybean.

Nitrogen (N) and phosphorus (P) are two essential plant macronutrients that can limit plant growth by different mechanisms. We aimed to shed light on how soybean respond to low nitrogen (LN), low phosphorus (LP) and their combined deficiency (LNP). Generally, these conditions triggered changes in gene expression of the same processes, including cell wall organization, defense response, response to oxidative stress, and photosynthesis, however, response was different in each condition. A typical primary response to LN and LP was detected also in soybean, i.e., the enhanced uptake of N and P, respectively, by upregulation of genes for the corresponding transporters. The regulation of genes involved in cell wall organization showed that in LP roots tended to produce more casparian strip, in LN more secondary wall biosynthesis occurred, and in LNP reduction in expression of genes involved in secondary wall production accompanied by cell wall loosening was observed. Flavonoid biosynthesis also showed distinct pattern of regulation in different conditions: more anthocyanin production in LP, and more isoflavonoid production in LN and LNP, which we confirmed also on the metabolite level. Interestingly, in soybean the nutrient deficiencies reduced defense response by lowering expression of genes involved in defense response, suggesting a role of N and P nutrition in plant disease resistance. In conclusion, we provide detailed information on how LN, LP, and LNP affect different processes in soybean roots on the molecular and physiological levels.

Cytokinin modulates the metabolic network of sulfur and glutathione.

The phytohormone cytokinin is implicated in a range of growth, developmental, and defense processes. A growing body of evidence supports a crosstalk between cytokinin and nutrient signaling pathways, such as nitrate availability. Cytokinin signaling regulates sulfur-responsive gene expression, but the underlying molecular mechanisms and their impact on sulfur-containing metabolites have not been systematically explored. Using a combination of genetic and pharmacological tools, we investigated the interplay between cytokinin signaling and sulfur homeostasis. Exogenous cytokinin triggered sulfur starvation-like gene expression accompanied by a decrease in sulfate and glutathione content. This process was uncoupled from the activity of the major transcriptional regulator of sulfate starvation signaling SULFUR LIMITATION 1 and an important glutathione-degrading enzyme, γ-glutamyl cyclotransferase 2;1, expression of which was robustly up-regulated by cytokinin. Conversely, glutathione accumulation was observed in mutants lacking the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 3 and in cytokinin-deficient plants. Cytokinin-deficient plants displayed improved root growth upon exposure to glutathione-depleting chemicals which was attributed to a higher capacity to maintain glutathione levels. These results shed new light on the interplay between cytokinin signaling and sulfur homeostasis. They position cytokinin as an important modulator of sulfur uptake, assimilation, and remobilization in plant defense against xenobiotics and root growth.

Measurement of flux through sulfate assimilation using [35S]sulfate.

Sulfur metabolism provides a number of compounds that are essential for plant survival and fitness and that affect the yield and quality of crops. Sulfur metabolism is a dynamic process, responding to a number of external cues. Because of this dynamics and rapid turnover, steady-state levels of sulfur-containing compounds do not always fully reflect plant responses to such cues. Therefore, measurements of the flux through sulfate assimilation may give a more precise estimate of the effects of environmental stimuli or metabolic disturbances on sulfur metabolism. The flux can be determined after feeding plants with sulfate labelled with an isotopic tracer. Here we describe a protocol for using [35S]sulfate to measure flux through sulfate assimilation in Arabidopsis. The protocol can be adapted for any plant species and growth conditions, and does not require any special equipment beyond a standard high performance liquid chromatograph. We hope that the protocol will support our colleagues in a more frequent use of flux measurements to answer new biological questions in plant sulfur metabolism.

Sulfur signaling and starvation response in Arabidopsis.

As sessile organisms, plants have developed sophisticated mechanism to sense and utilize nutrients from the environment, and modulate their growth and development according to the nutrient availability. Research in the past two decades revealed that nutrient assimilation is not occurring spontaneously, but nutrient signaling networks are complexly regulated and integrate sensing and signaling, gene expression, and metabolism to ensure homeostasis and coordination with plant energy conversion and other processes. Here, we review the importance of the macronutrient sulfur (S) and compare the knowledge of S signaling with other important macronutrients, such as nitrogen (N) and phosphorus (P). We focus on key advances in understanding sulfur sensing and signaling, uptake and assimilation, and we provide new analysis of published literature, to identify core genes regulated by the key transcriptional factor in S starvation response, SLIM1/EIL3, and compare the impact on other nutrient deficiency and stresses on S-related genes.

Plant secondary metabolites altering root microbiome composition and function.

Plants share their natural environment with numerous microorganisms, commensal as well as harmful. Plant fitness and performance are thus dependent on an efficient communication with such microbiota. The primary means of communication are metabolites exuded from roots, primarily diverse secondary metabolites. The exuded metabolites trigger changes in composition and function of plant associated microbiome. In the last few years, many metabolites were uncovered that are part of this communication network and modulate specific functions of the root microbiota. Here, we describe the progress in identification of such metabolites and their functions and outline the most significant knowledge gaps for future research.

Characterization of the amidoxime reducing components ARC1 and ARC2 from Arabidopsis thaliana.

Five molybdenum-dependent enzymes are known in eukaryotes. While four of them are under investigation since decades, the most recently discovered, (mitochondrial) amidoxime reducing component ((m)ARC), has only been characterized in mammals and the green algae Chlamydomonas reinhardtii. While mammalian mARCs have been shown to be involved in various signalling pathways, Chlamydomonas ARC was shown to be a nitric oxide (NO)-forming nitrite reductase. Similar to mammals, higher plants possess two ARC proteins. To test whether plant ARCs have a similar function in NO production to the function they have in C. reinhardtii, we analysed the enzymes from the model plant Arabidopsis thaliana. Both ARC1 and ARC2 from Arabidopsis could reduce N-hydroxylated compounds, while nitrite reduction to form NO could only be demonstrated for ARC2. Searching for physiological electron donors, we found that both ARC enzymes accept electrons from NADH via cytochrome b5 reductase and cytochrome b5 , but only ARC2 is able to accept electrons from nitrate reductase at all. Furthermore, arc-deficient mutant plants were similar to wildtype plants regarding growth and also nitrite-dependent NO-formation. Altogether, our results did not confirm the hypothesis that either ARC1 or ARC2 from Arabidopsis are involved in physiologically relevant nitrite-dependent NO-formation. In contrast, our data suggest that ARC1 and ARC2 have distinct, yet unknown physiological roles in higher plants.

Glutathione contributes to plant defence against parasitic cyst nematodes.

Cyst nematodes (CNs) are an important group of root-infecting sedentary endoparasites that severely damage many crop plants worldwide. An infective CN juvenile enters the host's roots and migrates towards the vascular cylinder, where it induces the formation of syncytial feeding cells, which nourish the CN throughout its parasitic stages. Here, we examined the role of glutathione (l-γ-glutamyl-l-cysteinyl-glycine) in Arabidopsis thaliana on infection with the CN Heterodera schachtii. Arabidopsis lines with mutations pad2, cad2, or zir1 in the glutamate-cysteine ligase (GSH1) gene, which encodes the first enzyme in the glutathione biosynthetic pathway, displayed enhanced CN susceptibility, but susceptibility was reduced for rax1, another GSH1 allele. Biochemical analysis revealed differentially altered thiol levels in these mutants that was independent of nematode infection. All glutathione-deficient mutants exhibited impaired activation of defence marker genes as well as genes for biosynthesis of the antimicrobial compound camalexin early in infection. Further analysis revealed a link between glutathione-mediated plant resistance to CN infection and the production of camalexin on nematode infection. These results suggest that glutathione levels affect plant resistance to CN by fine-tuning the balance between the cellular redox environment and the production of compounds related to defence against infection.

Natural Variation in OASC Gene for Mitochondrial O-Acetylserine Thiollyase Affects Sulfate Levels in Arabidopsis.

Sulfur plays a vital role in the primary and secondary metabolism of plants, and carries an important function in a large number of different compounds. Despite this importance, compared to other mineral nutrients, relatively little is known about sulfur sensing and signalling, as well as about the mechanisms controlling sulfur metabolism and homeostasis. Sulfur contents in plants vary largely not only among different species, but also among accessions of the same species. We previously used associative transcriptomics to identify several genes potentially controlling variation in sulfate content in the leaves of Brassica napus, including an OASC gene for mitochondrial O-acetylserine thiollyase (OAS-TL), an enzyme involved in cysteine synthesis. Here, we show that loss of OASC in Arabidopsis thaliana lowers not only sulfate, but also glutathione levels in the leaves. The reduced accumulation is caused by lower sulfate uptake and translocation to the shoots; however, the flux through the pathway is not affected. In addition, we identified a single nucleotide polymorphism in the OASC gene among A. thaliana accessions that is linked to variation in sulfate content. Both genetic and transgenic complementation confirmed that the exchange of arginine at position 81 for lysine in numerous accessions resulted in a less active OASC and a lower sulfate content in the leaves. The mitochondrial isoform of OAS-TL is, thus, after the ATPS1 isoform of sulfurylase and the APR2 form of APS reductase 2, the next metabolic enzyme with a role in regulation of sulfate content in Arabidopsis.