From biosynthesis and beyond-Loss or overexpression of the cytokinin synthesis gene, iptA, alters cytokinesis and mitochondrial and amino acid metabolism in Dictyostelium discoideum.

Cytokinins (CKs) are a class of growth-promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA-IPTs). In this study, the social amoeba, Dictyostelium discoideum, was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate-IPT gene, iptA. The life cycle of D. discoideum is unique and possesses both single- and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum. To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA-deficiency results in cytokinesis defects, and both iptA-deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.

Impact of glyphosate and glyphosate-based herbicides on phyllospheric Methylobacterium.

Symbiotic Methylobacterium comprise a significant portion of the phyllospheric microbiome, and are known to benefit host plant growth, development, and confer tolerance to stress factors. The near ubiquitous use of the broad-spectrum herbicide, glyphosate, in farming operations globally has necessitated a more expansive evaluation of the impacts of the agent itself and formulations containing glyphosate on important components of the plant phyllosphere, including Methylobacterium.This study provides an investigation of the sensitivity of 18 strains of Methylobacterium to glyphosate and two commercially available glyphosate-based herbicides (GBH). Nearly all strains of Methylobacterium showed signs of sensitivity to the popular GBH formulations WeatherMax® and Transorb® in a modified Kirby Bauer experiment. However, exposure to pure forms of glyphosate did not show a significant effect on growth for any strain in both the Kirby Bauer test and in liquid broth, until polysorbate-20 (Tween20) was added as a surfactant. Artificially increasing membrane permeability through the introduction of polysorbate-20 caused a 78-84% reduction in bacterial cell biomass relative to controls containing glyphosate or high levels of surfactant only (0-9% and 6-37% reduction respectively). Concentrations of glyphosate as low as 0.05% w/v (500 µg/L) from both commercial formulations tested, inhibited the culturability of Methylobacterium on fresh nutrient-rich medium.To better understand the compatibility of important phyllospheric bacteria with commercial glyphosate-based herbicides, this study endeavours to characterize sensitivity in multiple strains of Methylobacterium, and explore possible mechanisms by which toxicity may be induced.

Silicon ameliorates clubroot responses in canola (Brassica napus): A "multi-omics"-based investigation into possible mechanisms.

Clubroot disease, caused by Plasmodiophora brassicae Woronin, results in severe yield losses in Brassica crops, including canola. Silicon (Si) mitigates several stresses and enhances plant resistance to phytopathogens. We investigated the effects of Si on clubroot disease symptoms in canola at two concentrations of Si, Si: soil in 1: 100 w/w (Si1.0) and Si: soil in 1:200 w/w (Si0.5) under greenhouse conditions. In addition, the effects of Si on P. brassicae-induced gene expression, endogenous levels of phytohormones and metabolites were studied using "omics" approaches. Si application reduced clubroot symptoms and improved plant growth parameters. Gene expression analysis revealed increased transcript-level responses in Si1.0 compared to Si0.5 plants at 7-, 14-, and 21-days post-inoculation (dpi). Pathogen-induced transcript-level changes were affected by Si treatment, with genes related to antioxidant activity (e.g., POD, CAT), phytohormone biosynthesis and signalling (e.g., PDF1.2, NPR1, JAZ, IPT, TAA), nitrogen metabolism (e.g., NRT, AAT), and secondary metabolism (e.g., PAL, BCAT4) exhibiting differential expression. Endogenous levels of phytohormones (e.g., auxin, cytokinin), a majority of the amino acids and secondary metabolites (e.g., glucosinolates) were increased at 7 dpi, followed by a decrease at 14- and 21-dpi due to Si-treatment. Stress hormones such as abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) also decreased at the later time points in Si0.5, and Si1.0 treated plants. Si appears to improve clubroot symptoms while enhancing plant growth and associated metabolic processes, including nitrogen metabolism and secondary metabolite biosynthesis.

A survey of Methylobacterium species and strains reveals widespread production and varying profiles of cytokinin phytohormones.

BACKGROUND: Symbiotic Methylobacterium strains comprise a significant part of plant microbiomes. Their presence enhances plant productivity and stress resistance, prompting classification of these strains as plant growth-promoting bacteria (PGPB). Methylobacteria can synthesize unusually high levels of plant hormones, called cytokinins (CKs), including the most active form, trans-Zeatin (tZ). RESULTS: This study provides a comprehensive inventory of 46 representatives of Methylobacterium genus with respect to phytohormone production in vitro, including 16 CK forms, abscisic acid (ABA) and indole-3-acetic acid (IAA). High performance-liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analyses revealed varying abilities of Methylobacterium strains to secrete phytohormones that ranged from 5.09 to 191.47 pmol mL-1 for total CKs, and 0.46 to 82.16 pmol mL-1 for tZ. Results indicate that reduced methanol availability, the sole carbon source for bacteria in the medium, stimulates CK secretion by Methylobacterium. Additionally, select strains were able to transform L-tryptophan into IAA while no ABA production was detected. CONCLUSIONS: To better understand features of CKs in plants, this study uncovers CK profiles of Methylobacterium that are instrumental in microbe selection for effective biofertilizer formulations.

Beyond transport: cytokinin ribosides are translocated and active in regulating the development and environmental responses of plants.

MAIN CONCLUSION: Riboside type cytokinins are key components in cytokinin metabolism, transport, and sensitivity, making them important functional signals in plant growth and development and environmental stress responses. Cytokinin (CKs) are phytohormones that regulate multiple processes in plants and are critical for agronomy, as they are involved in seed filling and plant responses to biotic and abiotic stress. Among the over 30 identified CKs, there is uncertainty about the roles of many of the individual CK structural forms. Cytokinin free bases (CKFBs), have been studied in great detail, but, by comparison, roles of riboside-type CKs (CKRs) in CK metabolism and associated signaling pathways and their distal impacts on plant physiology remain largely unknown. Here, recent findings on CKR abundance, transport and localization, are summarized, and their importance in planta is discussed. The history of CKR analyses is reviewed, in the context of the determination of CK metabolic pathways, and research on CKR affinity for CK receptors, all of which yield essential insights into their functions. Recent studies suggest that CKR forms are a lot more than a group of transport CKs and, beyond this, they play important roles in plant development and responses to environmental stress. In this context, this review discusses the involvement of CKRs in plant development, and highlight the less anticipated functions of CKRs in abiotic stress tolerance. Based on this, possible mechanisms for CKR modes of action are proposed and experimental approaches to further uncover their roles and future biotechnological applications are suggested.

Isopentenyltransferases as master regulators of crop performance: their function, manipulation, and genetic potential for stress adaptation and yield improvement.

Isopentenyltransferase (IPT) in plants regulates a rate-limiting step of cytokinin (CTK) biosynthesis. IPTs are recognized as key regulators of CTK homeostasis and phytohormone crosstalk in both biotic and abiotic stress responses. Recent research has revealed the regulatory function of IPTs in gene expression and metabolite profiles including source-sink modifications, energy metabolism, nutrient allocation and storage, stress defence and signalling pathways, protein synthesis and transport, and membrane transport. This suggests that IPTs play a crucial role in plant growth and adaptation. In planta studies of IPT-driven modifications indicate that, at a physiological level, IPTs improve stay-green characteristics, delay senescence, reduce stress-induced oxidative damage and protect photosynthetic machinery. Subsequently, these improvements often manifest as enhanced or stabilized crop yields and this is especially apparent under environmental stress. These mechanisms merit consideration of the IPTs as 'master regulators' of core cellular metabolic pathways, thus adjusting plant homeostasis/adaptive responses to altered environmental stresses, to maximize yield potential. If their expression can be adequately controlled, both spatially and temporally, IPTs can be a key driver for seed yield. In this review, we give a comprehensive overview of recent findings on how IPTs influence plant stress physiology and yield, and we highlight areas for future research.

Cytokinin activity during early kernel development corresponds positively with yield potential and later stage ABA accumulation in field-grown wheat (Triticum aestivum L.).

MAIN CONCLUSION: Early cytokinin activity and late abscisic acid dynamics during wheat kernel development correspond to cultivars with higher yield potential. Cytokinins represent prime targets for marker development for wheat breeding programs. Two major phytohormone groups, abscisic acid (ABA) and cytokinins (CKs), are of crucial importance for seed development. Wheat (Triticum aestivum L.) yield is, to a high degree, determined during the milk and dough stages of kernel development. Therefore, understanding the hormonal regulation of these early growth stages is fundamental for crop-improvement programs of this important cereal. Here, we profiled ABA and 25 CK metabolites (including active forms, precursors and inactive conjugates) during kernel development in five field-grown wheat cultivars. The levels of ABA and profiles of CK forms varied greatly among the tested cultivars and kernel stages suggesting that several types of CK metabolites are involved in spatiotemporal regulation of kernel development. The seed yield potential was associated with the elevated levels of active CK levels (tZ, cZ). Interestingly, the increased kernel cZ levels were followed by higher ABA production, suggesting there is an interaction between these two phytohormones. Furthermore, we analyzed the expression patterns of representatives of the four main CK metabolic gene families. The unique transcriptional patterns of the IPT (biosynthesis) and ZOG (reversible inactivation) gene family members (GFMs) in the high and low yield cultivars additionally indicate that there is a significant association between CK metabolism and yield potential in wheat. Based on these results, we suggest that both CK metabolites and their associated genes, can serve as important, early markers of yield performance in modern wheat breeding programs.

The Role of Phytohormones in Enhancing Metal Remediation Capacity of Algae.

Heavy metal (HM) contamination of the environment is a major issue worldwide, creating an ever-increasing demand for remediation techniques. Remediation with algae offers an ecologically safe, cost-effective, and efficient option for HM removal. Similar to plants, growth and development of algae are controlled by the hormonal system, where phytohormones are involved in HM tolerance and thus can regulate remediation ability; however, the underlying mechanisms of phytohormone function remain elusive. This review aims to draw a comprehensive model of phytohormone contributions to algal performance under HM stress. We focus on the mechanisms of HM biosorption, uptake and intracellular storage, and on how phytohormones interact with algal defence systems under HM exposure. We provide examples of successful utilization of algae in remediation, and of post-processing of algal materials. Finally, we discuss the advantages and risks of using algae for remediation. An in-depth understanding of these processes can inform effective HM remediation techniques.

Quantification of Cytokinins Using High-Resolution Accurate-Mass Orbitrap Mass Spectrometry and Parallel Reaction Monitoring (PRM).

Cytokinins (CKs) are adenine derivatives that act as phytohormones. These signaling molecules control plant cell division and differentiation, organ growth, and senescence, and they orchestrate plant interactions with biotic and abiotic environments. While CKs are predominately recognized as plant-based substances, CKs have been found across different domains of life, including microorganisms, insects, mammals, and humans. In plants, CKs act at trace, often low femtomolar concentrations; therefore, sensitive and precise analytical techniques are required to accurately detect and quantify them from complex biological matrices. Here, we report the first comprehensive CK quantification method using a QExactive Orbitrap mass spectrometer in high-resolution with a parallel reaction monitoring (PRM)-based approach. The current method progresses upon multiple reaction monitoring (MRM) methods, previously used for CK profiling on triple quadrupole mass spectrometers. This method offers improved mass accuracy and the complete product ion mass spectra (MS/MS) for compound determination with increased specificity, and sensitivity comparable with triple quadrupole instruments. The presented PRM approach was successfully applied to quantify 32 CKs in several biological samples.

Elevated carbon dioxide decreases the adverse effects of higher temperature and drought stress by mitigating oxidative stress and improving water status in Arabidopsis thaliana.

MAIN CONCLUSION: This study revealed that elevated carbon dioxide increases Arabidopsis tolerance to higher temperature and drought stress by mitigating oxidative stress and improving water status of plants. Few studies have considered multiple aspects of plant responses to key components of global climate change, including higher temperature, elevated carbon dioxide (ECO2), and drought. Hence, their individual and combinatorial effects on plants need to be investigated in the context of understanding climate change impact on plant growth and development. We investigated the interactive effects of temperature, CO2, watering regime, and genotype on Arabidopsis thaliana (WT and ABA-insensitive mutant, abi1-1). Plants were grown in controlled-environment growth chambers under two temperature regimes (22/18 °C and 28/24 °C, 16 h light/8 h dark), two CO2 concentrations (400 and 700 µmol mol-1), and two watering regimes (well-watered and water-stressed) for 18 days. Plant growth, anatomical, physiological, molecular, and hormonal responses were determined. Our study provided valuable information about plant responses to the interactive effects of multiple environmental factors. We showed that drought and ECO2 had larger effects on plants than higher temperatures. ECO2 alleviated the detrimental effects of temperature and drought by mitigating oxidative stress and plant water status, and this positive effect was consistent across multiple response levels. The WT plants performed better than the abi1-1 plants; the former had higher rosette diameter, total dry mass, leaf and soil water potential, leaf moisture, proline, ethylene, trans-zeatin, isopentyladenine, and cis-zeatin riboside than the latter. The water-stressed plants of both genotypes accumulated more abscisic acid (ABA) than the well-watered plants; however, higher temperatures decreased the ability of WT plants to produce ABA in response to drought. We conclude that drought strongly, while higher temperature to a lesser extent, affects Arabidopsis seedlings, and ECO2 reduces the adverse effects of these stressors more efficiently in the WT plants than in the abi1-1 plants. Findings from this study can be extrapolated to other plant species that share similar characteristics and/or family with Arabidopsis.

Canis familiaris tissues are characterized by different profiles of cytokinins typical of the tRNA degradation pathway.

Cytokinins (CKs) are a group of phytohormones essential to plant growth and development. The presence of these N6-modified adenine derivatives has also been documented in other groups of organisms, including bacteria, fungi, and insects. Thus far, however, only a single CK, N6-(Δ2-isopentenyl) adenine-9-riboside (iPR), has been identified in mammals. In plants, the nucleotide form of isopentenyladenine [iPR (either mono-, di-, or tri-) phosphate (iPRP)] is the first form of CK synthesized, and it is further modified to produce other CK types. To determine if a similar biosynthesis pathway exists in mammals, we tested for the presence of 27 CKs in a wide selection of canine organs using HPLC electrospray ionization-tandem mass spectrometry. Seven forms of CK were detected in the majority of the analyzed samples, including iPR, iPRP, cis-zeatin-9-riboside, cis-zeatin-9-riboside-5' (either mono-, di-, or triphosphate), 2-methylthio-N6-isopentenyladenine, 2-methylthio-N6-isopentenyladenosine, and 2-methylthio-zeatin. Total CK concentrations ranged from 1.96 pmol/g fresh weight (adrenal glands) to 1.40 × 103 pmol/g fresh weight (thyroid). The results of this study provide evidence that mammalian cells, like plant cells, can synthesize and process a diverse set of CKs including cis- and methylthiol-type CKs.-Seegobin, M., Kisiala, A., Noble, A., Kaplan, D., Brunetti, C., Emery, R. J. N. Canis familiaris tissues are characterized by different profiles of cytokinins typical of tRNA degradation pathway.

Cytokinin Production by the Rice Blast Fungus Is a Pivotal Requirement for Full Virulence.

Plants produce cytokinin (CK) hormones for controlling key developmental processes like source/sink distribution, cell division or programmed cell-death. Some plant pathogens have been shown to produce CKs but the function of this mimicry production by non-tumor inducing pathogens, has yet to be established. Here we identify a gene required for CK biosynthesis, CKS1, in the rice blast fungus Magnaporthe oryzae. The fungal-secreted CKs are likely perceived by the plant during infection since the transcriptional regulation of rice CK-responsive genes is altered in plants infected by the mutants in which CKS1 gene was deleted. Although cks1 mutants showed normal in vitro growth and development, they were severely affected for in planta growth and virulence. Moreover, we showed that the cks1 mutant triggered enhanced induction of plant defenses as manifested by an elevated oxidative burst and expression of defense-related markers. In addition, the contents of sugars and key amino acids for fungal growth were altered in and around the infection site by the cks1 mutant in a different manner than by the control strain. These results suggest that fungal-derived CKs are key effectors required for dampening host defenses and affecting sugar and amino acid distribution in and around the infection site.

DELLA1-Mediated Gibberellin Signaling Regulates Cytokinin-Dependent Symbiotic Nodulation.

In legume plants, low-nitrogen soils promote symbiotic interactions with rhizobial bacteria, leading to the formation of nitrogen-fixing root nodules. Among critical signals regulating this developmental process are bacterial Nod Factors (NFs) and several plant hormones, including cytokinins (CKs) and gibberellins (GAs). Here, we show in Medicago truncatula that GA signaling mediated by DELLA1 decreases the amount of bioactive CKs in roots and negatively impacts the Cytokinin Response1 (CRE1)-dependent NF activation of a subset of CK-signaling genes as well as of the CK-regulated Nodulation Signaling Pathway2 and Ethylene Response Factor Required for Nodulation1 early nodulation genes. Consistently, a dominant-active DELLA1 protein can partially rescue the reduced nodulation of the cre1 mutant and triggers the formation of nodule-like structures when expressed in the root cortex or in the root epidermis. This suggests a model where the DELLA1-mediated GA signaling interplays with the CRE1-dependent CK pathway to regulate early nodulation in response to both NF and CK signals critical for this symbiotic interaction.

A Laser Dissection-RNAseq Analysis Highlights the Activation of Cytokinin Pathways by Nod Factors in the Medicago truncatula Root Epidermis.

Nod factors (NFs) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4 and 24 h of treatment) in the root epidermis of the model legume Medicago truncatula Tissue-specific transcriptome analysis was achieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detected in the epidermis, among which 1,070 were found to be regulated by NF addition, including previously characterized NF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, with numerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways was indicated, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:ß-glucuronidase fusions with the MtCRE1 CK receptor gene and a CK response reporter (TWO COMPONENT SIGNALING SENSOR NEW). A CK pretreatment reduced the NF induction of the EARLY NODULIN11 (ENOD11) symbiotic marker, while a CK-degrading enzyme (CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased NF induction of ENOD11 and nodulation. Therefore, CK may play both positive and negative roles in M. truncatula nodulation.

Soybean Seed Development: Fatty Acid and Phytohormone Metabolism and Their Interactions.

Vegetable oil utilization is determined by its fatty acid composition. In soybean and other grain crops, during the seed development oil accumulation is important trait for value in food or industrial applications. Seed development is relatively short and sensitive to unfavorable abiotic conditions. These stresses can lead to a numerous undesirable qualitative as well as quantitative changes in fatty acid production. Fatty acid manipulation which targets a higher content of a specific single fatty acid for food or industrial application has gained more attention. Despite several successes in modifying the ratio of endogenous fatty acids in most domesticated oilseed crops, numerous obstacles in FA manipulation of seed maturation are yet to be overcome. Remarkably, connections with plant hormones have not been well studied despite their critical roles in the regulation and promotion of a plethora of processes in plant growth and development. While activities of phytohormones during the reproductive phase have been partially clarified in seed physiology, the biological role of plant hormones in oil accumulation during seed development has not been investigated. In this review seed development and numerous effects of abiotic stresses are discussed. After describing fatty acid and phytohormone metabolism and their interactions, we postulate that the endogenous plant hormones play important roles in fatty acid production in soybean seeds.

Biochemical characterization of a unique cytokinin and nucleotide phosphoribohydrolase Lonely Guy protein from Dictyostelium discoideum.

Lonely guy (LOG) proteins are phosphoribohydrolases (PRHs) that are key cytokinin (CK)-activating enzymes in plant and non-plant CK-producing organisms. During CK biosynthesis, LOGs catalyze the conversion of precursor CK-nucleotides (CK-NTs) to biologically active free base forms. LOG/PRH activity has been detected in bacteria, archaea, algae, and fungi. However, in these organisms, the LOG/PRH activity for CK-NTs and non-CK-NTs (e.g., adenine-NTs) has not been assessed simultaneously, which leaves limited knowledge about the substrate specificity of LOGs. Thus, we performed bioinformatic analyses and a biochemical characterization of a LOG ortholog from Dictyostelium discoideum, a soil-dwelling amoeba, which produces CKs during unicellular growth and multicellular development. We show that DdLog exhibits LOG/PRH activity on two CK-NTs, N 6 -isopentenyladenosine-5'-monophosphate (iPMP) and N 6 -benzyladenosine-5'-monophosphate (BAMP), and on adenosine 5'-monophosphate (AMP) but not on 3', 5'-cyclic adenosine-monophosphate (cAMP). Additionally, there were higher turnover rates for CK-NTs over AMP. Together, these findings confirm that DdLog acts as a CK-activating enzyme; however, in contrast to plant LOGs, it maintains a wider specificity for other substrates (e.g., AMP) reflecting it has maintained its original, non-CK related role even after diversifying into a CK-activating enzyme.

Proteome- and metabolome-level changes during early stages of clubroot infection in Brassica napus canola.

Clubroot is a destructive root disease of canola (Brassica napus L.) caused by Plasmodiophora brassicae Woronin. Despite extensive research into the molecular responses of B. napus to P. brassicae, there is limited information on proteome- and metabolome-level changes in response to the pathogen, especially during the initial stages of infection. In this study, we have investigated the proteome- and metabolome- level changes in the roots of clubroot-resistant (CR) and -susceptible (CS) doubled-haploid (DH) B. napus lines, in response to P. brassicae pathotype 3H at 1-, 4-, and 7-days post-inoculation (DPI). Root proteomes were analyzed using nanoflow liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS). Comparisons of pathogen-inoculated and uninoculated root proteomes revealed 2515 and 1556 differentially abundant proteins at one or more time points (1-, 4-, and 7-DPI) in the CR and CS genotypes, respectively. Several proteins related to primary metabolites (e.g., amino acids, fatty acids, and lipids), secondary metabolites (e.g., glucosinolates), and cell wall reinforcement-related proteins [e.g., laccase, peroxidases, and plant invertase/pectin methylesterase inhibitors (PInv/PMEI)] were identified. Eleven nucleotides and nucleoside-related metabolites, and eight fatty acids and sphingolipid-related metabolites were identified in the metabolomics study. To our knowledge, this is the first report of root proteome-level changes and associated alterations in metabolites during the early stages of P. brassicae infection in B. napus.

Bioactive cytokinins are selectively secreted by Sinorhizobium meliloti nodulating and nonnodulating strains.

Bacteria present in the rhizosphere of plants often synthesize phytohormones, and these signals can consequently affect root system development. In legumes, plants adapt to nitrogen starvation by forming lateral roots as well as a new organ, the root nodule, following a symbiotic interaction with bacteria collectively referred to as rhizobia. As cytokinin (CK) phytohormones were shown to be necessary and sufficient to induce root nodule organogenesis, the relevance of CK production by symbiotic rhizobia was questioned. In this study, we analyzed quantitatively, by liquid chromatography-tandem mass spectrometry, the production of 25 forms of CK in nine rhizobia strains belonging to four different species. All bacterial strains were able to synthesize a mix of CK, and bioactive forms of CK, such as iP, were notably found to be secreted in bacterial culture supernatants. Use of a mutant affected in extracellular polysaccharide (EPS) production revealed a negative correlation of EPS production with the ability to secrete CK. In addition, analysis of a nonnodulating Sinorhizobium meliloti strain revealed a similar pattern of CK production and secretion when compared with a related nodulating strain. This indicates that bacterially produced CK are not sufficient to induce symbiotic nodulation.

Abscisic Acid: A Potential Secreted Effector Synthesized by Phytophagous Insects for Host-Plant Manipulation.

Abscisic acid (ABA) is an isoprenoid-derived plant signaling molecule involved in a wide variety of plant processes, including facets of growth and development as well as responses to abiotic and biotic stress. ABA had previously been reported in a wide variety of animals, including insects and humans. We used high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-(ESI)-MS/MS) to examine concentrations of ABA in 17 species of phytophagous insects, including gall- and non-gall-inducing species from all insect orders with species known to induce plant galls: Thysanoptera, Hemiptera, Lepidoptera, Coleoptera, Diptera, and Hymenoptera. We found ABA in insect species in all six orders, in both gall-inducing and non-gall-inducing species, with no tendency for gall-inducing insects to have higher concentrations. The concentrations of ABA in insects often markedly exceeded those typically found in plants, suggesting it is highly improbable that insects obtain all their ABA from their host plant via consumption and sequestration. As a follow-up, we used immunohistochemistry to determine that ABA localizes to the salivary glands in the larvae of the gall-inducing Eurosta solidaginis (Diptera: Tephritidae). The high concentrations of ABA, combined with its localization to salivary glands, suggest that insects are synthesizing and secreting ABA to manipulate their host plants. The pervasiveness of ABA among both gall- and non-gall-inducing insects and our current knowledge of the role of ABA in plant processes suggest that insects are using ABA to manipulate source-sink mechanisms of nutrient allocation or to suppress host-plant defenses. ABA joins the triumvirate of phytohormones, along with cytokinins (CKs) and indole-3-acetic acid (IAA), that are abundant, widespread, and localized to glandular organs in insects and used to manipulate host plants.

Profiling of adenine-derived signaling molecules, cytokinins, in myotubes reveals fluctuations in response to lipopolysaccharide-induced cell stress.

Cytokinins (CTKs) are a diverse collection of evolutionarily conserved adenine-derived signaling molecules classically studied as phytohormones; however, their roles and production have been less studied in mammalian systems. Skeletal muscles are sensitive to cellular cues such as inflammation and in response, alter their secretome to regulate the muscle stem cell and myofiber niche. Using cultured C2C12 muscle cells, we profiled CTK levels to understand (1) whether CTKs are part of the muscle secretome and (2) whether CTKs are responsive to cellular stress. To induce cellular stress, C2C12 myotubes were treated with lipopolysaccharides (LPS) for 24 h and then media and cell fractions were collected for ultra high-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-(ESI+)-HRMS/MS) for metabolomics and CTK profiling. Across LPS-treated and control cells, 11 CTKs were detected in the extracellular space while 6 were detected intracellularly. We found that muscle cells are enriched in isopentenyladenine (iP) species (from free base, riboside to nucleotide forms), and that extracellular levels are increased after LPS treatment. Our study establishes that muscle cells express various forms of CTKs, and that CTK levels are responsive to LPS-induced cell stress, suggesting a role for CTKs in intra- and extracellular signaling of mammalian cells.