CPK28 is a modulator of purinergic signaling in plant growth and defense.

Extracellular ATP (eATP) is a key signaling molecule that plays a pivotal role in plant growth and defense responses. The receptor P2K1 is responsible for perceiving eATP and initiating its signaling cascade. However, the signal transduction mechanisms downstream of P2K1 activation remain incompletely understood. We conducted a comprehensive analysis of the P2K1 interactome using co-immunoprecipitation-coupled tandem mass spectrometry, leading to the identification of 121 candidate proteins interacting with P2K1. In silico analysis narrowed down the candidates to 47 proteins, including Ca2+-binding proteins, ion transport-related proteins, and receptor kinases. To investigate their involvement in eATP signaling, we employed a screening strategy based on changes in gene expression in response to eATP in mutants of the identified interactors. This screening revealed several Ca2+-dependent protein kinases (CPKs) that significantly affected the expression of eATP-responsive genes, suggesting their potential roles in eATP signaling. Notably, CPK28 and CPK6 showed physical interactions with P2K1 both in yeast and plant systems. Calcium influx and gene expression studies demonstrated that CPK28 perturbed eATP-induced Ca2+ mobilization and some early transcriptional responses. Overexpression of CPK28 resulted in an antagonistic physiological response to P2K1-mediated eATP signaling during both plant growth and defense responses to the necrotrophic pathogen Botrytis cinerea. Our findings highlight CPK28, among other CPKs, as a modulator of P2K1-mediated eATP signaling, providing valuable insights into the coordination of eATP signaling in plant growth and immunity.

The intrinsically disordered C-terminus of purinoceptor P2K1 fine-tunes plant responses to extracellular ATP.

P2K1 is a plant-specific purinoceptor that perceives extracellular ATP (eATP), a signaling molecular implicated in various physiological processes. Interestingly, P2K1 harbors a C-terminal intrinsically disordered region (IDR). When we overexpressed a truncated P2K1 (P2K1t ) lacking the IDR, primary root growth completely ceased in response to eATP. We investigated the functional roles of the IDR in P2K1 using a combination of molecular genetics, calcium imaging, gene expression analysis, and histochemical approaches. We found that the P2K1t variant gave rise to an amplified response to eATP, through accumulation of superoxide, altered cell wall integrity, and ultimate cell death in the primary root tip. Together, these observations underscore the significant involvement of the C-terminal tail of P2K1 in root growth regulation.

Nanopore Sequencing with GraphMap for Comprehensive Pathogen Detection in Potato Field Soil.

Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, such as soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests of three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs further experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.

Distinct Molecular Pattern-Induced Calcium Signatures Lead to Different Downstream Transcriptional Regulations via AtSR1/CAMTA3.

Plants encrypt the perception of different pathogenic stimuli into specific intracellular calcium (Ca2+) signatures and subsequently decrypt the signatures into appropriate downstream responses through various Ca2+ sensors. Two microbe-associated molecular patterns (MAMPs), bacterial flg22 and fungal chitin, and one damage-associated molecular pattern (DAMP), AtPep1, were used to study the differential Ca2+ signatures in Arabidopsis leaves. The results revealed that flg22, chitin, and AtPep1 induced distinct changes in Ca2+ dynamics in both the cytosol and nucleus. In addition, Flg22 and chitin upregulated the expression of salicylic acid-related genes, ICS1 and EDS1, whereas AtPep1 upregulated the expression of jasmonic acid-related genes, JAZ1 and PDF1.2, in addition to ICS1 and EDS1. These data demonstrated that distinct Ca2+ signatures caused by different molecular patterns in leaf cells lead to specific downstream events. Furthermore, these changes in the expression of defense-related genes were disrupted in a knockout mutant of the AtSR1/CAMTA3 gene, encoding a calmodulin-binding transcription factor, in which a calmodulin-binding domain on AtSR1 was required for deciphering the Ca2+ signatures into downstream transcription events. These observations extend our knowledge regarding unique and intrinsic roles for Ca2+ signaling in launching and fine-tuning plant immune response, which are mediated by the AtSR1/CAMTA3 transcription factor.

Transcriptomic perspective on extracellular ATP signaling: a few curious trifles.

Extracellular ATP is perceived by the purinoceptor P2K1, leading to induction of defense response in plants. Previously, we described the transcriptomic response to extracellular ATP in wild-type Arabidopsis seedlings and mutants of classical defense hormone signaling pathways (Jewell et al., 2019, Plant Physiol. 179: 1144-58), in which extracellular ATP was found to induce defense-related genes independently and also along with other defense signaling pathways. In the present study, we provide further analysis and discussion of the data that we neglected to describe in the previous transcriptomics report. Briefly, we describe transcriptomic differences between a P2K1 knockout mutant (dorn1) and wild-type seedlings in the absence of exogenous ATP as well as an analysis of genes more responsive to extracellular ATP in a P2K1 overexpression line. Finally, we describe an exaggerated response to extracellular ATP in the ein2 mutant and suggest testable explanations of this phenomenon.

Extracellular ATP Shapes a Defense-Related Transcriptome Both Independently and along with Other Defense Signaling Pathways.

ATP is not only an essential metabolite of cellular biochemistry but also acts as a signal in the extracellular milieu. In plants, extracellular ATP is monitored by the purinergic receptor P2K1. Recent studies have revealed that extracellular ATP acts as a damage-associated molecular pattern in plants, and its signaling through P2K1 is important for mounting an effective defense response against various pathogenic microorganisms. Biotrophic and necrotrophic pathogens attack plants using different strategies, to which plants respond accordingly with salicylate-based or jasmonate/ethylene-based defensive signaling, respectively. Interestingly, defense mediated by P2K1 is effective against pathogens of both lifestyles, raising the question of the level of interplay between extracellular ATP signaling and that of jasmonate, ethylene, and salicylate. To address this issue, we analyzed ATP-induced transcriptomes in wild-type Arabidopsis (Arabidopsis thaliana) seedlings and mutant seedlings defective in essential components in the signaling pathways of jasmonate, ethylene, and salicylate (classic defense hormones) as well as a mutant and an overexpression line of the P2K1 receptor. We found that P2K1 function is crucial for faithful ATP-induced transcriptional changes and that a subset of genes is more responsive in the P2K1 overexpression line. We also found that more than half of the ATP-responsive genes required signaling by one or more of the pathways for the classical defense hormones, with the jasmonate-based signaling being more critical than others. By contrast, the other ATP-responsive genes were unaffected by deficiencies in signaling for any of the classical defense hormones. These ATP-responsive genes were highly enriched for defense-related Gene Ontology terms. We further tested the ATP-induced genes in knockout mutants of transcription factors, demonstrating that MYCs acting downstream of the jasmonate receptor complex and calmodulin-binding transcription activators are nuclear transducers of P2K1-mediated extracellular ATP signaling.

Trimethylguanosine Synthase1 (TGS1) Is Essential for Chilling Tolerance.

Chilling stress is a major factor limiting plant development and crop productivity. Because the plant response to chilling is so complex, we are far from understanding the genes important in the response to chilling. To identify new genes important in chilling tolerance, we conducted a novel mutant screen, combining a confirmed SALK T-DNA insertion collection with traditional forward genetics. We screened a pool of more than 3700 confirmed homozygous SALK T-DNA insertion lines for visible defects under prolonged growth at 5°C. Of the chilling-sensitive mutants we observed, mutations at one locus were characterized in detail. This gene, At1g45231, encodes an Arabidopsis (Arabidopsis thaliana) trimethylguanosine synthase (TGS1), previously uncharacterized in the plant kingdom. We confirmed that Arabidopsis TGS1 is a functional ortholog of other trimethylguanosine synthases based both on its in vitro methyltransferase activity and on its ability to rescue the cold-growth inhibition of a Saccharomyces cerevisiae tgs1Δ mutant in vivo. While tgs1 mutant plants grew normally at 22°C, their vegetative and reproductive growth was severely compromised under chilling conditions. When we transgenically expressed TGS1 in the mutant plants, the chilling-sensitive phenotype was relieved, demonstrating that TGS1 is required for chilling tolerance.

Control of Carbon Assimilation and Partitioning by Jasmonate: An Accounting of Growth-Defense Tradeoffs.

Plant growth is often constrained by the limited availability of resources in the microenvironment. Despite the continuous threat of attack from insect herbivores and pathogens, investment in defense represents a lost opportunity to expand photosynthetic capacity in leaves and absorption of nutrients and water by roots. To mitigate the metabolic expenditure on defense, plants have evolved inducible defense strategies. The plant hormone jasmonate (JA) is a key regulator of many inducible defenses. Synthesis of JA in response to perceived danger leads to the deployment of a variety of defensive structures and compounds, along with a potent inhibition of growth. Genetic studies have established an important role for JA in mediating tradeoffs between growth and defense. However, several gaps remain in understanding of how JA signaling inhibits growth, either through direct transcriptional control of JA-response genes or crosstalk with other signaling pathways. Here, we highlight recent progress in uncovering the role of JA in controlling growth-defense balance and its relationship to resource acquisition and allocation. We also discuss tradeoffs in the context of the ability of JA to promote increased leaf mass per area (LMA), which is a key indicator of leaf construction costs and leaf life span.

Epidermal jasmonate perception is sufficient for all aspects of jasmonate-mediated male fertility in Arabidopsis.

Jasmonate (JA) signaling is essential for several environmental responses and reproductive development in many plant species. In Arabidopsis thaliana, the most obvious phenotype of JA biosynthetic and perception mutants is profound sporophytic male sterility characterized by failure of stamen filament elongation, severe delay of anther dehiscence and pollen inviability. The site of action of JA in the context of reproductive development has been discussed, but the ideas have not been tested experimentally. To this end we used targeted expression of a COI1-YFP transgene in the coi1-1 mutant background. As COI1 is an essential component of the JA co-receptor complex, the null coi1-1 mutant is male sterile due to lack of JA perception. We show that expression of COI1-YFP in the epidermis of the stamen filament and anther in coi1 mutant plants is sufficient to rescue filament elongation, anther dehiscence and pollen viability. In contrast, filament expression alone or expression in the tapetum do not restore dehiscence and pollen viability. These results demonstrate that epidermal JA perception is sufficient for anther function and pollen viability, and suggest the presence of a JA-dependent non-autonomous signal produced in the anther epidermis to synchronize both anther dehiscence and pollen maturation.

Hormone crosstalk in wound stress response: wound-inducible amidohydrolases can simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana.

Jasmonate (JA) and auxin are essential hormones in plant development and stress responses. While the two govern distinct physiological processes, their signaling pathways interact at various levels. Recently, members of the Arabidopsis indole-3-acetic acid (IAA) amidohydrolase (IAH) family were reported to metabolize jasmonoyl-isoleucine (JA-Ile), a bioactive form of JA. Here, we characterized three IAH members, ILR1, ILL6, and IAR3, for their function in JA and IAA metabolism and signaling. Expression of all three genes in leaves was up-regulated by wounding or JA, but not by IAA. Purified recombinant proteins showed overlapping but distinct substrate specificities for diverse amino acid conjugates of JA and IAA. Perturbed patterns of the endogenous JA profile in plants overexpressing or knocked-out for the three genes were consistent with ILL6 and IAR3, but not ILR1, being the JA amidohydrolases. Increased turnover of JA-Ile in the ILL6- and IAR3-overexpressing plants created symptoms of JA deficiency whereas increased free IAA by overexpression of ILR1 and IAR3 made plants hypersensitive to exogenous IAA conjugates. Surprisingly, ILL6 overexpression rendered plants highly resistant to exogenous IAA conjugates, indicating its interference with IAA conjugate hydrolysis. Fluorescent protein-tagged IAR3 and ILL6 co-localized with the endoplasmic reticulum-localized JA-Ile 12-hydroxylase, CYP94B3. Together, these results demonstrate that in wounded leaves JA-inducible amidohydrolases contribute to regulate active IAA and JA-Ile levels, promoting auxin signaling while attenuating JA signaling. This mechanism represents an example of a metabolic-level crosstalk between the auxin and JA signaling pathways.

Predicting gene function from uncontrolled expression variation among individual wild-type Arabidopsis plants.

Gene expression profiling studies are usually performed on pooled samples grown under tightly controlled experimental conditions to suppress variability among individuals and increase experimental reproducibility. In addition, to mask unwanted residual effects, the samples are often subjected to relatively harsh treatments that are unrealistic in a natural context. Here, we show that expression variations among individual wild-type Arabidopsis thaliana plants grown under the same macroscopic growth conditions contain as much information on the underlying gene network structure as expression profiles of pooled plant samples under controlled experimental perturbations. We advocate the use of subtle uncontrolled variations in gene expression between individuals to uncover functional links between genes and unravel regulatory influences. As a case study, we use this approach to identify ILL6 as a new regulatory component of the jasmonate response pathway.