Similar to animals, both the 3’,5- and the 2,’3’-cAMP isomers are present in plants. The former is the enzymatic product of adenylate cyclases (ACs), the latter is an RNA degradation product. While there is increasing evidence that both isomers can elicit or modulate a broad range of physiological responses, the question of isomer specificity of responses has remained largely unresolved. To delineate isomer-specific responses in Arabidopsis thaliana at the systems level, we have combined a comparative proteomics and electrophysiological approaches. Both isomers cause distinct systemic effects on the proteome, with the 2’,3’ isomer notably affecting systems-level functions like transcriptional regulation. None of the isomers affects net ion fluxes in the root under control conditions, but both were able to attenuate the magnitude of oxidative stress-induced K+ net loss and Ca2+ uptake by 2-fold. Isomer-specific responses of single molecular targets were assessed in the cyclic nucleotide-gated channels 2 and 18 (CNGC2 and CNGC18). Both channels are gated by the 3’,5’-cAMP isomer only, suggesting that the gating is isomer-specific and this implies that gating in vivo depends on catalytically active ACs. Highlights Distinct Arabidopsis responses to 3’,5’- and 2’,3’-cAMP uncover isomer-specific molecular targets and physiological effects in cyclic nucleotide signaling. ### Competing Interest Statement The authors have declared no competing interest. This work was supported by the University of Insubria grant Fondo di Ateneo per la Ricerca 2024 and 2025

cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP–dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular “sponge” that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

Increasing numbers of reports have revealed novel catalytically active cryptic guanylate cyclases (GCs) and adenylate cyclases (ACs) operating within complex proteins in prokaryotes and eukaryotes. Here we review the structural and functional aspects of some of these cyclases and provide examples that illustrate their roles in the regulation of the intramolecular functions of complex proteins, such as the phytosulfokine receptor (PSKR), and reassess their contribution to signal generation and tuning. Another multidomain protein, Arabidopsis thaliana K+ uptake permease (AtKUP5), also harbors multiple catalytically active sites including an N-terminal AC and C-terminal phosphodiesterase (PDE) with an abscisic acid-binding site. We argue that this architecture may enable the fine-tuning and/or sensing of K+ flux and integrate hormone responses to cAMP homeostasis. We also discuss how searches with motifs based on conserved amino acids in catalytic centers led to the discovery of GCs and ACs and propose how this approach can be applied to discover hitherto masked active sites in bacterial, fungal, and animal proteomes. Finally, we show that motif searches are a promising approach to discover ancient biological functions such as hormone or gas binding.

In addition to defensins, plants possess an array of defensin-like peptides that share many of their characteristics, as well as a role in plant’s innate immunity. Their involvement in the response to pathogens is well-known but the contribution in the plant response to abiotic stimuli is not fully understood. We have undertaken an in silico analysis to characterize all defensin family genes hitherto found in Arabidopsis, including genes encoding for defensin-like peptides, by detecting several peptides as candidates for further studies aiming to decipher specific responses to biotic and abiotic stresses, as well as to their crosstalk. We performed several analyses, including co-expression and cis-regulatory elements analyses, using transcriptomic data obtained from the ARS database, which integrates more than 20,000 Arabidopsis RNA-seq libraries.In silico analysis showed that jasmonates and ABA, together with transcription factors belonging to WRKY and AP2/EREBP families, modulate defensin and defensin-like gene expression. Indeed, the analysis performed in this study allowed to extract and organize omics data, which finally supported the inducible nature of defensins under both abiotic and biotic stresses. Moreover, in vivo expression analyses confirmed the heat and drought responsiveness of PDF1.4, ATTI1, PDF1.1, DEFL 206, defensin family genes selected for being upregulated by several abiotic conditions, at transcriptional level. Finally, the co-expression analysis provided information on other biological processes that may be correlated to the defensin induction, such as maintaining ROS homeostasis. Combining the comprehensive analysis of different transcriptional datasets with the integration of in vivo analyses emerged as a robust methodological approach to assess the proposed multi-stress responsive nature of defensin family genes.

Immersive virtual reality (VR) simulations are increasingly being used in diverse educational and training contexts to supplement traditional learning methods. The high versatility of virtual laboratories allows students to take advantage of many benefits, like experiencing dangerous reactions, time-consuming protocols, or expensive equipment without the necessity of a real science laboratory. However, little research is currently available to support the efficacy and efficiency of this new learning tool. In this context, the main objective of this study was to assess the influence of biotechnology training by using immersive VR technologies on the student’s motivational and learning outcomes as compared with learning with conventional methods only. To this aim we tested two diverse strategies, respectively VR simulations were used in place of or in addition to the teacher’s introductory lesson of a hands-on laboratory experience. Aligned questionaries were administered before and after the proposed activities to assess theoretical knowledge, self-efficacy, interest in biotechnology, and engagement of the participants. We found that when the introductory lesson to a biotechnology hands-on laboratory is replaced with an equivalent immersive VR simulation, the student’s learning outcomes are lower with respect to the traditional approach. On the contrary, when VR simulations are integrated as an additional tool to the existing learning methods, higher learning outcomes were observed demonstrating a deeper understanding of the learning contents. Furthermore, our study showed that learning with immersive VR simulations motivates students more than the traditional methods, thus, using this new technology in addition to the existing educational methods in biotechnology could be considered as a win-win strategy to raise the attention of the students while increasing the learning outcomes.

We analyzed the changes in the volatilome, proteome, stomatal conductance, salicylic and jasmonic acid contents of a susceptible and a moderately resistant genotype of common bean, Phaseoulus vulgaris L., challenged with Colletotrichum lindemuthianum, the causal agent of fungal anthracnose. Our results indicate differences at both proteome and volatilome levels between the two genotypes, before and after the infection, and different defense strategies. The moderately resistant genotype hindered pathogen infection, invasion, and replication mainly by maintaining epidermal and cell wall structure. The susceptible genotype was not able to limit the early stages of pathogen infection. Rather, stomatal conductance increased in the infected susceptible genotype, and enhanced synthesis of Green Leaf Volatiles and salicylic acid was observed, together with a strong hypersensitive response. Proteomic investigation provided a general framework for physiological changes, whereas observed variations in the volatilome suggested that volatile organic compounds may principally represent stress markers rather than defensive compounds per se.

The beneficial symbiosis between plants and arbuscular mycorrhizal (AM) fungi leads to a deep reprogramming of plant metabolism, involving the regulation of several molecular mechanisms, many of which are poorly characterized. In this regard, proteomics is a powerful tool to explore changes related to plant-microbe interactions. This study provides a comprehensive proteomic meta-analysis conducted on AM-modulated proteins at local (roots) and systemic (shoots/leaves) level. The analysis was implemented by an in-depth study of root membrane-associated proteins and by a comparison with a transcriptome meta-analysis. A total of 4262 differentially abundant proteins were retrieved and, to identify the most relevant AM-regulated processes, a range of bioinformatic studies were conducted, including functional enrichment and protein-protein interaction network analysis. In addition to several protein transporters which are present in higher amounts in AM plants, and which are expected due to the well-known enhancement of AM-induced mineral uptake, our analysis revealed some novel traits. We detected a massive systemic reprogramming of translation with a central role played by the ribosomal translational apparatus. On one hand, these new protein-synthesis efforts well support the root cellular re-organization required by the fungal penetration, and on the other they have a systemic impact on primary metabolism.

3',5'-cyclic adenosine monophosphate (cAMP) is finally recognized as an essential signaling molecule in plants where cAMP-dependent processes include responses to hormones and environmental stimuli. To better understand the role of 3',5'-cAMP at the systems level, we have undertaken a phosphoproteomic analysis to elucidate the cAMP-dependent response of tobacco BY-2 cells. These cells overexpress a molecular "sponge" that buffers free intracellular cAMP level. The results show that, firstly, in vivo cAMP dampening profoundly affects the plant kinome and notably mitogen-activated protein kinases, receptor-like kinases, and calcium-dependent protein kinases, thereby modulating the cellular responses at the systems level. Secondly, buffering cAMP levels also affects mRNA processing through the modulation of the phosphorylation status of several RNA-binding proteins with roles in splicing, including many serine and arginine-rich proteins. Thirdly, cAMP-dependent phosphorylation targets appear to be conserved among plant species. Taken together, these findings are consistent with an ancient role of cAMP in mRNA processing and cellular programming and suggest that unperturbed cellular cAMP levels are essential for cellular homeostasis and signaling in plant cells.

How temperate trees respond to drier summers strongly depends on the drought susceptibility and the starch reserve of the very-fine roots (<0.5 mm in diameter). We performed morphological, physiological, chemical, and proteomic analyses on very-fine roots of Fagus sylvatica seedlings grown under moderate- and severe drought conditions. Moreover, to reveal the role of the starch reserves, a girdling approach was adopted to interrupt the flux of photosynthates toward the downstream sinks. Results show a seasonal sigmoidal growth pattern without evident mortality under moderate drought. After the severe-drought period, intact plants showed lower starch concentration and higher growth than those subjected to moderate drought, highlighting that very-fine roots rely on their starch reserves to resume growth. This behavior caused them to die with the onset of autumn, which was not observed under moderate drought. These findings indicated that extreme dry soil conditions are needed for significant root death in beech seedlings and that mortality mechanisms are defined within individual compartments. The girdling treatment showed that the physiological responses of very-fine roots to severe drought stress are critically related to the altered load or the reduced transport velocity of the phloem and that the changes in starch allocation critically alter the distribution of biomass. Proteomic evidence revealed that the phloem flux-dependent response was characterized by the decrease of carbon enzymes and the establishment of mechanisms to avoid the reduction of the osmotic potential. The response independent from the aboveground mainly involved the alteration of primary metabolic processes and cell wall-related enzymes.

Lentinula edodes (Shiitake) is one of the most heavily cultivated mushrooms in the world with proven antioxidant and antibacterial properties, among others. Evidence indicates that the choice of mushroom cultivation technique strongly influences the production of bioactive compounds, but to date the nature of many of these compounds has not been fully established. This work focuses on the proteomic characterization of L. edodes to highlight the main active processes two days after harvest and elucidates the proteins involved in the known antioxidant and antibacterial proprieties of Shiitake fruit bodies cultivated on oak logs. A label-free approach allowed us to identify a total of 2702 proteins which were mainly involved in carbohydrate and protein metabolism, cell growth and replication, indicating that several developmental processes remain active in fruit bodies post-harvest. Proteins with antioxidant activities were identified, indicating the contribution of proteins to the antioxidant properties of L. edodes extracts. Antibacterial assays also reveal the activity of a serine protease inhibitor that strongly accumulates in the post-harvest fruit body grown on oak logs. Overall, this study contributes to the understanding of the impact of the log cultivation method on the production of Shiitake mushrooms richest in high-value bioactive compounds.

Isoprene is a small lipophilic molecule synthesized in plastids and abundantly released into the atmosphere. Isoprene-emitting plants are better protected against abiotic stresses, but the mechanism of action of isoprene is still under debate. In this study, we compared the physiological responses and proteomic profiles of Arabidopsis which express the isoprene synthase (ISPS) gene and emit isoprene with those of non-emitting plants under both drought-stress (DS) and well-watered (WW) conditions. We aimed to investigate whether isoprene-emitting plants displayed a different proteomic profile that is consistent with the metabolic changes already reported. Only ISPS DS plants were able to maintain the same photosynthesis and fresh weight of WW plants. LC–MS/MS-based proteomic analysis revealed changes in protein abundance that were dependent on the capacity for emitting isoprene in addition to those caused by the DS. The majority of the proteins changed in response to the interaction between DS and isoprene emission. These include proteins that are associated with the activation of secondary metabolisms leading to ABA, trehalose, and proline accumulations. Overall, our proteomic data suggest that isoprene exerts its protective mechanism at different levels: under drought stress, isoprene affects the abundance of chloroplast proteins, confirming a strong direct or indirect antioxidant action and also modulates signaling and hormone pathways, especially those controlling ABA synthesis. Unexpectedly, isoprene also alters membrane trafficking.

Plants rely on their microbiota for improving the nutritional status and environmental stress tolerance. Previous studies mainly focused on bipartite interactions (a plant challenged by a single microbe), while plant responses to multiple microbes have received limited attention. Here, we investigated local and systemic changes induced in wheat by two plant growth-promoting bacteria (PGPB), Azospirillum brasilense and Paraburkholderia graminis, either alone or together with an arbuscular mycorrhizal fungus (AMF). We conducted phenotypic, proteomic, and biochemical analyses to investigate bipartite (wheat-PGPB) and tripartite (wheat-PGPB-AMF) interactions, also upon a leaf pathogen infection. Results revealed that only AMF and A. brasilense promoted plant growth by activating photosynthesis and N assimilation which led to increased glucose and amino acid content. The bioprotective effect of the PGPB-AMF interactions on infected wheat plants depended on the PGPB-AMF combinations, which caused specific phenotypic and proteomic responses (elicitation of defense related proteins, immune response and jasmonic acid biosynthesis). In the whole, wheat responses strongly depended on the inoculum composition (single vs. multiple microbes) and the investigated organs (roots vs. leaf). Our findings showed that AMF is the best-performing microbe, suggesting its presence as the crucial one for synthetic microbial community development.

Among all the food-related nanoparticles consumed every day, silver nanoparticles (AgNPs) have become one of the most commonly utilized because of their antimicrobial properties. Despite their common use, the effects of sublethal concentrations of AgNPs, especially on gut biofilms, have been poorly investigated. To address this issue, we investigated in vitro the proteomic response of a monospecies Escherichia coli gut biofilm to chronic and acute exposures in sublethal concentrations of AgNPs. We used a new gel- and label-free proteomic approach based on shotgun nanoflow liquid chromatography-tandem mass spectrometry. This approach allows a quantification of the whole proteome at a dynamic range that is higher than the traditional proteomic investigation. To assess all different possible exposure scenarios, we compared the biofilm proteome of four treatments: (i) untreated cells for the control treatment, (ii) cells treated with 1 μg/ml AgNPs for 24 h for the acute treatment, (iii) cells grown with 1 μg/ml AgNPs for 96 h for the chronic treatment, and (iv) cells grown in the presence of 1 μg/ml AgNPs for 72 h and then further treated for 24 h with 10 μg/ml AgNPs for the chronic + acute treatment. Among the 1,917 proteins identified, 212 were significantly differentially expressed proteins. Several pathways were altered including biofilm formation, bacterial adhesion, stress response to reactive oxygen species, and glucose utilization.

Silver nanoparticles (Ag NPs) can easily leak into the aquatic environment and, depending on the environmental conditions, readily transform. Since ionic silver is considered one of the most toxic ions for aquatic organisms, there is a growing concern about the effects of Ag NPs on the aquatic ecosystem. Aquatic organisms are very sensitive to pollutants, but the fate and concentrations of Ag NPs in water are still unknown. Data shows that even low concentrations of Ag NPs could affect aquatic photosynthetic organisms, and the size and shape of nanoparticles plays a central role in Ag NP toxicity. Ag NP toxicity is not fully attributable to released ions: many studies show that in photosynthetic organisms silver ions and Ag NPs caused similar effects, although silver ions were often active at lower concentrations. Toxicity of Ag NPs may be caused by a Trojan-horse type of mechanism, in which nanoparticles are internalized within cells where they can release silver ions. In photosynthetic aquatic organisms, one of the main targets of Ag NPs is the photosynthetic apparatus. AgNPs can also lead to an increase in oxidative stress through reactive oxygen species production. Ag NPs can modify the composition of a natural community of aquatic microorganisms, such as phytoplankton, and accumulate in the food chain, even if the potential for their biomagnification remains unclear.

Proteasome activity is essential for pollen tube emergence and growth; nevertheless, little is known about proteasome function at the molecular level. The objective of this study was to identify molecular targets and pathways which are directly/indirectly controlled by the proteasome during pollen germination. To this aim, changes in the proteome and phosphoproteome of Actinidia pollen, germinated in the presence of the proteasome inhibitor MG132, were investigated. Phosphoproteins were enriched by metal oxide/hydroxide affinity chromatography and phosphopeptides were further isolated using titanium ion (Ti4+) functional magnetic microparticles prior to liquid chromatography-tandem mass spectrometry analysis. Our results show that proteasome inhibition affects the phosphoproteome more profoundly than the proteome. Accordingly, the steady-state abundance of some kinases and phosphatases was changed and/or their phosphorylation status altered. Notably, affected proteins are involved in processes that are fundamental to pollen germination such as cytoskeletal organization, vesicular transport, cell wall synthesis and remodeling, protein synthesis, folding and degradation as well as energetic metabolism. Our data provide a molecular framework for the structural alterations observed when the proteasome is inhibited, contribute to the understanding of how proteasome activity regulates pollen germination, show the cross-talk between phosphorylation and proteasomal degradation and are a resource for further functional analyses. SIGNIFICANCE: Pollen germination and tube growth are fundamental to successful fertilization in seed plants. These events are based on dramatic remodeling and the dismantling of existing programs, which are replaced by new ones. Degradation plays a prominent role in reshaping the protein repertoire, also cross talking with the bulk of post-translational modifications. At present, phosphorylation is the only modification studied in germinating pollen on a large scale. The proteasome has been universally recognized as one of the most important sites for protein degradation and its function has been shown to be essential for pollen tube emergence and elongation. Upon proteasome inhibition structural alterations and dysregulation of pivotal processes governing pollen germination have been described; however, a mechanistic framework for the proteasome function at the molecular level is still lacking. In this investigation we provide the very first view of the global impact of the proteasome in remodeling the proteome and phosphoproteome during germination and tube growth. Our results show how proteasome inhibition alters the levels, and profoundly affects the phosphorylation status of many proteins involved, controlling energetic and synthetic pathways and signaling cascades.

Besides improved mineral nutrition, plants colonised by arbuscular mycorrhizal (AM) fungi often display increased biomass and higher tolerance to biotic and abiotic stresses. Notwithstanding the global importance of wheat as an agricultural crop, its response to AM symbiosis has been poorly investigated. We focused on the role of an AM fungus on mineral nutrition of wheat, and on its potential protective effect against Xanthomonas translucens. To address these issues, phenotypical, molecular and metabolomic approaches were combined. Morphological observations highlighted that AM wheat plants displayed an increased biomass and grain yield, as well as a reduction in lesion area following pathogen infection. To elucidate the molecular mechanisms underlying the mycorrhizal phenotype, we investigated changes of transcripts and proteins in roots and leaves during the double (wheat-AM fungus) and tripartite (wheat-AM fungus-pathogen) interaction. Transcriptomic and proteomic profiling identified the main pathways involved in enhancing plant biomass, mineral nutrition and in promoting the bio-protective effect against the leaf pathogen. Mineral and amino acid contents in roots, leaves and seeds, and protein oxidation profiles in leaves, supported the omics data, providing new insight into the mechanisms exerted by AM symbiosis to confer stronger productivity and enhanced resistance to X. translucens in wheat.

The molecular details of local plant response against Xanthomonas translucens infection is largely unknown. Moreover, there is no knowledge about effects of the pathogen on the root's transcriptome and proteome. Therefore, we investigated the global gene and protein expression changes both in leaves and roots of wheat (Triticum aestivum) 24 h post leaf infection of X. translucens. This simultaneous analysis allowed us to obtain insight into possible metabolic rearrangements in above-and belowground tissues and to identify common responses as well as specific alterations. At the site of infection, we observed the implication of various components of the recognition, signaling, and amplification mechanisms in plant response to the pathogen. Moreover, data indicate a massive down-regulation of photosynthesis and confirm the chloroplast as crucial signaling hub during pathogen attack. Notably, roots responded as well to foliar attack and their response significantly differed from that locally triggered in infected leaves. Data indicate that roots as a site of energy production and synthesis of various secondary metabolites may actively influence the composition and colonisation level of root-associated microbes. Finally, our results emphasize the accumulation of jasmonic acid, pipecolic acid and/or the downstream mediator of hydrogen peroxide as long distal signals from infected leaves to roots.

The aim of this study was to investigate the impact of elevated concentration of carbon dioxide (CO2), as expected over coming decades, on yield and quality of winter bread wheat (Triticum aestivum L.). Plants (cv. Bologna) were grown by using the free-air CO2 enrichment (FACE) system at Fiorenzuola d’Arda under ambient (control) and elevated (570 ppm, e[CO2]) CO2 concentrations for two growing seasons. We addressed whether there would be a response of wheat grains to elevated CO2 concentration in terms of the contents of nitrogen (N), micro- and macronutrients, proteins and free amino acids. Under e[CO2], total wheat biomass and grain yield increased in both years of the study. Grain N percentage was reduced under e[CO2], but grain N yield (kg ha–1) was increased. Among macro- and micronutrients, a decrease in zinc concentration was observed. The proteome pattern was significantly different in grains grown at the two different CO2 levels, but the observed changes were highly dependent on interactions with prevailing environmental conditions. Finally, a negative trend was observed in the early germination rates of seeds from plants grown under e[CO2] compared with the controls. The results suggest that the expected increase in CO2 levels and their interactive effects with environmental variables may influence agronomic performance by increasing yield and negatively affecting quality.

In the present study, the protective role of inulin against lipopolysaccharide (LPS)-induced oxidative stress was evaluated on human colonic mucosa using a proteomic approach. Human colonic mucosa and submucosa were sealed between two chambers, with the luminal side facing upwards and overlaid with Krebs (control), LPS or LPS+ inulin IQ solution. The solutions on the submucosal side (undernatants) were collected following 30 min of mucosal exposure. iTRAQ based analysis was used to analyze the total soluble proteomes from human colonic mucosa and submucosa treated with different undernatants. Human colonic muscle strips were exposed to the undernatants to evaluate the response to acetylcholine. Inulin exposure was able to counteract, in human colonic mucosa, the LPS-dependent alteration of some proteins involved in the intestinal contraction (myosin light chain kinase (MLCK), myosin regulatory subunit (MYL)), to reduce the up-regulation of two proteins involved in the radical-mediated oxidative stress (the DNA-apurinic or apyrimidinic site) lyase) APEX1 and the T-complex protein 1 subunit eta (CCT7) and to entail a higher level of some detoxification enzymes (the metallothionein-2 MT2A, the glutathione-S-transferase K GSTk, and two UDP- glucuronosyltransferases UGT2B4, UGT2B17). Inulin exposure was also able to prevent the LPS-dependent intestinal muscle strips contraction impairment and the mucosa glutathione level alterations. Exposure of colonic mucosa to inulin seems to prevent LPS-induced alteration in expression of some key proteins, which promote intestinal motility and inflammation, reducing the radical-mediated oxidative stress.