Glucosinolates (GS) are sulfur-containing compounds that serve as a deterrentto herbivores, though are tolerated by some specialized insects. The correlation of GS with floral traits such as colour and scent might represent a signaling of defense status, a pattern rarely recorded in natural populations. We used Raphanus raphanistrum to discover GS signaling patterns and their effects on plant fitness. We recorded fitness, floral colour, GS, and floral Volatile Organic Compounds (fVOCs) from five natural populations. We then tested correlations among traits at the species level and at the population level. Plant fitness was not correlated with any of the GS and fVOCs analysed. At the species level, flower colour was correlated with GS levels in leaves indicating that there might be visual signaling of defense status, while there was a weak correlation between minor GS and fVOCs. However, at the population level, we found multiple and discordant significant correlations, mainly negative, between GS and fVOCs. Our study indicates that signaling channels used by R. raphanistrum to advertise its chemical defense are complex and variable among populations suggesting that they can be shaped by biotic networks of pollinators and herbivores varying in space.

The emission of isoprene from plants was first discovered in the 1950s but was relatively unknown in the plant science community until the 1990s. Isoprene is the five-carbon branched molecule that is the root member of the extensive family of isoprenoids. The amount of isoprene from plants exceeds all other hydrocarbon inputs to the atmosphere. Plant-emitted isoprene can affect ozone formation and often increases (but can decrease) growth of aerosols (particles in the atmosphere). The rate of isoprene emission is estimated using empirical or mechanistic modeling has been used to predict global emissions. Beyond its atmospheric role, isoprene can improve plant resilience to biotic and abiotic stress, likely through interactions with transcriptional networks that regulate plant growth and defense. Isoprene has all the properties of the five compounds classically described as plant hormones. These and an additional four molecules are now called small molecule plant growth regulators, and we propose that isoprene also belongs to this group. Plants previously thought to lack the capacity for isoprene emission have now been found that make isoprene in response to leaf damage. This discovery suggests that many plants once classified as non-emitters likely have the capacity to emit isoprene under specific conditions. This review summarizes past and current understanding of the biosynthesis and regulatory mechanisms, atmospheric significance, and physiological roles of isoprene emitted from plants.

State transition (ST) is a key regulatory mechanism that balances excitation energy between photosystem II (PSII) and photosystem I (PSI). Although its general principles have been known for decades, methodological constraints have limited precise quantification of ST kinetics and of the associated changes in photosystem absorption cross sections. We present a PCA‑assisted fluorescence spectral analysis that enables in vivo separation of PSI and PSII chlorophyll fluorescence at physiological temperatures. Applied to intact Nicotiana tabacum leaves undergoing ST, this method provides more accurate estimates of both the rates and the extent of the process than earlier approaches. The half‑time of the State 1 to State 2 (St1→St2) transition was approximately 1 min, nearly twice as fast as values inferred from traditional Ffar‑red/Fred fluorescence ratios measured at room temperature or 77 K. The analysis also clarifies how PSI and PSII fluorescence changes relate to variations in their absorption cross sections. Accounting for PSII‑to‑PSI spillover, we found that the mobile LHCII fraction associating with PSI during the St1→St2 transition increased the PSI absorption cross section by 31

High ozone (O3) and nitrogen (N) deposition are commonly co-occurring consequences of environmental changes. They have profound and contrasting impacts on plant growth and productivity, as well as on emission of isoprene (ISO), the main biogenic volatile organic compound driving biosphere-atmosphere interactions. Previous studies have mainly characterized responses of ISO to either elevated O3 (EO3) and N deposition in potted seedlings. Here, we investigated the responses of two field-grown poplar clones with different O3-sensitivity to the combined effects of realistic O3 increase and N deposition at both leaf and canopy levels, in a Free-Air O3 Concentration Enrichment (O3-FACE) facility. ISO emission was similar in the two clones, and strongly influenced by seasonality and leaf position in the canopy, being higher in June than in September, and topping in the upper leaves of the canopy. Photosynthesis and the intercellular CO2 concentration were respectively positively and negatively correlated to ISO emission. Whereas, ISO emission was not strictly controlled by availability of ISO specific substrate (dimethylallyl pyrophosphate) or enzyme (isoprene synthase). Small but significant negative effects of EO3 were only detected on photosynthesis and ISO emission of leaves positioned in the low and medium part of the canopy of both clones, and confirmed by canopy-level ISO estimations. Our results suggest that, in field conditions, realistic exposure to EO3 and N deposition will have an overall minor effect on ISO and plant physiology. They also indicate that leaf-level ISO emission proxies canopy-level ISO emissions, and could be used to upscale ISO emission responses at community, ecosystem or regional level.

Isoprene is the most abundant biogenic volatile organic compound emitted by terrestrial vegetation. Here we report the impact of isoprene on root-associated microbiomes. Using isoprene-emitting (IE) transgenic tobacco and isogenic non-emitting (NE) controls, we performed co-cultivation experiments in natural soil and analysed plant phenotypes and growth alongside bacterial and fungal communities across root, rhizosphere, and soil niches. NE plants co-cultivated with IE neighbours displayed increased shoot and root biomass, suggesting interactive belowground functions of isoprene. Amplicon sequencing revealed more growth-promoting microbiota in root and rhizosphere of IE plants than NE plants. Both bacterial and fungal growth-promoting microbiota were enriched in IE and NE plants grown in the same pot. However, isoprene-fumigated plant-free soils did not replicate these shifts, indicating that plant-microbe interactions are required for the modulation of the soil microbiome. Our results suggest that isoprene acts as a belowground cue influencing microbiome assembly and indirectly enhancing growth in neighbouring plants. This work uncovers a potential ecological role for isoprene, highlighting how plant-derived isoprene can mediate plant-plant-microbiome interactions and contribute to community-level processes in the rhizosphere.

Mimosa pudica is a plant known for its ability to fold leaves in response to mechanical disturbances, which serves as a visible phenotypic stress marker. Leaf folding occurs with a response timing and intensity that varies depending on the stimulus. This adaptive behavior may function as a defense mechanism, helping plant resist herbivores and environmental stressors. However, the molecular and genetic mechanisms that are involved in leaf folding are still not totally understood. In this study, the gene regulatory networks underlying M. pudica leaf closure following single and multiple mechanical disturbances (whole pot drops) were investigated. Chlorophyll fluorescence was measured as fast phenotypic indicator of transient or permanent photochemical damage, and transcriptional responses were measured to identify the key genes regulating phenotypic changes after single or multiple drops. A progressive reduction of the quantum yield of photosystem 2 revealed a lower electron transport rate in leaves subjected to one or more drops, which may indicate the onset of energy shortage, perhaps caused by low ATP availability, limiting both leaf movement and photosynthesis. The transcriptomic profiles revealed larger differences when plants were subjected to multiple drops than to a single drop, with respect to unstressed controls. Interestingly, following a single drop, the majority of up-regulated genes were associated with the flavonoid biosynthetic pathway. After multiple drops, however, genes associated with biotic and abiotic stress resistance pathways were predominantly up-regulated. These results provide a basis for developing a gene regulatory network model of stress-induced movements in M. pudica leaves, which may help design sustainable strategies of plant stress defense. Main Conclusions Repeated stress in Mimosa pudica reduces photosystem efficiency, alters gene expression, shifting from flavonoid biosynthesis to stress resistance pathways, offering insights for sustainable plant stress defense strategies. ### Competing Interest Statement The authors have declared no competing interest. ministero della ricerca e dell'università, Fore-VOC (PRIN 2022 PNRR), LEGU-MED (PRIMA 2019)

Repeated stress in Mimosa pudica reduces photosystem efficiency, alters gene expression, shifting from flavonoid biosynthesis to stress resistance pathways, offering insights for sustainable plant stress defense strategies. Mimosa pudica is a plant known for its ability to fold leaves in response to mechanical disturbances, which serves as a visible phenotypic stress marker. Leaf folding response occurs with a timing and an intensity that vary depending on the stimulus. This adaptive behavior may function as a defense mechanism, helping plant resist herbivores and environmental stressors. In this study, we investigated the gene regulatory networks underlying M. pudica leaf closure following single and multiple mechanical disturbances (whole pot drops). Chlorophyll fluorescence was measured as fast phenotypic indicator of transient or permanent photochemical damage, and transcriptional responses were measured to identify the key genes regulating phenotypic changes after single or multiple drops. A progressive reduction of the quantum yield of PSII revealed a lower electron transport rate in leaves subjected to one or more drops, which may indicate the onset of energy shortage, potentially caused by limited ATP availability that constrains both leaf movement and photosynthesis. The transcriptomic profiles revealed larger differences when plants were subjected to multiple drops than to a single drop, with respect to unstressed controls. Interestingly, following a single drop, the majority of up-regulated genes were associated with the flavonoid biosynthetic pathway. After multiple drops, however, genes associated with biotic and abiotic stress resistance pathways were predominantly up-regulated. These findings provide new insights into the gene regulatory networks driving stress-induced movements in M. pudica leaves and lay the groundwork for developing sustainable strategies for plant stress defense.

Drought limits the productivity of fast-growing woody crops, although the metabolic adjustments conferring water stress tolerance remain poorly understood. We investigated the responses of Populus nigra seedlings to water stress by integrating daily physiological measurements and NMR metabolomic analyses. Our aims were to: (i) determine key metabolic and biochemical responses in leaves subjected to moderate (WS1) and severe (WS2) water stress and (ii) identify the metabolites responsible for dissipating excess photochemical energy and maintaining cellular turgor. Despite the reduction in electron transport rate, photoprotective mechanisms were activated. A threefold increase in isoprene emission was observed at midday in WS1 plants, while ascorbic acid and other antioxidants were higher under WS2 conditions. Relative water content and osmotic potential decreased throughout the day, suggesting that passive osmotic adjustments were primarily driven by soluble sugar accumulation. Organic acid reduction in WS1 (-9%) and WS2 (-17%) plants suggested an inhibition of the tricarboxylic acid cycle. Additionally, amino acids were twofold higher in water-stressed plants compared to controls, likely reflecting an increased demand for primary and secondary metabolite biosynthesis. Our results provide new insights into the daily response of P. nigra to water stress, highlighting a delicate balance between metabolic and physiological adjustments.

Soil is a depletable and non-renewable resource essential for food production, crop growth, and supporting ecosystem services, such as the retaining and cycling of various elements, including water. Therefore characterization and preservation of soil biological health is a key point for the development of sustainable agriculture. We conducted a comprehensive review of the use of Artificial Intelligence (AI) techniques to develop forecasting models based on soil microbiota data able to monitor and predict soil health. We also investigated the potentiality of AI-based Decision Support Systems (DSSs) for improving the use of microorganisms to enhance soil health and fertility. While available studies are limited, potential applications of AI seem relevant to develop predictive models for soil fertility, based on its biological properties and activities, and implement sustainable precision agriculture, safeguarding ecosystems, bolstering soil resilience, and ensuring the production of high-quality food.

Tomato (Solanum lycopersicum L.) is a species of high economic value, an essential food source, and a model organism for both applied and basic research in crop science. Tomato plants also produce and emit a wide variety of volatile organic compounds (VOCs), which are thought to play a prominent role in multitrophic interactions. This review aims to provide a comprehensive overview of the extensive literature about tomato VOCs emitted by leaves. We explored the role of VOCs in the interactions of tomato plants with the environment, focusing on VOCs that provide plant protection against herbivores, pathogen vectors, pathogens, and abiotic stresses. VOC functions in plant-plant communication and defence are less known, but new evidence is now being collected showing that VOCs sent by plants can inform neighbour plants about impending stresses. Overall, improved knowledge on VOC biochemistry and functions may soon allow their use for sustainable protection practices of tomato crops. Remaining gaps and promising areas for future research are also examined.

Seed deterioration remains a major constraint in agriculture, limiting crop productivity and sustainability. While volatile organic compounds (VOCs) have been primarily studied in the context of seed ageing, little is known about VOCs emissions associated with seed priming treatments. Therefore, this work aims to identify and compare quality related VOCs in legume seeds subjected to hydropriming (HP) and artificial ageing (AA). HP, a cost-effective priming method involving water soaking and dry-back, enhances germination and stress resilience, while AA induces seed deterioration after exposure to high temperature and humidity. Using Proton Transfer Reaction - Quadrupole ion guide - Time of Flight - Mass Spectrometry (PTR-Qi-ToF-MS), VOC emissions from four legume species, including both commonly cultivated and neglected crops, were measured. A total of 395 m/ z values were detected, with different emission patterns depending on treatments and accession. Among the putatively identified VOCs, 2(3H)-furanone/2(5H)-furanone (m/z 85.0266) and benzene (m/z 79.0517) were associated with low-quality AA seeds across all tested varieties. In contrast, methanethiol (m/z 49.0098), acetaldehyde (m/z 45.0328), and ethanol (m/z 47.0482) were predominantly emitted by high-quality HP seeds, suggesting their potential role as indicators of germination efficiency. However, the occurrence of these same compounds in a few accessions of both the AA and CTRL groups highlights a partial overlap between metabolic pathways triggered by priming and those associated with ageing. This overlap introduces a novel yet problematic aspect in interpreting VOC emission patterns, indicating the need for further investigation to differentiate shared and distinct metabolic responses underlying seed priming and deterioration processes.

To unravel the complex interactions between microplastics (MPs), plants, and pathogens, Arabidopsis thaliana plants were grown for 3 weeks in soils containing polyethylene terephthalate (PET) or polyvinyl chloride (PVC) MPs (0.2% and 0.5% w/w), and leaves were then exposed to the PAMP (Pathogen-Associated Molecular Pattern) protein cerato-platanin (CP) or Botrytis cinerea conidia. PET caused a stimulation of stomatal conductance, and PVC decreased the aboveground biomass of A. thaliana plants. PVC (0.2%) triggered a primed state in A. thaliana, enhancing its response to B. cinerea infection and cerato-platanin. This was demonstrated by decreased lesion size, enhanced ROS generation, and elevated camalexin synthesis following PAMP elicitation, and increased levels of defensive isothiocyanate and phenylpropanoid metabolites. Our results indicate that MPs also affect soil structure, ionome balance, and specialised metabolite accumulation. However, MPs did not provide an unambiguous response, underscoring challenges in formulating a model of plant response to MPs when exposed to pathogens.

Bryophytes desiccate rapidly when relative humidity decreases. The capacity to withstand dehydration depends on several ecological and physiological factors. Volatile organic compounds (VOCs) may have a role in enhancing tolerance to desiccating bryophytes. However, the functions of VOCs in bryophytes have received little attention so far. We aimed to investigate the impact of a dehydration-rehydration treatment on primary carbon metabolism and volatile terpenes (VTs) in three bryophytes with contrasting ecological traits: Vessicularia dubyana, Porella platyphylla and Pleurochaete squarrosa. First, we confirmed the desiccation sensitivity gradient of the species. Under fully hydrated conditions, the photosynthetic rate (A) was inversely associated with stress tolerance, with a lower rate in more tolerant species. The partial recovery of A in P. platyphylla and P. squarrosa after rehydration confirmed the desiccation tolerance of these two species. On the other hand, A did not recover after rehydration in V. dubyana. Regarding VT, each species exhibited a distinct VT profile under optimum hydration, with the highest VT pool found in the more desiccation-sensitive species (V. dubyana). However, the observed species-specific VT pattern could be associated with the ecological habitat of each species. P. squarrosa, a moss of dry habitats, may synthesize mainly non-volatile secondary metabolites as stress-defensive compounds. On the other hand, V. dubyana, commonly found submerged, may need to invest photosynthetically assimilated carbon to synthesize a higher amount of VTs to cope with transient water stress occurrence. Further research on the functions of VTs in bryophytes is needed to deepen our understanding of their ecological significance.

This study investigates the use of exploratory data analysis and supervised learning techniques to analyze plant phenotyping traits, with a specific focus on: i) genetic diversity (wild type vs mutant tomato plants); ii) plant-plant interactions (primed vs non-primed plants using volatiles emitted by other stressed plants); and iii) plant stress response (using drought stress and comparing droughted plants with controls). The analyzed data consisted of high-throughput imaging at multiple wavelengths, which allowed for the examination of various morphological traits. The dataset contained the phenotypic characteristics of both wildtype and mutated tomato plants exposed to water stress. Machine learning algorithms were used to identify significant phenotypic indicators and predict plant stress responses. The use of techniques such as K-means clustering and Bayesian classifiers provided valuable insights into the temporal dynamics of plant traits under a variety of experimental conditions. This research emphasizes the importance of employing advanced statistical and machine learning methods to improve the precision and efficacy of phenotypic analysis in plant sciences.

Isoprene, a volatile hydrocarbon, is typically emitted from the leaves of many plant species. Given its well-known function in plant growth and defense aboveground, we examined its effects on root physiology. We used isoprene-emitting (IE) lines and a non-emitting (NE) line of Arabidopsis and investigated their performance by analyzing root phenotype, hormone levels, transcriptome, and metabolite profiles under both normal and salt stress conditions. We show that IE lines emitted tiny amounts of isoprene from roots and showed an increased root/shoot ratio compared with NE line. Isoprene emission exerted a noteworthy influence on hormone profiles related to plant growth and stress response, promoting root development and salt-stress resistance. Methyl erythritol 4-phosphate pathway metabolites, precursors of isoprene and hormones, were higher in the roots of IE lines than in the NE line. Transcriptome data indicated that the presence of isoprene increased the expression of key genes involved in hormone metabolism/signaling. Our findings reveal that constitutive root isoprene emission sustains root growth under saline conditions by regulating and/or priming hormone biosynthesis and signaling mechanisms and expression of key genes relevant to salt stress defense.

The increase in soil salinization represents a current challenge for plant productivity, as most plants, including crops, are mainly salt-sensitive species. The identification of molecular traits underpinning salt tolerance represents a primary goal for breeding programs. In this scenario, the study of intraspecific variability represents a valid tool for investigating natural genetic resources evolved by plants in different environmental conditions. As a model system, Arabidopsis thaliana, including over 750 natural accessions, represents a species extensively studied at phenotypic, metabolic, and genomic levels under different environmental conditions. Two haplogroups showing opposite root architecture (shallow or deep roots) in response to auxin flux perturbation were identified and associated with EXO70A3 locus variations. Here, we studied the influence of these genetic backgrounds on plant salt tolerance. Eight accessions belonging to the two haplogroups were tested for salt sensitivity by exposing them to moderate (75 mM NaCl) or severe (150 mM NaCl) salt stress. Salt-tolerant accessions were found in both haplogroups, and all of them showed efficient ROS-scavenging ability. Even if an exclusive relation between salt tolerance and haplogroup membership was not observed, the modulation of root system architecture might also contribute to salt tolerance.

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.

Climate change, population growth and resource scarcity are putting increasing pressure on agriculture. In this perspective, progress has been made to replace traditional methods for plant phenotyping, which are destructive, time-consuming, inefficient and high-cost, with high-throughput, time-saving and highly efficient phenotyping technologies able to evaluate complex plant traits, assess plant responses to stresses, and screen desirable new genotypes. Volatile Organic Compounds (VOCs), are now deeply investigated as promising markers in high-throughput plant phenotyping. An overview is given of current state-of-the-art technologies to phenotype VOC emissions at leaf, whole plant, and plant community (ecosystem) levels.

Distinct photosynthetic physiologies are found within the Moricandia genus, both C3-type and C2-type representatives being known. As C2-physiology is an adaptation to drier environments, a study of physiology, biochemistry and transcriptomics was conducted to investigate whether plants with C2-physiology are more tolerant of low water availability and recover better from drought. Our data on Moricandia moricandioides (Mmo, C3), M. arvensis (Mav, C2) and M. suffruticosa (Msu, C2) show that C3 and C2-type Moricandias are metabolically distinct under all conditions tested (well-watered, severe drought, early drought recovery). Photosynthetic activity was found to be largely dependent upon the stomatal opening. The C2-type M. arvensis was able to secure 25–50% of photosynthesis under severe drought as compared to the C3-type M. moricandioides. Nevertheless, the C2-physiology does not seem to play a central role in M. arvensis drought responses and drought recovery. Instead, our biochemical data indicated metabolic differences in carbon and redox-related metabolism under the examined conditions. The cell wall dynamics and glucosinolate metabolism regulations were found to be major discriminators between M. arvensis and M. moricandioides at the transcription level.