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Although microalgae have only recently been recognized as part of the plant and soil microbiome, their application as biofertilizers has a tradition in sustainable crop production. Under consideration of their ability to produce the plant growth-stimulating hormone cytokinin (CK), known to also induce pathogen resistance, we have assessed the biocontrol ability of CK-producing microalgae. All pro- and eukaryotic CK-producing microalgae tested were able to enhance the tolerance of tobacco against Pseudomonas syringae pv. tabaci (PsT) infection. Since Chlamydomonas reinhardtii (Cre) proved to be the most efficient, we functionally characterized its biocontrol ability. We employed the CRISPR-Cas9 system to generate the first knockouts of CK biosynthetic genes in microalgae. Specifically, we targeted Cre Lonely Guy (LOG) and isopentenyltransferase (IPT) genes, the key genes of CK biosynthesis. While Cre wild-type exhibits a strong protection, the CK-deficient mutants have a reduced ability to induce plant defence. The degree of protection correlates with the CK levels, with the IPT mutants showing less protection than the LOG mutants. Gene expression analyses showed that Cre strongly stimulates tobacco resistance through defence gene priming. This study functionally verifies that Cre primes defence responses with CK, which contributes to the robustness of the effect. This work contributes to elucidate microalgae-mediated plant defence priming and identifies the role of CKs. In addition, these results underscore the potential of CK-producing microalgae as biologicals in agriculture by combining biofertilizer and biocontrol ability for sustainable and environment-friendly crop management.

The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including ‘Candidiatus Phytoplasma solani’ are unknown. Six putative pathogenicity factors/effectors from six different strains of ‘Ca. P. solani’ were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate–glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate–glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed., The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including ‘Candidiatus Phytoplasma solani’ are unknown. Six putative pathogenicity factors/effectors from six different strains of ‘Ca. P. solani’ were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate–glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate–glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.

Alterations in plant metabolism play a key role in the complex plant-pathogen interactions. However, there is still a lack of knowledge about the connection between changes in primary and specialized metabolism and the plant defense against diseases that impact crops. Thus, we aim to study the metabolic reprograming in Brassica oleracea plants upon infection by Xanthomonas campestris pv. campestris (Xcc). To accomplish this, we utilized a combination of untargeted and targeted metabolomics, through UPLC-Q-TOF-MS/MS and H-NMR, in two crop lines differing in resistance that were evaluated at two- and four-week intervals following inoculation (T1 and T2, respectively). Besides, to depict the physiological status of the plant during infection, enzymatic activities related to the carbohydrate pathway and oxidative stress were studied. Our results revealed different temporal dynamics in the responses of the susceptible vs. resistant crops lines. Resistant B. oleracea line suppresses carbohydrate metabolism contributing to limit nutrient supplies to the bacterium and prioritizes the induction of defensive compounds such as indolic glucosinolates, salicylic acid, phenylpropanoids and phytoalexins precursors at early infection stages. In contrast, the susceptible line invests in carbohydrate me

Alterations in plant metabolism play a key role in the complex plant–pathogen interactions. However, there is still a lack of knowledgeabout the connection between changes in primary and specialized metabolism and the plant defense against diseases that impact crops.Thus, we aim to study the metabolic reprograming in Brassica oleracea plants upon infection by Xanthomonas campestris pv. campestris (Xcc).To accomplish this, we utilized a combination of untargeted and targeted metabolomics, through UPLC-Q-TOF-MS/MS and 1H-NMR, intwo crop lines differing in resistance that were evaluated at two- and four-week intervals following inoculation (T1 and T2, respectively).Besides, to depict the physiological status of the plant during infection, enzymatic activities related to the carbohydrate pathwayand oxidative stress were studied. Our results revealed different temporal dynamics in the responses of the susceptible vs. resistantcrops lines. Resistant B. oleracea line suppresses carbohydrate metabolism contributing to limit nutrient supplies to the bacterium andprioritizes the induction of defensive compounds such as indolic glucosinolates, salicylic acid, phenylpropanoids and phytoalexinsprecursors at early infection stages. In contrast, the susceptible line invests in carbohydrate metabolism, including enzymatic activitiesrelated to the hexoses turnover, and activates defense signaling related to reactive oxygen species. Thus, each line triggers a differentmetabolic strategy that will affect how the plant overcomes the disease in terms of resistance and growth. This work provides firstinsights of a fine-tuned metabolic regulation during Xcc infection in B. oleracea that will contribute to develop new strategies for plantdisease management., Alterations in plant metabolism play a key role in the complex plant-pathogen interactions. However, there is still a lack of knowledge about the connection between changes in primary and specialized metabolism and the plant defense against diseases that impact crops. Thus, we aim to study the metabolic reprograming in Brassica oleracea plants upon infection by Xanthomonas campestris pv. campestris (Xcc). To accomplish this, we utilized a combination of untargeted and targeted metabolomics, through UPLC-Q-TOF-MS/MS and 1H-NMR, in two crop lines differing in resistance that were evaluated at two- and four-week intervals following inoculation (T1 and T2, respectively). Besides, to depict the physiological status of the plant during infection, enzymatic activities related to the carbohydrate pathway and oxidative stress were studied. Our results revealed different temporal dynamics in the responses of the susceptible vs. resistant crops lines. Resistant B. oleracea line suppresses carbohydrate metabolism contributing to limit nutrient supplies to the bacterium and prioritizes the induction of defensive compounds such as indolic glucosinolates, salicylic acid, phenylpropanoids and phytoalexins precursors at early infection stages. In contrast, the susceptible line invests in carbohydrate metabolism, including enzymatic activities related to the hexoses turnover, and activates defense signaling related to reactive oxygen species. Thus, each line triggers a different metabolic strategy that will affect how the plant overcomes the disease in terms of resistance and growth. This work provides first insights of a fine-tuned metabolic regulation during Xcc infection in B. oleracea that will contribute to develop new strategies for plant disease management.

Leaf rust caused by Puccinia triticina Erikss. can have devastating effects on wheat (Triticum aestivum L.), causing severe economic losses. This comprehensive study serves to facilitate our understanding of the impact of carbohydrate and antioxidant metabolism in association with sensor-based phenotyping and leaf rust stress responses in wheat seedlings. After 24 h of inoculation (hai) very susceptible variety to leaf rust (Ficko) increased cell-wall invertase (cwInv; EC 3.2.1.26), compared to other varieties that significantly increased cwInv later. This could mean that the Ficko variety cannot defend itself from leaf rust infections once symptoms have started to develop. Also, Ficko had significantly decreased amounts of cytoplasmic invertase (cytInv; EC 3.2.1.26) at 8 hai. The downregulation of cytInv in susceptible plants may facilitate the maintenance of elevated apoplastic sucrose availability favoring the pathogen. The significant role of vacuolar invertase (vacInv; EC 3.2.1.26) in moderately resistant varieties was recorded. Also, a significant decrease of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) in moderately resistant varieties might restrict normal development of leaf rust due to reduced sugar. During plant-pathogen interaction, when the invader spreads systemically throughout the plant, the main role of ascorbate peroxidase (APX; EC 1.11.1.11) activity in one moderately resistant variety (Olimpija) and catalase (CAT; EC 1.11.1.6) activity in another moderately resistant variety (Alka) is to protect the plant against oxidative damage in the early stages of infection. Non-invasive phenotyping with a sensor-based technique could be used as a rapid method for pre-symptomatic determination of wheat leaf rust resistance or susceptibility.

Endophytic fungi that colonize the plant root live in an environment with relative high concentrations of different sugars. Analyses of genome sequences indicate that such endophytes can secrete carbohydrate-related enzymes to compete for these sugars with the surrounding plant cells. We hypothesized that typical plant sugars can be used as carbon source by root endophytes and that these sugars also serve as signals to induce the expression and secretion of glycolytic enzymes. The plant-growth-promoting endophytes Serendipita indica and Serendipita herbamans were selected to first determine which sugars promote their growth and biomass formation. Secondly, particular sugars were added to liquid cultures of the fungi to induce intracellular and extracellular enzymatic activities which were measured in mycelia and culture supernatants. The results showed that both fungi cannot feed on melibiose and lactose, but instead use glucose, fructose, sucrose, mannose, arabinose, galactose and xylose as carbohydrate sources. These sugars regulated the cytoplasmic activity of glycolytic enzymes and also their secretion. The levels of induction or repression depended on the type of sugars added to the cultures and differed between the two fungi. Since no conventional signal peptide could be detected in most of the genome sequences encoding the glycolytic enzymes, a non-conventional protein secretory pathway is assumed. The results of the study suggest that root endophytic fungi translocate glycolytic activities into the root, and this process is regulated by the availability of particular plant sugars., Peer Reviewed

Interannual and local fluctuations in wheat crop yield are majorly explained by abiotic constraints. Heatwaves and drought, which are among the top stressors, commonly co-occur, and their frequency is increasing with global climate change. High-throughput methods were optimised to phenotype wheat plants under controlled water deficit and high temperature, with the aim to identify phenotypic traits conferring adaptative stress responses. Wheat plants of 10 genotypes were grown in a fully automated plant facility under 25/18ºC day/night for 30 days, and then the temperature was increased for seven days (38/31ºC day/night) while maintaining half of the plants well irrigated and half at 30% field capacity. Thermal and multispectral images and pot weights were registered twice daily. At the end of the experiment, key metabolites and enzyme activities from the carbohydrate and antioxidant metabolisms were quantified. Regression machine learning models were successfully established to predict plant biomass using image-extracted parameters. Evapotranspiration traits expressed significant genotype-environment interactions (GxE) when acclimatization to stress was continuously monitored. Consequently, transpiration efficiency was essential to maintain the balance between water-saving strategies and biomass production in wheat under water deficit and high temperature. Stress tolerance included changes in the carbohydrate metabolism, particularly in the sucrolytic and glycolytic pathways, and in the antioxidant metabolism. The observed genetic differences in sensitivity to high temperature and water deficit can be exploited in breeding programs to improve wheat resilience to climate change. [Abstract copyright: © The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.]

Interannual and local fluctuations in wheat crop yield are mostly explained by abiotic constraints. Heatwaves and drought, which are among the top stressors, commonly co-occur, and their frequency is increasing with global climate change. High-throughput methods were optimized to phenotype wheat plants under controlled water deficit and high temperature, with the aim to identify phenotypic traits conferring adaptative stress responses. Wheat plants of 10 genotypes were grown in a fully automated plant facility under 25/18 °C day/night for 30 d, and then the temperature was increased for 7 d (38/31 °C day/night) while maintaining half of the plants well irrigated and half at 30% field capacity. Thermal and multispectral images and pot weights were registered twice daily. At the end of the experiment, key metabolites and enzyme activities from carbohydrate and antioxidant metabolism were quantified. Regression machine learning models were successfully established to predict plant biomass using image-extracted parameters. Evapotranspiration traits expressed significant genotype–environment interactions (G×E) when acclimatization to stress was continuously monitored. Consequently, transpiration efficiency was essential to maintain the balance between water-saving strategies and biomass production in wheat under water deficit and high temperature. Stress tolerance included changes in carbohydrate metabolism, particularly in the sucrolytic and glycolytic pathways, and in antioxidant metabolism. The observed genetic differences in sensitivity to high temperature and water deficit can be exploited in breeding programmes to improve wheat resilience to climate change.

The genus Pseudomonas harbours numerous strains that positively affect plant growth and defence through diverse mechanisms such as nutrient solubilisation and production of phytohormones or secondary metabolites. The aim of this study was to compare the impact of six plant-beneficial Pseudomonas strains on tomato (Solanum lycopersicum) growth and holobiont physiology. The physiological impact was determined by profiling the activities of key enzymes in the central carbohydrate and antioxidant metabolism. Root inoculation of tomato seedlings with Pseudomonas strains in a greenhouse experiment induced plant growth, measured as biomass and plant height promotion. The bacterial strains also increased leaf chlorophyll content and caused distinct carbohydrate and antioxidative metabolism enzyme activity profiles in leaf and root tissue respectively. For the carbohydrate metabolism, the activities of several key enzymes involved in assimilate partitioning from source to sink and processing of the transport sugar sucrose for catabolism and anabolism were stimulated. For the antioxidative metabolism, both enzymes involved in detoxification of reactive oxygen species and redox buffering were increased. These increased enzyme activities in response to bacterial inoculation could contribute to balancing plant growth and defence. Importantly, positive correlations between plant growth parameters and distinct enzyme activities suggest that host plant biosignatures may be predicting bacteria with plant growth-promoting potential. These findings offer new perspectives for integrating physiological fingerprinting in the screening of microbes during early developmental stages of the host plant. In addition, determining plant metabolic biosignatures could be a rapid tool for predicting the potential and improvement of stress resiliency.

High-throughput profiling of key enzyme activities of carbon, nitrogen, and antioxidant metabolism is emerging as a valuable approach to integrate cell physiological phenotyping into a holistic functional phenomics approach. However, the analyses of the large datasets generated by this method represent a bottleneck, often keeping researchers from exploiting the full potential of their studies. We address these limitations through the exemplary application of a set of data evaluation and visualisation tools within a case study. This includes the introduction of multivariate statistical analyses which can easily be implemented in similar studies, allowing researchers to extract more valuable information to identify enzymatic biosignatures. Through a literature meta-analysis, we demonstrate how enzyme activity profiling has already provided functional information on the mechanisms regulating plant development and response mechanisms to abiotic stress and pathogen attack. The high robustness of the distinct enzymatic biosignatures observed during developmental processes and under stress conditions underpins the enormous potential of enzyme activity profiling for future applications both in basic and applied research. Enzyme activity profiling will complement molecular -omics approaches to contribute to the mechanistic understanding required to narrow the genotype-to-phenotype knowledge gap and to identify predictive biomarkers for plant breeding to develop climate-resilient crops.

The five Nordic countries span the most northern region for field cultivation in the world. This presents challenges per se with short growing seasons, long days and a need for frost tolerance. Climate change has additionally increased risks for micro-droughts and water logging as well as pathogens and pests expanding northwards. Thus, Nordic agriculture demands crops that are adapted to the special Nordic growth conditions and future climate scenarios. A focus on crop varieties and traits important to Nordic agriculture, including the unique resource of nutritious wild crops, can meet these needs. In fact, with a future longer growing season due to climate change the region could contribute proportionally more to the global agricultural production. This also applies to other northern regions, including the Arctic. To address current growth conditions, mitigate impacts of climate change and meet market demands, the adaptive capacity of crops that both perform well in northern latitudes and are more climate resilient has to be increased, and better crop management systems be built. This requires functional phenomics approaches that integrate versatile high-throughput phenotyping, physiology and bioinformatics. This review stresses key target traits, the opportunities of latitudinal studies and infrastructure needs for phenotyping to support Nordic agriculture.

Agriculture has always been the most susceptible sector to climate change, as environmental stresses severely impact plant development and final yield. The interest in the application of plant growth promoting rhizobacteria (PGPR) in agriculture is exponentially increasing, as it provides a sustainable solution against hazardous agrochemicals. A vast amount of research has indicated that PGPRs can have bio stimulatory activity and function as biocontrol agents, providing protection against biotic and abiotic stresses and therefore offering a less environmentally invasive approach to food supply. This study was conducted to enlighten the effects of two PGPRs, the Enterobacter spp., and the commercially applied Bacillus amyloliquefaciens FZB42, on root growth and architecture as well as on carbohydrate-metabolic and antioxidant enzyme activity in barley (Hordeum vulgare cv Guld). Preliminary experimental results have shown that the impact of Enterobacter spp. is very similar to the effect of cytokinins on root growth. The bacterial inoculation response of the plants was initially characterized by phenotypical and biochemical analysis. There was no distinct effect on the root system and plant metabolism with FZB42 treatment, therefore further focus was given only to the impact of Enterobacter spp. Enterobacter spp. caused a strong inhibitory impact on root growth and affected the carbohydrate and antioxidant enzyme activity of plants grown in vitro. This specific effect on roots did not appear under soil conditions, in greenhouse experiments. The study around Enterobacter spp. was complemented with analysis of the available RNA Sequencing data. Expression profiling of the roots of barley plants identified the number of differentially regulated genes. Functional annotation of these differentially expressed genes was performed using Gene Ontology (GO) and the Kyoto Encyclopedia

Plant growth-promoting rhizobacteria (PGPR) are known for exerting beneficial effects on plant growth and tolerance to plant pathogens. However, their specific role in mediating protection against abiotic stress remains underexplored. The aim of this study was to characterise the ability of the cytokinin-producing beneficial bacterium Pseudomonas fluorescens G20-18 to enhance tomato growth and boost tolerance to drought stress. Tomato seedlings were root inoculated and their growth and physiological and molecular responses assessed under well-watered conditions and also in response to progressive drought stress and a subsequent recovery period. Root inoculation with G20-18 had a significant positive impact on tomato growth. Furthermore, G20-18 inoculated and drought-stressed plants showed higher leaf chlorophyll and abscisic acid (ABA) content and stomatal closure than non-inoculated controls. Root inoculation also increased the activity of different carbohydrate metabolism enzymes, which are important for root and leaf growth and development in drought stressed plants. A significant increase in the activity of different antioxidant enzymes and total antioxidant capacity correlated with elevated levels of relevant secondary metabolites, such as phenolics, anthocyanins and flavonoids. RNA sequencing revealed distinct qualitative and quantitative differences in gene regulation in response to G20-18. Notably, the number of genes differentially regulated in response to G20-18 was approximately sevenfold higher during drought stress, indicating that root inoculation with the bacteria primed the plants for a much stronger transcriptionally regulated systemic drought stress response. The regulated genes are related to phenylalanine metabolism and other key processes linked to plant growth, development and drought stress resilience. A role of the ability of G20-18 to produce the plant hormone cytokinin for interaction with tomato was established by the cytokinin-deficient b