Plant defence and stress acclimation : regulation by protein phosphatase 2A

Environmental alterations challenge the plant growth and reproduction in nature and in crop fields. Plants harbour acclimatory and defensive mechanisms that become activated under unfavourable conditions to ensure the plant survival. Recognition of stress factors triggers signalling cascades in plant cell that activate changes in gene expression, induce hormonal signals and modulate plant metabolism via posttranslational modification of enzymes. Reversible protein phosphorylation, carried out by counteracting pairs of protein kinases and phosphatases, presents a major mechanism in signal transduction as well as in the control of metabolic enzymes in plant defence and stress acclimation. Protein phosphatase 2A (PP2A) is a trimeric phosphatase composed of a scaffold subunit A, catalytic subunit C and regulatory subunit B, all of which are encoded by multiple genes in the model plant Arabidopsis thaliana. PP2A regulatory subunit B’γ (PP2A-B’γ) has been previously identified as a negative regulator of salicylic acid associated defence and cell death in Arabidopsis leaves. In this PhD thesis, PP2A-B’γ target proteins and its role in regulation of plant acclimation and metabolism were further studied. PP2A-B’γ and its closest homologue PP2A-B’ζ were found to modulate the plant growth and stress acclimation under normal growth conditions and under severe abiotic stress. Moreover, PP2A-B’γ was shown to regulate enzymes in plant primary and secondary metabolism. PP2A-B’γ interacted with ACONITASE 3 (ACO3) and controlled its phosphorylation. ACO3 phosphorylation was further connected to accumulation of ACO3 protein. Both PP2A-B’γ and ACO3 abundance were discovered to affect the accumulation of mitochondrial alternative oxidase at post-translational level thus contributing to the control of cell redox balance. In addition to modulation of primary metabolism, PP2A-B’γ was found to influence the formation of 4-methoxyindole- 3-yl-methly glucosinolate (4MO-I3M GSL), a defence compound with antimicrobial activities. Moreover, PP2A-B’γ interacted with activated methyl cycle (AMC) enzymes linked to production of 4MO-I3M GSL. PP2A-B’γ regulated the protein complex formation of AMC enzymes exerting its control over the cell methylation capacity. These findings provide new information of plant acclimation under abiotic stress and regulation of stress associated adjustments in plant metabolism. Detailed knowledge of plant acclimatory and defensive mechanisms and stress induced adjustments in plant metabolism is valuable in the development of more tolerant and nutritious crops.Vaihtelevat ympäristöolot vaikuttavat kasvien kasvuun ja lisääntymiseen sekä luonnossa että viljelmillä. Kasveilla onkin puolustus- ja sopeutumismekanismeja, jotka käynnistyvät epäsuotuisissa oloissa ja auttavat kasvia selviytymään. Stressitekijöiden havaitseminen aktivoi kasvisolussa viestejä, jotka aikaansaavat muutoksia geenien ilmenemisessä, käynnistävät hormonaalisia signaaleja ja säätelevät kasvin aineenvaihduntaa translaation jälkeisen säätelyn avulla. Vastavaikuttavien kinaasifosfataasiparien katalysoima palautuva proteiinifosforylaatio on tärkeä sekä viestinvälitystä ajava että aineenvaihdunnan entsyymejä ohjaava mekanismi kasvin puolustuksessa ja sopeutumisessa ympäristönmuutoksiin. Proteiinifosfataasi 2A (PP2A) on trimeerinen fosfataasi, joka koostuu rakennealayksiköstä A, katalyyttisestä alayksiköstä C ja säätelyalayksiköstä B. Jokaista alayksikköä koodaa usea geeni mallikasvi lituruohon (Arabidopsis thaliana) genomissa. PP2A:n säätelyalayksikön B’γ (PP2A-B’γ) on aiemmin havaittu estävän salisyylihaposta riippuvaisia puolustusvasteita ja solukuolemaa lituruoholla. Tässä väitöskirjassa on tutkittu tarkemmin PP2A-B’γ:n kohdeproteiineja ja merkitystä kasvin stressisopeutumisessa ja aineenvaihdunnan säätelyssä. PP2A-B’γ:n ja tämän homologin PP2A-B’ζ:n osoitettiin ohjaavan kasvin kasvua ja stressisopeutumista normaaleissa kasvuolosuhteissa ja vakavan abioottisen ympäristöstressin aikana. Lisäksi PP2A-B’γ:n havaittiin säätelevän entsyymejä sekä kasvin perusaineenvaihdunnassa että puolustusaineenvaihdunnassa. PP2A-B’γ:n osoitettiin vuorovaikuttavan akonitaasin ACO3 kanssa ja säätelevän tämän fosforylaatiota. Lisäksi ACO3:n fosforylaation havaittiin olevan kytköksissä ACO3 proteiinin kertymiseen. Sekä PP2A-B’γ:n että ACO3:n todettiin vaikuttivat vaihtoehtoisen oksidaasin määrään translaation jälkeisellä tasolla, ja sitä kautta solun hapetus-pelkistystasapainoon. Perusaineenvaihdunnan ohella PP2A-B’γ:n havaittiin säätelevän taudinaiheuttajille haitallisen 4-metoksi-indoli-3-yyli-metyyliglukosinolaattin (4MO-I3M glukosinolaatti) muodostusta. Lisäksi PP2A-B’γ vuorovaikutti 4MO-I3M glukosinolaatin muodostusta edesauttavan aktiivisen metyylikierron entsyymien kanssa. PP2A-B’γ:n osoitettiin säätelevän aktiivisen metyylikierron entsyymien muodostamia proteiinikomplekseja ja solun metylaatiokykyä. Tämä tutkimus lisää tietämystämme kasvien sopeutumisesta abioottiseen stressiin sekä ympäristöstressin aikaansaamien aineenvaihdunnan muutosten säätelystä. Yksityiskohtainen tieto kasvin sopeutumis- ja puolustusmekanismeista sekä ympäristöstressin aikaansaamista muutoksista kasvin aineenvaihdunnassa on tärkeää puolustuskykyisempien ja ravinteikkaampien viljelykasvien jalostuksessa

Protein Phosphatase 2A in the Regulatory Network Underlying Biotic Stress Resistance in Plants

Biotic stress factors pose a major threat to plant health and can significantly deteriorate plant productivity by impairing the physiological functions of the plant. To combat the wide range of pathogens and insect herbivores, plants deploy converging signaling pathways, where counteracting activities of protein kinases and phosphatases form a basic mechanism for determining appropriate defensive measures. Recent studies have identified Protein Phosphatase 2A (PP2A) as a crucial component that controls pathogenesis responses in various plant species. Genetic, proteomic and metabolomic approaches have underscored the versatile nature of PP2A, which contributes to the regulation of receptor signaling, organellar signaling, gene expression, metabolic pathways, and cell death, all of which essentially impact plant immunity. Associated with this, various PP2A subunits mediate post-translational regulation of metabolic enzymes and signaling components. Here we provide an overview of protein kinase/phosphatase functions in plant immunity signaling, and position the multifaceted functions of PP2A in the tightly inter-connected regulatory network that controls the perception, signaling and responding to biotic stress agents in plants.Peer reviewe

Alternative proteins and EU food law

We ask how European food law impacts the transformative potential of alternative proteins, including single-cell proteins, plant-based novel proteins, cultured meat,macroalgae, and insects. The Novel Food Regulation may prove insurmountable for small companies, and it is demanding and time-consuming even for larger companies, dampening the transformative potential of all novel foods and traditional foods from third countries. Several microalgae and macroalgae are non-novel in the EU, which eases their way into the markets. The unclear novel food status of some potential green macroalgae species is a hindrance. All insects are novel, and none has EU-level authorization yet, although some Member States allow insect food. The GM Food Regulation is proce-durally and scientifically demanding, and it forces GM labelling. The Regulation dampens the transformative potential of food GM technology. In addition to crops and fruit, GM Food Regulation applies to genetically modified or edited microbes,microalgae, cultured meat, and insects. The naming and labelling rules of plant -based products have caused controversy. From the business perspective, the health claims process is similarly challenging as the novel food process. EU food law must guarantee food safety and consumer rights while applying the principles of nondiscrimination and proportionality

Alternative proteins and EU food law

Highlights • EU food law impacts the transformative potential of alternative proteins. • Insects and cultured meat are novel foods; several microalgae and macroalgae are not. • The GM Food Regulation applies to all genetically modified or edited foods. • The names of vegan products have caused controversy. • The principles of non-discrimination and proportionality are important for fairness.We ask how European food law impacts the transformative potential of alternative proteins, including single-cell proteins, plant-based novel proteins, cultured meat,macroalgae, and insects. The Novel Food Regulation may prove insurmountable for small companies, and it is demanding and time-consuming even for larger companies,dampening the transformative potential of all novel foods and traditional foods from third countries. Several microalgae and macroalgae are non-novel in the EU, which eases their way into the markets. The unclear novel food status of some potential green macroalgae species is a hindrance. All insects are novel, and none has EU-level authorization yet, although some Member States allow insect food. The GM Food Regulation is procedurally and scientifically demanding, and it forces GM labelling. The Regulation dampens the transformative potential of food GM technology. In addition to crops and fruit, GM Food Regulation applies to genetically modified or edited microbes,microalgae, cultured meat, and insects. The naming and labelling rules of plant-based products have caused controversy. From the business perspective, the health claims process is similarly challenging as the novel food process. EU food law must guarantee food safety and consumer rights while applying the principles of nondiscrimination and proportionality

Trans-methylation reactions in plants: focus on the activated methyl cycle

Trans-methylation reactions are vital in basic metabolism, epigenetic regulation, RNA metabolism, and posttranslational control of protein function and therefore fundamental in determining the physiological processes in all living organisms. The plant kingdom is additionally characterized by the production of secondary metabolites that undergo specific hydroxylation, oxidation and methylation reactions to obtain a wide array of different chemical structures. Increasing research efforts have started to reveal the enzymatic pathways underlying the biosynthesis of complex metabolites in plants. Further engineering of these enzymatic machineries offers significant possibilities in the development of bio-based technologies, but necessitates deep understanding of their potential metabolic and regulatory interactions. Trans-methylation reactions are tightly coupled with the so-called activated methyl cycle (AMC), an essential metabolic circuit that maintains the trans-methylation capacity in all living cells. Tight regulation of the AMC is crucial in ensuring accurate trans-methylation reactions in different subcellular compartments, cell types, developmental stages and environmental conditions. This review addresses the organization and posttranslational regulation of the AMC and elaborates its critical role in determining metabolic regulation through modulation of methyl utilization in stress-exposed plants.</p

PP2A Phosphatase as a Regulator of ROS Signaling in Plants

Reactive oxygen species (ROS) carry out vital functions in determining appropriate stress reactions in plants, but the molecular mechanisms underlying the sensing, signaling and response to ROS as signaling molecules are not yet fully understood. Recent studies have underscored the role of Protein Phosphatase 2A (PP2A) in ROS-dependent responses involved in light acclimation and pathogenesis responses in Arabidopsis thaliana. Genetic, proteomic and metabolomic studies have demonstrated that trimeric PP2A phosphatases control metabolic changes and cell death elicited by intracellular and extracellular ROS signals. Associated with this, PP2A subunits contribute to transcriptional and post-translational regulation of pro-oxidant and antioxidant enzymes. This review highlights the emerging role of PP2A phosphatases in the regulatory ROS signaling networks in plants. </p

The impact of light intensity on metabolomic profile of Arabidopsis thaliana wild type and reticulata mutant by NMR spectroscopy

Light acclimation involves biochemical, metabolic and developmental adjustments that allow plants to cope with a vast range of growth environments. Arabidopsis thaliana mutants with photoperiod-dependent defects in leaf development and metabolism have been instrumental in deciphering the interlinked regulatory networks in plants. The reticulata (re) mutant displays dark green veins and pale green mesophyll tissues when grown under long day conditions. RE is a chloroplast envelope membrane protein of unknown function and is required for accurate primary metabolism and leaf development under long photoperiod, while its functional significance under short photoperiods has remained poorly understood. In the present study we assessed whether RE impacts primary metabolism or leaf development when Arabidopsis plants acclimate to different light intensities under short photoperiod. We show that growth under short day conditions annuls the metabolic and developmental defects of re mutants, suggesting that RE does not significantly modulate leaf development or primary metabolism under short photoperiod. Based on proton nuclear magnetic resonance spectroscopy (H-1 NMR) and statistical analysis, however, the metabolite profiles of differentially light-acclimated short-day-grown plants differ with respect to sugars (glucose, fructose and sucrose), TCA cycle intermediates (fumaric, malic, citric and succinic acids) and fatty acids, which become more abundant under high light. Moreover, in contrast to isoleucine, leucine, valine, threonine, serine, tyrosine and phenylalanine, which show increased abundance in highlight-acclimated plants, the contents of alanine, glutamine, glutamic acid and aspartic acid are higher when plants grow under normal growth light. These findings indicate that NMR can detect high-light-induced metabolic adjustments that arise upon plant acclimation to light stress

Protein Phosphatase 2A in the Regulatory Network Underlying Biotic Stress Resistance in Plants

Biotic stress factors pose a major threat to plant health and can significantly deteriorate plant productivity by impairing the physiological functions of the plant. To combat the wide range of pathogens and insect herbivores, plants deploy converging signaling pathways, where counteracting activities of protein kinases and phosphatases form a basic mechanism for determining appropriate defensive measures. Recent studies have identified Protein Phosphatase 2A (PP2A) as a crucial component that controls pathogenesis responses in various plant species. Genetic, proteomic and metabolomic approaches have underscored the versatile nature of PP2A, which contributes to the regulation of receptor signaling, organellar signaling, gene expression, metabolic pathways, and cell death, all of which essentially impact plant immunity. Associated with this, various PP2A subunits mediate post-translational regulation of metabolic enzymes and signaling components. Here we provide an overview of protein kinase/phosphatase functions in plant immunity signaling, and position the multifaceted functions of PP2A in the tightly inter-connected regulatory network that controls the perception, signaling and responding to biotic stress agents in plants

Evolutionary conservation and post-translational control of S-adenosyl-L-homocysteine hydrolase in land plants

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.Peer reviewe

PP2A-B’γ modulates foliar trans-methylation capacity and the formation of 4-methoxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves

Glucosinolates (GSL) of cruciferous plants comprise a major group of structurally diverse secondary compounds which act as deterrents against aphids and microbial pathogens and have large commercial and ecological impacts. While the transcriptional regulation governing the biosynthesis and modification of GSL is now relatively well understood, post-translational regulatory components that specifically determine the structural variation of indole glucosinolates have not been reported. We show that the cytoplasmic protein phosphatase 2A regulatory subunit B'gamma (PP2A-B'gamma) physically interacts with indole glucosinolate methyltransferases and controls the methoxylation of indole glucosinolates and the formation of 4-meth-oxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves. By taking advantage of proteomic approaches and metabolic analysis we further demonstrate that PP2A-B'gamma is required to control the abundance of oligomeric protein complexes functionally linked with the activated methyl cycle and the trans-methylation capacity of leaf cells. These findings highlight the key regulatory role of PP2A-B'gamma in methionine metabolism and provide a previously unrecognized perspective for metabolic engineering of glucosinolate metabolism in cruciferous plants.Peer reviewe