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.

These authors contributed equally to this work. SUMMARYGlucosinolates (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 0 c (PP2A-B 0 c) physically interacts with indole glucosinolate methyltransferases and controls the methoxylation of indole glucosinolates and the formation of 4-methoxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves. By taking advantage of proteomic approaches and metabolic analysis we further demonstrate that PP2A-B 0 c 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 0 c in methionine metabolism and provide a previously unrecognized perspective for metabolic engineering of glucosinolate metabolism in cruciferous plants.

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.

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.

Mitochondria are tightly embedded within metabolic and regulatory networks that optimize plant performance in response to environmental challenges. The best-known mitochondrial retrograde signaling pathway involves stress-induced activation of the transcription factor NAC DOMAIN CONTAINING PROTEIN 17 (ANAC017), which initiates protective responses to stress-induced mitochondrial dysfunction in Arabidopsis (Arabidopsis thaliana). Post-translational control of the elicited responses, however, remains poorly understood. Previous studies linked protein phosphatase 2A subunit PP2A-B’γ, a key negative regulator of stress responses, with reversible phosphorylation of ACONITASE 3 (ACO3). Here we report on ACO3 and its phosphorylation at Ser91 as key components of stress regulation that are induced by mitochondrial dysfunction. Targeted mass spectrometry-based proteomics revealed that the abundance and phosphorylation of ACO3 increased under stress, which required signaling through ANAC017. Phosphomimetic mutation at ACO3-Ser91 and accumulation of ACO3S91D-YFP promoted the expression of genes related to mitochondrial dysfunction. Furthermore, ACO3 contributed to plant tolerance against UV-B or antimycin A-induced mitochondrial dysfunction. These findings demonstrate that ACO3 is both a target and mediator of mitochondrial dysfunction signaling, and critical for achieving stress tolerance in Arabidopsis leaves.

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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.

Although responses to UV stress have been characterised at system and cellular levels, the dynamics of the nuclear proteome triggered in this situation are still unknown, despite its essential role in regulating gene expression and in last term plant physiology. To fill this gap, we characterised the variations in the nuclear proteome after 2 h and 16 h (8 h/day) of UV irradiation by using state-of-the-art mass spectrometry-based shotgun proteomics methods combined with novel bioinformatics workflows that were employed in the manuscript entitled “The variations in the nuclear proteome reveal new transcription factors and mechanisms involved in UV stress response in Pinus radiata” (Pascual et al., 2016) [1]. We employed in-gel digestion followed by a 120 min gradient prior to MS analysis. Data was processed following two approaches: a database dependent employing the SEQUEST algorithm and custom databases, and a database independent by mass accuracy precursor alignment (MAPA). 388 proteins were identified by SEQUEST search and 9094 m/z were quantified by MAPA. Significant m/z were de novo sequenced using the Novor algorithm. We present here the complete datasets and the analysis workflow.