Systems biology reveals key tissue-specific metabolic and transcriptional signatures involved in the response of Medicago truncatula plant genotypes to salt stress.

Salt stress is an important factor limiting plant productivity by affecting plant physiology and metabolism. To explore salt tolerance adaptive mechanisms in the model legume Medicago truncatula, we used three genotypes with differential salt-sensitivity: TN6.18 (highly sensitive), Jemalong A17 (moderately sensitive), and TN1.11 (tolerant). Cellular damage was monitored in roots and leaves 48 h after 200 mM NaCl treatment by measuring lipid peroxidation, nitric oxide, and hydrogen peroxide contents, further supported by leaf stomatal conductance and chlorophyll readings. The salt-tolerant genotype TN1.11 displayed the lowest level of oxidative damage, in contrast to the salt sensitive TN6.18, which showed the highest responses. Metabolite profiling was employed to explore the differential genotype-related responses to stress at the molecular level. The metabolic data in the salt tolerant TN1.11 roots revealed an accumulation of metabolites related to the raffinose pathway. To further investigate the sensitivity to salinity, global transcriptomic profiling using microarray analysis was carried out on the salt-stressed sensitive genotypes. In TN6.18, the transcriptomic analysis identified a lower expression of many genes related to stress signalling, not previously linked to salinity, and corresponding to the TIR-NBS-LRR gene class. Overall, this global approach contributes to gaining significant new insights into the complexity of stress adaptive mechanisms and to the identification of potential targets for crop improvement.

Involvement of Polyamine Metabolism in the Response of Medicago truncatula Genotypes to Salt Stress.

Salinity constitutes one of the most important causes leading to severe reduction in plant yield. Several reports correlate the accumulation of polyamines in plants with tolerance to abiotic stress cues. The present study examined three Medicago truncatula genotypes with differing sensitivities to salinity (TN1.11, tolerant; Jemalong A17, moderately sensitive; TN6.18, sensitive), with the aim of examining the genotype-specific involvement of the polyamine metabolic pathway in plant response to salinity. The study was carried out with leaves harvested 48 h after watering plants with 200 mM NaCl. A comprehensive profile of free polyamines was determined using high performance liquid chromatography. All genotypes showed spermidine and spermine as the most abundant polyamines under control conditions. In salinity conditions, spermine levels increased at the expense of putrescine and spermidine, indicating a drift of polyamine metabolism towards the synthesis of increasing polycationic forms as a stress response. The increasing balance between high and low polycationic forms was clearly diminished in the salt-sensitive genotype TN6.18, showing a clear correlation with its sensitive phenotype. The polyamine metabolic profile was then supported by molecular evidence through the examination of polyamine metabolism transcript levels by RT-qPCR. General suppression of genes that are involved upstream in the PA biosynthetic pathway was determined. Contrarily, an induction in the expression of genes involved in the biosynthesis of spermine and spermidine was observed, in agreement with the metabolic analysis. A significant induction in diamino oxidase expression, involved in the catabolism of putrescine, was specifically found in the sensitive genotype ΤΝ6.18, indicating a distinct metabolic response to stress. Present findings highlight the involvement of polyamines in the defense response of Medicago genotypes showing sensitivity to salt stress.

Polyamines: Small Amines with Large Effects on Plant Abiotic Stress Tolerance.

In recent years, climate change has altered many ecosystems due to a combination of frequent droughts, irregular precipitation, increasingly salinized areas and high temperatures. These environmental changes have also caused a decline in crop yield worldwide. Therefore, there is an urgent need to fully understand the plant responses to abiotic stress and to apply the acquired knowledge to improve stress tolerance in crop plants. The accumulation of polyamines (PAs) in response to many abiotic stresses is one of the most remarkable plant metabolic responses. In this review, we provide an update about the most significant achievements improving plant tolerance to drought, salinity, low and high temperature stresses by exogenous application of PAs or genetic manipulation of endogenous PA levels. We also provide some clues about possible mechanisms underlying PA functions, as well as known cross-talks with other stress signaling pathways. Finally, we discuss about the possible use of PAs for seed priming to induce abiotic stress tolerance in agricultural valuable crop plants.

Putrescine elicits ROS-dependent activation of the salicylic acid pathway in Arabidopsis thaliana.

Polyamines are small amines that accumulate during stress and contribute to disease resistance through as yet unknown signaling pathways. Using a comprehensive RNA-sequencing analysis, we show that early transcriptional responses triggered by each of the most abundant polyamines (putrescine, spermidine, spermine, thermospermine and cadaverine) exhibit specific quantitative differences, suggesting that polyamines (rather than downstream metabolites) elicit defense responses. Signaling by putrescine, which accumulates in response to bacteria that trigger effector triggered immunity (ETI) and systemic acquired resistance (SAR), is largely dependent on the accumulation of hydrogen peroxide, and is partly dependent on salicylic acid (SA), the expression of ENHANCED DISEASE SUSCEPTIBILITY (EDS1) and NONEXPRESSOR of PR GENES1 (NPR1). Putrescine elicits local SA accumulation as well as local and systemic transcriptional reprogramming that overlaps with SAR. Loss-of-function mutations in arginine decarboxylase 2 (ADC2), which is required for putrescine synthesis and copper amine oxidase (CuAO), which is involved in putrescine oxidation, compromise basal defenses, as well as putrescine and pathogen-triggered systemic resistance. These findings confirm that putrescine elicits ROS-dependent SA pathways in the activation of plant defenses.

Lipid kinases PIP5K7 and PIP5K9 are required for polyamine-triggered K+ efflux in Arabidopsis roots.

Polyamines, such as putrescine, spermidine and spermine (Spm), are low-molecular-weight polycationic molecules present in all living organisms. Despite their implication in plant cellular processes, little is known about their molecular mode of action. Here, we demonstrate that polyamines trigger a rapid increase in the regulatory membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2 ), and that this increase is required for polyamine effects on K+ efflux in Arabidopsis roots. Using in vivo 32 Pi -labelling of Arabidopsis seedlings, low physiological (µm) concentrations of Spm were found to promote a rapid PIP2 increase in roots that was time- and dose-dependent. Confocal imaging of a genetically encoded PIP2 biosensor revealed that this increase was triggered at the plasma membrane. Differential 32 Pi -labelling suggested that the increase in PIP2 was generated through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity rather than inhibition of a phospholipase C or PIP2 5-phosphatase activity. Systematic analysis of transfer DNA insertion mutants identified PIP5K7 and PIP5K9 as the main candidates involved in the Spm-induced PIP2 response. Using non-invasive microelectrode ion flux estimation, we discovered that the Spm-triggered K+ efflux response was strongly reduced in pip5k7 pip5k9 seedlings. Together, our results provide biochemical and genetic evidence for a physiological role of PIP2 in polyamine-mediated signalling controlling K+ flux in plants.

The Polyamine Putrescine Contributes to H2O2 and RbohD/F-Dependent Positive Feedback Loop in Arabidopsis PAMP-Triggered Immunity.

Polyamines are involved in defense against pathogenic microorganisms in plants. However, the role of the polyamine putrescine (Put) during plant defense has remained elusive. In this work, we studied the implication of polyamines during pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in the model species Arabidopsis thaliana. Our data indicate that polyamines, particularly Put, accumulate in response to non-pathogenic Pseudomonas syringae pv. tomato DC3000 hrcC and in response to the purified PAMP flagellin22. Exogenously supplied Put to Arabidopsis seedlings induces defense responses compatible with PTI activation, such as callose deposition and transcriptional up-regulation of several PTI marker genes. Consistent with this, we show that Put primes for resistance against pathogenic bacteria. Through chemical and genetic approaches, we find that PTI-related transcriptional responses induced by Put are hydrogen peroxide and NADPH oxidase (RBOHD and RBOHF) dependent, thus suggesting that apoplastic ROS mediates Put signaling. Overall, our data indicate that Put amplifies PTI responses through ROS production, leading to enhanced disease resistance against bacterial pathogens.

Extracellular Spermine Triggers a Rapid Intracellular Phosphatidic Acid Response in Arabidopsis, Involving PLDδ Activation and Stimulating Ion Flux.

Polyamines, such as putrescine (Put), spermidine (Spd), and spermine (Spm), are low-molecular-weight polycationic molecules found in all living organisms. Despite the fact that they have been implicated in various important developmental and adaptative processes, their mode of action is still largely unclear. Here, we report that Put, Spd, and Spm trigger a rapid increase in the signaling lipid, phosphatidic acid (PA) in Arabidopsis seedlings but also mature leaves. Using time-course and dose-response experiments, Spm was found to be the most effective; promoting PA responses at physiological (low µM) concentrations. In seedlings, the increase of PA occurred mainly in the root and partly involved the plasma membrane polyamine-uptake transporter (PUT), RMV1. Using a differential 32Pi-labeling strategy combined with transphosphatidylation assays and T-DNA insertion mutants, we found that phospholipase D (PLD), and in particular PLDδ was the main contributor of the increase in PA. Measuring non-invasive ion fluxes (MIFE) across the root plasma membrane of wild type and pldδ-mutant seedlings, revealed that the formation of PA is linked to a gradual- and transient efflux of K+. Potential mechanisms of how PLDδ and the increase of PA are involved in polyamine function is discussed.

Analysis of Polyamines Conjugated with Hydroxycinnamoyl Acids by High-Performance Liquid Chromatography Coupled to Electrospray Ionization Tandem Mass Spectrometry.

Polyamines conjugated with hydroxycinnamic acids are phenolic compounds, which are widespread in the plant kingdom playing important roles in development and defence responses. This chapter describes the methodology employed to analyze these phenolamides in plant material by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS-MS). These compounds are not always in sufficient concentration in plant tissues for analysis by more conventional methods such as UV detection of HPLC. Owing to their particular molecular structure, they cannot be analyzed as free polyamines. Thus, described herein is an extraction method for hydroxycinnamic acid amides in plant tissues such as leaves, and their analysis by LC-MS-MS, including identification and quantification protocols.

Determination of Arginine and Ornithine Decarboxylase Activities in Plants.

In plants, putrescine is synthesized directly from the decarboxylation of ornithine and/or by the alternative arginine decarboxylase pathway. The prevalence of one or the other depends on the tissue and stress conditions. In both amino acid decarboxylation reactions, the corresponding enzymes use pyridoxal phosphate (PLP) as co-factor. PLP combines with the α-amino acid to form a Schiff base, which acts as substrate in the carboxyl group removal and CO2 formation. We describe the methodology employed for the determination of ODC and ADC activities in plant tissues by detecting the release of (C14) CO2 using (C14) labelled substrates (ornithine or arginine).

Determination of S-Adenosylmethionine Decarboxylase Activity in Plants.

The synthesis of spermidine, spermine and thermospermine requires the addition of aminopropyl groups from decarboxylated S-adenosyl-methionine (dSAM). The synthesis of dSAM is catalyzed by S-adenosylmethionine decarboxylase. dSAM levels are usually low, which constitutes a rate-limiting factor in the synthesis of polyamines. In this chapter, we provide a protocol for the determination of SAMDC activity in plants through the detection of radiolabelled CO2 released during the SAMDC reaction.

Generation of EMS-Mutagenized Populations of Arabidopsis thaliana for Polyamine Genetics.

In the recent years, genetic engineering of polyamine biosynthetic genes has provided evidence for their involvement in plant stress responses and different aspects of plant development. Such approaches are being complemented with the use of reverse genetics, in which mutants affected on a particular trait, tightly associated with polyamines, are isolated and the causal genes mapped. Reverse genetics enables the identification of novel genes in the polyamine pathway, which may be involved in downstream signaling, transport, homeostasis, or perception. Here, we describe a basic protocol for the generation of ethyl methanesulfonate (EMS) mutagenized populations of Arabidopsis thaliana for its use in reverse genetics applied to polyamines.

Determination of Posttranslational Modifications by 2D PAGE: Applications to Polyamines.

Polyamines not only affect transcription and translation but also may induce a number of posttranslational modifications. The identification of polyamine-induced posttranslational modifications can be performed by 2D PAGE analyses. Here, we provide a protocol for 2D-gel electrophoresis that has been optimized for plants. The combined use of this protocol with epitope-tagged proteins expressed in plants enables the detailed analysis of posttranslational modifications induced by different polyamines in vivo.

Potential Applications of Polyamines in Agriculture and Plant Biotechnology.

The polyamines putrescine, spermidine and spermine have been implicated in a myriad of biological functions in many organisms. Research done during the last decades has accumulated a large body of evidence demonstrating that polyamines are key modulators of plant growth and development. Different experimental approaches have been employed including the measurement of endogenous polyamine levels and the activities of polyamine metabolic enzymes, the study of the effects resulting from exogenous polyamine applications and chemical or genetic manipulation of endogenous polyamine titers. This chapter reviews the role of PAs in seed germination, root development, plant architecture, in vitro plant regeneration, flowering and plant senescence. Evidence presented here indicates that polyamines should be regarded as plant growth regulators with potential applications in agriculture and plant biotechnology.

Metabolic Flexibility Underpins Growth Capabilities of the Fastest Growing Alga.

The factors rate-limiting growth of photosynthetic organisms under optimal conditions are controversial [1-8]. Adaptation to extreme environments is usually accompanied by reduced performance under optimal conditions [9, 10]. However, the green alga Chlorella ohadii, isolated from a harsh desert biological soil crust [11-17], does not obey this rule. In addition to resistance to photodamage [17, 18], it performs the fastest growth ever reported for photosynthetic eukaryotes. A multiphasic growth pattern (very fast growth [phase I], followed by growth retardation [phase II] and additional fast growth [phase III]) observed under constant illumination and temperature indicates synchronization of the algal population. Large physiological changes at transitions between growth phases suggest metabolic shifts. Indeed, metabolome analyses at points along the growth phases revealed large changes in the levels of many metabolites during growth with an overall rise during phase I and decline in phase II. Multivariate analysis of the metabolome data highlighted growth phase as the main factor contributing to observed metabolite variance. The analyses identified putrescine as the strongest predictive metabolite for growth phase and a putative growth regulator. Indeed, extracellular additions of polyamines strongly affected the growth rate in phase I and the growth arrest in phase II, with a marked effect on O2 exchange. Our data implicate polyamines as the signals harmonizing metabolic shifts and suggest that metabolic flexibility enables the immense growth capabilities of C. ohadii. The data provide a new dimension to current models focusing on growth-limiting processes in photosynthetic organisms where the anabolic and catabolic metabolisms must be strictly regulated.

Polyamine oxidase 5 loss-of-function mutations in Arabidopsis thaliana trigger metabolic and transcriptional reprogramming and promote salt stress tolerance.

The family of polyamine oxidases (PAO) in Arabidopsis (AtPAO1-5) mediates polyamine (PA) back-conversion, which reverses the PA biosynthetic pathway from spermine and its structural isomer thermospermine (tSpm) into spermidine and then putrescine. Here, we have studied the involvement of PA back-conversion in Arabidopsis salinity tolerance. AtPAO5 is the Arabidopsis PAO gene member most transcriptionally induced by salt stress. Two independent loss-of-function mutants (atpao5-2 and atpao5-3) were found to exhibit constitutively higher tSpm levels, with associated increased salt tolerance. Using global transcriptional and metabolomic analyses, the underlying mechanisms were studied. Stimulation of abscisic acid and jasmonate (JA) biosynthesis and accumulation of important compatible solutes, such as sugars, polyols and proline, as well as TCA cycle intermediates were observed in atpao5 mutants under salt stress. Expression analyses indicate that tSpm modulates the transcript levels of several target genes, including many involved in the biosynthesis and signalling of JA, some of which are already known to promote salinity tolerance. Transcriptional modulation by tSpm is isomer-dependent, thus demonstrating the specificity of this response. Overall, we conclude that tSpm triggers metabolic and transcriptional reprogramming that promotes salt stress tolerance in Arabidopsis.

Genome Wide Association Mapping for the Tolerance to the Polyamine Oxidase Inhibitor Guazatine in Arabidopsis thaliana.

Guazatine is a potent inhibitor of polyamine oxidase (PAO) activity. In agriculture, guazatine is used as non-systemic contact fungicide efficient in the protection of cereals and citrus fruits against disease. The composition of guazatine is complex, mainly constituted by a mixture of synthetic guanidated polyamines (polyaminoguanidines). Here, we have studied the effects from exposure to guazatine in the weed Arabidopsis thaliana. We report that micromolar concentrations of guazatine are sufficient to inhibit growth of Arabidopsis seedlings and induce chlorosis, whereas germination is barely affected. We observed the occurrence of quantitative variation in the response to guazatine between 107 randomly chosen Arabidopsis accessions. This enabled us to undertake genome-wide association (GWA) mapping that identified a locus on chromosome one associated with guazatine tolerance. CHLOROPHYLLASE 1 (CLH1) within this locus was studied as candidate gene, together with its paralog (CLH2). The analysis of independent clh1-2, clh1-3, clh2-3, clh2-2, and double clh1-2 clh2-3 mutant alleles indicated that CLH1 and/or CLH2 loss-of-function or expression down-regulation promote guazatine tolerance in Arabidopsis. We report a natural mechanism by which Arabidopsis populations can overcome toxicity by the fungicide guazatine.

Global Metabolic Profiling of Arabidopsis Polyamine Oxidase 4 (AtPAO4) Loss-of-Function Mutants Exhibiting Delayed Dark-Induced Senescence.

Early and more recent studies have suggested that some polyamines (PAs), and particularly spermine (Spm), exhibit anti-senescence properties in plants. In this work, we have investigated the role of Arabidopsis Polyamine Oxidase 4 (PAO4), encoding a PA back-conversion oxidase, during dark-induced senescence. Two independent PAO4 (pao4-1 and pao4-2) loss-of-function mutants have been found that accumulate 10-fold higher Spm, and this associated with delayed entry into senescence under dark conditions. Mechanisms underlying pao4 delayed senescence have been studied using global metabolic profiling by GC-TOF/MS. pao4 mutants exhibit constitutively higher levels of important metabolites involved in redox regulation, central metabolism and signaling that support a priming status against oxidative stress. During senescence, interactions between PAs and oxidative, sugar and nitrogen metabolism have been detected that additively contribute to delayed entry into senescence. Our results indicate the occurrence of metabolic interactions between PAs, particularly Spm, with cell oxidative balance and transport/biosynthesis of amino acids as a strategy to cope with oxidative damage produced during senescence.

The roles of polyamines during the lifespan of plants: from development to stress.

Compelling evidence indicates that free polyamines (PAs) (mainly putrescine, spermidine, spermine, and its isomer thermospermine), some PA conjugates to hydroxycinnamic acids, and the products of PA oxidation (hydrogen peroxide and γ-aminobutyric acid) are required for different processes in plant development and participate in abiotic and biotic stress responses. A tight regulation of PA homeostasis is required, since depletion or overaccumulation of PAs can be detrimental for cell viability in many organisms. In plants, homeostasis is achieved by modulation of PA biosynthesis, conjugation, catabolism, and transport. However, recent data indicate that such mechanisms are not mere modulators of PA pools but actively participate in PA functions. Examples are found in the spermidine-dependent eiF5A hypusination required for cell division, PA hydroxycinnamic acid conjugates required for pollen development, and the involvement of thermospermine in cell specification. Recent advances also point to implications of PA transport in stress tolerance, PA-dependent transcriptional and translational modulation of genes and transcripts, and posttranslational modifications of proteins. Overall, the molecular mechanisms identified suggest that PAs are intricately coordinated and/or mediate different stress and developmental pathways during the lifespan of plants.