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.

EARLY RESPONSE TO DEHYDRATION 7 Remodels Cell Membrane Lipid Composition during Cold Stress in Arabidopsis.

Plants adjust to unfavorable conditions by altering physiological activities, such as gene expression. Although previous studies have identified multiple stress-induced genes, the function of many genes during the stress responses remains unclear. Expression of ERD7 (EARLY RESPONSE TO DEHYDRATION 7) is induced in response to dehydration. Here, we show that ERD7 plays essential roles in both plant stress responses and development. In Arabidopsis, ERD7 protein accumulated under various stress conditions, including exposure to low temperature. A triple mutant of Arabidopsis lacking ERD7 and two closely related homologs had an embryonic lethal phenotype, whereas a mutant lacking the two homologs and one ERD7 allele had relatively round leaves, indicating that the ERD7 gene family has essential roles in development. Moreover, the importance of the ERD7 family in stress responses was evidenced by the susceptibility of the mutant lines to cold stress. ERD7 protein was found to bind to several, but not all, negatively charged phospholipids and was associated with membranes. Lipid components and cold-induced reduction in PIP2 in the mutant line were altered relative to wild type. Furthermore, membranes from the mutant line had reduced fluidity. Taken together, ERD7 and its homologs are important for plant stress responses and development and associated with the modification in membrane lipid composition.

AUTOPHAGY-RELATED14 and Its Associated Phosphatidylinositol 3-Kinase Complex Promote Autophagy in Arabidopsis.

Phosphatidylinositol 3-phosphate (PI3P) is an essential membrane signature for both autophagy and endosomal sorting that is synthesized in plants by the class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of the VPS34 kinase, together with ATG6, VPS15, and either VPS38 or ATG14 as the fourth subunit. Although Arabidopsis (Arabidopsis thaliana) plants missing the three core subunits are infertile, vps38 mutants are viable but have aberrant leaf, root, and seed development, Suc sensing, and endosomal trafficking, suggesting that VPS38 and ATG14 are nonredundant. Here, we evaluated the role of ATG14 through a collection of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and T-DNA insertion mutants disrupting the two Arabidopsis paralogs. atg14a atg14b double mutants were relatively normal phenotypically but displayed pronounced autophagy defects, including reduced accumulation of autophagic bodies and cargo delivery during nutrient stress. Unexpectedly, homozygous atg14a atg14b vps38 triple mutants were viable but showed severely compromised rosette development and reduced fecundity, pollen germination, and autophagy, consistent with a need for both ATG14 and VPS38 to fully actuate PI3P biology. However, the triple mutants still accumulated PI3P, but they were hypersensitive to the PI3K inhibitor wortmannin, indicating that the ATG14/VPS38 component is not essential for PI3P synthesis. Collectively, the ATG14/VPS38 mutant collection now permits the study of plants altered in specific aspects of PI3P biology.

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.

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.

Role for Arabidopsis PLC7 in Stomatal Movement, Seed Mucilage Attachment, and Leaf Serration.

Phospholipase C (PLC) has been suggested to play important roles in plant stress and development. To increase our understanding of PLC signaling in plants, we have started to analyze knock-out (KO), knock-down (KD) and overexpression mutants of Arabidopsis thaliana, which contains nine PLCs. Earlier, we characterized PLC2, PLC3 and PLC5. Here, the role of PLC7 is functionally addressed. Promoter-GUS analyses revealed that PLC7 is specifically expressed in the phloem of roots, leaves and flowers, and is also present in trichomes and hydathodes. Two T-DNA insertion mutants were obtained, i.e., plc7-3 being a KO- and plc7-4 a KD line. In contrast to earlier characterized phloem-expressed PLC mutants, i.e., plc3 and plc5, no defects in primary- or lateral root development were found for plc7 mutants. Like plc3 mutants, they were less sensitive to ABA during stomatal closure. Double-knockout plc3 plc7 lines were lethal, but plc5 plc7 (plc5/7) double mutants were viable, and revealed several new phenotypes, not observed earlier in the single mutants. These include a defect in seed mucilage, enhanced leaf serration, and an increased tolerance to drought. Overexpression of PLC7 enhanced drought tolerance too, similar to what was earlier found for PLC3-and PLC5 overexpression. In vivo 32Pi-labeling of seedlings and treatment with sorbitol to mimic drought stress, revealed stronger PIP2 responses in both drought-tolerant plc5/7 and PLC7-OE mutants. Together, these results show novel functions for PLC in plant stress and development. Potential molecular mechanisms are discussed.

Knock-Down of Arabidopsis PLC5 Reduces Primary Root Growth and Secondary Root Formation While Overexpression Improves Drought Tolerance and Causes Stunted Root Hair Growth.

Phospholipase C (PLC) is a well-known signaling enzyme in metazoans that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate and diacylglycerol as second messengers involved in mutiple processes. Plants contain PLC too, but relatively little is known about its function there. The model system Arabidopsis thaliana contains nine PLC genes. Reversed genetics have implicated several roles for PLCs in plant development and stress signaling. Here, PLC5 is functionally addressed. Promoter-ß-glucuronidase (GUS) analyses revealed expression in roots, leaves and flowers, predominantly in vascular tissue, most probably phloem companion cells, but also in guard cells, trichomes and root apical meristem. Only one plc5-1 knock-down mutant was obtained, which developed normally but grew more slowly and exhibited reduced primary root growth and decreased lateral root numbers. These phenotypes could be complemented by expressing the wild-type gene behind its own promoter. Overexpression of PLC5 (PLC5-OE) using the UBQ10 promoter resulted in reduced primary and secondary root growth, stunted root hairs, decreased stomatal aperture and improved drought tolerance. PLC5-OE lines exhibited strongly reduced phosphatidylinositol 4-monophosphate (PIP) and PIP2 levels and increased amounts of phosphatidic acid, indicating enhanced PLC activity in vivo. Reduced PIP2 levels and stunted root hair growth of PLC5-OE seedlings could be recovered by inducible overexpression of a root hair-specific PIP 5-kinase, PIP5K3. Our results show that PLC5 is involved in primary and secondary root growth and that its overexpression improves drought tolerance. Independently, we provide new evidence that PIP2 is essential for the polar tip growth of root hairs.

Arabidopsis Phospholipase C3 is Involved in Lateral Root Initiation and ABA Responses in Seed Germination and Stomatal Closure.

Phospholipase C (PLC) is well known for its role in animal signaling, where it generates the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), by hydrolyzing the minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), upon receptor stimulation. In plants, PLC's role is still unclear, especially because the primary targets of both second messengers are lacking, i.e. the ligand-gated Ca2+ channel and protein kinase C, and because PIP2 levels are extremely low. Nonetheless, the Arabidopsis genome encodes nine PLCs. We used a reversed-genetic approach to explore PLC's function in Arabidopsis, and report here that PLC3 is required for proper root development, seed germination and stomatal opening. Two independent knock-down mutants, plc3-2 and plc3-3, were found to exhibit reduced lateral root densities by 10-20%. Mutant seeds germinated more slowly but were less sensitive to ABA to prevent germination. Guard cells of plc3 were also compromised in ABA-dependent stomatal closure. Promoter-ß-glucuronidase (GUS) analyses confirmed PLC3 expression in guard cells and germinating seeds, and revealed that the majority is expressed in vascular tissue, most probably phloem companion cells, in roots, leaves and flowers. In vivo 32Pi labeling revealed that ABA stimulated the formation of PIP2 in germinating seeds and guard cell-enriched leaf peels, which was significantly reduced in plc3 mutants. Overexpression of PLC3 had no effect on root system architecture or seed germination, but increased the plant's tolerance to drought. Our results provide genetic evidence for PLC's involvement in plant development and ABA signaling, and confirm earlier observations that overexpression increases drought tolerance. Potential molecular mechanisms for the above observations are discussed.

Vacuolar Trafficking Protein VPS38 Is Dispensable for Autophagy.

Phosphatidylinositol 3-P (PI3P) is a signaling molecule that controls a variety of processes in endosomal, autophagic, and vacuolar/lysosomal trafficking in yeasts and mammals. Vacuolar protein sorting 34 (Vps34) is a conserved PI3K present in multiple complexes with specific functions and regulation. In yeast, the PI3K complex II consists of Vps34p, Vps15p, Vps30p/Atg6p, and Vps38p, and is essential for vacuolar protein sorting. Here, we describe the Arabidopsis (Arabidopsis thaliana) homolog of yeast Vps38p and human UV radiation resistance-associated gene protein. Arabidopsis VPS38 interacts with VPS30/ATG6 both in yeast and in planta. Although the level of PI3P in Arabidopsis vps38 mutants is similar to that in wild type, vps38 cells contain enlarged multivesicular endosomes and fewer organelles enriched in PI3P than the wild type. The vps38 mutants are defective in the trafficking of vacuolar cargo and its receptor VACUOLAR SORTING RECEPTOR2;1. The mutants also exhibit abnormal cytoplasmic distributions of endocytic cargo, such as auxin efflux carriers PINFORMED1 (PIN1) and PIN2. Constitutive autophagy is normal in the mutants but starvation-induced autophagy was slightly inhibited. We conclude that Arabidopsis VPS38 is dispensable for autophagy but essential for efficient targeting of biosynthetic and endocytic cargo to the vacuole.

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.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Analyzing plant signaling phospholipids through 32Pi-labeling and TLC.

Lipidomic analyses through LC-, GC-, and ESI-MS/MS can detect numerous lipid species based on headgroup and fatty acid compositions but usually miss the minor phospholipids involved in cell signaling because of their low chemical abundancy. Due to their high turnover, these signaling lipids are, however, readily picked up by labeling plant material with (32)P-orthophosphate and subsequent analysis of the lipid extracts by thin layer chromatography. Here, protocols are described for suspension-cultured tobacco BY-2 cells, young Arabidopsis seedlings, Vicia faba roots, and Arabidopsis leaf disks, which can easily be modified for other plant species and tissues.

Polyamines under Abiotic Stress: Metabolic Crossroads and Hormonal Crosstalks in Plants.

Polyamines are essential compounds for cell survival and have key roles in plant stress protection. Current evidence points to the occurrence of intricate cross-talks between polyamines, stress hormones and other metabolic pathways required for their function. In this review we integrate the polyamine metabolic pathway in the context of its immediate metabolic network which is required to understand the multiple ways by which polyamines can maintain their homeostasis and participate in plant stress responses.

Integration of polyamines in the cold acclimation response.

Temperature is one of the most important environmental factors limiting the geographical distribution of plants and accounts for significant reductions in the yield of agriculturally important crops. Low temperature damages many plant species, especially those adapted to tropical climates. In contrast, some species from temperate regions are able to develop freezing tolerance in response to low-non-freezing temperature, an adaptive process named cold acclimation. Numerous molecular, biochemical and physiological changes occur during cold acclimation, most of them being associated with significant changes in gene expression and metabolite profiles. During recent years, transcriptomic and metabolomic approaches have allowed the identification of cold-responsive genes and main metabolites which accumulate in plants exposed to cold. The obtained data support the previously held idea that polyamines (PAs) are involved in plant responses to cold, although their specific role is still not well understood. In this review, we synthesize published data regarding PA-responses to cold stress and integrate them with global transcriptional and metabolic changes. The potential of PA genetic engineering for the development of plants resistant to cold and freezing temperatures, and their plausible mechanisms of action are also discussed.

Putrescine accumulation confers drought tolerance in transgenic Arabidopsis plants over-expressing the homologous Arginine decarboxylase 2 gene.

In Arabidopsis, a model genus missing a functional ornithine decarboxylase pathway, most of the key genes involved in polyamine biosynthesis are duplicated. This gene redundancy has been related to the involvement of certain gene isoforms in the response to specific environmental stimuli. We have previously shown that drought stress induces Arginine decarboxlase 2 expression, while transcript levels for Arginine decarboxlase 1 remain constant. Accumulation of putrescine and increased arginine decarboxlase activity (EC 4.1.1.19) levels in response to different abiotic stresses have been reported in many different plant systems, but the biological meaning of this increase remains unclear. To get a new insight into these questions, we have studied the response to drought of transgenic Arabidopsis thaliana lines constitutively expressing the homologous Arginine decarboxlase 2 gene. These lines contain high levels of putrescine with no changes in spermidine and spermine content even under drought stress. Drought tolerance experiments indicate that the different degree of resistance to dehydration correlates with Put content. Although no significant differences were observed in the number of stomata between wild-type and transgenic plants, a reduction in transpiration rate and stomata conductance was observed in the ADC2 over-expressor lines. These results indicate that one of the mechanisms involved in the drought tolerance of transgenic plants over-producing Put is related to a reduction of water loss by transpiration.

Putrescine is involved in Arabidopsis freezing tolerance and cold acclimation by regulating abscisic acid levels in response to low temperature.

The levels of endogenous polyamines have been shown to increase in plant cells challenged with low temperature; however, the functions of polyamines in the regulation of cold stress responses are unknown. Here, we show that the accumulation of putrescine under cold stress is essential for proper cold acclimation and survival at freezing temperatures because Arabidopsis (Arabidopsis thaliana) mutants defective in putrescine biosynthesis (adc1, adc2) display reduced freezing tolerance compared to wild-type plants. Genes ADC1 and ADC2 show different transcriptional profiles upon cold treatment; however, they show similar and redundant contributions to cold responses in terms of putrescine accumulation kinetics and freezing sensitivity. Our data also demonstrate that detrimental consequences of putrescine depletion during cold stress are due, at least in part, to alterations in the levels of abscisic acid (ABA). Reduced expression of NCED3, a key gene involved in ABA biosynthesis, and down-regulation of ABA-regulated genes are detected in both adc1 and adc2 mutant plants under cold stress. Complementation analysis of adc mutants with ABA and reciprocal complementation tests of the aba2-3 mutant with putrescine support the conclusion that putrescine controls the levels of ABA in response to low temperature by modulating ABA biosynthesis and gene expression.