Lipid signaling and proline catabolism are activated in barley roots (Hordeum vulgare L.) during recovery from cold stress.

Previous findings have shown that phospholipase D (PLD) contributes to the response to long-term chilling stress in barley by regulating the balance of proline (Pro) levels. Although Pro accumulation is one of the most prominent changes in barley roots exposed to this kind of stress, the regulation of its metabolism during recovery from stress remains unclear. Research has mostly focused on the responses to stress per se, and not much is known about the dynamics and mechanisms underlying the subsequent recovery. The present study aimed to evaluate how PLD, its product phosphatidic acid (PA), and diacylglycerol pyrophosphate (DGPP) modulate Pro accumulation in barley during recovery from long-term chilling stress. Pro metabolism involves different pathways and enzymes. The rate-limiting step is mediated by pyrroline-5-carboxylate synthetase (P5CS) in its biosynthesis, and by proline dehydrogenase (ProDH) in its catabolism. We observed that Pro levels decreased in recovering barley roots due to an increase in ProDH activity. The addition of 1-butanol, a PLD inhibitor, reverted this effect and altered the relative gene expression of ProDH. When barley tissues were treated with PA before recovery, the fresh weight of roots increased and ProDH activity was stimulated. These data contribute to our understanding of how acidic membrane phospholipids like PA help to control Pro degradation during recovery from stress.

Biophysical Properties of Lipid Membranes from Barley Roots during Low-Temperature Exposure and Recovery.

Glycerolipid remodeling, a dynamic mechanism for plant subsistence under cold stress, has been posited to affect the biophysical properties of cell membranes. In barley roots, remodeling has been observed to take place upon exposure to chilling stress and to be partially reverted during stress relief. In this study, we explored the biophysical characteristics of membranes formed with lipids extracted from barley roots subjected to chilling stress, or during a subsequent short- or long-term recovery. Our aim was to determine to what extent barley roots were able to offset the adverse effects of temperature on their cell membranes. For this purpose, we analyzed the response of the probe Laurdan inserted in bilayers of different extracts, the zeta potential of liposomes, and the behavior of Langmuir monolayers upon compression. We found important changes in the order of water molecules, which is in agreement with the changes in the unsaturation index of lipids due to remodeling. Regarding Langmuir monolayers, we found that films from all the extracts showed a reorganization at a surface pressure that depends on temperature. This reorganization occurred with an increase in entropy for extracts from control plants and without entropy changes for extracts from acclimated plants. In summary, some membrane properties were recovered after the stress, while others were not, suggesting that the membrane biophysical properties play a role in the mechanism of plant acclimation to chilling. These findings contribute to our understanding of the impact of lipid remodeling on biophysical modifications in plant roots.

Using MetaboAnalyst to introduce undergraduates to lipidomic analysis and lipid remodeling in barley roots.

Lipidomics is a discipline that focuses on the identification and quantification of lipids. Although a part of the larger omics field, lipidomics requires specific approaches for the analysis and biological interpretation of datasets. This article presents a series of activities for introducing undergraduate microbiology students to lipidomic analysis through tools from the web-based platform MetaboAnalyst. The students perform a complete lipidomic workflow, which includes experiment design, data processing, data normalization, and statistical analysis of molecular phospholipid species obtained from barley roots exposed to Fusarium macroconidia. The input data are provided by the teacher, but students also learn about the methods through which they were originally obtained (untargeted liquid chromatography coupled with mass spectrometry). The ultimate aim is for students to understand the biological significance of phosphatidylcholine acyl editing. The chosen methodology allows users who are not proficient in statistics to make a comprehensive analysis of quantitative lipidomic datasets. We strongly believe that virtual activities based on the analysis of such datasets should be incorporated more often into undergraduate courses, in order to improve students' data-handling skills for omics sciences.

Recovery from chilling modulates the acyl-editing of phosphatidic acid molecular species in barley roots (Hordeum vulgare L.).

In plants, lipid metabolism and remodelling are key mechanisms for survival under temperature stress. The present study attempted to compare the lipid profile in barley roots both under chilling stress treatment and in the subsequent recovery to stress. Lipids were obtained through a single-extraction method with a polar solvent mixture, followed by mass spectrometry analysis. The results indicate that lipid metabolism was significantly affected by chilling. Most of the glycerolipids analysed returned to control values during short- and long-term recovery, whereas several representative phosphatidic acid (PA) molecular species were edited during long-term recovery. Most of the PA molecular species that increased in the long-term had the same acyl chains as the phosphatidylcholine (PC) species that decreased. C34:2 and C36:4 underwent the most remarkable changes. Given that the mechanisms underlying the acyl-editing of PC in barley roots remain elusive, we also evaluated the contribution of lysophosphatidylcholine acyltransferases (HvLPCAT) and phospholipase A (HvPLA). In line with the aforementioned results, the expression of the HvLPCAT and HvPLA genes was up-regulated during recovery from chilling. The differential acyl-editing of PA during recovery, which involves the remodelling of PC, might therefore be a regulatory mechanism of cold tolerance in barley.

Deepening the knowledge on the removal of Cr(VI) by L. minuta Kunth: removal efficiency and mechanisms, lipid signaling pathways, antioxidant response, and toxic effects.

Lemna minuta Kunth was used to remove Cr(VI) from aqueous solutions, and some of the mechanisms involved in this process were analyzed. In addition, the cellular signaling mediated by phospholipase D activity as well as antioxidant responses was also evaluated during the process. Cr(VI) removal efficiencies were 40% for 0.5 mg/L, after 24 h, and up to 18% at metal concentrations as high as 5 mg/L. Removal mechanisms displayed by these macrophytes include bioadsorption to cell surfaces and, to a greater extent, Cr internalization and bioaccumulation within cells. Inside of them, Cr(VI) was reduced to Cr(III), a less toxic form of this metal. At the first hours of Cr(VI) exposure, plants were able to sense chromium, activating membrane signal transduction pathways mediated by phospholipase D and phosphatidic acid. Moreover, an increase in the activity of antioxidant enzymes such as superoxide dismutases and peroxidases was observed in the same time. These and other components of the antioxidant defense system would help to reduce the stress generated by the metal. The toxicity of the products formed during the removal process was assessed through Lactuca sativa L. and AMPHIAGU test. It was evidenced that Cr(VI) phytoremediation process by L. minuta plants did not generate acute toxicity neither for L. sativa seeds nor for embryos of Rhinella arenarum (Hensel, 1876). Thus, L. minuta plants could be considered as valuable species for the treatment of waters contaminated with Cr(VI).

Influence of Ca2+ on the surface behavior of phosphatidic acid and its mixture with diacylglycerol pyrophosphate at different pHs.

The signaling lipids phosphatidic acid (PA) and diacylglycerol pyrophosphate (DGPP) are involved in regulating the stress response in plants. PA and DGPP are anionic lipids consisting of a negatively charged phosphomonoester or pyrophosphate group attached to diacylglycerol, respectively. Changes in the pH modulate the protonation of their head groups modifying the interaction with other effectors. Here, we examine in a controlled system how the presence of Ca2+ modulates the surface organization of dioleyl diacylglycerol pyrophosphate (DGPP) and its interaction with dioleoyl phosphatidic acid (DOPA) at different pHs. Both lipids formed expanded monolayers at pH 5 and 8. At acid and basic pHs, monolayers formed by DOPA or DGPP became denser when Ca2+ was added to the subphase. At pH 5, Ca2+ also induced an increase of surface potential of both lipids. Conversely, at pH 8 the effects induced by the presence of Ca2+ on the surface potential were reversed. Mixed monolayers of DOPA and DGPP showed a non-ideal behavior. The addition of even tiny amounts of DGPP to DOPA films caused a reduction of the mean molecular area. This effect was more evident at pH 8 compared to pH 5. Our finding suggests that low amounts of DGPP in an film enriched in DOPA could lead to a local increase in film packing with a concomitant change in the local polarization, further regulated by local pH. This fact may have implications for the assigned role of PA as a pH-sensing phospholipid or during its interaction with proteins.

Differential phosphatidic acid metabolism in barley leaves and roots induced by chilling temperature.

Phosphatidic acid (PA) is an important bioactive lipid that mediates chilling responses in barley. Modifications in the lipid composition of cellular membranes during chilling are essential to maintain their integrity and fluidity. First, we investigated the molecular species of PA present in leaves and roots by ESI-MS/MS, to evaluate the modifications that occur in response to chilling. We demonstrated that PA pools in leaves differ from PA fatty acid composition in roots. Compared with plants grown at 25 °C, the short-term and long-term chilling for 3 h and 36 h at 4 °C not produced significant changes in PA molecular species. The endogenous DAG and PA phosphorylation by in vitro DAG and PA kinase activities showed higher activity in leaves compared with that in root, and they showed contrasting responses to chilling. Similarly, PA removal by phosphatidate phosphohydrolase was tested, showing that this activity was specifically increased in response to chilling in roots. The findings presented here may be helpful to understand how the PA signal is modulated between tissues, and may serve to highlight the importance of knowing the basal PA pools in different plant organs.

Lipid signalling mediated by PLD/PA modulates proline and H2O2 levels in barley seedlings exposed to short- and long-term chilling stress.

Phospholipase D (PLD) hydrolyses phospholipids to yield phosphatidic acid (PA) and a head group, and is involved in responses to a variety of environmental stresses, including chilling and freezing stress. Barley responses to chilling stress (induced by incubating seedlings at 4 °C) are dynamic and the duration of stress, either short (0-180 min) or long-term (24-36 h) had a significant impact on the response. We investigated the roles of PLD/PA in responses of barley (Hordeum vulgare) seedlings to short and long-term chilling stress, based on regulation of proline and reactive oxygen species (ROS) levels. Short-term chilling stress caused rapid and transient increases in PLD activity, proline level, and ROS levels in young leaves. PLD has the ability to catalyse the transphosphatidylation reaction leading to formation of phosphatidylalcohol (preferentially, to PA). Pre-treatment of seedlings with 1-butanol significantly increased proline synthesis but decreased ROS (H2O2) formation. These observations suggest that PLD is a negative regulator of proline synthesis, whereas PA/PLD promote ROS signals. Exogenous PA pre-treatment reduced the proline synthesis but enhanced H2O2 formation. Effects of long-term chilling stress on barley seedlings differed from those of short-term chilling stress. E.g., PLD activity was significantly reduced in young leaves and roots, whereas proline synthesis and ROS signals were increased in roots. Exogenous ROS application enhanced proline level while exogenous proline application reduced ROS level and modulated some effects of long-term chilling stress. Our findings suggest that PLD contributes to signalling pathways in responses to short-term chilling stress in barley seedling, through regulation of the balance between proline and ROS levels. In contrast, reduced PLD activity in the response to long-term chilling stress did not affect proline level. Increased ROS levels may reflect an antioxidant system that is affected by chilling stress and positively compensated by changes in proline level. Implications of our findings are discussed in regard to adaptation strategies of barley seedlings to low temperatures.

Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone.

The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone.

Differences in phosphatidic acid signalling and metabolism between ABA and GA treatments of barley aleurone cells.

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [(32)P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.

Arabidopsis thaliana lipid phosphate phosphatase 2 is involved in abscisic acid signalling in leaves.

Lipid phosphate phosphatases (LPPs, E.C. 3.1.3.4) catalyse the dephosphorylation of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA), which are secondary messengers in abscisic acid (ABA) signalling. In this study, we investigated the effect of ABA on the expression of AtLPP genes as they encode putative ABA-signalling partners. We observed that AtLPP2 expression was down-regulated by ABA and we performed experiments on Atlpp2-2, an AtLPP2 knockout mutant, to determine whether AtLPP2 was involved in ABA signalling. We observed that Atlpp2-2 plantlets contained about twice as much PA as the wild-type Col-0 and exhibited higher PA kinase (PAK) activity than Col-0 plants. In addition, we showed that ABA stimulated diacylglycerol kinase (DGK) activity independently of AtLPP2 activity but that the ABA-stimulation of PAK activity recorded in Col-0 was dependent on AtLPP2. In order to evaluate the involvement of AtLPP2 activity in guard cell function, we measured the ABA sensitivity of Atlpp2-2 stomata. The inhibition of stomatal opening was less sensitive to ABA in Atlpp2-2 than in Col-0. Watered and water-stressed plants of the two genotypes accumulated ABA to the same extent, thus leading us to consider Atlpp2-2 an ABA-signalling mutant. Taken together our observations show that AtLPP2 is a part of ABA signalling and participate to the regulation of stomatal movements.

The surface organization of diacylglycerol pyrophosphate and its interaction with phosphatidic acid at the air-water interface.

Diacylglycerol pyrophosphate (DGPP), a phosphorylated form of phosphatidic acid (PA), gained attention recently due to its role as signaling lipid. However, little is known about its surface organization and potential impact on membrane-mediated function. In this work we investigated the interfacial behavior of Langmuir monolayers formed with pure DGPP and of its mixtures with PA. We found that changes of the subphase pH affect the surface behavior of DGPP. At pH 8, DGPP forms liquid expanded monolayers with a compressibility modulus of about 60mNm⁻¹ at collapse. On acidic solutions, the compressibility modulus increases to 90mNm⁻¹ and the average molecular area is smaller. At pH 8, DGPP and its precursor PA form thermodynamically favored topographically homogeneous non-ideal mixtures. The interaction among these lipids leads to a non-ideal diminution of the mean molecular area and consequently, to an increase of the compressibility modulus, with variations of the surface electrostatics. The favorable interaction of PA and DGPP, leading to changes of the film packing suggest that DGPP may act as a structural signal transducer in membrane-mediated cellular processes.

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels.

BACKGROUND AND AIMS: Experimental evidence in the literature suggests that O(2)(*-) produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O(2)(*-). METHODS: Stress treatments were imposed using 150 mm NaCl or 300 mM sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O(2)(*-) was determined using nitro blue tetrazolium, and H(2)O(2) was determined using 2', 7'-dichlorofluorescin. KEY RESULTS: In non-stressed plants, the distribution of accelerating growth and highest O(2)(*-) levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O(2)(*-) levels increased in sorbitol-treated roots and decreased in NaCl-treated roots. CONCLUSIONS: The lack of association between apoplastic O(2)(*-) levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O(2)(*-) may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.

Phosphatidylinositol kinases as regulators of GA-stimulated alpha-amylase secretion in barley (Hordeum vulgare).

Phosphorylated derivatives of phosphatidylinositol, in association with phosphatidylinositol 3-kinase (PI3 kinase, EC 2.7.1.137) and phosphatidylinositol 4-kinase (PI4 kinase, EC 2.7.1.67), play a key role in regulation of fundamental cell processes. We present evidence for a relationship between alpha-amylase (EC 3.2.1.1) secretion regulated by GA and levels of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate (PtdIns(4)P) in barley (Hordeum vulgare). Microsomal membranes were incubated in the presence of [gamma-(32)P]ATP, and radiolabeled membrane lipids were extracted and separated by TLC using a boric acid system. Treatment of aleurone layers with GA for short or long periods of time increased PI4 kinase activity. To evaluate the effect of PtdIns(4)P levels on GA signaling, we used phenylarsine oxide (PAO), an inhibitor of PI4 kinase activity. PAO reversibly reduced the alpha-amylase secretion and protoplast cell vacuolation in a dose-dependent manner. Wortmannin showed a similar inhibitory effect on alpha-amylase secretion and PI4 kinase activity. GA evoked only a long-term increase in PI3 kinase activity, which was also affected by PAO. The effect of PAO was suppressed by the reducing agent 2,3-dimercapto-1-propanol (BAL), leading to restoration of secretion, vacuolation and PI4 kinase activity. In contrast, the effect of PAO on PI3 kinase activity was not abolished by BAL, suggesting that PI3 kinase is not involved in the secretion process. Likewise, the compound LY294002 inhibited PI3 kinase but had no effect on the secretion process. These findings indicate that PI4 kinase acts as a positive regulator of early GA signaling in aleurone.