Phospholipid Signaling Research Articles

Mlf mediates proteotoxic response via formation of cellular foci for protein folding and degradation in Giardia.

Myeloid leukemia factor 1 (Mlf1) was identified as a proto-oncoprotein that affects hematopoietic differentiation in humans. However, its cellular function remains elusive, spanning roles from cell cycle regulation to modulation of protein aggregate formation and participation in ciliogenesis. Given that structurally conserved homologs of Mlf1 can be found across the eukaryotic tree of life, we decided to characterize its cellular role underlying this phenotypic pleiotropy. Using a model of the unicellular eukaryote Giardia intestinalis, we demonstrate that its Mlf1 homolog (GiMlf) mainly localizes to two types of cytosolic foci: microtubular structures, where it interacts with Hsp40, and ubiquitin-rich, membraneless compartments, found adjacent to mitochondrion-related organelles known as mitosomes, containing the 26S proteasome regulatory subunit 4. Upon cellular stress, GiMlf either relocates to the affected compartment or disperses across the cytoplasm, subsequently accumulating into enlarged foci during the recovery phase. In vitro assays suggest that GiMlf can be recruited to membranes through its affinity for signaling phospholipids. Importantly, cytosolic foci diminish in the gimlf knockout strain, which exhibits extensive proteomic changes indicative of compromised proteostasis. Consistent with data from other cellular systems, we propose that Mlf acts in the response to proteotoxic stress by mediating the formation of function-specific foci for protein folding and degradation.

The Delayed Turnover of Proteasome Processing of Myocilin upon Dexamethasone Stimulation Introduces the Profiling of Trabecular Meshwork Cells' Ubiquitylome.

Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis.

How coat proteins shape autophagy in plant cells.

Autophagy is a membrane trafficking pathway through which eukaryotic cells target their own cytoplasmic constituents for degradation in the lytic compartment. Proper biogenesis of autophagic organelles requires a conserved set of autophagy-related (ATG) proteins and their interacting factors, such as signalling phospholipid phosphatidylinositol 3-phosphate (PI3P) and coat complex II (COPII). The COPII machinery, which was originally identified as a membrane coat involved in the formation of vesicles budding from the endoplasmic reticulum, contributes to the initiation of autophagic membrane formation in yeast, metazoan, and plant cells; however, the exact mechanisms remain elusive. Recent studies using the plant model species Arabidopsis thaliana have revealed that plant-specific PI3P effectors are involved in autophagy. The PI3P effector FYVE2 interacts with the conserved PI3P effector ATG18 and with COPII components, indicating an additional role for the COPII machinery in the later stages of autophagosome biogenesis. In this Update, we examined recent research on plant autophagosome biogenesis and proposed working models on the functions of the COPII machinery in autophagy, including its potential roles in stabilizing membrane curvature and sealing the phagophore.

Phospholipid production and signaling by a plant defense inducer against Podosphaera xanthii is genotype-dependent.

Biotrophic phytopathogenic fungi such as Podosphaera xanthii have evolved sophisticated mechanisms to adapt to various environments causing powdery mildews leading to substantial yield losses. Today, due to known adverse effects of pesticides, development of alternative control means is crucial and can be achieved by combining plant protection products with resistant genotypes. Using plant defense inducers, natural molecules that stimulate plant immune system mimicking pathogen attack is sustainable, but information about their mode of action in different hosts or host genotypes is extremely limited. Reynoutria sachalinensis extract, a known plant defense inducer, especially through the Salicylic acid pathway in Cucurbitaceae crops against P. xanthii, was employed to analyze the signaling cascade of defense activation. Here, we demonstrate that R. sachalinensis extract enhances phospholipid production and signaling in a Susceptible to P. xanthii courgette genotype, while limited response is observed in an Intermediate Resistance genotype due to genetic resistance. Functional enrichment and cluster analysis of the upregulated expressed genes revealed that inducer application promoted mainly lipid- and membrane-related pathways in the Susceptible genotype. On the contrary, the Intermediate Resistance genotype exhibited elevated broad spectrum defense pathways at control conditions, while inducer application did not promote any significant changes. This outcome was obvious and at the metabolite level. Main factor distinguishing the Intermediate Resistance form the Susceptible genotype was the epigenetic regulated increased expression of a G3P acyltransferase catalyzing phospholipid production. Our study provides evidence on phospholipid-based signaling after plant defense inducer treatment, and the selective role of plant's genetic background.

Diacylglycerol kinase is a keystone regulator of signaling relevant to the pathophysiology of asthma.

Signal transduction by G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immunoreceptors converge at the activation of phospholipase C (PLC) for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This is a point for second-messenger bifurcation where DAG via protein kinase C (PKC) and IP3 via calcium activate distinct protein targets and regulate cellular functions. IP3 signaling is regulated by multiple calcium influx and efflux proteins involved in calcium homeostasis. A family of lipid kinases belonging to DAG kinases (DGKs) converts DAG to phosphatidic acid (PA), negatively regulating DAG signaling and pathophysiological functions. PA, through a series of biochemical reactions, is recycled to produce new molecules of PIP2. Therefore, DGKs act as a central switch in terminating DAG signaling and resynthesis of membrane phospholipids precursor. Interestingly, calcium and PKC regulate the activation of α and ζ isoforms of DGK that are predominantly expressed in airway and immune cells. Thus, DGK forms a feedback and feedforward control point and plays a crucial role in fine-tuning phospholipid stoichiometry, signaling, and functions. In this review, we discuss the previously underappreciated complex and intriguing DAG/DGK-driven mechanisms in regulating cellular functions associated with asthma, such as contraction and proliferation of airway smooth muscle (ASM) cells and inflammatory activation of immune cells. We highlight the benefits of manipulating DGK activity in mitigating salient features of asthma pathophysiology and shed light on DGK as a molecule of interest for heterogeneous diseases such as asthma.

Phospholipid Signaling in Crop Plants: A Field to Explore.

In plant models such as Arabidopsis thaliana, phosphatidic acid (PA), a key molecule of lipid signaling, was shown not only to be involved in stress responses, but also in plant development and nutrition. In this article, we highlight lipid signaling existing in crop species. Based on open access databases, we update the list of sequences encoding phospholipases D, phosphoinositide-dependent phospholipases C, and diacylglycerol-kinases, enzymes that lead to the production of PA. We show that structural features of these enzymes from model plants are conserved in equivalent proteins from selected crop species. We then present an in-depth discussion of the structural characteristics of these proteins before focusing on PA binding proteins. For the purpose of this article, we consider RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), the most documented PA target proteins. Finally, we present pioneering experiments that show, by different approaches such as monitoring of gene expression, use of pharmacological agents, ectopic over-expression of genes, and the creation of silenced mutants, that lipid signaling plays major roles in crop species. Finally, we present major open questions that require attention since we have only a perception of the peak of the iceberg when it comes to the exciting field of phospholipid signaling in plants.

Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

Class II diacylglycerol kinases participate in the basal immune responses of Nicotiana benthamiana to Ralstonia solanacearum

Phospholipid signaling plays an important role in Nicotiana benthamiana immune responses to phytopathogenic bacteria. In this study, we isolated three orthologs encoding class II diacylglycerol kinase (NbDGK4-1, NbDGK4-2 and NbDGK7) in the N. benthamiana genome. In contrast to the undetectable expression of NbDGK4-2, the NbDGK4-1 and NbDGK7 expression levels increased in response to Ralstonia solanacearum and the flg22 peptide. The induction of the hypersensitive response was not affected, but bacterial growth increased and the onset of bacterial wilt symptoms was accelerated in the NbDGK4-1-silenced and NbDGK7-silenced plants challenged with R. solanacearum. The expression levels of NbPR-1 and NbPR-4 (marker genes for salicylic acid and jasmonic acid signaling, respectively) decreased in the NbDGK4-1-silenced and NbDGK7-silenced plants inoculated with R. solanacearum. The expression of pathogen-associated molecular pattern-triggered immunity (PTI) marker genes were also inhibited in the NbDGK4-1-silenced and NbDGK7-silenced plants infiltrated with the type III secretion system (T3SS)-deficient R. solanacearum mutant or flg22. Accordingly, the T3SS-deficient R. solanacearum bacterial population increased in the NbDGK4-1-silenced and NbDGK7-silenced plants. Moreover, flg22-induced resistance and callose deposition were also compromised in the NbDGK4-1-silenced and NbDGK7-silenced plants. Considered together, the study results indicate class II diacylglycerol kinases may be important for N. benthamiana basal disease resistance (e.g., PTI responses).

Constitutive activation mechanism of a class C GPCR.

Class C G-protein-coupled receptors (GPCRs) are activated through binding of agonists to the large extracellular domain (ECD) followed by rearrangement of the transmembrane domains (TMDs). GPR156, a class C orphan GPCR, is unique because it lacks an ECD and exhibits constitutive activity. Impaired GPR156-Gi signaling contributes to loss of hearing. Here we present the cryo-electron microscopy structures of human GPR156 in the Go-free and Go-coupled states. We found that an endogenous phospholipid molecule is located within each TMD of the GPR156 dimer. Asymmetric binding of Gα to the phospholipid-bound GPR156 dimer restructures the first and second intracellular loops and the carboxy-terminal part of the elongated transmembrane 7 (TM7) without altering dimer conformation. Our findings reveal that GPR156 is a transducer for phospholipid signaling. Constant binding of abundant phospholipid molecules and the G-protein-induced reshaping of the cytoplasmic face provide a basis for the constitutive activation of GPR156.

Enhanced Fc Receptor Expression In Sea Urchin Spermatozoa By Platelet-Activating Factor

Platelet-activating factor (PAF) is a signaling phospholipid that has a variety of reproductive roles, e.g. sperm capacitation and the acrosome reaction (an exocytotic process). PAF’s mechanism action involves G-protein receptor activation induction of intracellular calcium, which enhances sperm motility. In nonreproductive cells, the Fc receptor (FcR) functions in ligand-triggered transmission signals across the membrane which alters secretion, exocytosis, and increased intracellular calcium. This study looked at the impact of PAF on FcR expression in sea urchin spermatozoa. Sea urchin sperm were exposed to exogenous PAF (10-7M; 15 minutes) and evaluated for FcR activity via flow cytometry. A significant (P=0.01) difference (64.30%) in FcR wave activity between the PAF (mean 3.286) and control (2.0) groups was observed. Within waves, there were more (83.31%) high points (crests) in the PAF (mean 1.571) group than the control (mean 0.857) group. Similarly, there were more (49.96%) low (troughs) points in the PAF (mean 1.714) group than the control (mean 1.143) group. This is the first report to demonstrate a relative positive change in spermatozoa FcR expression when exposed to PAF. The FcR functions in ligand-triggered transmission of PAF via a G-protein coupled receptor mediated pathway leading to physiological alterations. Provided the known mechanism of action of PAF on spermatozoa physiology, the data suggests that the interaction of PAF and FcR play a pivotal role in sperm activity and fertilization potential.

Gut microbiota analyses of cutaneous T-cell lymphoma patients undergoing narrowband ultraviolet B therapy reveal alterations associated with disease treatment.

Recent studies have shown a close relationship between cutaneous T-cell lymphoma (CTCL) and its microbiome. CTCL disease progression is associated with gut dysbiosis and alterations in bacterial taxa parallel those observed in immunologically similar atopic dermatitis. Moreover, the microbial profile of lesional skin may predict response to narrowband ultraviolet B (nbUVB), a common skin-directed therapy. However, the relationship between the gut microbiome, an immunologically vital niche, and nbUVB remains unexplored in CTCL. Herein, we performed 16S rRNA sequencing and PICRUSt2 predictive metagenomics on DNA extracted from stool swabs of 13 CTCL patients treated with nbUVB, 8 non-treated patients, and 13 healthy controls. Disease response was assessed with modified Severity Weighted Assessment Tool (mSWAT); of nbUVB-treated patients, 6 improved (decreased mSWAT), 2 remained stable, and 5 worsened (increased mSWAT). Protective commensal bacteria including Lactobacillaceae and Erysipelatoclostridiaceae were significantly less abundant in CTCL patients compared to controls. With treatment, the CTCL gut microbiome exhibited decreased phylogenetic diversity and lower relative abundance of pro-inflammatory Sutterellaceae. Sutterellaceae was also significantly more abundant in patients who worsened, and Eggerthellaceae and Erysipelotrichaceae trended higher in patients who improved. Finally, PICRUSt2 functional predictions based on shifts in abundance of bacterial sequences repeatedly identified alterations in inositol degradation, which plays a key role in host immunomodulation, including inositol phospholipid signaling relevant to T-cell survival and proliferation. Our results bolster the paradigm of gut dysbiosis in CTCL and its functional implications in disease pathogenesis, and further delineate bacterial taxa associated with nbUVB response and with nbUVB treatment itself.

Hypertensive Pressure Mechanosensing Alone Triggers Lipid Droplet Accumulation and Transdifferentiation of Vascular Smooth Muscle Cells to Foam Cells.

Arterial Vascular smooth muscle cells (VSMCs) play a central role in the onset and progression of atherosclerosis. Upon exposure to pathological stimuli, they can take on alternative phenotypes that, among others, have been described as macrophage like, or foam cells. VSMC foam cells make up >50% of all arterial foam cells and have been suggested to retain an even higher proportion of the cell stored lipid droplets, further leading to apoptosis, secondary necrosis, and an inflammatory response. However, the mechanism of VSMC foam cell formation is still unclear. Here, it is identified that mechanical stimulation through hypertensive pressure alone is sufficient for the phenotypic switch. Hyperspectral stimulated Raman scattering imaging demonstrates rapid lipid droplet formation and changes to lipid metabolism and changes are confirmed in ABCA1, KLF4, LDLR, and CD68 expression, cell proliferation, and migration. Further, a mechanosignaling route is identified involving Piezo1, phospholipid, and arachidonic acid signaling, as well as epigenetic regulation, whereby CUT&Tag epigenomic analysis confirms changes in the cells (lipid) metabolism and atherosclerotic pathways. Overall, the results show for the first time that VSMC foam cell formation can be triggered by mechanical stimulation alone, suggesting modulation of mechanosignaling can be harnessed as potential therapeutic strategy.

Improving the Signal Intensity of Cryosections Using a Conductive Adhesive Film in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to obtain the molecular images of cryosections without labeling. Although MALDI-MSI has been widely used to detect small molecules from biological tissues, issues remain due to the technical process of cryosectioning and limited mass spectrometry parameters. The use of a conductive adhesive film is a unique method to obtain high-quality sections from cutting tissue, such as bone, muscle, adipose tissue, and whole body of mice or fish, and we have reported the utilization of the film for MALDI-MSI in previous. However, some signal of the small molecules using the conductive adhesive films was still lower than on the indium tin oxide (ITO) glass slide. Here, the sample preparation and analytical conditions for MALDI-MSI using an advanced conductive adhesive film were optimized to obtain strong signals from whole mice heads. The effects of tissue thickness and laser ionization power on signal intensity were verified using MALDI-MSI. The phospholipid signal intensity was measured for samples with three tissue thicknesses (5, 10, and 20 μm); compared to the signals from the samples on the ITO glass slides, the signals with conductive adhesive films exhibited significantly higher intensities when a laser with a higher range of power was used to ionize the small molecules. Thus, the technique using the advanced conductive adhesive film showed an improvement in MALDI-MSI analysis.

Biomolecular condensates create phospholipid-enriched microenvironments.

Proteins and RNA can phase separate from the aqueous cellular environment to form subcellular compartments called condensates. This process results in a protein-RNA mixture that is chemically different from the surrounding aqueous phase. Here, we use mass spectrometry to characterize the metabolomes of condensates. To test this, we prepared mixtures of phase-separated proteins and extracts of cellular metabolites and identified metabolites enriched in the condensate phase. Among the most condensate-enriched metabolites were phospholipids, due primarily to the hydrophobicity of their fatty acyl moieties. We found that phospholipids can alter the number and size of phase-separated condensates and in some cases alter their morphology. Finally, we found that phospholipids partition into a diverse set of endogenous condensates as well as artificial condensates expressed in cells. Overall, these data show that many condensates are protein-RNA-lipid mixtures with chemical microenvironments that are ideally suited to facilitate phospholipid biology and signaling.

Lysophosphatidic acid exerts protective effects on HEI-OC1 cells against cytotoxicity of cisplatin by decreasing apoptosis, excessive autophagy, and accumulation of ROS

Lysophosphatidic acid (LPA) is an active phospholipid signaling molecule that binds to six specific G protein-coupled receptors (LPA1-6) on the cell surface and exerts a variety of biological functions, including cell migration and proliferation, morphological changes, and anti-apoptosis. The earliest study from our group demonstrated that LPA treatment could restore cochlear F-actin depolymerization induced by noise exposure, reduce hair cell death, and thus protect hearing. However, whether LPA could protect against cisplatin-induced ototoxicity and which receptors play the major role remain unclear. To this end, we integrated the HEI-OC1 mouse cochlear hair cell line and zebrafish model, and found that cisplatin exposure induced a large amount of reactive oxygen species accumulation in HEI-OC1 cells, accompanied by mitochondrial damage, leading to apoptosis and autophagy. LPA treatment significantly attenuated autophagy and apoptosis in HEI-OC1 cells after cisplatin exposure. Further investigation revealed that all LPA receptors except LPA3 were expressed in HEI-OC1 cells, and the mRNA expression level of LPA1 receptor was significantly higher than that of other receptors. When LPA1 receptor was silenced, the protective effect of LPA was reduced and the proportion of apoptosis cells was increased, indicating that LPA-LPA1 plays an important role in protecting HEI-OC1 cells from cisplatin-induced apoptosis. In addition, the behavioral trajectory and in vivo fluorescence imaging results showed that cisplatin exposure caused zebrafish to move more actively, and the movement speed and distance were higher than those of the control and LPA groups, while LPA treatment reduced the movement behavior. Cisplatin caused hair cell death and loss in zebrafish lateral line, and LPA treatment significantly protected against hair cell death and loss. LPA has a protective effect on hair cells in vitro and in vivo against the cytotoxicity of cisplatin, and its mechanism may be related to reducing apoptosis, excessive autophagy and ROS accumulation.

TIPE proteins control directed migration of human T cells by directing GPCR and lipid second messenger signaling.

Tissue infiltration by circulating leukocytes via directed migration (also referred to as chemotaxis) is a common pathogenic mechanism of inflammatory diseases. G protein-coupled receptors (GPCRs) are essential for sensing chemokine gradients and directing the movement of leukocytes during immune responses. The tumor necrosis factor α-induced protein 8-like (TIPE or TNFAIP8L) family of proteins are newly described pilot proteins that control directed migration of murine leukocytes. However, how leukocytes integrate site-specific directional cues, such as chemokine gradients, and utilize GPCR and TIPE proteins to make directional decisions are not well understood. Using both gene knockdown and biochemical methods, we demonstrated here that 2 human TIPE family members, TNFAIP8 and TIPE2, were essential for directed migration of human CD4+ T cells. T cells deficient in both of these proteins completely lost their directionality. TNFAIP8 interacted with the Gαi subunit of heterotrimeric (α, β, γ) G proteins, whereas TIPE2 bound to PIP2 and PIP3 to spatiotemporally control immune cell migration. Using deletion and site-directed mutagenesis, we established that Gαi interacted with TNFAIP8 through its C-terminal amino acids, and that TIPE2 protein interacted with PIP2 and PIP3 through its positively charged amino acids on the α0 helix and at the grip-like entrance. We also discovered that TIPE protein membrane translocation (i.e. crucial for sensing chemokine gradients) was dependent on PIP2. Collectively, our work describes a new mechanistic paradigm for how human T cells integrate GPCR and phospholipid signaling pathways to control directed migration. These findings have implications for therapeutically targeting TIPE proteins in human inflammatory and autoimmune diseases.

Stress- and phospholipid signalling responses in Arabidopsis PLC4-KO and -overexpression lines under salt- and osmotic stress

Several drought and salt tolerant phenotypes have been reported when overexpressing (OE) phospholipase C (PLC) genes across plant species. In contrast, a negative role for Arabidopsis PLC4 in salinity stress was recently proposed, showing that roots of PLC4-OE seedlings were more sensitive to NaCl while plc4 knock-out (KO) mutants were more tolerant. To investigate this apparent contradiction, and to analyse the phospholipid signalling responses associated with salinity stress, we performed root growth- and phospholipid analyses on plc4-KO and PLC4-OE seedlings subjected to salinity (NaCl) or osmotic (sorbitol) stress and compared these with wild type (WT). Only very minor differences between PLC4 mutants and WT were observed, which even disappeared after normalization of the data, while in soil, PLC4-OE plants were clearly more drought tolerant than WT plants, as was found earlier when overexpressing Arabidopsis PLC2, -3, -5, -7 or -9. We conclude that PLC4 plays no opposite role in salt-or osmotic stress and rather behaves like the other Arabidopsis PLCs.

Synthesis of a photocleavable bola-phosphatidylcholine

Phosphatidylinositol transfer proteins (PITPs) are ubiquitous in eukaryotes and are involved in the regulation of phospholipid metabolism, membrane trafficking, and signal transduction. Sec14 is a yeast PITP that has been shown to transfer phosphatidylinositol (PI) or phosphatidylcholine (PC) from the endoplasmic reticulum to the Golgi. It is now believed that Sec14 may play a greater role than just shuttling PI and PC throughout the cell. Genetic evidence suggests that retrieval of membrane-bound PI by Sec14 also manages to present PI to the phosphatidylinositol-4-kinase, Pik1, to generate phosphatidylinositol-4-phosphate, PI(4)P. To test this hypothetical model, we designed a photocleavable bolalipid to span the entire membrane, having one phosphatidylcholine or phosphatidylinositol headgroup on each leaflet connected by a photocleavable diacid. Sec14 should not be able to present the bola-PI to Pik1 for phosphorylation as the head group will be difficult to lift from the bilayer as it is tethered on the opposite leaflet. After photocleavage the two halves would behave as a normal phospholipid, thus phosphorylation by Pik1 would resume. We report here the synthesis of a photocleavable bola-PC, a precursor to the desired bola-PI. The mono-photocleavable bola-PC lipid was designed to contain two glycerol molecules with choline head groups connected through a phosphodiester bond at the sn3 position. Each glycerol was acylated with palmitic acid at the sn1 position. These two glycerol moieties were then connected through their respective sn2 hydroxyls via a photocleavable dicarboxylic acid containing a nitrophenyl ethyl photolabile protecting group. The bola-PC and its precursors were found to undergo efficient photocleavage when irradiated in solution or in vesicles with 365 nm light for two minutes. Treatment of the bola-PC with a mutant phospholipase D and myo-inositol produced a mono-inositol bola-PC-PI.

Analysis of N6-methyladenosine reveals a new important mechanism regulating the salt tolerance of sugar beet (Beta vulgaris)

Salinity is an important environmental factor in crop growth and development. N6-methyladenosine (m6A) is an essential epigenetic modification that regulates plant-environment interaction. Sugar beet is a major sugar-yielding crop that has a certain tolerance to salt, but the dynamic response elicited by the m6A modification of transcripts under salt stress remains unknown. In this study, sugar beet was exposed to 300 mM NaCl to investigate its physiological response to high salinity and transcriptome-wide m6A modification profile. After the salt treatment, 7737 significantly modified m6A sites and 4981 differentially expressed genes (DEGs) were identified. Among the 312 m6A-modified DEGs, 113 hypomethylated DEGs were up-regulated and 99 hypermethylated DEGs were down-regulated, indicating a negative correlation between m6A modification and gene expression. Well-known salt tolerance genes (e.g., sodium/hydrogen exchanger 1, choline monooxygenase, and nucleoredoxin 2) and phospholipid signaling pathway genes (phosphoinositol-specific phospholipase C, phospholipase D, diacylglycerol kinase 1, etc.) were also among the m6A-modified genes. Further analysis showed that m6A modification may regulate salt-tolerant related gene expression by controlling mRNA stability. Therefore, changes in m6A modification may negatively regulate the expression of the salt-resistant genes in sugar beet, at least in part by modulating the stability of the mRNA via demethylase BvAlkbh10B. These findings could provide a better understanding of the epigenetic mechanisms of salt tolerance in sugar beets and uncover new candidate genes for improving the production of sugar beets planted in high-salinity soil.

Phosphatidylinositol-phospholipase C3 negatively regulates the hypersensitive response via complex signaling with MAP kinase, phytohormones and reactive oxygen in Nicotiana benthamiana.

Phospholipid signaling plays important roles in plant immune responses. Here, we focused on two phospholipase C3 (PLC3) orthologs in the Nicotiana benthamiana genome, NbPLC3-1 and NbPLC3-2. We created NbPLC3-1 and NbPLC3-2-double-silenced plants (NbPLC3s-silenced plants). In NbPLC3s-silenced plants challenged with Ralstonia solanacearum 8107, the induction of the hypersensitive response (HR), including HR-related cell death and bacterial population reduction, was accelerated, the expression level of Nbhin1, an HR marker gene was enhanced, the expression levels of genes involved in salicylic acid and jasmonic acid signaling drastically increased, the reactive oxygen species hyper-production, was accelerated, and NbMEK2-induced HR-related cell death were also enhanced. Accelerated HR-cell death was also observed by bacterial pathogens Pseudomonas cichorii and P. syringae and bacterial AvrA, oomycete INF1 and TMGMV-CP with L1 in NbPLC3s-silenced plants. Although HR-related cell death was accelerated, the bacterial population was not reduced in double NbPLC3s and NbCoi1 suppressed plants nor in NbPLC3s-silenced NahG plants. The HR-related cell death acceleration and bacterial population reduction resulting from NbPLC3s-silencing were compromised by the concomitant suppression of either NbPLC3s and NbrbohB or NbPLC3s and NbMEK2. Thus, NbPLC3s may negatively regulate both HR-related cell death and disease resistance through MAP kinase- and reactive oxygen species-dependent signaling. Disease resistance was also regulated by NbPLC3s through jasmonic acid- and salicylic acid-dependent pathways.