Pollen germination and subsequent pollen tube elongation are essential for successful land plant reproduction. These processes are achieved through well-documented activation of membrane trafficking and cell metabolism. Despite this, our knowledge of the dynamics of cellular phospholipids remains scarce. Here we present the turnover of the glycerolipid composition during the establishment of cell polarity and elongation processes in tobacco pollen and show the lipid composition of pollen plasma membrane-enriched fraction for the first time. To achieve this, we have combined several techniques, such as lipidomics, plasma membrane isolation, and live-cell microscopy, and performed a study with different time points during the pollen germination and pollen tube growth. Our results showed that tobacco pollen tubes undergo substantial changes in their whole-cell lipid composition during the pollen germination and growth, finding differences in most of the glycerolipids analyzed. Notably, while lysophospholipid levels decrease during germination and growth, phosphatidic acid increases significantly at cell polarity establishment and continues with similar abundance in cell elongation. We corroborated these findings by measuring several phospholipase activities in situ. We also observed that lysophospholipids and phosphatidic acid are more abundant in the plasma membrane-enriched fraction than that in the whole cell. Our results support the important role for the phosphatidic acid in the establishment and maintenance of cellular polarity in tobacco pollen tubes and indicate that plasma membrane lysophospholipids may be involved in pollen germination.

• Keywords
• lipidomics, phosphatidic acid, phospholipid, plasma membrane, pollen, pollen tube, tip growth, tobacco,
• Publication type
• Journal Article MeSH

Reactive oxygen species (ROS) are produced in the olive reproductive organs as the result of intense metabolism. ROS production and pattern of distribution depend on the developmental stage, supposedly playing a broad panel of functions, which include defense and signaling between pollen and pistil. Among ROS-producing mechanisms, plasma membrane NADPH-oxidase activity is being highlighted in plant tissues, and two enzymes of this type have been characterized in Arabidopsis thaliana pollen (RbohH and RbohJ), playing important roles in pollen physiology. Besides, pollen from different species has shown distinct ROS production mechanism and patterns of distribution. In the olive reproductive tissues, a significant production of superoxide has been described. However, the enzymes responsible for such generation are unknown. Here, we have identified an Rboh-type gene (OeRbohH), mainly expressed in olive pollen. OeRbohH possesses a high degree of identity with RbohH and RbohJ from Arabidopsis, sharing most structural features and motifs. Immunohistochemistry experiments allowed us to localize OeRbohH throughout pollen ontogeny as well as during pollen tube elongation. Furthermore, the balanced activity of tip-localized OeRbohH during pollen tube growth has been shown to be important for normal pollen physiology. This was evidenced by the fact that overexpression caused abnormal phenotypes, whereas incubation with specific NADPH oxidase inhibitor or gene knockdown lead to impaired ROS production and subsequent inhibition of pollen germination and pollen tube growth.

• Keywords
• NADPH oxidase, NOX, Rboh, olive, pollen, sexual plant reproduction,
• Publication type
• Journal Article MeSH

Phospholipases (PLs) are lipid-hydrolyzing enzymes known to have diverse signaling roles during plant abiotic and biotic stress responses. They catalyze lipid remodeling, which is required to generate rapid responses of plants to environmental cues. Moreover, they produce second messenger molecules, such as phosphatidic acid (PA) and thus trigger or modulate signaling cascades that lead to changes in gene expression. The roles of phospholipases in plant abiotic and biotic stress responses have been intensively studied. Nevertheless, emerging evidence suggests that they also make significant contributions to plants' cellular and developmental processes. In this mini review, we summarized recent advances in the study of the cellular and developmental roles of phospholipases in plants.

• Keywords
• cellular functions, phosphatidic acid, phospholipase A, phospholipase C, phospholipase D, phospholipases, phytohormones, plant development,
• Publication type
• Journal Article MeSH
• Review MeSH

Phytohormone salicylic acid (SA) is a crucial component of plant-induced defense against biotrophic pathogens. Although the key players of the SA pathway are known, there are still gaps in the understanding of the molecular mechanism and the regulation of particular steps. In our previous research, we showed in Arabidopsis suspension cells that n-butanol, which specifically modulates phospholipase D activity, significantly suppresses the transcription of the pathogenesis related (PR-1) gene, which is generally accepted as the SA pathway marker. In the presented study, we have investigated the site of n-butanol action in the SA pathway. We were able to show in Arabidopsis plants treated with SA that n-butanol inhibits the transcription of defense genes (PR-1, WRKY38). Fluorescence microscopy of Arabidopsis thaliana mutants expressing 35S::NPR1-GFP (nonexpressor pathogenesis related 1) revealed significantly decreased nuclear localization of NPR1 in the presence of n-butanol. On the other hand, n-butanol did not decrease the nuclear localization of NPR1 in 35S::npr1C82A-GFP and 35S::npr1C216A-GFP mutants constitutively expressing NPR1 monomers. Mass spectrometric analysis of plant extracts showed that n-butanol significantly changes the metabolic fingerprinting while t-butanol had no effect. We found groups of the plant metabolites, influenced differently by SA and n-butanol treatment. Thus, we proposed several metabolites as markers for n-butanol action.

• Keywords
• NPR1, PR-1, metabolome, n-butanol, phospholipase D, salicylic acid, signaling,
• Publication type
• Journal Article MeSH

UNLABELLED: Invasive cell growth and migration is usually considered a specifically metazoan phenomenon. However, common features and mechanisms of cytoskeletal rearrangements, membrane trafficking and signalling processes contribute to cellular invasiveness in organisms as diverse as metazoans and plants - two eukaryotic realms genealogically connected only through the last common eukaryotic ancestor (LECA). By comparing current understanding of cell invasiveness in model cell types of both metazoan and plant origin (invadopodia of transformed metazoan cells, neurites, pollen tubes and root hairs), we document that invasive cell behavior in both lineages depends on similar mechanisms. While some superficially analogous processes may have arisen independently by convergent evolution (e.g. secretion of substrate- or tissue-macerating enzymes by both animal and plant cells), at the heart of cell invasion is an evolutionarily conserved machinery of cellular polarization and oriented cell mobilization, involving the actin cytoskeleton and the secretory pathway. Its central components - small GTPases (in particular RHO, but also ARF and Rab), their specialized effectors, actin and associated proteins, the exocyst complex essential for polarized secretion, or components of the phospholipid- and redox- based signalling circuits (inositol-phospholipid kinases/PIP2, NADPH oxidases) are aparently homologous among plants and metazoans, indicating that they were present already in LECA. REVIEWER: This article was reviewed by Arcady Mushegian, Valerian Dolja and Purificacion Lopez-Garcia.

• MeSH
• Actins metabolism   MeSH
• Cytoskeleton metabolism   MeSH
• Humans MeSH
• Cell Movement physiology   MeSH
• Pollen Tube cytology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Actins MeSH

Formins (FH2 proteins) are an evolutionarily conserved family of eukaryotic proteins, sharing the common FH2 domain. While they have been, until recently, understood mainly as actin nucleators, formins are also engaged in various additional aspects of cytoskeletal organization and signaling, including, but not limited to, the crosstalk between the actin and microtubule networks. A surprising diversity of domain organizations has been discovered among the FH2 proteins, and specific domain setups have been found in plants. Seed plants have two clades of formins, one of them (Class I) containing mostly transmembrane proteins, while members of the other one (Class II) may be anchored to membranes via a putative membrane-binding domain related to the PTEN antioncogene. Thus, plant formins present good candidates for possible mediators of coordination of the cortical actin and microtubule cytoskeletons, as well as their attachment to the plasma membrane, that is, aspects of cell cortex organization likely to be important for cell and tissue morphogenesis. Although experimental studies of plant formin function are hampered by the large number of formin genes and their functional redundancy, recent experimental work has already resulted in some remarkable insights into the function of FH2 proteins in plants.

• Publication type
• Journal Article MeSH
• Review MeSH

Phosphatidic acid (PA) is an important intermediate in membrane lipid metabolism that acts as a key component of signaling networks, regulating the spatio-temporal dynamics of the endomembrane system and the cytoskeleton. Using tobacco pollen tubes as a model, we addressed the signaling effects of PA by probing the functions of three most relevant enzymes that regulate the production and degradation of PA, namely, phospholipases D (PLD), diacylglycerol kinases (DGKs), and lipid phosphate phosphatases (LPPs). Phylogenetic analysis indicated a highly dynamic evolution of all three lipid-modifying enzymes in land plants, with many clade-specific duplications or losses and massive diversification of the C2-PLD family. In silico transcriptomic survey revealed increased levels of expression of all three PA-regulatory genes in pollen development (particularly the DGKs). Using specific inhibitors we were able to distinguish the contributions of PLDs, DGKs, and LPPs into PA-regulated processes. Thus, suppressing PA production by inhibiting either PLD or DGK activity compromised membrane trafficking except early endocytosis, disrupted tip-localized deposition of cell wall material, especially pectins, and inhibited pollen tube growth. Conversely, suppressing PA degradation by inhibiting LPP activity using any of three different inhibitors significantly stimulated pollen tube growth, and similar effect was achieved by suppressing the expression of tobacco pollen LPP4 using antisense knock-down. Interestingly, inhibiting specifically DGK changed vacuolar dynamics and the morphology of pollen tubes, whereas inhibiting specifically PLD disrupted the actin cytoskeleton. Overall, our results demonstrate the critical importance of all three types of enzymes involved in PA production and degradation, with strikingly different roles of PA produced by the PLD and DGK pathways, in pollen tube growth.

• Keywords
• diacylglycerol kinase, lipid phosphate phosphatase, phosphatidic acid, phospholipase D, pollen tube, signaling, tip growth, tobacco,
• Publication type
• Journal Article MeSH

Pollen tube cell volume changes rapidly in response to perturbation of the extracellular osmotic potential. This report shows that specific phospholipid signals are differentially stimulated or attenuated during osmotic perturbations. Hypo-osmotic stress induces rapid increases in phosphatidic acid (PA). This response occurs starting at the addition of 25% (v/v) water to the pollen tube cultures and peaks at 100% (v/v) water. Increased levels of PA were detected within 30 s and reached maximum by 15 to 30 min after treatment. The pollen tube apical region undergoes a 46% increase in cell volume after addition of 100% water (v/v), and there is an average 7-fold increase in PA. This PA increase appears to be generated by phospholipase D because concurrent transphosphatidylation of n-butanol results in an average 8-fold increase in phosphatidylbutanol. Hypo-osmotic stress also induces an average 2-fold decrease in phosphatidylinositol phosphate; however, there are no detectable changes in the levels of phosphatidylinositol bisphosphates. In contrast, salt-induced hyperosmotic stress from 50 to 400 mm NaCl inhibits phospholipase D activity, reduces the levels of PA, and induces increases in the levels of phosphatidylinositol bisphosphate isomers. The pollen tube apical region undergoes a 41% decrease in cell volume at 400 mm NaCl, and there is an average 2-fold increase in phosphatidylinositol 3,5-bisphosphate and 1.4-fold increase in phosphatidylinositol 4,5-bisphosphate. The phosphatidylinositol 3,5-bisphosphate increase is detected within 30 s and reaches maximum by 15 to 30 min after treatment. In summary, these results demonstrate that hypo-osmotic versus hyperosmotic perturbation and the resultant cell swelling or shrinking differentially activate specific phospholipid signaling pathways in tobacco (Nicotiana tabacum) pollen tubes.

• MeSH
• Sodium Chloride pharmacology   MeSH
• Phosphatidylinositol 4,5-Diphosphate biosynthesis   MeSH
• Phosphatidylinositol Phosphates biosynthesis   MeSH
• Phospholipase D drug effects   metabolism   MeSH
• Phospholipids metabolism   MeSH
• Flowers growth & development   metabolism   MeSH
• Phosphatidic Acids biosynthesis   MeSH
• Osmotic Pressure drug effects   MeSH
• Signal Transduction drug effects   physiology   MeSH
• Nicotiana growth & development   metabolism   MeSH
• Water pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Sodium Chloride MeSH
• Phosphatidylinositol 4,5-Diphosphate MeSH
• Phosphatidylinositol Phosphates MeSH
• Phospholipase D MeSH
• Phospholipids MeSH
• Phosphatidic Acids MeSH
• phosphatidylinositol 3,5-diphosphate MeSH Browser
• Water MeSH