Metabolomics and transcriptomics to decipher molecular mechanisms underlying ectomycorrhizal root colonization of an oak tree.

Mycorrhizas are known to have a positive impact on plant growth and ability to resist major biotic and abiotic stresses. However, the metabolic alterations underlying mycorrhizal symbiosis are still understudied. By using metabolomics and transcriptomics approaches, cork oak roots colonized by the ectomycorrhizal fungus Pisolithus tinctorius were compared with non-colonized roots. Results show that compounds putatively corresponding to carbohydrates, organic acids, tannins, long-chain fatty acids and monoacylglycerols, were depleted in ectomycorrhizal cork oak colonized roots. Conversely, non-proteogenic amino acids, such as gamma-aminobutyric acid (GABA), and several putative defense-related compounds, including oxylipin-family compounds, terpenoids and B6 vitamers were induced in mycorrhizal roots. Transcriptomic analysis suggests the involvement of GABA in ectomycorrhizal symbiosis through increased synthesis and inhibition of degradation in mycorrhizal roots. Results from this global metabolomics analysis suggest decreases in root metabolites which are common components of exudates, and in compounds related to root external protective layers which could facilitate plant-fungal contact and enhance symbiosis. Root metabolic pathways involved in defense against stress were induced in ectomycorrhizal roots that could be involved in a plant mechanism to avoid uncontrolled growth of the fungal symbiont in the root apoplast. Several of the identified symbiosis-specific metabolites, such as GABA, may help to understand how ectomycorrhizal fungi such as P. tinctorius benefit their host plants.

In vitro functional characterization of missense mutations in the LDLR gene.

BACKGROUND: Mutations in the LDL receptor gene are the major cause of familial hypercholesterolaemia (FH) but it has been previously shown that the simple finding of a variation in the coding sequence of the LDLR does not confirm that it is the actual cause of FH. The pathogenicity of five missense alterations in the LDLR gene coding sequence found in a previous epidemiologic study was investigated. METHODS: The effects of the different sequence variants on LDLR expression and activity were analysed in vitro stably transfected CHO-ldlA7 cells by immunobloting of cell extracts, by uptake and degradation rates of (125)I-labelled LDL and immunofluorescence microscopy of whole cells. Analysis in silico was also performed. RESULTS: LDLR functional assays showed that variants p.V429L, p.W490R and p.S648P of the LDLR coding sequence severely impaired receptor function, while variant p.P685S had a milder effect and cells carrying p.V859M variant had LDL clearance rates comparable to cells expressing normal LDLR. In silico analysis failed to predict correctly the effect of 4/5 alterations. CONCLUSION: Assessing the pathogenicity of the different variants found in patients with clinical diagnosis of FH is of great importance to distinguish pathogenic mutations from rare silent variants and has clinical implications for determining the associated cardiovascular risk.

Signalling pathways in pollen germination and tube growth.

Signalling is an integral component in the establishment and maintenance of cellular identity. In plants, tip-growing cells represent an ideal system to investigate signal transduction mechanisms, and among these, pollen tubes (PTs) are one of the favourite models. Many signalling pathways have been identified during germination and tip growth, namely, Ca(2+), calmodulin, phosphoinositides, protein kinases, cyclic AMP, and GTPases. These constitute a large and complex web of signalling networks that intersect at various levels such as the control of vesicle targeting and fusion and the physical state of the actin cytoskeleton. Here we discuss some of the most recent advances made in PT signal transduction cascades and their implications for our future research. For reasons of space, emphasis was given to signalling mechanisms that control PT reorientation, so naturally many other relevant works have not been cited.

Modulation of endocytosis in pollen tube growth by phosphoinositides and phospholipids.

In plants, tip-growing cells represent an ideal system to investigate signal transduction mechanisms, and among those, pollen tubes are one of the favourite models. Many signalling pathways have been identified during germination and tip growth, namely, Ca2+, calmodulin, phosphoinositides, cyclic AMP, and GTPases. Not surprisingly, the apical secretory machinery, essential for tip growth, seems to be an intersection point for all these pathways. Recently, the phospholipid phosphatidic acid was also suggested to actively participate in the control of endo- and exocytosis and to interfere with the correct positioning of the actin cytoskeleton. Phosphatidic acid seems to act concertedly with the phosphoinositides phosphatidylinositol 4,5-bisphosphate and D-myo-inositol 1,4,5-trisphosphate. Here we review previous data and discuss additional evidence that these three molecules have a combined action modulating both the actin cytoskeleton and the apical secretory machinery. We further discuss how these findings can be integrated into a working model for pollen tube apical secretion that contemplates the existence of a rapid endocytosis mechanism.

cAMP acts as a second messenger in pollen tube growth and reorientation.

Pollen tube growth and reorientation is a prerequisite for fertilization and seed formation. Here we report imaging of cAMP distribution in living pollen tubes microinjected with the protein kinase A-derived fluorosensor. Growing tubes revealed a uniform distribution of cAMP with a resting concentration of approximately 100-150 nM. Modulators of adenylyl cyclase (AC), forskolin, and dideoxyadenosine could alter these values. Transient elevations in the apical region could be correlated with changes in the tube-growth axis, suggesting a role for cAMP in polarized growth. Changes in cAMP arise through the activity of a putative AC identified in pollen. This signaling protein shows homology to functional motifs in fungal AC. Expression of the cDNA in Escherichia coli resulted in cAMP increase and complemented a catabolic defect in the fermentation of carbohydrates caused by the absence of cAMP in a cyaA mutant. Antisense assays performed with oligodeoxynucleotide probes directed against conserved motifs perturbed tip growth, suggesting that modulation of cAMP concentration is vital for tip growth.

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells.

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.

Relocation of a Ca2+-dependent protein kinase activity during pollen tube reorientation

Pollen tube reorientation is a dynamic cellular event that is crucial for successful fertilization. We have shown previously that pollen tube orientation is regulated by cytosolic free calcium ([Ca2+]c). In this paper, we studied the activity of a Ca2+-dependent protein kinase during reorientation. The kinase activity was assayed in living cells by using confocal ratio imaging of BODIPY FL bisindolylmaleimide. We found that growing pollen tubes exhibited higher protein kinase activity in the apical region, whereas nongrowing cells showed uniform distribution. Modification of growth direction by diffusion of inhibitors/activators from a micropipette showed the spatial redistribution of kinase activity to predict the new growth orientation. Localized increases in [Ca2+]c induced by photolysis of caged Ca2+ that led to reorientation also increased kinase activity. Molecular and immunological assays suggest that this kinase may show some functional homology with protein kinase C. We suggest that the tip-localized gradient of kinase activity promotes Ca2+-mediated exocytosis and may act to regulate Ca2+ channel activity.

Ca2+ signalling in plant cells: the big network!

Significant advances in Ca2+ and calmodulin signalling in whole plants and individual cells have been recently reported. Particular relevant contributions have been made to the study of the modification of gene expression by osmotic, light and gravity signals and the growth of root hairs and pollen tubes.

Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells.

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/-0.03 and +/-0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.

Localized Apical Increases of Cytosolic Free Calcium Control Pollen Tube Orientation.

To reach the ovule, pollen tubes must undergo many changes in growth direction. We have shown in previous work that elevation of cytosolic free calcium ([Ca2+]c) can manipulate orientation in growing pollen tubes, but our results suggested that [Ca2+]c changes either in the tip or in more distal regions might regulate the critical orienting mechanism. To identify the spatial location of the orienting motor, we combined the techniques of ion imaging with confocal microscopy and localized photoactivation of loaded caged Ca2+ (nitr-5) and diazo-2 (a caged Ca2+ chelator) to manipulate [Ca2+]c in different pollen tube domains. We found that increasing [Ca2+]c on one side of the pollen tube apex induced reorientation of the growth axis toward that side. Similarly, a decrease in [Ca2+]c promoted bending toward the opposite side. These effects could be mimicked by imposing localized external gradients of an ionophore (A23187) or a Ca2+ channel blocker (GdCl3); the pollen tubes bend toward the highest concentration of A23187 and away from GdCl3. Manipulation of [Ca2+]c in regions farther back from the apical zone also induced changes in growth direction, but the new orientation was at random. We observed communication of these distal events to the tip through a slow-moving [Ca2+]c wave. These data show that localized changes of [Ca2+]c in the tip, which could result from asymmetric channel activity, control the direction of pollen tube growth.

Calcium Channel Activity during Pollen Tube Growth and Reorientation.

We have shown previously that the inhibition of pollen tube growth and its subsequent reorientation in Agapanthus umbellatus are preceded by an increase in cytosolic free calcium ([Ca2+]c), suggesting a role for Ca2+ in signaling these processes. In this study, a novel procedure was used to measure Ca2+ channel activity in living pollen tubes subjected to various growth reorienting treatments (electrical fields and ionophoretic microinjection). The method involves adding extracellular Mn2+ to quench the fluorescence of intracellular Indo-1 at its ca2+-insensitive wavelength (isosbestic point). The spatial and temporal kinetics of Ca2+ channel activity correlated well with measurements of [Ca2+]c dynamics obtained by fluorescence ratio imaging of Indo-1. Tip-focused gradients in Ca2+ channel activity and [Ca2+]c were observed and quantified in growing pollen tubes and in swollen pollen tubes before reoriented growth. In nongrowing pollen tubes, Ca2+ channel activity was very low and [Ca2+]c gradients were absent. Measurements of membrane potential indicated that the growth reorienting treatments induced a depolarization of the plasma membrane, suggesting that voltage-gated Ca2+ channels might be activated.