Laura Zonia

Essential Role of Urease in Germination of Nitrogen-Limited Arabidopsis thaliana Seeds

In Arabidopsis thaliana, urease transcript levels increased sharply between 2 and 4 d after germination (DAG) and were maintained at maximal levels until at least 8 DAG. Seed urease specific activity declined upon germination but began to increase in seedlings 2 DAG, reaching approximately 75% of seed activity by 8 DAG. Urea levels showed a small transient increase 1 DAG and then approximately paralleled urease activity, reaching maximal levels at approximately 9 DAG. Urease inhibition with phenylphosphorodiamidate resulted in a 2- to 4-fold increase in urea levels throughout seedling development. Arginine pools (0–8 DAG) changed approximately in parallel with the urea pool. Consistent with arginine being a major source of urea, arginase activity increased 10-fold in the interval 0 to 6 DAG. Allopurinol, a xanthine dehydrogenase inhibitor, had no effect on urea levels up to 3 DAG but reduced the urea pool by 30 to 40% during the interval 5 to 8 DAG, suggesting that purine degradation contributed to the urea pool well after germination, if at all. In aged Arabidopsis seeds, there was a correlation between phenylphosphorodiamidate inactivation of urease and germination inhibition, the latter overcome by NH4NO3 or amino acids. Since urease activity, urea precursor, and urea increase in young seedlings, and since urease inactivation results in a nitrogen-reversible inhibition of germination, we propose that urease recycles urea-nitrogen in the seedling.

Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes

Pollen tubes are one of the fastest growing eukaryotic cells. Rapid anisotropic growth is supported by highly active exocytosis and endocytosis at the plasma membrane, but the subcellular localization of these sites is unknown. To understand molecular processes involved in pollen tube growth, it is crucial to identify the sites of vesicle localization and trafficking. This report presents novel strategies to identify exocytic and endocytic vesicles and to visualize vesicle trafficking dynamics, using pulse-chase labelling with styryl FM dyes and refraction-free high-resolution time-lapse differential interference contrast microscopy. These experiments reveal that the apex is the site of endocytosis and membrane retrieval, while exocytosis occurs in the zone adjacent to the apical dome. Larger vesicles are internalized along the distal pollen tube. Discretely sized vesicles that differentially incorporate FM dyes accumulate in the apical, subapical, and distal regions. Previous work established that pollen tube growth is strongly correlated with hydrodynamic flux and cell volume status. In this report, it is shown that hydrodynamic flux can selectively increase exocytosis or endocytosis. Hypotonic treatment and cell swelling stimulated exocytosis and attenuated endocytosis, while hypertonic treatment and cell shrinking stimulated endocytosis and inhibited exocytosis. Manipulation of pollen tube apical volume and membrane remodelling enabled fine-mapping of plasma membrane dynamics and defined the boundary of the growth zone, which results from the orchestrated action of endocytosis at the apex and along the distal tube and exocytosis in the subapical region. This report provides crucial spatial and temporal details of vesicle trafficking and anisotropic growth.

Osmotically Induced Cell Swelling versus Cell Shrinking Elicits Specific Changes in Phospholipid Signals in Tobacco Pollen Tubes

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.

Uncovering hidden treasures in pollen tube growth mechanics

The long-standing model of tip growth in pollen tubes considers that exocytosis and growth occur at the apex and that the pool of very small vesicles in the apical dome contains secretory (exocytic) vesicles. However, recent work on vesicle trafficking dynamics in tobacco pollen tubes shows that exocytosis occurs in the subapical region. Taking these and other new results into account, we set out to resolve specific problems that are endemic in current models and present a two-part ACE (apical cap extension)-H (hydrodynamics) growth model. The ACE model involves delivery and recycling of materials required for new cell synthesis and the H model involves mechanisms that integrate and regulate key cellular pathways and drive cell elongation during growth.

Cracking the Green Paradigm: Functional Coding of Phosphoinositide Signals in Plant Stress Responses

Plant form and function represent unique adaptations to life on earth. Although many fundamental elements of biochemistry and cell biology are shared among mammals, plants, fungi and protists, each has the potential to express these elements differently. Thus it should not be surprising that while plant cells contain many of the same elements of phosphoinositide signaling systems as other organisms, the functional coding of these elements may differ because both the information signaled as well as the responses elicited are different. The goal of this review is to provide a critical overview of what is currently known about phosphoinositide signaling in plants and in plant stress responses, to focus on a specific system in which differential coding of phosphatidylinositol signals has been elucidated, and to indicate directions for future research.

Spatial and temporal integration of signalling networks regulating pollen tube growth

The overall function of a cell is determined by its contingent of active signal transduction cascades interacting on multiple levels with metabolic pathways, cytoskeletal organization, and regulation of gene expression. Much work has been devoted to analysis of individual signalling cascades interacting with unique cellular targets. However, little is known about how cells integrate information across hierarchical signalling networks. Recent work on pollen tube growth indicates that several key signalling cascades respond to changes in cell hydrodynamics and apical volume. Combined with known effects on cytoarchitecture and signalling from other cell systems, hydrodynamics has the potential to integrate and synchronize the function of the broader signalling network in pollen tubes. This review will explore recent work on cell hydrodynamics in a variety of systems including pollen, and discuss hydrodynamic regulation of cell signalling and function including exocytosis and endocytosis, actin cytoskeleton reorganization, cell wall deposition and assembly, phospholipid and inositol polyphosphate signalling, ion flux, small G-proteins, fertilization, and self-incompatibility. The combined data support a newly emerging model of pollen tube growth.

Understanding pollen tube growth: the hydrodynamic model versus the cell wall model

Scientific progress stimulates the evolution of models used to understand and conceptualize biological behaviors. The widely accepted cell wall model of pollen tube growth explains stochastic growth of the apical pectin wall, but fails to explain the mechanism driving oscillations in growth and cell signaling. Recent advances led to the formulation of a new hydrodynamic model that explains the mechanism that drives both stochastic and oscillatory growth, as well as oscillations in cell signaling and ion fluxes. A critical analysis of evidence that has been used to challenge the validity of the hydrodynamic model yields new information on turgor pressure, cell mechanical properties and nonlinear dynamics in pollen tube growth. These results may have broader significance for plant cell growth.

Hydrodynamics and Cell Volume Oscillations in the Pollen Tube Apical Region are Integral Components of the Biomechanics of Nicotiana tabacum Pollen Tube Growth.

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H2O with 2H2O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing 2H2O with H2O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.

Swimming patterns and dynamics of simulated Escherichia coli bacteria.

A spatially and temporally realistic simulation of Escherichia coli chemotaxis was used to investigate the swimming patterns of wild-type and mutant bacteria within a rectangular arena in response to chemoattractant gradients. Swimming dynamics were analysed during long time series with phase-space trajectories, power spectra and estimations of fractal dimensions (FDs). Cell movement displayed complex trajectories in the phase space owing to interaction of multiple attractors that captured runs and tumbles. Deletion of enzymes responsible for adaptation (CheR and CheB) restricted the pattern of bacterial swimming in the absence of a gradient. In the presence of a gradient, there was a strong increase in trajectories arising from runs and attenuation of those arising from tumbles. Similar dynamics were observed for mutants lacking CheY, which are unable to tumble. The deletion of CheR, CheB and CheY also caused significant shifts in chemotaxis spectral frequencies. Rescaled range analysis and estimation of FD suggest that wild-type bacteria display characteristics of fractional Brownian motion with positive correlation between past and future events. These results reveal an underlying order in bacterial swimming dynamics, which enables a chemotactic search strategy conforming to a fractal walk.