Spatial distribution of growth rates and of epidermal cell lengths in the elongation zone during leaf development in Lolium perenne L.

Relative elemental growth rates (REGR) and lengths of epidermal cells along the elongation zone of Lolium perenne L. leaves were determined at four developmental stages ranging from shortly after emergence of the leaf tip to shortly before cessation of leaf growth. Plants were grown at constant light and temperature. At all developmental stages the length of epidermal cells in the elongation zone of both the blade and sheath increased from 12 m at the leaf base to about 550 m at the distal end of the elongation zone, whereas the length of epidermal cells within the joint region only increased from 12 to 40 m. Throughout the developmental stages elongation was confined to the basal 20 to 30 mm of the leaf with maximum REGR occurring near the center of the elongation zone. Leaf elongation rate (LER) and the spatial distributions of REGR and epidermal cell lengths were steady to a first approximation between emergence of the leaf tip and transition from blade to sheath growth. Elongation of epidermal cells in the sheath started immediately after the onset of elongation of the most proximal blade epidermal cells. During transition from blade to sheath growth the length of the blade and sheath portion of the elongation zone decreased and increased, respectively, with the total length of the elongation zone and the spatial distribution of REGR staying near constant, with exception of the joint region which elongated little during displacement through the elongation zone. Leaf elongation rate decreased rapidly during the phase when only the sheath was growing. This was associated with decreasing REGR and only a small decrease in the length of the elongation zone. Data on the spatial distributions of growth rates and of epidermal cell lengths during blade elongation were used to derive the temporal pattern of epidermal cell elongation. These data demonstrate that the elongation rate of an epidermal cell increased for days and that cessation of epidermal cell elongation was an abrupt event with cell elongation rate declining from maximum to zero within less than 10 h.Abbreviations LER leaf elongation rate - REGR relative elemental growth rates     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.     

A kinematic analysis of tepal growth in Lilium longiflorum

Time-lapse marking experiments indicate that the growth of tepals in Lilium longiforum Thunb. from 3.7 mm to maturity is triphasic. Phase I (tepal lengths 3.7–10 mm) is characterized by spatial and temporal variation in growth rate and, in the epidermis, a random distribution of mitoses with an acropetal increase in cell area. During phase II (10–90 mm) cell elongation and (later) cell division is restricted largely to basal regions. Cell division ceases when tepals are less than one-third of their mature length of 155 mm. Phase III (90–155 mm) is characterized by the gradual transition from basal to apical growth, and a modification of epidermal cell shape. A sharp peak in growth at the extreme tip of the tepal coincides with anthesis.Abbreviations LRGR local relative growth rate - RER relative elemental rate of growth     

KINEMATICS OF HYPOCOTYL CURVATURE

Time lapse photographs were analyzed for curvature, κ, of the plumular hook as a function of distance from the apex, s, of seedlings of lettuce. Lactuca sativa cv ‘Grand Rapids,‘ during hook maintenance (red light) and hook opening (white light). Curvature of the inner edge of the photographic projection of the hook increases from 0.14 mm^–1 near the apex to 12.7 mm^–1 at the hook bisector (about 1 mm from the apex) and decreases to approximately zero below the hook (2 mm from the apex). Using concepts from fluid dynamics we relate growth rates to curvature changes. For a material element of stem cross section located at s we predict that where M(s,o) and M(s,i) are the relative elemental growth rates at the outer and inner edges of the hypocotyl cross section, w is element width, t is time, and u(s) is velocity of departure of s from the apex. During hook maintenance ∂[ln(1 + κw)]/∂t is approximately zero if the apex is taken as origin. As each hypocotyl element is displaced from the apex it becomes increasingly curved; then, after displacement past the hook bisector, the element straightens. Growth rates, determined by measurements of the displacement of epidermal hairs, show the pattern expected from the equation: On the apical side of the hook, relative growth rates of the outer edge exceed growth of the inner edge, M(s,o) > M(s,i), while on the basal side of the hook. M(s,o) < M(s,i). The constraint for hook maintenance is that [L(s,o) – L(s,i)] /u(s) be constant in time, where L(s,o) and L(s,i) are the relative elemental growth rates at s. During hook opening ∂[ln(1 + κw)]/∂t becomes important.     

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL-SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm^?2, whereas that of the exoplasmic fracture face was 325 μm^?2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm^?2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm² of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.     

Laser ablation ICP-MS reveals patterns of copper differing from zinc in growth zones of cucumber roots

Laser ablation coupled with inductively coupled plasma-mass spectrometry was used to find Cu and Zn concentration in surface tissue along a longitudinal developmental gradient with meristem, rapidly elongating tissue, and nongrowing tissue in a model system of seedling roots of Cucumis sativus L. (cucumber). Tissue metal accumulation was determined for roots of seedlings growing on cellulosic germination paper treated with nutrient solution (controls), and also treated with concentrations of Zn (40 ppm) and Cu (10 ppm) that reduced growth. Cu content of all roots is highest at the apex and falls sharply to lower values by 2 mm from the root tip. In contrast, at moderate Zn availability (0.07 ppm), Zn content rises from the apex to 2 mm then falls throughout the remainder of the growth zone. At high external Zn the spatial pattern resembles that of Cu. Cucumber root growth zones accumulate more of each metal with higher external availability. Metal deposition rates were calculated using a continuity equation with data on local metal content and growth velocity. Deposition rates of both metals are generally highest in the rapidly elongating region, 1.5–3.5 mm, even where metal concentration is decreasing with position and root age and even when the accumulation is inhibitory to growth.     

The kinetics of tip growth in the Nitella rhizoid

A detailed observation of the growth kinetics of the rhizoidalcell in Nitella was made possible by application of artificialmarkers on the cell surface. The elongation of the cell is linearat the rate of 1.7 µm/min (2.5 mm/day), and the activityof the elongation is limited to the cell's apical dome (tipgrowth) whose shape is unchanged during the elongation. Thegreatest relative expansion, 0.15 per min (15% per min), occursat the tip region of the dome where the expansion is roughlyisotropic. In the remaining area of the upper half of the domethe expansion is apparently longitudinal stretching (meridionalanisotropy), and in the lower half, transverse stretching (latitudinalanisotropy). The observed directionality of the expansion iscompared with the expansion of some theoretical cases in whicha perfect hemispherical shape and a certain degree of the directionality(described by an allometric coefficient) are maintained duringthe expansion, and with a similar observation made from theapical cell of the Nitella shoot. (Received January 9, 1973; )     

The fine structure ofPhycomyces

Summary The cytological organization of the apices of sporangiophores and hyphae ofPhycomyces Blakesleeanus was studied by means of light- and electron microscopy. The sporangiophore apex in growth stage I contains a mass of cytoplasm in which is embedded a cluster of lipid globules. Within the plug several zones are differentiated by the grouping of organelles. These zones are not separated by membranes. The most apical zone is low in nuclei and vesicles but rich in mitochondria and dense bodies. Below this zone lies a compact group containing up to several hundred nuclei. Along the midline of the cell, below these nuclei, lies an ovoid region from which vesicles, nuclei and mitochondria are excluded. In this ovoid exclusion zone lies the cluster of lipid globules mentioned above. Lateral to the exclusion zone (i.e. in the peripheral region of the cell) the cytoplasm is rich in nuclei, mitochondria, dense bodies, and especially in developing autophagic vesicles. Of these vesicles, the most mature are found farthest from the cell apex. The region between the exclusion zone and the upper end of the cell's large central vacuole is occupied largely by mature, swollen autophagic vesicles. In addition to the zonal organization described above, microtubules are found to run along the cylindrical cell's axis at a distance from the cell wall, and extend to the extreme apex of the cell. Similar tubules occur in growing hyphae, together with dense bodies, and the hyphal apex contains non-autophagic vesicles that increase in size with distance from the hyphal tip. The hyphae lack the zonation shown by sporangiophore apices. Perinuclear masses of cisternae are described and related to the dictyosomes of higher plants. The findings are discussed in relation to the function of the apices in tip growth and sporulation.This work was supported in part by a National Science Foundation Graduate Fellowship to the author, and in part by grant No. GB 3241 from the National Science Foundation to ProfessorKenneth V.Thimann.     

Cell surface expansion in polarly growing root hairs of Medicago truncatula

Fluorescent microspheres were used as material markers to investigate the relative rates of cell surface expansion at the growing tips of Medicago truncatula root hairs. From the analysis of tip shape and microsphere movements, we propose three characteristic zones of expansion in growing root hairs. The center of the apical dome is an area of 1- to 2- microm diameter with relatively constant curvature and high growth rate. Distal to the apex is a more rapidly expanding region 1 to 2 microm in width exhibiting constant surges of off-axis growth. This middle region forms an annulus of maximum growth rate and is visible as an area of accentuated curvature in the tip profile. The remainder of the apical dome is characterized by strong radial expansion anisotropy where the meridional rate of expansion falls below the radial expansion rate. Data also suggest possible meridional contraction at the juncture between the apical dome and the cell body. The cell cylinder distal to the tip expands slightly over time, but only around the circumference. These data for surface expansion in the legume root hair provide new insight into the mechanism of tip growth and the morphogenesis of the root hair.     

The tip growth apparatus of Aspergillus nidulans

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1–2 μm behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.     

Localization of arabidopsis SYP125 syntaxin in the plasma membrane sub-apical and distal zones of growing pollen tubes

Tip growth in pollen tubes occurs by continuous vesicle secretion and delivery of new wall material, but the exact sub-cellular location of endocytic and exocytic domains remains unclear. Here we studied the localization of the Arabidopsis thaliana pollen specific syntaxin SYP125 using GFP-fusion constructs expressed in Nicotiana tobaccum pollen tubes. In agreement with the predicted role for syntaxins, SYP125 was found to be associated with the plasma membrane and apical vesicles in growing cells. At the plasma membrane, SYP125 was asymmetrically localized with a higher labeling 20–35 µm behind the apex, a distribution which is distinct from SYP124, another pollen-specific syntaxin. Competition with a related dominant negative mutant affected the specific distribution of SYP125 but not tip growth. Co-expression of the phosphatidylinositol-4-monophosphate-5-kinase 4 (PIP5K4) or of the small GTPase Rab11 perturbed polarity and the normal distribution of GFP-SYP but did not inhibit the accumulation in vesicles or at the plasma membrane.Taken together, our results corroborates previous observations that in normal growing pollen tubes, the asymmetric distribution of syntaxins helps to define exocytic sub-domains but requires the involvement of additional signaling and functional mechanisms, namely phosphoinositides and small GTPases. The localization of syntaxins at different membrane domains likely depends on the interaction with specific partners not yet identified.Key words: [Ca²⁺]_c, endocytosis, exocytosis, secretion, syntaxins, tip 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.     

Effect of cadmium on the distribution of hydroxyl radical, superoxide and hydrogen peroxide in barley root tip

In the present study, we investigated the alteration of reactive oxygen species production along the longitudinal axis of barley root tips during Cd treatment. In unstressed barley root tips, H₂O₂ production decreased from the root apex towards the differentiation zone where again, a slight increase was observed towards the more mature region of root. An opposite pattern was observed for O ₂ ^?? and OH^? generation. The amount of both O ₂ ^?? and OH^? was highest in the elongation zone, decreased in the root apex and at the differentiation zone of root, then increased again towards the more mature region of root. An elevated Cd-induced O ₂ ^?? production started in the elongation zone and increased further along the differentiation zone of barley root tip. In contrast, Cd-induced H₂O₂ production was localised to the root elongation zone and to the beginning of the differentiation zone. In contrast to Cd-induced H₂O₂ and O ₂ ^?? production, Cd reduced OH^? production along the whole barley root tip. Our results suggest that not only an increase but also the spatial distribution of reactive oxygen species production is involved in the Cd-induced stress response of barley root tip.     

Co-regulation of root hair tip growth by ROP GTPases and nitrogen source modulated pH fluctuations

Growth of plant cells involves tight regulation of the cytoskeleton and vesicle trafficking by processes including the action of the ROP small G proteins together with pH-modulated cell wall modifications. Yet, little is known on how these systems are coordinated. In a paper recently published in Plant Cell and Environment¹ we show that ROPs/RACs function synergistically with NH₄NO₃-modulated pH fluctuations to regulate root hair growth. Root hairs expand exclusively at their apical end in a strictly polarized manner by a process known as tip growth. The highly polarized secretion at the apex is maintained by a complex network of factors including the spatial organization of the actin cytoskeleton, tip-focused ion gradients and by small G proteins. Expression of constitutively active ROP mutants disrupts polar growth, inducing the formation of swollen root hairs. Root hairs are also known to elongate in an oscillating manner, which is correlated with oscillatory H⁺ fluxes at the tip. Our analysis shows that root hair elongation in wild type plants and swelling in transgenic plants expressing a constitutively active ROP11 (rop11^CA) is sensitive to the presence of NH₄⁺ at concentrations higher than 1 mM and on NO₃⁻. The NH₄⁺ and NO₃⁻ ions did not affect the localization of ROP in the membrane but modulated pH fluctuations at the root hair tip. Actin organization and reactive oxygen species distribution were abnormal in rop11^CA root hairs but were similar to wild-type root hairs when seedlings were grown on medium lacking NH₄⁺ and/or NO₃⁻. These observations suggest that the nitrogen source-modulated pH fluctuations may function synergistically with ROP regulated signaling during root hair tip growth. Interestingly, under certain growth conditions, expression of rop11^CA suppressed ammonium toxicity, similar to auxin resistant mutants. In this short review we discuss these findings and their implications.Key words: ROP, RAC, nitrogen, root hair, cell polarity, ammoniumIn Arabidopsis, root hairs grow out at the basal, rootward region (closer to root tip) of specialized root epidermal cells and expand exclusively at their apical end in a strictly polarized manner by a process known as tip growth. Tip growth is facilitated by Rho of Plants (ROP)-regulated processes such as maintenance of longitudinally-oriented actin cables in the shank of the root hair that are required for myosin-mediated organelle transport through the cytoplasm. ROPs also play a role in sustaining fine F-actin structures at the root hair tip, which promote the transport of secretory vesicles to sites of their fusion with the plasma membrane.^2,3 In addition, the polar growth of root hairs involves an oscillatory tip-focused Ca²⁺ gradient⁴ and tip-localized reactive oxygen species (ROS).⁵ Tip growth is also associated with oscillatory fluxes of H⁺ at the apex that correlate with the periodicity of growth.^6,7 These oscillations in extracellular pH and ROS have been shown to modulate tip growth and are predicted to act in a coordinated and complementary mode to regulate root hair elongation. Growth accelerates following reduction of apoplastic pH and slows upon apoplastic ROS increase and a coincident pH increase.⁷ROPs are small G proteins that localize to the plasma membrane at the apex of growing root hairs, where they activate a range of downstream pathways required for tip growth.^8,9 ROP activity is regulated by its cycling between a GTP-bound, active and GDP-bound, inactive state. Ectopic expression of constitutively active mutants of ROPs (dominant mutations in conserved residues that abolish the GTPase activity) depolarizes the growth of root hairs.^8–10 Downstream pathways activated by such ROP GTPases include the regulation of cytoskeletal dynamics and vesicular trafficking, production of ROS, maintenance of intracellular Ca²⁺ gradients and accumulation of signaling lipids, features all related to the regulation of apical growth.^11,12 For example, ectopic expression of constitutively active ROP11 (Atrop11^CA) depolarizes root hair growth, leading to the formation of swollen root hairs. This bulging root hair phenotype was associated with altered actin organization and inhibition of endocytosis.¹⁰It is well known that root hair development is highly plastic and regulated by environmental signals.^13,14 Yet, despite the known function of ROP GTPases and their regulatory proteins in root hair growth there is no data in the literature describing the relationship between ROP signaling and environmental factors in this process. Our results¹ show that induction of root hair swelling by rop11^CA occurs only under specific growth conditions, indicating that there is an interplay between ROP activity and the external environment, particularly nitrogen supply. We demonstrated that high external concentrations of ammonium are essential for the induction of depolarized root hair growth and activation of downstream pathways by rop11^CA. Depletion of ammonium did not affect the membrane localization and expression of GFP-rop11^CA, implying that NH₄⁺ was required in addition to ROP activity to cause root hair swelling. In agreement with this idea, normal actin organization and ROS localization were detected in rop11^CA root hairs when NH₄⁺ was depleted, suggesting that ammonium functions downstream of, or in parallel to ROP signaling (Fig. 1).Open in a separate windowFigure 1A model for regulation of root hair tip growth by ROP GTPases and pH oscillations dependent on nitrogen supply. GTP bound ROPs activate downstream effectors which directly affect actin organization, vesicular trafficking and localized ROS production as well as indirectly affecting the localization of membrane proteins involved in ion/proton fluxes. High concentrations of nitrogen ions in the growth medium increase pH oscillations at the apex of growing root hairs. In turn downstream ROP effectors sense the changes in pH and adjust their function accordingly. pH oscillations affect tip growth independent of ROPs via changes of wall pH and possibly through additional unknown factors. Dashed lines indicate that these effects were not confirmed experimentally.Plants can absorb and use various forms of nitrogen from soils, primarily the inorganic ions ammonium and nitrate. The concentrations of these ions are highly heterogeneous around the plant and can vary across several orders of magnitude among different soils and as a result of seasonal changes.¹⁵ Thus, plants would be expected to display highly plastic, N-regulated developmental responses and to employ a range of nitrogen uptake transport systems to optimize exploitation of local N resources. Transport systems that mediate NH₄ fluxes across the plasma membrane of root cells are divided into two categories: high affinity transport systems (HATS) that mediate uptake from relatively dilute solutions at relatively low rates and low affinity transport systems (LATS) that operate at high rates and higher external concentrations.¹⁶ The HATS are plasma membrane localized NH₄⁺-specific transporters (AMTs) that are most likely proton-coupled and their expression and function are repressed at external ammonium concentrations of 1 mM or higher.^17–19 In contrast, ammonium uptake by LATS is believed to take place through non-specific cation channels.^17,20 The NH₄⁺ concentration in the 0.5× Murashige Skoog (MS) medium is 10.3 mM, exceeding by an order of magnitude the concentration at which the high affinity NH₄⁺ uptake system is repressed. The root hair swelling in Atrop11_CA plants and inhibition of root hair elongation in wild type plants occurred primarily at external ammonium concentrations greater than 1 mM, and thus is most likely associated with uptake by the LATS.As noted above, root hair elongation is associated with oscillations of cytoplasmic and apoplastic pH that have been linked to growth control. Simultaneous fluorescence ratio imaging of internal and external pH revealed that application of 10 mM NH₄NO₃ enhanced the amplitude of these pH oscillations at the extreme apex of wild type root hairs1 and Figure 2. These oscillations are thought to modulate tip growth through altering the extensibility of the wall.⁴ Additional measurements (Fig. 2) show that similar to the effects of NH₄NO₃, addition of NH₄Cl induced increase in the apoplastic pH fluctuations and reduced the pH. However, the effects of NH₄Cl on cytoplasmic pH fluctuations seem subtler compared to the effects of NH₄NO₃. Thus, one possible explanation for the observed swelling of the root hair apex in rop11_CA expressing plants in media containing NH₄NO₃ is that rop11_CA root hairs are affected in their ability to re-establish the normal proton gradient across the plasma membrane in response to ammonium transport. The altered proton gradient would then prevent the normal localized oscillatory changes in pH-dependent wall properties required to restrict expansion to the very tip of the elongating root hair.Open in a separate windowFigure 2Changes in apoplastic and cytoplasmic pH fluctuations, following application of NH₄NO₃, NH₄Cl or KNO₃. (A) Apolplastic pH (pH_ex) following treatments with either NH₄NO₃, NH₄Cl or KNO₃. Note the increase pH fluctuations induced by either NH₄NO₃ and NH₄Cl but not by KNO₃. (B) Cytoplasmic pH (pH_cyt) following treatments as above. Note the changes in pH fluctuations induced by NH₄NO₃ and the subtler effects of NH₄Cl.Concurrent absorption of NH₄⁺ and NO₃⁻- maintains the cation-anion balance within both the rooting medium and the root, and thus potentially has an important function in maintaining intracellular and extracellular pH.^21,22 In agreement, application of these ions affected the amplitude of pH oscillations¹ and Figure 2. Interestingly, treatments of WT seedlings with 10 mM NH₄NO₃ causes increase in root hair pH oscillations and often tip bursting. Yet, prolonged exposure of WT root hairs to NH₄NO₃ is accompanied by adaptation (our unpublished data). This adaptation does not occur in rop11_CA mutants, suggesting that cycling of ROPs between active and inactive states maybe important in adaptation to changing environment. These data strongly suggest that NH₄⁺-dependent root hair swelling in the plants expressing activated ROP resulted from physiological changes in ion balance rather than a direct effect of ammonium on enzymatic activities required for root hair growth (Fig. 1). Application of NH₄⁺ and NO₃⁻, in the absence of other ions, induced formation of additional growth tips, in which the membrane localized GFP-rop11^CA was concentrated. This observation suggests that interplay between the regulation of ROP localization and activity and the regulation of nitrogen fluxes may have an important function in the maintenance of unidirectional growth. As root hair elongation is coupled to spatially distinct regulation of extracellular pH oscillations and ROS production,⁷ it seems likely that there is a mechanism that can adjust the fluxes of nitrogen ions relative to these pH fluxes. This system would then maintain the oscillations in pH such that polarized growth is continued. One possible mechanism for this coordination is through the highly localized ROP cycling between active and inactive states that has an important role in the spatial activation of cell polarization machinery.^23–27 Due to the function of ROP GTPases in vesicle trafficking, actin organization and maintenance of ROS and Ca²⁺ gradients,^{2,8,9,23,24,28–33} expression of activated ROP11 may indirectly influence cell wall properties by altering the localization and/or recycling of cation and anion transporters/channels or plasma membrane H⁺-ATPases delivered to the growing tip of the hair and in this way affect the maintenance of the proton gradients. In agreement with a possible effect of activated ROPs on localization and/or recycling of membrane transporters we discovered that rop11^CA plants were resistant to ammonium toxicity when grown in the presence of NH₄NO₃ and several micronutrients.¹We propose a model (Fig. 1) in which spatial regulation of ROP activity creates a positive feedback loop with pH oscillations around the growing apex of root hairs. According to this model ROP cycling between active and inactive states spatially and temporally activates the downstream signaling cascades essential for the tip-growth of root hairs. At the same time, localization of membrane proteins involved in maintenance of normal nitrogen fluxes across the plasma membrane is indirectly affected by ROP signaling. Alternatively, ROP signaling is modulated to adapt to altered nitrogen fluxes. NH₄⁺ fluxes increase the amplitude of pH oscillations at the root hair apex and in turn affect cell-wall properties. Thus, when the ROP activity is upregulated by dominant mutations, the synergistic effects of pH changes and constant activation of ROP downstream effectors result in the uncontrolled cell expansion seen as root hair bulging. Previous studies have suggested that feedback between oscillatory pH change and ROS distribution is required to support tip growth.⁷ However, the factors that may integrate these processes are unknown. Our results suggest that spatial regulation of ROP activity in response to changing environments is one of the key elements that may coordinate the pH and ROS oscillations during the root hair tip growth.It will be interesting to examine whether ROP function is coordinated with apoplastic pH fluctuation in other cell types. Recently, it has been suggested that the effects of auxin on pavement cell structure in leaf epidermis require Auxin Binding Protein 1 (ABP1) dependent ROP activation.³⁴ It is well known that auxin induces changes in apoplastic pH. Possibly, like nitrogen source in root hairs, auxin dependent apolplastic pH fluctuations in the leaf epidermis may function coordinately with ROP in the regulation of cell growth. Consistent with this idea, it has been shown that auxin inhibits clathrin-dependent endocytosis through ABP1 reinforcing a possible role in modulating membrane flux/membrane properties.³⁵ Some auxin resistant mutants also display resistance to ammonium toxicity³⁶ further suggesting a link between auxin and membrane transport. Hence, auxin and ROPs may indeed function synergistically to modulate plasma membrane properties, in turn affecting ion balance in the apoplast and so modulating cell wall properties and growth.     

The pulse current pattern generated by developing fucoid eggs

Using a newly developed extracellular vibrating electrode, we have made the first study of the spatial distribution of the growth currents around a single developing egg. This pattern was studied during the current pulses wihic traverse two-celled Pelvetia embryos. These pulses can be stimulated to occur with a periodicity of 70 min by mild acidification of the dea water medium. Current enters only at the growing rhizoid's tip while leaving both the base of the rhizoid cell and the whole outer membrane of the thallus cell. The field in front of the rhizoid cell falls off as the inverse cube of the distance from the rhizoid cell's center in the manner of a dipole field. The total inward and outward currents are equal, agreeing with theory. The current density at the rhizoid cell's base is twice that at the top of the thallus cell and this probably represents a change in the outer membrane's properties. There are no significant differences in the durrent density over the thallus cell. These results suggest a model in which the pulse current leaks in through newly opened channels in the growing tip and leaks out elsewhere due to the resultant fall in the membrane potential.     

Longitudinal variation in cadmium influx in sunflower (Helianthus annuus L.) roots as depending on the growth substrate,root age and root order

Aims

In a previous work, we observed a longitudinal decrease in Cd²⁺ influx starting from the root tip in first order lateral roots of sunflower (Helianthus annuus L.) grown in hydroponics. This variable influx was expected to impact the total Cd²⁺ uptake depending on the root system architecture and on how steep was the decrease of the influx. Here, we examined the influence of the culture substrate, of the age and order of lateral roots on the longitudinal variation of Cd²⁺ influx.

Methods

By using short-term exposures to ¹⁰⁹Cd-labelled solution (5 to 200 nM), we compared the longitudinal variations in Cd²⁺ roots influx depending on the growth substrate (hydroponics or sand), on the root age and order.

Results

In second order laterals, Cd²⁺ influx decreased from the apex to the root base, as for first order laterals. For sand cultures compared to hydroponics, the mean Cd²⁺ influx was lower and decreased more steeply with the distance from the apex. The influx also decreased with increasing root age and order, markedly in hydroponics but less for sand cultures.

Conclusion

Results suggested that for a given root surface area, the Cd²⁺ uptake by a root system should increase with increasing number of root tips and decreasing individual root length.     

Lateral diffusion anisotropy and membrane lipid/skeleton interaction in outer hair cells

The organization of the plasma membrane of cells in lipid domains affects the way the membrane interacts with the underlying protein skeleton, which in turn affects the lateral mobility of lipid and protein molecules in the membrane. Membrane fluidity properties can be monitored by various approaches, the most versatile of which is fluorescence recovery after photobleaching (FRAP). We extended previous FRAP experiments on isolated cochlear outer hair cells (OHCs) by analyzing the two-dimensional pattern of lipid diffusion in the lateral membrane of these cells. We found that membrane lipid mobility in freshly isolated OHCs is orthotropic, diffusion being faster in the axial direction of the cell and slower in the circumferential direction. Increasing the cell's turgor pressure by osmotic challenge reduced the axial diffusion constant, but had only a slight effect on circumferential diffusion. Our results suggest that lipid mobility in the OHC plasma membrane is affected by the presence of the cell's orthotropic membrane skeleton. This effect could reflect interaction with spectrin filaments or with other membrane skeletal proteins. We also performed a number of FRAP measurements in temporal bone preparations preserving the structural integrity of the hearing organ. The diffusion rates measured for OHCs in this preparation were in good agreement with those obtained in isolated OHCs, and comparable to the mobility rates measured on the sensory inner hair cells. These observations support the idea that the plasma membranes of both types of hair cells share similar highly fluid phases in the intact organ. Lipid mobility was significantly slower in the membranes of supporting cells of the organ of Corti, which could reflect differences in lipid phase or stronger hindrance by the cytoskeleton in these membranes.     

Ca2+-Activated Reactive Oxygen Species Production by Arabidopsis RbohH and RbohJ Is Essential for Proper Pollen Tube Tip Growth

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca²⁺ binding EF-hand motifs, possessed Ca²⁺-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca²⁺-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca²⁺-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca²⁺. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca²⁺-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.