Short-term boron deprivation inhibits endocytosis of cell wall pectins in meristematic cells of maize and wheat root apices

By using immunofluorescence microscopy, we observed rapidly altered distribution patterns of cell wall pectins in meristematic cells of maize (Zea mays) and wheat (Triticum aestivum) root apices. This response was shown for homogalacturonan pectins characterized by a low level (up to 40%) of methylesterification and for rhamnogalacturonan II pectins cross-linked by a borate diol diester. Under boron deprivation, abundance of these pectins rapidly increased in cell walls, whereas their internalization was inhibited, as evidenced by a reduced and even blocked accumulation of these cell wall pectins within brefeldin A-induced compartments. In contrast, root cells of species sensitive to the boron deprivation, like zucchini (Cucurbita pepo) and alfalfa (Medicago sativa), do not internalize cell wall pectins into brefeldin A compartments and do not show accumulation of pectins in their cell walls under boron deprivation. For maize and wheat root apices, we favor an apoplastic target for the primary action of boron deprivation, which signals deeper into the cell via endocytosis-mediated pectin signaling along putative cell wall-plasma membrane-cytoskeleton continuum.     

Osmotic induction of fluid-phase endocytosis in onion epidermal cells

A transient plasmolysis/deplasmolysis (plasmolytic cycle) of onion epidermal cells has been shown to induce the formation of fluid-phase endocytic vesicles. Plasmolysis in the presence of the membrane-impermeant fluorescent probes Lucifer Yellow CH (LYCH) and Cascade Blue hydrazide resulted in the uptake of these probes by fluid-phase endocytosis. Following deplasmolysis, many of the dye-containing vesicles left their parietal positions within the cell and underwent vigorous streaming in the cytoplasm. Vesicles were observed to move within transvacuolar strands and their movements were recorded over several hours by video-microscopy. Within 2 h of deplasmolysis several of the larger endocytic vesicles had clustered around the nuclear membrane, apparently lodged in the narrow zone of cytoplams surrounding the nucleus. In further experiments LYCH was endocytically loaded into the cells during the first plasmolytic cycle and Cascade Blue subsequently loaded during a second plasmolytic cycle. This resulted in the introduction of two populations of endocytic vesicles into the cells, each containing a different probe. Both sets of vesicles underwent cytoplasmic streaming. The data are discussed in the light of previous observations of fluid-phase endocytosis in plant cells.     

Tissue-specific subcellular immunolocalization of a myosin-like protein in maize root apices

Summary Using a heterologous myosin antibody raised against the whole molecule of bovine muscle myosin, we have identified a myosin-like protein in maize. Immunoblots of subcellular fractions isolated from roots identified one distinct band at about 210 kDa in the microsomal protein fraction and one band at about 180 kDa in the soluble protein fraction. Indirect immunofluorescence was performed using maize root apex sections to reveal endocellular distributions of the myosin-like protein. Both diffuse and particulate labelling patterns were observed throughout the cytoplasm of all root cells. In mitotic cells, myosin-like protein was excluded from spindle regions. Amyloplast surfaces were labelled prominently in cells of the root cap statenchyma and in all root cortex cells. On the other hand, myosin-like protein was prominently enriched at cellular peripheries in cells of the pericycle and outer stele in the form of continuous peripheral labelling. From all root apex tissues, phloem elements showed the most abundant presence of myosinlike protein.Abbreviations AFs actin filaments - MTs microtubules Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Pharmacological attenuation of Paramecium fluid-phase endocytosis

Spectrophotometric quantification of fluid phase endocytosis in the presence of different pharmacological compounds was performed in the model unicellular eukaryote Paramecium. The kinetics of Lucifer Yellow Carbohydrazide (LY) uptake in cells exposed to forskolin and isoproterenol--known to stimulate phagocytosis in this cell--was analyzed. Reduction in both the rate of endocytosis and total accumulation of fluid phase marker was observed following the treatment. Forskolin diminished total LY accumulation by 11% and 21% after 5 min and 25 min of incubation, respectively, whereas the rate of uptake was lowered by 21% in comparison to control cells. The inhibitory effect ofisoproterenol was less pronounced than that of forskolin. The total accumulation of LY was decreased by 11% in 5 min as compared to the untreated cells and this effect was persistent upon further exposition to this reagent up to 25 min. To better understand these observations, the effect of inhibitors of PKA and cAMP phosphodiesterase on fluid phase uptake was tested. 3-isobutyl-1-methyl xanthine (IBMX) caused 12% decrease in LY accumulation after 5 min of incubation. In combination with isoproterenol or forskolin, IBMX enhanced their inhibitory effect on fluid endocytosis, which was lowered by 25% and 29%, respectively. The strongest inhibitory effect on fluid endocytosis was exerted by the 10 microM PKA inhibitor, which diminished endocytosis by 35% in 5 min. These results suggest that Paramecium fluid phase uptake may be regulated through activation of PKA, although the precise mechanism of this process has not yet been elucidated.     

Mastoparan alters subcellular distribution of profilin and remodels F-actin cytoskeleton in cells of maize root apices

Indirect immunofluorescence localization of profilin in cells of maize root apices revealed that this abundant protein was present both in the cytoplasm and within nuclei. Nucleo-cytoplasmic partitioning of profilin exhibits tissue-specific and developmental features. Mastoparan-mediated activation of heterotrimeric G-proteins, presumably through triggering a phosphoinositide-signaling pathway based on phosphatidylinositol-4,5-bisphosphate (PIP(2)), induced relocalization of profilin from nuclei into the cytoplasm of root apex cells. In contrast, PIP(2) accumulated within nuclei of mastoparan-treated root cells. Intriguingly, cytoplasmic accumulation of profilin was associated with remodeling of F-actin arrays in root apex cells. Specifically, dense F-actin networks were dismantled and distinct actin patches became associated with the periphery of small vacuoles. On the other hand, disruption of F-actin with the G-actin sequestering agent latrunculin B does not affect the subcellular distribution of profilin or PIP(2). These data suggest that nuclear profilin can mediate a stimulus-response action on the actin cytoskeleton which is somehow linked to a phosphoinositide-signaling cascade.     

A multicompartmental model of fluid-phase endocytosis in rabbit liver parenchymal cells.

Fluid-phase endocytosis was studied in isolated rabbit liver parenchymal cells by using 125I-poly(vinylpyrrolidone) (PVP) as a marker. First, uptake of 125I-PVP by cells was determined. Also, cells were loaded with 125I-PVP for 20, 60 and 120 min, and release of marker was monitored for 120-220 min. Then we used the Simulation, Analysis and Modeling (SAAM) computer program and the technique of model-based compartmental analysis to develop a mechanistic model for fluid-phase endocytosis in these cells. To fit all data simultaneously, a model with three cellular compartments and one extracellular compartment was required. The three kinetically distinct cellular compartments are interpreted to represent (1) early endosomes, (2) a prelysosomal compartment equivalent to the compartment for uncoupling of receptor and ligand (CURL) and/or multivesicular bodies (MVB), and (3) lysosomes. The model predicts that approx. 80% of the internalized 125I-PVP was recycled to the medium from the early-endosome compartment. The apparent first-order rate constant for this recycling was 0.094 min-1, thus indicating that an average 125I-PVP molecule is recycled in 11 min. The model also predicts that recycling to the medium occurs from all three intracellular compartments. From the prelysosomal compartment, 40% of the 125I-PVP molecules are predicted to recycle to the medium and 60% are transferred to the lysosomal compartment. The average time for recycling from the prelysosomal compartment to the medium was estimated to be 66 min. For 125I-PVP in the lysosomal compartment, 0.3%/min was transferred back to the medium. These results, and the model developed to interpret the data, predict that there is extensive recycling of material endocytosed by fluid-phase endocytosis to the extracellular environment in rabbit liver parenchymal cells.     

Reactive oxygen species mediate shear stress-induced fluid-phase endocytosis in vascular endothelial cells

To elucidate the role of shear stress in fluid-phase endocytosis of vascular endothelial cells (EC), we used a rotating-disk shearing apparatus to investigate the effects of shear stress on the uptake of lucifer yellow (LY) by cultured bovine aortic endothelial cells (BAEC). Exposure of EC to shear stress (area-mean value of 10 dynes/cm2) caused an increase in LY uptake that was abrogated by the antioxidant, N-acetyl-L-cysteine (NAC), the NADPH oxidase inhibitor, acetovanillone, and two inhibitors of protein kinase C (PKC), calphostin C and GF109203X. These results suggest that fluid-phase endocytosis is regulated by both reactive oxygen species (ROS) and PKC. Shear stress increased both ROS production and PKC activity in EC, and the increase in ROS was unaffected by calphostin C or GF109203X, whereas the activation of PKC was reduced by NAC and acetovanillone. We conclude that shear stress-induced increase in fluid-phase endocytosis is mediated via ROS generation followed by PKC activation in EC.     

A comparative study of fluid-phase and adsorptive endocytosis of horseradish peroxidase in lymphoid cells

Adsorptive and fluid-phase endocytosis of horseradish peroxidase (PO) was studied in lymph node cells depleted of macrophages, taken from popliteal lymph nodes of rats immunized against PO (anti-PO cells) and against rabbit IgG (anti-rIgG cells) respectively. The enzymatic activity of PO enabled us to measure the amount of PO endocytosed in the cells and to determine its subcellular localization by means of light and electron microscopy. Uptake of PO by anti-PO cells was a saturable process which reached a plateau at approx. 50 μg/ml of PO. After exposure for 3 h to 50–100 μg/ml of PO, anti-PO cells had endocytosed 5–6 ng of PO per 10⁷ cells. Internalized PO was distributed in cells carrying surface receptors for PO, representing about 6% of the total cell population and consisting mainly of large immunocytes (lymphoblasts, plasma cells). Anti-rIgG cells cultured for 3 h with 100 μ/ml of PO endocytosed a very minute, barely detectable amount of PO. The fluid-phase endocytosis of PO was observed by increasing the PO concentration in the culture medium of anti-rIgG cells. Anti-rIgG cells cultured for 3 h with 500 μg/ml of PO endocytosed about 6 ng of PO/10⁷ cells, but no (or very few) stained cells were found. A large number of PO-internalizing anti-rlgG cells were observed only after culture with high PO concentrations (2 or 5 mg/ml), being both large immunocytes and small-to-medium lymphocytes. Endocytic sites of PO in anti-PO large immunocytes and in anti-rIgG small lymphocytes or large immunocytes were the same and consisted of vesicles, tubules or cisternae located near the Golgi apparatus and round or oval bodies scattered throughout the cytoplasm or localized near the Golgi apparatus (lysosomes?). After exposure to PO, anti-PO and anti-rIgG cells were transferred into PO-free medium. The level of intracellular peroxidase activity did not change during the first 6 h of culture. Then a decrease in enzymatic activity occurred, most probably due to a degradation of PO, at the same rate in anti-PO as in anti-rIgG cells. In conclusion, our results show that intracellular pathways of endocytosis and rates of inactivation of PO entering lymphoid cells are the same, whether specific receptors are present or not. This suggests that endocytosis of antigen in antigen-binding cells could reflect a native membrane recycling event.     

Architectural remodeling of the tonoplast during fluid-phase endocytosis

During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration.     

Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes

We have found that hypertonic medium inhibited the receptor-mediated uptake of the chemotactic peptide N-formylnorleucylleucylphenylalanine without affecting fluid-phase endocytosis by polymorphonuclear leukocytes (PMNs). Morphological and biochemical evidence demonstrated that cells in hypertonic medium did not accumulate peptide in a receptor-mediated manner. However, the cells continued to form endosomes containing fluid-phase markers. Furthermore, the content of these endosomes was processed normally, i.e., both digested and intact material were released into the medium. The inhibition of receptor- mediated uptake was a function of the tonicity. Partial inhibition occurred in 0.45 and 0.6 osmolar medium and maximal inhibition occurred in 0.75 osmolar medium. The inhibition was independent of the solute used to increase the tonicity: sodium chloride, sucrose, and lactose all inhibited uptake to similar extents. Hypertonic medium had little effect on saturable peptide binding. However, it did prevent the clustering of surface molecules as indicated by the inhibition of capping of fluorescent concanavalin A. In addition, hypertonic medium prevented the peptide-stimulated increase in cytosolic calcium levels as measured by quin 2 fluorescence. The tonicity dependence of the inhibition of quin 2 fluorescence paralleled the inhibition of receptor- mediated uptake.     

The study of the process of fluid-phase endocytosis in cervical squamous cells using fluorescent microspheres

Physiological processes in cervical squamous epithelium have not been extensively studied. Perhaps understandably, most of the research has concentrated on the pathology of the cervix, in particular dysplasia and malignancy. Fluid-phase endocytosis is a physiological process which has been demonstrated to be important in understanding disease development at other squamous epithelial sites, e.g. oesophagus. In this study, we have demonstrated by a new methodology developed in our laboratory using fluorescent microspheres and flow cytometry that fluid-phase endocytosis occurs in cervical squamous cells. The process has been shown to be dose- and time-dependent. This novel approach provides a means to improve our understanding of the physiological functions of the cervix and may provide insight into the pathogenesis of cervical neoplastic and non-neoplastic disease.     

Impaired secretion and fluid-phase endocytosis in the End4 mutant of Chinese hamster ovary cells

Mutant V.24.1 defines the End4 complementation group of temperature-sensitive Chinese hamster ovary cell mutants selected for resistance to protein toxins. We investigated the secretory pathway in the mutant cells and found: 1) The hemagglutinin of influenza virus failed to reach the plasma membrane and was retained in a form sensitive to endoglycosidase H at the restrictive temperature. 2) Transferrin receptors synthesized at the restrictive temperature remained sensitive to endoglycosidase H. 3) Secretion of total soluble protein into the medium was strongly reduced at high temperature. These data indicate that V.24.1 cells are defective in secretion at the restrictive temperature. To see what effect the lesion had on the endocytic pathway, we measured the accumulation and recycling of the fluid-phase marker horseradish peroxidase. Accumulation was inhibited by 50% while recycling was barely affected, suggesting that the rate of fluid-phase endocytosis was reduced. We previously showed that the clathrin-coated pit pathway of endocytosis was not affected in the mutant, indicated by a normal transferrin cycle (Colbaugh, P. A., Stookey, M., and Draper, R. K. (1989) J. Cell Biol. 108, 2211-2219). Thus, the secretory lesion correlates with reduced fluid-phase endocytosis without impairing the clathrin-dependent pathway of receptor-mediated endocytosis. We also investigated the delivery of endocytosed material to lysosomes and found that delivery was partially, but not completely, impaired in the mutant. This suggests that endocytosed material can enter lysosomes, although slowly, in the absence of a functional secretory pathway.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

Endocytosis in maize root cap cells

Summary Primary roots of maize seedlings have been treated with solutions of lanthanum and lead salts in an attempt to demonstrate endocytosis. Subsurface cells in the root cap reveal deposits of these heavy metals in coated pits in the plasma membrane and in coated vesicles. In addition lead deposits were observed in coated evaginations (pits) on large (secretory) vesicles present at the trans-pole of the Golgi apparatus and on small vacuoles. Lead was also found in the peripheral regions of individual cisternae throughout the dictyosomal stack. We interpret our results as providing evidence for coated pit/coated vesicle-mediated endocytosis and for the direct recycling of plasma membrane to the Golgi apparatus.     

Modeling the formation of root apices

The formation of new root apices from small groups of cells with different cellular patterns has been simulated using an existing model based on growth tensors. To generate an apex, a steady growth field was used. The pattern of cells evolved to approach the steady state. Two extreme types of progressions have been obtained : one leading to an apex with a single or a few apical cells, and the other to an apex with a quiescent centre. The change of structure while applying a steady growth tensor indicates that development may involve a succession of discrete growth tensors.     

Regions of differential cell elongation and mitosis, and root meristem morphology in different tissues of geotropically stimulated maize root apices

We examined cell length, mitosis, and root meristem “cuticle” in different tissues of geostimulated, red light-exposed primary roots of corn (Zea Mays, Wisconsin hybrid 64A × 22R). The examination was done at 15-minute intervals for a period of 240 minutes. Differences in cell elongation between the upper and lower sides were most prominent between 1.5 and 2.5 mm from the root meristem; the outer cortex had the greatest elongation growth, and the upper cells showed a significant increase in length compared to the lower. A differential mitosis was also found, with the lower tissue being greater. We infer that the mitotic activity is indicative of cell division, and this division occurs strictly in the first 1.5 mm of the root meristem. The combined effect of differential cell elongation and cell division results in the localization of the geotropic curvature in the 1.5- to 2.5-mm region from the root meristem. Mitosis that occurs primarily in the cortex and stele were asynchronous; the peak of cortical division preceded that of the stele. Both peaks occurred before the peak of geotropism. A densely stained layer separates the cap from the root meristem. This layer is thinner at the apex of the root meristem. The area of the thin region increased with time and peaked at 180 minutes after geostimulation, which was coincidental with the peak of the geotropic response.     

Mannitol-enhanced, fluid-phase endocytosis in storage parenchyma cells of celery (Apium graveolens; Apiaceae) petioles

We recently demonstrated the occurrence of a sucrose-enhanced, fluid-phase endocytic (FPE) mechanism of nutrient uptake in heterotrophic cells. In the present work, the possible enhancement/induction of FPE by photoassimilates other than sucrose was investigated by measuring the incorporation of the fluorescent endocytosis marker d-TR (dextran-Texas red, 3000 mw) into celery (Apium graveolens) petiole storage parenchyma (CSP), a tissue that transports and accumulates mannitol. Mannitol uptake in these cells is biphasic, with a hyperbolic phase at concentrations below 20 mM and a linear phase above 20 mM external solute concentration. In the absence of mannitol, or in its presence at concentrations within the hyperbolic phase, CSP cells accumulated low levels of d-TR. Conversely, d-TR accumulation by CSP cells was greatly enhanced in the presence of mannitol at concentrations within the linear phase. At high external mannitol concentration, d-TR accumulation was prevented by the endocytic inhibitors LY294002 and latrunculin B. In addition, d-TR uptake was temperature dependent under high mannitol concentration. Microscopic observations revealed that d-TR accumulated in the vacuole. These data support the occurrence of an FPE mechanism in CSP cells that participates in trapping and transport of photoassimilates to the vacuole. The FPE mechanism is enhanced by high mannitol concentrations.     

Reassessment of fluid-phase endocytosis and diacytosis in monolayer cultures of human fibroblasts

We have investigated the kinetics of fluid-phase endocytosis and diacytosis in confluent monolayers of human fibroblasts by comparing the behavior of three markers that have been previously used to study this process: [14C]sucrose, 125I-labeled polyvinylpyrrolidone ([125I]PVP), and Lucifer Yellow. Three distinct kinetic compartments were observed with all markers. The first was relatively large (10-60 fl/cell), reached steady state within 15 min at 37 degrees C, and was rapidly lost from monolayers after removing the markers at 37 degrees C but not at 0 degree C. These properties indicate that this compartment is the same as that previously proposed to be the major intracellular compartment involved in diacytosis. However, this compartment is probably extracellular fluid trapped between cells since it is rapidly lost into the medium when the cells are either scraped or enzymatically removed from the culture dishes at 0 degree C. In addition, it very slowly undergoes both filling and emptying at 0 degree C. However, we did observe a second, much smaller, kinetic compartment (approximately 2 fl/cell) undergoing rapid diacytosis that does seem to be intracellular. A third compartment that we observed accumulates markers at a linear rate (10-20 fl cell-1 hr-1) and is not lost from cells even after incubation periods greater than 6 hr. The markers [14C]sucrose and [125I]PVP displayed very similar behavior with respect to all three compartments and yielded nearly linear long-term uptake rates, thus indicating that there is little if any absorbed component in their uptake. However, Lucifer Yellow displayed significantly higher incorporation rates and its uptake rate was strongly nonlinear, indicating its uptake in fibroblasts is predominantly adsorptive. Our observations indicate that the rate of fluid-phase endocytosis in fibroblasts is significantly less than previously reported and that any compartment involved in diacytosis is very small and turns over very rapidly. Significantly, we estimate that the constitutive internalization of clathrin-coated pits is sufficient to account for the majority of fluid-phase endocytosis and thus represents a major mechanism of membrane retrieval in these cells.     

Short-term boron deprivation enhances levels of cytoskeletal proteins in maize, but not zucchini, root apices

By using indirect immunofluorescence microscopy and Western blot analysis, we have demonstrated increased levels of actin and tubulin proteins as well as an altered polymerization pattern of their cytoskeletal assemblies in maize, but not zucchini root apices, as a response to early boron deprivation. Northern blot analysis, however, did not show significant increases in the amount of steady-state mRNAs of actin and tubulin. This finding indicates that these rapid cytoskeletal responses to boron removal are very likely regulated at translational/post-translational levels. Interestingly, these increased levels of cytoskeletal proteins coincided well with a reduction in the water-extractable, but not with the cell wall-bound, fraction of boron. This implicates that free boric acid, or other more labile boron complexes, might be involved in the activation of cytoskeletal responses in maize root apices. In fact, our experimental approach revealed that maize was suffering from boron deprivation as early as zucchini. This was evidenced by its slightly reduced root elongation rate recorded within 3–5 h of boron deprivation. Importantly, however, maize roots can recover from this early inhibition indicating an effective adaptation mechanism. In contrast, zucchini roots apparently lack this boron-deprivation response pathway and suffer extensively when exposed to boron-free environment. This leads to the tentative working hypothesis for an adaptive mechanism of maize roots to boron deprivation by enhancing its cytoskeletal protein levels and altering their polymerization patterns in order to mechanically reinforce the cell periphery complex of their cells. This testable hypothesis requires further experimental verification.     

Transport and dynamics of molecules dissolved in maize root cortex membranes

Translational diffusion of a fluorescent sterol probe was measured in the plasma membranes of protoplasts isolated from cortical cells of the primary root of maize seedlings. The apparent lateral diffusion coefficient was typically observed to be nearly insensitive to temperature, while the mobile fraction increased with increasing temperature. These fluorescence photobleaching recovery (FPR) measurements were compared with the electron paramagnetic resonance (EPR) spectra of the methyl ester of 13-doxyl palmitic acid in membranes of corn root tissue in situ. The complex spectra observed with this probe were analyzed as weighted sums of simpler spectra of various order parameters and rotational correlation times. The reconstituted spectra calculated from the model show that EPR also detects a mobile (less ordered, fluid) fraction, distinguished by the order parameter S=0.1 to 0.2, which becomes more abundant as temperature increases and is qualitatively comparable to the mobile fraction determined by the FPR method. The observed results on the mobile fractions and the diffusion rates for translational (FPR) as well as rotational (EPR) motions are interpreted in terms of membrane organization, thus providing information on the population and structural patterns of the coexisting domains with a special emphasis on the response of the membrane to temperature changes.This work was supported in part by grants from the Ministry of Science and Technology of the Republic of Slovenia and the International Research Program of the U.S. Department of Agriculture (USDA-JF 814-51) to M.S., and by grants from the Competitive Grants Program of the U.S. Department of Agriculture (88-37264-3807 and 90-37264-5471) to E.A.N.