The Role of Water in Fast Plant Movements.

Plants moved onto land ∼450 million years ago and faced their biggest challenge: living in a dry environment. Over the millennia plants have become masters of regulating water flow and the toolkit they have developed has been co-opted to effect rapid movements. Since plants are rooted, these fast movements are used to disperse reproductive propagules including spores, pollen, and seeds. We compare five plants to demonstrate three ways, used alone or in combination, that water powers rapid movements: the direct capture of the kinetic energy of a falling raindrop propels gemmae from the splash cups of the liverwort, Marchantia; the loss of water powers the explosive dispersal of the spores of Sphagnum moss; the alternate loss and gain of water in the bilayer of the elaters of Equisetum drive the walk, jump, and glide of spores; the gain of water in the inner layer of the arils of Oxalis drive the eversion of the aril that jettisons seeds from the capsule; and the buildup of turgor pressure in the petals and stamens of bunchberry dogwood (Cornus canadensis) explosively propels pollen. Each method is accompanied by morphological features, which facilitate water movement as a power source. The urn shaped splash cups of Marchantia allow dispersal of gemmae by multiple splashes. The air gun design of Sphagnum capsules results in a symmetrical impulse creating a vortex ring of spores. The elaters of Equisetum can unfurl while they are dropping from the plant, so that they capture updrafts and glide to new sites. The arils of Oxalis are designed like miniature toy "poppers." Finally, in bunchberry, the softening of stamen filament tissue where it attaches to the anther allows them to function as miniature hinged catapults or trebuchets.

Differential effects of methyl jasmonate on growth and division of etiolated zucchini cotyledons.

The jasmonates are well studied in the context of plant defence but increasingly are also recognised as playing roles in development. In many systems, jasmonates antagonise the effects of cytokinins. The aim of the present work was to elucidate interactions between methyl jasmonate and cytokinin (benzyladenine) in regulating growth of zucchini (Cucurbita pepo L., cv. Cocozelle, var. Tripolis) cotyledons, taking advantage of the ability to simultaneously quantify cell enlargement and division from paradermal sections of the first palisade layer. Growth regulators were applied to cotyledons, excised from dry seeds and grown in darkness. Cytokinin stimulated expansion and division whereas, surprisingly, jasmonate stimulated expansion but inhibited division. Jasmonate antagonised the stimulating effect of cytokinin on division but worked cooperatively with cytokinin in increasing expansion. However, expansion with jasmonate was more isotropic than with cytokinin. Jasmonate also stimulated the loss of cellular inclusions and soluble protein. Soluble proteins revealed a partial antagonism between jasmonate and cytokinin. These results illustrate the complex interplay between jasmonates and cytokinin in the regulatory network of cotyledon development following germination.

Morphology of rsw1, a cellulose-deficient mutant of Arabidopsis thaliana.

The rsw1 mutant of Arabidopsis thaliana is mutated in a gene encoding a cellulose synthase catalytic subunit. Mutant seedlings produce almost as much cellulose as the wild type at 21 degrees C but only about half as much as the wild type at 31 degrees C. We used this conditional phenotype to investigate how reduced cellulose production affects growth and morphogenesis in various parts of the plant. Roots swell in all tissues at 31 degrees C, and temperature changes can repeatedly switch them between swollen and slender growth patterns. Dark-grown hypocotyls also swell, whereas cotyledons and rosette leaf blades are smaller, their surfaces are more irregular and their petioles shorter. Leaf trichomes swell and branch abnormally. Plants readily initiate inflorescences at 31 degrees C which have shorter but not fatter bolts and stomata which bulge above the uneven surface of internodes. Bolts carry the normal number of flowers, but their stigmas protrude beyond the shortened sepals and petals. Anthers dehisce normally, but self-fertilisation is reduced because the stigma is well above the anthers. Anther filaments are short and show a crumpled surface. Viable pollen develops, but female reproductive competence and postpollination development are severely impaired. We conclude that the RSW1 gene is important for cellulose synthesis in many parts of the plant and that reduced cellulose synthesis suppresses organ expansion rather than organ initiation, causes radial swelling only in the root and hypocotyl, but makes the surfaces of many organs uneven. We discuss some possible reasons to explain why different organs vary in their responses. The morphological changes suggest that RSW1 contributes cellulose to primary walls but do not yet exclude a role during secondary-wall deposition.

On the alignment of cellulose microfibrils by cortical microtubules: a review and a model.

The hypothesis that microtubules align microfibrils, termed the alignment hypothesis, states that there is a causal link between the orientation of cortical microtubules and the orientation of nascent microfibrils. I have assessed the generality of this hypothesis by reviewing what is known about the relation between microtubules and microfibrils in a wide group of examples: in algae of the family Characeae, Closterium acerosum, Oocystis solitaria, and certain genera of green coenocytes and in land plant tip-growing cells, xylem, diffusely growing cells, and protoplasts. The salient features about microfibril alignment to emerge are as follows. Cellulose microfibrils can be aligned by cortical microtubules, thus supporting the alignment hypothesis. Alignment of microfibrils can occur independently of microtubules, showing that an alternative to the alignment hypothesis must exist. Microfibril organization is often random, suggesting that self-assembly is insufficient. Microfibril organization differs on different faces of the same cell, suggesting that microfibrils are aligned locally, not with respect to the entire cell. Nascent microfibrils appear to associate tightly with the plasma membrane. To account for these observations, I present a model that posits alignment to be mediated through binding the nascent microfibril. The model, termed templated incorporation, postulates that the nascent microfibril is incorporated into the cell wall by binding to a scaffold that is oriented; further, the scaffold is built and oriented around either already incorporated microfibrils or plasma membrane proteins, or both. The role of cortical microtubules is to bind and orient components of the scaffold at the plasma membrane. In this way, spatial information to align the microfibrils may come from either the cell wall or the cell interior, and microfibril alignment with and without microtubules are subsets of a single mechanism.

Temperature-sensitive alleles of RSW2 link the KORRIGAN endo-1,4-beta-glucanase to cellulose synthesis and cytokinesis in Arabidopsis.

An 8.5-kb cosmid containing the KORRIGAN gene complements the cellulose-deficient rsw2-1 mutant of Arabidopsis. Three temperature-sensitive alleles of rsw2 show single amino acid mutations in the putative endo-1,4-beta-glucanase encoded by KOR. The F1 from crosses between kor-1 and rsw2 alleles shows a weak, temperature-sensitive root phenotype. The shoots of rsw2-1 seedlings produce less cellulose and accumulate a short chain, readily extractable glucan resembling that reported for rsw1 (which is defective in a putative glycosyltransferase required for cellulose synthesis). The double mutant (rsw2-1 rsw1) shows further reductions in cellulose production relative to both single mutants, constitutively slow root growth, and enhanced temperature-sensitive responses that are typically more severe than in either single mutant. Abnormal cytokinesis and severely reduced birefringent retardation in elongating root cell walls of rsw2 link the enzyme to cellulose production for primary cell walls and probably cell plates. The Rsw2(-) phenotype generally resembles the Kor(-) and cellulose-deficient Rsw1(-) phenotypes, but anther dehiscence is impaired in Rsw2-1(-). The findings link a second putative enzyme activity to cellulose synthesis in primary cell walls of Arabidopsis and further increases the parallels to cellulose synthesis in Agrobacterium tumefaciens where the celA and celC genes are required and encode a putative glycosyltransferase and an endo-1,4-beta-glucanase related to RSW1 and KOR, respectively.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

Stunted plant 1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis.

Plants control organ growth rate by adjusting the rate and duration of cell division and expansion. Surprisingly, there have been few studies where both parameters have been measured in the same material, and thus we have little understanding of how division and expansion are regulated interdependently. We have investigated this regulation in the root meristem of the stunted plant 1 (stp1) mutation of Arabidopsis, the roots of which elongate more slowly than those of the wild type and fail to accelerate. We used a kinematic method to quantify the spatial distribution of the rate and extent of cell division and expansion, and we compared stp1 with wild type and with wild type treated with exogenous cytokinin (1 microM zeatin) or auxin (30 nM 2,4-dichlorophenoxyacetic acid). All treatments reduced average cell division rates, which reduced cell production by the meristem. Auxin lowered root elongation by narrowing the elongation zone and reducing the time spent by a cell in this zone, but did not decrease maximal strain rate. In addition, auxin increased the length of the meristem. In contrast, cytokinin reduced root elongation by lowering maximal strain rate, but did not change the time spent by a cell within the elongation zone; also, cytokinin blocked the increase in length and cell number of the meristem and elongation zone. The cytokinin-treated wild type phenocopied stp1 in nearly every detail, supporting the hypothesis that cytokinin affects root growth via STP1. The opposite effects of auxin and cytokinin suggest that the balance of these hormones may control the size of the meristem.

On the constancy of cell division rate in the root meristem.

This review examines under what circumstances the rate of cell division among cells of the root meristem is known to vary. First, methods are compared that have been used to quantify cell division rate. These can be grouped as being either cytological, in which the rate of accumulation of cells in a particular phase of the cell cycle is determined based on some form of cytological labeling, or kinematic, in which the rate of cell accumulation is determined from the net movement of cells. Then, evidence is reviewed as to whether cell division rates vary between different tissues or cell types, between different positions in the root, or finally between different environments. The evidence is consistent with cells dividing at a constant rate, and well documented examples where cell division rate changes substantially are rare. The constancy of cell division rate contrasts with the number of dividing cells, which varies extensively, and implies that a major point for cell cycle control is governing the exit from the proliferative state at the basal boundary of the meristem.

Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient-agar media.

We have characterized the growth responses of Arabidopsis thaliana seedlings to water deficit. To manipulate the water potential, we developed a method whereby the nutrient-agar medium could be supplemented with polyethylene glycol (PEG 8000); PEG was introduced into gelled media by diffusion, which produced media with water potential as low as -1.6 MPa. For dark-grown plants, hypocotyl growth had a hyperbolic dependence on water potential, and was virtually stopped by -1 MPa. In contrast, primary root elongation was stimulated by moderate deficit and even at -1.6 MPa was not significantly less than the control. That these results did not depend on a direct effect of PEG was attested by obtaining indistinguishable results when a dialysis membrane impermeable to PEG was placed between the medium and the seedlings. For light-grown seedlings, moderate deficit also stimulated primary root elongation and severe deficit reduced elongation only partially. These changes in elongation were paralleled by changes in root system dry weight. At moderate deficit, lateral root elongation and initiation were unaffected and at higher stress levels both were inhibited. Primary root diameter increased steadily with time in well-watered controls and under water deficit increased transiently before stabilizing at a diameter that was inversely proportional to the deficit. Along with stimulated primary root elongation, moderate water deficit also stimulated the rate of cell production. Thus, A. thaliana responds to water deficit vigorously, which enhances its use as a model to uncover mechanisms underlying plant responses to water deficit.

The Arabidopsis thaliana PPX/PP4 phosphatases: molecular cloning and structural organization of the genes and immunolocalization of the proteins to plastids.

The PPX/PP4 Ser/Thr protein phosphatases belong to the type 2A phosphatase subfamily and are present in most eukaryotic organisms. We have previously isolated two closely related DNAs encoding PPX isoforms (PPX-1 and PPX-2) of Arabidopsis thaliana. Here we report the molecular cloning of the genes encoding these proteins. The genes PPX-1 and PPX-2 are composed of eight exons and seven introns located at equivalent positions related to the coding sequences. Whereas the intron-exon organization of the PPX genes is completely different from that of the PP2A-3/PP2A-4 A. thaliana family, specific intron-exon boundaries are conserved among PPX genes from distantly related organisms. Based on GUS expression, both PPX genes show the same spatial and temporal pattern of expression: they are expressed in all the organs and tissues analyzed, and from the earliest stage of development. When PPX proteins were localized to the root in semi-thin methacrylate sections by immunofluorescence, staining was predominantly confined to small organelles, shown to be plastids by co-localization of PPX and ferredoxin. Interestingly, only some ferredoxin-positive plastids were also PPX-positive, and PPX staining was consistently brighter in the epidermis. The localization was confirmed with immunogold and electron microscopy. Our results suggest that, despite its strong sequence conservation, PPX in plants functions differently than in animals.

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 degreesC 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.

Inhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules.

To investigate molecular mechanisms controlling plant morphogenesis, we examined the morphology of primary roots of Arabidopsis thaliana and the organization of cortical microtubules in response to inhibitors of serine/threonine protein phosphatases and kinases. We found that cantharidin, an inhibitor of types 1 and 2A protein phosphatases, as previously reported for okadaic acid and calyculin A (R.D. Smith, J.E. Wilson, J.C. Walker, T.I. Baskin [1994] Planta 194: 516-524), inhibited elongation and stimulated radial expansion. Of the protein kinase inhibitors tested, chelerythrine, 6-dimethylaminopurine, H-89, K252a, ML-9, and staurosporine all inhibited elongation, but only staurosporine appreciably stimulated radial expansion. To determine the basis for the root swelling, we examined cortical microtubules in semithin sections of material embedded in butyl-methyl-methacrylate. Chelerythrine and 100 nM okadaic acid, which inhibited elongation without causing swelling, did not change the appearance of cortical arrays, but calyculin A, cantharidin, and staurosporine, which caused swelling, disorganized cortical microtubules. The stability of the microtubules in the aberrant arrays was not detectably different from those in control arrays, as judged by similar sensitivity to depolymerization by cold or oryzalin. These results identify protein phosphorylation and dephosphorylation as requirements in one or more steps that organize the cortical array of microtubules.

Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots (I. Spatial Distribution of Longitudinal, Radial, and Tangential Expansion Rates).

As a system to study the regulation of growth anisotropy, we studied thinning of the primary root of maize (Zea mays L.) occurring developmentally or induced by water stress. Seedlings were transplanted into vermiculite at a water potential of approximately -0.03 MPa (well-watered) or -1.6 MPa (water-stressed). The diameter of roots in both treatments decreased with time after transplanting; the water-stressed roots became substantially thinner than well-watered roots at steady state, showing that root thinning is a genuine response to water stress. To analyze the thinning responses we quantified cell numbers and the spatial profiles of longitudinal, radial, and tangential expansion rates separately for the cortex and stele. The results showed that there was no zone of isotropic expansion and the degree of anisotropy varied greatly with position and treatment. Thinning over time in well-watered roots was caused by rates of radial and tangential expansion being too low to maintain the shape of the root. In response to low water potential, cell number in both tissues was unchanged radially but increased tangentially, which shows that thinning was caused wholly by reduced cell expansion. Water stress substantially decreased rates of tangential and radial expansion in both the stele and cortex, but only in the apical 5 mm of the root; basal to this, rates were similar in well-watered and water-stressed roots. By contrast, as in previous studies, longitudinal expansion was identical between the treatments in the apical 3 mm but in water-stressed roots was inhibited at more basal locations. The results show that expansion in longitudinal and radial directions can be regulated independently.

Association of Plant p40 Protein with Ribosomes Is Enhanced When Polyribosomes Form during Periods of Active Tissue Growth.

p40s are acidic proteins of eukaryotic cells occurring either free in the cytoplasm or in association with ribosomes, the latter occurring in both monosomes and polysomes. p40s may play a role in the regulation of protein synthesis, although the exact mechanism is not known. Leaves of all 10 plant species examined here, including both monocots and dicots, contained proteins detected on immunoblots with Arabidopsis thaliana p40 antiserum. The number and apparent size of the protein bands were variable even among closely related species. Abundance of p40 relative to ribosomal content during soybean (Glycine max L.) seed germination and during seed and leaf development was examined. p40 abundance correlated with periods of active tissue growth and high polysome content. The plant growth regulator indole acetic acid caused an increase in polysome formation in etiolated pea (Pisum sativum L.) plants and a concomitant recruitment of p40 into polysomes. Subcellular localization at the microscopy level indicated that the pattern of p40 staining is very similar to that for RNA, except that p40 is excluded from the nucleus. These data suggest that p40 is an accessory protein of the ribosome that might play a role in plant growth and development.

Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy.

Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze-substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells. Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in plunge-frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge-frozen roots also had cells with more life-like cytoplasm than those of chemically fixed roots, as assessed with toluidine-blue staining or high-resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40-75 microns from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.

STUNTED PLANT 1, A Gene Required for Expansion in Rapidly Elongating but Not in Dividing Cells and Mediating Root Growth Responses to Applied Cytokinin.

To understand the control of spatial patterns of expansion, we have studied root growth in wild type and in the stunted plant 1 mutant, stp1, of Arabidopsis thaliana. We measured profiles of cell length and calculated the distribution of elongation rate. Slow growth of stp1 results both from a failure of dividing cell number to increase and from low elongation rates in the zone of rapid expansion. However, elongation of dividing cells was not greatly affected, and stp1 and wild-type callus grew at identical rates. Thus, rapid cellular expansion differs in mechanism from expansion in dividing cells and is facilitated by the STP1 gene. Additionally, there was no difference between stp1 and wild-type roots for elongation in response to abscisic acid, auxin, ethylene, or gibberellic acid or for radial expansion in response to ethylene; however, stp1 responded to cytokinin much less than wild type. In contrast, both genotypes responded comparably to hormones when explants were cultured; in particular, there was no difference between genotypes in shoot regeneration in response to cytokinin. Thus, effects on root expansion mediated by cytokinin, but not effects mediated by other hormones or effects on other cytokinin-mediated responses, require the STP1 locus.

Stimulation of radial expansion in arabidopsis roots by inhibitors of actomyosin and vesicle secretion but not by various inhibitors of metabolism.

Plant morphogenesis depends on accurate control over growth anisotropy. To learn to what extent the control of growth anisotropy depends on cellular metabolism, we surveyed the response of growing roots to a range of inhibitors. Seedlings of Arabidopsis thaliana L. (Heynh), 7-8 d old, were transplanted onto plates containing an inhibitor, and elongation and radial expansion of roots were measured over the subsequent 2-d period. Fourteen inhibitors of diverse metabolic processes inhibited root elongation but failed to stimulate radial expansion. These inhibitors were aluminum sulfate, aphidicolin (DNA synthesis), caffeine (cell-plate formation), cisplatin (DNA synthesis), cycloheximide (protein synthesis), 3,4-dehydro-L-proline (proline hydroxylation), 6-dimethylaminopurine (protein kinases), dinitrophenol (mitochondrial ATP synthesis), galactose (UDP-glucose formation), Lovastatin, formerly mevinolin (isoprenoid formation), methionine sulfoximine (glutamine synthetase), methotrexate (folate metabolism), XRD-489 (synthesis of branched-chain amino acids), and high or low calcium treatments. These results show that various types of metabolic disruption, although inhibitory to elongation, do not reduce the high degree of anisotropic growth of the root. However, five chemicals did stimulate radial expansion; namely, the detergent, digitonin; two inhibitors of vesicle secretion, monensin and brefeldin A; and two inhibitors of actomyosin, cytochalasin B and butanedione monoxime. The maximum radial expansion induced by these compounds (except butanedione monoxime) was greater than that caused by ethylene, and the morphology of treated roots did not resemble that of roots treated with ethylene. These results indicate that vesicle secretion and actomyosin play a role in controlling anisotropic expansion.

Morphology and microtubule organization in Arabidopsis roots exposed to oryzalin or taxol.

In roots of Arabidopsis thaliana, we examined the effects of low concentrations of microtubule inhibitors on the polarity of growth and on the organization of microtubule arrays. Intact 6 d old seedlings were transplanted onto plates containing inhibitors, and sampled 12 h, 24 h and 48 h later. Oryzalin, a compound that causes microtubule depolymerization, stimulates the radial expansion of roots. The amount of radial swelling is linearly proportional to the logarithm of the oryzalin concentration, from the response threshold, 170 nM, to 1 microM. Cells in the zone of division were slightly more sensitive to oryzalin than were cells in the zone of pure elongation. Radial swelling is also stimulated by taxol, a compound that causes microtubule polymerization. Taxol at 1 microM causes little swelling, but at 10 microM causes extensive radial swelling of cells in the elongation zone, and does not affect cells in the division zone. To examine the microtubules in these roots, we used methacrylate sections with immunofluorescence microscopy. At all concentrations of oryzalin, cortical arrays are disorganized and depleted of microtubules, and the microtubules themselves often appear fragmented. These effects increase in severity with concentration, but are unmistakable at 170 nM. In taxol, cortical arrays appear to be more intensely stained than those of controls. At 10 microM, many cells in growing regions of the stele have longitudinal microtubules, whereas many cells in the cortex appear to have transversely aligned microtubules. Taxol affects microtubules in cells of division and elongation zones to the same extent, despite the observed difference in growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa.

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5-2 µm) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(ß-aminoethyl ether)N,N,N',N'-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.