Nuclear centering in Spirogyra: force integration by microfilaments along microtubules

The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated in the charophycean green alga Spirogyracrassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and a web of F-actin. Bundles of microtubules, emerging from the nuclear rim, are organized into dividing fascicles within the strands and reach to the inner surface of the chloroplast envelope. Organelles are translocated in both directions along similarly arranged fascicles of microfilament bundles which extend from the nucleus to the peripheral actin cytoskeleton. Application of microtubule- and/or microfilament-depolymerizing drugs affected the position of the nucleus only slowly, but in distinct ways. The differential effects suggest that nuclear centering depends on the tensional integrity of the perinuclear scaffold, with microfilaments conveying tension along stabilized microtubules and the actin cytoskeleton integrating the translocation forces generated within the scaffold. Received: 9 December 1996 / Accepted: 29 April 1997     

Disruption of cellulose synthesis by isoxaben causes tip swelling and disorganizes cortical microtubules in elongating conifer pollen tubes

Summary.  In elongating pollen tubes of the conifer Picea abies (Norway spruce), microtubules form a radial array beneath the plasma membrane only at the elongating tip and an array parallel with elongation throughout the tube. Tips specifically swell following microtubule disruption. Here we test whether these radial microtubules coordinate cell wall deposition and maintain tip integrity as tubes elongate. Control pollen tubes contain cellulose throughout the walls, including the tip. Pollen tubes grown in the presence of isoxaben, which disrupts cellulose synthesis, are significantly shorter with a decrease in cellulose throughout the walls. Isoxaben also significantly increases the frequency of tip swelling, with no effect on tube width outside of the swollen tip. The decrease in cellulose is more pronounced in pollen tubes with swollen tips. The effects of isoxaben are reversible. Following isoxaben treatment, the radial array of microtubules persists beneath the plasma membrane of nonswollen tips, while this array is specifically disrupted in swollen tips. Microtubules instead form a random network throughout the tip. Growth in these pollen tubes is turgor driven, but the morphological changes due to isoxaben are not just the result of weakened cell walls since pollen tubes grown in hypoosmotic media are not significantly shorter but do have swollen tips and tubes are wider along their entire length. We conclude that the radial microtubules in the tip do maintain tip integrity and that the specific inhibition of cellulose microfibril deposition leads to the disorganization of these microtubules. This supports the emerging model that there is bidirectional communication across the plasma membrane between cortical microtubules and cellulose microfibrils. Received January 15, 2002; accepted August 3, 2002; published online March 11, 2003     

A stochastic model of brain cell differentiation in tissue culture

 The timing of cell differentiation can be controlled both by cell-intrinsic mechanisms and by cell-extrinsic signals. Oligodendrocyte type-2 astrocyte progenitor cells are known to be the precursor cells that give rise to oligodendrocytes. When stimulated to divide by purifed cortical astrocytes or by platelet-derived growth factor, these progenitor cells generate oligodendrocytes in vitro with a timing like that observed in vivo. The most widely accepted model of this process assumes a cell-intrinsic biological clock that resides in the progenitor cell. The intrinsic clock model originally proposed in 1986 remains as the dominant theoretical concept for the analysis of timed differentiation in this cell lineage. However, the results of a recent experimental study (Ibarrola et al., Developmental Biology, vol. 180, 1–21, 1996) are most consistent with the hypothesis that the propensity of a clone of dividing O-2A progenitor cells initially to generate at least one oligodendrocyte may be regulated by cell-intrinsic mechanisms, but that environmental signals regulate the extent of further oligodendrocyte generation. We propose a stochastic model of cell differentiation in culture to accommodate the most recent experimental findings. Our model is an age-dependent branching stochastic process with two types of cells. The model makes it possible to derive analytical expressions for the expected number of progenitor cells and of oligodendrocytes as functions of time. The model parameters were estimated by fitting these functions through data on the average (sample mean) number of both types of cells per colony at different time intervals from start of experiment. Using this method we provide a biologically meaningful interpretation of the observed pattern of oligodendrocyte generation in vitro and its modification in the presence of thyroid hormone. Received: 18 April 1997 / Revised version: 30 November 1997     

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: I. The effect of cadmium

Summary. The aim of the study was to elucidate the effect of cadmium ions on the arrangement of the actin and tubulin cytoskeleton, as well as the relationships between cytoskeletal changes and growth processes in the green filamentous alga Spirogyra decimina. Batch cultures of algae were carried out under defined conditions in the presence of various cadmium concentrations. In control cells, the cytoskeleton appeared to be a transversely oriented pattern of both microtubules and actin filaments of various thickness in the cell cortex; colocalization of cortical microtubules and actin filaments was apparent. Microtubules were very sensitive to the presence of cadmium ions. Depending on the cadmium concentration and the time of exposure, microtubules disintegrated into short rod-shaped fragments or they completely disappeared. A steep increase in cell width and a decrease in growth rate accompanied (and probably ensued) a very rapid disintegration of microtubules. Actin filaments were more stable because they were disturbed several hours later than microtubules at any cadmium concentration used. When cadmium ions were washed out, the actin cytoskeleton was rebuilt even in cells in which actin filaments were completely disintegrated at higher cadmium concentrations (40 or 100 μM). The much more sensitive microtubules were regenerated after treatment with lower cadmium concentrations (10 or 15 μM) only. Correspondence and reprints: Centre of Phycology, Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, 379 82 Třeboň, Czech Republic.     

Multiple activities of a novel substance,dictyopyrone C isolated from Dictyostelium discoideum,in cellular growth and differentiation

Summary.  We report that a novel substance named dictyopyrone C (DPC) has remarkable effects on growth and differentiation of Dictyostelium discoideum Ax-2 cells, in a dose-dependent manner. In the presence of 3–15 μM DPC, differentiation of starving Ax-2 (clone MS) cells was greatly enhanced in submerged culture, when vegetative MS cells were harvested at the mid-late-exponential growth phase (>3 × 10⁶ cells per ml) and starved. In contrast, DPC above 30 μM markedly impaired the progression of differentiation including cell aggregation, most of starved cells being round after 3–4 h of DPC application and then lysed during further incubation. In the presence of 30 μM DPC however, MS cells that had been harvested at the early exponential growth phase (<5 × 10⁵ cells per ml) and starved became neither round nor lysed and exhibited rather enhanced differentiation. Essentially the same results were obtained in cultures of starved cells on nonnutrient agar. With respect to the DPC effect on MS cells growing in axenic medium, cell lysis and growth inhibition by DPC at concentrations higher than 15 μM were realized in the mid-late-exponential-growth-phase cells (>3 × 10⁶ cells per ml) but not in the early-exponential-growth-phase cells (<5 × 10⁵ cells per ml). Moreover, analysis using synchronized MS cells has demonstrated that the DPC effect changes in a cell-cycle-dependent manner. In contrast to such unique DPC actions, the pyrone ring of DPC had no effects on growth and differentiation within the range of 3–120 μM tested. These findings strongly suggested the importance of the combined structure of the pyrone ring and the linear carbon chain in revelation of the DPC activities. Received August 5, 2002; accepted November 11, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan. E-mail: ymaeda@mail.cc.tohoku.ac.jp     

Chromosomal differentiation and genome size in three European mountain Lilium species

 Three related and taxonomically close species of the genus Lilium (L. pyrenaicum Gouan, L. pomponium L. and L. carniolicum Bernh.), all of them with 2n=24 chromosomes, have been studied for chromosomal differentiation, using fluorochrome banding and fluorescence in situhybridization (FISH), and for genome size and GC percentage using flow cytometry. The total DNA content of L. pomponium (2C=70.26 pg) was about 5% higher than that of L. pyrenaicum (2C=67.74) and L. carniolicum (2C=67.37 pg), while GC percentage was higher in this last species (36.60%) than in L. pomponium (35.56%) and lower than in L. pyrenaicum (37.92%). Silver staining, fluorochrome banding with chromomycin A₃ (CMA) and fluorescence in situ hybridization (FISH) clearly pointed out the number of nucleoli, the number and position of GC-rich bands and the number and location of rDNA sites thus permitting distinction of the three species at chromosomal level. Two families of ribosomal genes, 18S-5.8S-26S (18S) and 5S rRNA genes, were separated onto different pairs in chromosome complements of examined species. Chromosome regions containing both kinds of rRNA genes were also GC-rich regions. The results revealed a clear interspecific differentiation at the chromosomal level and permitted the discussion about relationships among the species. Received June 21, 2002; accepted October 4, 2002 Published online: Febraury 7, 2003     

The interaction of thin-film flow, bacterial swarming and cell differentiation in colonies of Serratia liquefaciens

 The rate of expansion of bacterial colonies of S. liquefaciens is investigated in terms of a mathematical model that combines biological as well as hydrodynamic processes. The relative importance of cell differentiation and production of an extracellular wetting agent to bacterial swarming is explored using a continuum representation. The model incorporates aspects of thin film flow with variable suspension viscosity, wetting, and cell differentiation. Experimental evidence suggests that the bacterial colony is highly sensitive to its environment and that a variety of mechanisms are exploited in order to proliferate on a variety of surfaces. It is found that a combination of effects are required to reproduce the variation of bacterial colony motility over a large range of nutrient availability and medium hardness. Received: 29 April 1999     

The SCHIZOID gene regulates differentiation and cell division in Arabidopsis thaliana shoots

 Cell division and cell differentiation are key processes in shoot development. The Arabidopsis thaliana (L.) Heynh. SCHIZOID (SHZ) gene appears to influence cell differentiation and cell division in the shoot. The shz-2 mutant is notable in that distinct phenotypes develop, depending on the environment in which the plants are grown. When shz-2 mutants are grown in petri dishes, callus develops from the petiole and hypocotyl. In contrast, when the mutants are grown on soil, shoots appear externally stunted with malformed leaves. However, detailed examination of soil-grown mutants shows that the two phenotypes are related. Soil-grown mutants form adventitious meristems, produce a large amount of vascular tissues and have aberrant cell divisions in the meristem. Cells with abnormal cell-division patterns were found in the apical and vascular meristems, suggesting SHZ influences cell division. Development of callus in petri dishes, development of adventitious meristems and aberrations in leaves on soil suggest that SHZ influences cell differentiation. The distinct, but related phenotypes on soil and in petri dishes suggests that SHZ normally functions to regulate differentiation and/or cell division in a manner that is responsive to environmental conditions. Received: 30 July 1999 / Accepted: 22 September 1999     

Phytochrome-dependent modulation and re-induction of growth of the first rhizoid in Dryopteris paleacea Sw

Phytochrome-dependent growth in Dryopteris paleacea Sw. was investigated in young, developing gametophytes with respect to formation and differentiation of rhizoids. Under continuous red light (Rc), the first rhizoids grew synchronously by tip elongation at a constant rate of 240 μm · d⁻¹ until formation and outgrowth of the second rhizoid. Cessation of growth of the first rhizoids and outgrowth of the second rhizoids showed a correlation in time assumed to be mediated by intercellular signaling. The first rhizoids showed two modes of response to actinic irradiations: (i) modulation of rhizoid growth, and (ii) re-induction of growth in non-growing rhizoids. In the former, the promotory effect of actinic irradiations on rhizoids pre-cultured under Rc determined both the time for which rhizoids continued to grow after transfer into darkness and the final rhizoid length. In the latter, re-induced growth was studied using non-growing rhizoids which were obtained after irradiation with a far-red light (FR) pulse at the end of the pre-culture in Rc and transfer into darkness for 3 d to stop growth. Re-induction of growth occurred with a lag phase of 36 to 48 h after formation of the FR-absorbing form of phytochrome (Pfr) by a red light (R) pulse. From the incomplete R/FR reversibility it is evident that, here, coupling of Pfr to signal transduction is possible within minutes. Re-induction of growth possesses the advantage that the effect of actinic irradiations can be studied as an all-or-none response at the level of single gametophytes in future experiments. The present results clearly indicate that the developmental stage of the whole gametophyte, i.e. temporal and spatial patterns undergone during development, affects the regulation of rhizoid growth by the external factor light. Received: 8 June 1998 / Accepted: 22 December 1998     

Protophloem differentiation in early Arabidopsis thaliana development

During Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell divisions, giving rise to vascular precursor cells (protophloem and protoxylem precursors). After germination, these cells terminally differentiate into specialized conducting cells, referred to as protophloem and protoxylem cells. Few readily identifiable markers of the onset of specification and differentiation are available, hampering the molecular genetic analysis of protophloem development. Confocal microscopy was used to investigate the patterning and differentiation of phloem cells during early plant development. Longitudinal divisions of phloem initials allowed the identification of protophloem precursor cells and adjacent metaphloem initials along the length of the plant. During germination, protophloem differentiation was observed at two independent locations, in the cotyledons and the hypocotyl. In both locations, differentiation was concomitant with cell elongation. We identified five gene-trap lines (PD1-PD5) with marker gene expression in immature protophloem elements. The spatio-temporal marker expression pattern of the lines divides them into two groups. The early specification markers PD4 and PD5 were expressed in developing organs before procambium formation and then became restricted to phloem initial cells. The protophloem precursor markers PD1-PD3 were expressed in differentiating protophloem cells at different stages of their development. All markers were expressed transiently and iteratively during the differentiation of protophloem in newly formed organs. Flanking genes were identified for four out of five gene-trap insertion lines. The possible function of these genes with respect to phloem differentiation is discussed.     

Cortical microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not

The plant cytoskeleton has been implicated in a variety of morphogenetic events in higher plants. Most of this work, however, has concentrated on epidermal cells or primary tissues. We have investigated the cortical microtubular (CMT) and microfilament (MF) components of the cytoskeleton in a secondary tissue  –  active vascular cambium of Aesculus hippocastanum L. (horse-chestnut)  –  and followed the changes in these components during the early stages of differentiation of fusiform cambial derivatives to axial elements of the secondary vascular system. A correlative approach was used employing indirect immunofluorescence microscopy of α-tubulin on 6 μm sections, and transmission electron microscopy of 60 nm sections. The study has demonstrated a rearrangement of the CMT cytoskeleton, from random to helical, as fusiform vascular cambial cells begin to differentiate as secondary phloem vascular tissue. A similar CMT rearrangement is seen as fusiform cambial cells begin to differentiate as secondary xylem fibres. This rearrangement is interpreted as evidence of determination of cambial derivatives towards vascular development. Axially-oriented MF bundles are present in fusiform cambial cells and their axial orientation is retained in the vascular derivatives at early stages of their development even though the CMTs have become rearranged. Received: 5 August 1996 /  Accepted: 23 September 1996     

Nuclear DNA contents of all species of Helleborus (Ranunculaceae) discriminate between species and sectional divisions

 Genome size (C-values) and pollen viability staining were applied as new criteria to investigate the species of the genus Helleborus Linnaeus (Ranunculaceae). All species have the same chromosome number (2n=32). However, the nuclear DNA content, as measured by flow cytometry with propidium iodide, could be demonstrated to range between 19 pg to 35.7 pg. The different genome sizes of the species coincided to a large extent with earlier determined section boundaries based on morphology. Flow cytometry can be a convenient method to discriminate between some species. Received April 17, 2001 Accepted May 7, 2001     

The pattern of acropetal and basipetal cytoplasmic streaming velocities in Chara rhizoids and protonemata, and gravity effect on the pattern as measured by laser-Doppler-velocimetry

 The spatial pattern of acropetal and basipetal cytoplasmic streaming velocities has been studied by laser-Doppler-velocimetry (LDV) in the positively gravitropic (downward growing) rhizoids of Chara globularis Thuill. and for the first time in the negatively gravitropic (upward growing) protonemata. The LDV method proved to be precise and yielded reproducible results even when tiny differences in velocities were measured. In the apical parts of the streaming regions of both cell types, acropetal streaming was faster than basipetal streaming. Starting at the apical reversal point of streaming, the velocity increased basipetally with the distance from that point and became fairly constant close to the basal reversal point; subsequently, the velocity decreased slightly acropetally as the apical reversal point was again approached. There was no change in velocity at the basal reversal point. However, at the apical reversal point there was an abrupt decrease in velocity. The pattern of the ratio of acropetal to basipetal streaming velocity (VR) was a function of the relative distance of the site of measurement from the apical reversal point rather than a function of the absolute distance. Upon inversion of the rhizoids, the VR decreased on average by 3.8% (±0.4%), indicating that the effect of gravity on the streaming velocity was merely physical and without a physiological amplification. Rhizoids that had developed on the slowly rotating horizontal axis of a clinostat, and had never experienced a constant gravity vector, were similar to normally grown rhizoids with respect to VR pattern. In protonemata, the VR pattern was not significantly different from that in rhizoids although the direction of growth was inverse. In rhizoids, oryzalin caused the polar organization of the cell to disappear and nullified the differences in streaming velocities, and cytochalasin D decreased the velocity of basipetal streaming slightly more than that of acropetal streaming. Cyclopiazonic acid, known as an inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum, also reduced the streaming velocities in rhizoids, but had slightly more effect on the acropetal stream. It is possible that the endogenous difference in streaming velocities in both rhizoids and protonemata is caused by differences in the cytoskeletal organization of the opposing streams and/or loading of inhibitors (like Ca²⁺) from the apical/subapical zone into the basipetally streaming endoplasm. Received: 4 October 1999 / Accepted: 4 November 1999     

Reversible protein phosphorylation regulates the dynamic organization of the pollen tube cytoskeleton: effects of calyculin A and okadaic acid

We investigated the cytoskeleton of Lilium longiflorum pollen tubes and examined the effects of the type 2A protein phosphatase (PP2A) inhibitors calyculin A and okadaic acid. An improved method for actin visualization, the simultaneous fixation and staining with rhodamine-labelled phalloidin during microscopical observation, revealed abundant actin filaments of no preferential orientation in the apical clear zone. Microtubules, visualized by indirect immunofluorescence, were mostly absent from the apices of straight-growing pollen tubes but present in those with irregular shape. Double labelling showed that both actin bundles and microtubules had a similar longitudinal or slightly helical orientation in the pollen tube shaft. In the presence of 30 nM calyculin A or okadaic acid, pollen tubes grew very slowly, branched frequently, and contained isolated, randomly oriented, curved actin bundles and microtubules. Treating pollen tubes with calyculin A or okadaic acid after germination arrested growth immediately, reversibly altered the alignment of actin bundles from axial to transverse, and disassembled microtubules. The changes in actin organization caused by the PP2A inhibitors were similar to those observed upon overexpression of AtRop1 (Y. Fu, G. Wu, Z. Yang, Journal of Cell Biology 152: 1019-1032, 2001), suggesting that hyperphosphorylation interferes with the signalling pathway of small GTPases. The effects of the PP2A inhibitors could be ameliorated with nanomolar concentrations of latrunculin B.     

The generic placement of a morphologically enigmatic species in Asteraceae: evidence from ITS sequences

 Bidens cordylocarpa is a high polyploid species restricted in distribution to stream sides in the mountains of Jalisco, Mexico. The morphologically enigmatic species was originally described as a member of the genus Coreopsis, but later transferred to Bidens, largely because the involucral bracts appear most similar to Bidens. Characters of the cypselae, often useful in generic placement, are of no value for this species because the fruits have features not detected in either Bidens or Coreopsis. Sequences from the internal transcribed spacer region of nuclear ribosomal DNA (ITS) were used to assess the relationships of Bidens cordylocarpa. The molecular phylogeny places B. cordylocarpa in a strongly supported clade of Mexican and South American Bidens, and provides more definitive evidence of relationships than morphology, chromosome number, or secondary chemistry. Molecular, morphological, and chromosomal data suggest that B. cordylocarpa is an ancient polyploid, perhaps the remnant of a polyploid complex. Received August 28, 2000 Accepted February 11, 2001     

The Involvement of Sphingolipids in Multidrug Resistance

Administration of most chemotherapeutic agents eventually results in the onset of apoptosis, despite the agents' variety in structure and molecular targets. Ceramide, the central molecule in cellular glycosphingolipid metabolism, has recently been identified as an important mediator of this process. Indeed, one of the events elicited by application of many cytotoxic drugs is an accumulation of this lipid. Treatment failure in cancer chemotherapy is largely attributable to multidrug resistance, in which tumor cells are typically cross-resistant to multiple chemotherapeutic agents. Different cellular mechanisms underlying this phenomenon have been described. Of these the drug efflux pump activity of P-glycoprotein and the multidrug resistance-associated proteins are the most extensively studied examples. Recently, an increased cellular capacity for ceramide glycosylation has been recognized as a novel multidrug resistance mechanism. Indeed, virtually all multidrug-resistant cells exhibit a deviating sphingolipid composition, most typically, increased levels of glucosylceramide. On the other hand, several direct molecular interactions between sphingolipids and drug efflux proteins have been described. Therefore, in addition to a role in the multidrug resistance phenotype by which ceramide accumulation and, thus, the onset of apoptosis are prevented, an indirect role for sphingolipids might be envisaged, by which the activity of these efflux proteins is modulated. In this review, we present an overview of the current understanding of the interesting relations that exist between sphingolipid metabolism and multidrug resistance. Received: 16 June 2000/Revised: 16 August 2000     

Approach to the early sporangial development in angiosperms considering meiosis control and cellular differentiation

 Applying current data on cell differentia- tion and meiosis control to the early sporangial development in angiosperms, a strict relationship between cell lineage and its differentiation fate is rejected. An evaluation of cytological features indicative of a meiotic (sporogenous) fate discards the sterilization phenomenon and introduces the premeiotic cellular differentiation (PCD) concept. The early sporangial development comprises 5 basic steps and 4 cellular stages, where PCD and meiosis extension and gradient are related to mechanisms of spore mother cell selection. Concepts here discussed explain the exceptions to the normal early sporangial development and allow a precise definition of archesporium and archespore. PCD and meiotic extension and gradient recover more information of the early sporangial development, distinguishing different developmental patterns leading to the same final result and retaining slight developmental differences. However, there are no early developmental characteristics distinctive of andro- or gynosporangia. Therefore, the heterosporic condition is not related to early developmental changes. Received January 5, 2001 Accepted August 29, 2001     

Excessive expression of the plant kinesin TBK5 converts cortical and perinuclear microtubules into a radial array emanating from a single focus

TBK5 is a plant-specific kinesin constantly expressed in tobacco BY-2 cells. An analysis of the distribution of green fluorescent protein-tagged TBK5 (GFP-TBK5) transiently expressed in BY-2 protoplasts revealed that TBK5 could associate with microtubules in vivo. GFP-TBK5 often assembled to form a single particle when accumulated in cells. The particle was located in close proximity to the nucleus, and its formation was accompanied by the development of a radial array of microtubules emanating from it and the loss of cortical microtubules. Microtubule depolymerization by treatment with propyzamide inhibited particle formation and stimulated the formation of dispersed aggregates of GFP-TBK5. Through expression of different TBK5 mutants as GFP fusions, the motor domain, two separated coiled-coil domains and the C-terminal domain of TBK5 were identified as the domains playing essential roles in particle formation. Mutants with putatively non-motile motor domains or lacking the C-terminal domain were localized to cortical and perinuclear microtubules, whereas those lacking either of the coiled-coil domains were preferentially distributed around the nucleus and along perinuclar microtubules. Further, the deletion of one of the coiled-coil domains or the C-terminal domain was sufficient to inhibit the propyzamide-induced formation of dispersed aggregates, whereas the mutation in the motor domain was not. These results led us to propose a model in which the particle is formed through the microtubule-based movement of GFP-TBK5 toward the nucleus and subsequent microtubule-independent aggregation based on coiled-coil interactions. The dramatic microtubule rearrangement would be explained if GFP-TBK5 relocated and gathered newly formed microtubules and/or microtubule-nucleating units.     

Allozyme variation in Mexican species and classification of Datura (Solanaceae)

 Fifty natural Datura populations, belonging to eleven species (D. ceratocaula, D. discolor, D. inoxia, D. kymatocarpa, D. lanosa, D. metel, D. pruinosa, D. quercifolia, D. reburra, D. stramonium, D. wrightii) from Mexico and adjacent USA, were investigated using starch gel electrophoresis. A total of 64 alleles were scored at 17 loci (DIA1, DIA2, GOT1, GOT2, G6PDH, IDH, MDH1, MDH2, MDH3, ME, PGD1, PGD2, PGM1, PGM2, PHI, SAD, SOD). The heterozygosity among the species ranged from 0.166 (D. ceratocaula) to 0.276 (D. wrightii). Most genetic diversity was found within populations (average H_s=0.242), while values between populations are relatively low (average Dst=0.066, Gst=0.171). The analysis of the genetic distance suggested new taxonomic relationships among the species. Rather than supporting the conventional infrageneric classification with three sections, the results revealed that the herbaceous members of the genus Datura form four groups. One group included four of the eight species of the section Dutra and was more similar to the section Ceratocaulis than it was to the other group that contained the remaining taxa of Dutra. Received February 13, 2001 Accepted December 25, 2001     

Positional differentiation for development along the sporangiophore in <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis>

 Small segments of Phycomyces sporangiophores regenerate various structures on incubation in a moist chamber. We tested the regeneration capacities of middle sporangiophore segments whose protoplasm had been totally or partially removed and replaced with protoplasm from various segments of a genetically different strain. The structures that were regenerated depended on the source of the injected protoplasm (sporangia of various sizes and segments from middle and basal parts of the sporangiophores), implying a positional differentiation of protoplasm along the sporangiophore axis. Protoplasm from various sources showed a high affinity; that is, they mixed successfully and led, in most cases, to the formation of heterokaryotic regenerating structures. The highest affinity was seen when mixing protoplasm from the middle segments of two different strains. Received: November 29, 2000 / Accepted: March 19, 2002